COMMITTEE ON TOXICITY OF CHEMICALS IN FOOD, CONSUMER

THIS IS A DRAFT PAPER FOR DISCUSSION. IT SHOULD NOT BE QUOTED, CITED OR REPRODUCED

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TOX/2012/26 Annex 5 COMMITTEE ON TOXICITY OF CHEMICALS IN FOOD, CONSUMER PRODUCTS AND THE ENVIRONMENT EVALUATION OF NEUROPHYSIOLOGICAL EFFECTS OVERVIEW OF COT 1999 REPORT 1. The Committee noted that one of the limitations of neuropsychology testing is the possibility that performance of test subjects is influenced by their knowledge that they have been exposed or poisoned to OPs. Neurophysiological testing can provide a more objective index of central nervous system function. There were two studies cited in the COT 1999 report which provided information on EEG abnormalities following acute poisoning by OPs. One study of workers acutely exposed to sarin at an industrial plant reported significant abnormalities in waking and sleeping EEG, whilst the other study of patients with acute OP poisoning due to agricultural pesticides reported no significant effects on EEG. No clear conclusions could be reached with regard to these data. No studies of EEG in subjects with chronic low-level exposure to OPs were retrieved for the COT 1999 report. OVERVIEW OF LITERATURE PUBLISHED AFTER COT 1999 REPORT Acute Exposure with Chronic Neurophysiological Changes 2. One case-control study of auditory evoked potentials (auditory EPs) in subjects with acute OP poisoning was identified along with a further case-control study which pre-dated 1999 (investigating Tokyo subway victims six-eight months after the incident) identified by checking the reference lists of published papers. A separate cross-sectional study of auditory EPs in Tokyo subway incident victims (5-6 years post incident) was retrieved. A cross-sectional study of EEG was identified for Gulf War Khamisiyah incident veterans 8-9 years post incident. Chronic Exposure with Chronic Neurophysiological Changes 3. A cross-sectional study of EEG in an agricultural community In Israel where OPs were used regularly was retrieved. Cross-sectional studies of auditory ERPs in vegetable farmers in Sri Lanka who had applied OPs for at least 5 years and in tobacco workers (from Malaysia, 1->20 years use) were identified. A further cross-sectional study of auditory EPs of pesticide workers engaged in spraying fenthion, which predated 1999 was identified by checking the reference lists of published papers.


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STUDIES OF EEG Acute Exposure with Chronic Neurophysiological Data Study of Gulf War veterans 4. In this cross-sectional study, Spencer recruited 3219 Gulf war veterans (923 in Khamisiyah group (present within a 50 Km radius of Khamisiyah Coalition Occupied Iraq during the first two weeks of March 1991)) (Spencer et al 2001) (Annex 5, Table 6.A.3). There were 927 non-Khamisiyah veterans and 1369 non-deployed. Of these, 98 underwent neurophysiological testing (from 211 attempts to recruit for testing). Forty-three were classified as exposed to sarin at Khamisiyah, 26 non-Khamisiyah, 29 non-deployed. Neurophysiological testing was undertaken between Jan 26, 1999 to June 14, 2000 (approximately 8-9 years after the Khamisiyah incident). No EEG abnormalities were reported for any of the groups of veterans examined. There were uncertainties regarding the extent of exposure of Khamisiyah veterans and the neurophysiological testing was undertaken on a small selected sample of veterans (Annex 5, Table 7.A.3). Chronic Low- Level Exposure with Chronic Neurophysiological Data Study of agricultural community (Israel) 5. In this cross-sectional study Browne et al 2006, identified 291 subjects (149 males, 142 females) who lived 25-150 m from fields where spraying with OPs was reported (Browne et al 2006). Of these subjects, only 60 gave consent to participation. EEG data were available for 19 exposed subjects. EEG data were obtained from 9 control (urban) subjects of similar age and gender distribution. Details of how control subjects were selected were not reported. Low Resolution brain Electromagnetic Tomography (LORETA) was used for localisation of presumptive cortical sources of EEG (Annex 5, Table 6.B.3). Significantly reduced serum cholinesterase (41% of control P<0.001) was reported for exposed subjects (30 subjects, including 10 agricultural workers) compared to controls (91 urban subjects). EEG abnormalities were reported in exposed subjects comprising significantly lower power was found in the theta band and increased power in the beta 3 band (P=0.04 and P=0.03 respectively). LORETA showed sources of changed brain activity mainly in the limbic structures and frontal cortices (Annex 5, Table 7.B.3). In this study, a small selected group of exposed and controls underwent neurophysiological testing. Reduced serum cholinesterase was reported in exposed subjects but it is unclear whether this related to subjects who underwent neurophysiological testing. The role of potential confounding factors was not considered. Conclusion Studies of EEG 6. The available data do not permit any definitive conclusions to drawn regarding either acute or chronic exposure to OPs and EEG changes.


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STUDIES OF EVOKED RELATED POTENTIALS 7. Auditory evoked potentials (auditory EPs) have been measured in several studies as an objective measure of CNS neurophysiology. Visual evoked potentials (visual EPs) have been recorded in one study. The extent of control for potential confounding factors for EPs varies between the studies considered. Acute Exposure with Chronic Neurophysiological Data OP-poisoned subjects 8. In this case-control study, Dassanayake et al 2008, published the results of auditory evoked potentials (auditory EPs) for nine OP-poisoned subjects compared to same sized matched healthy and paracetamol over-dose control groups (Annex 5, Table 6.A.3) (Dassanayake et al 2008). A significant prolongation of P300 latency was reported in OP poisoned subjects at the first test (from 1-45 days post poisoning at day of discharge) (P=0.004). A comparison of the initial and six-month follow-up data for 21 poisoned subjects (testing at 124-402 days, median 182 days after poisoning) for auditory EP tests showed P300 latency was still prolonged at the follow-up test (Annex 5, Table 7.A.3). This study was limited by its small size and lack of control data for the six-month follow-up. Tokyo subway sarin incident 9. Auditory EPs were recorded in 18 subjects exposed to sarin in the Tokyo subway incident compared to 18 age/sex matched controls (5-6 years after the incident) in a case-control study (Murata et al 1997). Tests were undertaken in September to November 1995 (6-8 months post incident). No direct measure of exposure was reported but subjects had signs and symptoms of cholinergic poisoning and reduced serum cholinesterase at admission (Annex 5, Table 6.A.3). The P300 and P100 latencies in cases were significantly prolonged. (P<0.001 and P<0.05 respectively. Data presented in graphical form only). This study presented information on auditory EPs in acutely poisoned subjects who had recovered from the acute cholinergic phase of toxicity (Annex 5, Table 7.A.3). The study was limited by the small number of subjects investigated which were selected from a potential 150 victims at a time period approximately 6-8 months after exposure. 10. In this cross-sectional study, Araki et al (2005), found a significant increase in the amplitude of P300 auditory EP in a small group of Tokyo subway victims who had Post Traumatic Stress Disorder (PTSD) compared to victims who did not exhibit PTSD symptoms (Araki et al (2005)). There was no difference between the two groups for the P300 latency (Annex 5, Table 7.A.3). This study did not specifically investigate the effect of OP exposure.


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Chronic Low-Level Exposure with Chronic Neurophysiological Data Study of Sri-Lankan vegetable farmers 11. Auditory EPs were recorded for 35 vegetable farmers (mean age 50.1±7.6 years) who had applied OP pesticides for more than 5 years compared to 38 controls (hospital labourers mean age 49.0±7.2 years) in a cross-sectional study (Dassanayake et al 2009). No direct measure of exposure was provided, although vegetable farmers had applied OP pesticides for at least five years (mean duration 26.0±10.4y) and had applied pesticides at least 3 times in the 6 months prior to testing (Annex 5, Table 6.B.3). A significant increase in P300 latency was reported (P=0.0006) (Annex 5, Table 7.B.3). Study of Malaysian tobacco farmers 12. Auditory EPs (65 subjects) and Visual EPs (64 subjects) were recorded in Malaysian tobacco farmers who applied a number of different pesticides (including α-cypermethrin, methamidophos, butralin, chlorothalonil, mancozeb) in a cross-sectional study (Kimura et al 2005). Thirty-two male (age-matched) officers from the National Tobacco Board who had no exposure to pesticides were used as controls. Twenty-six of the initial 76 tobacco workers recruited used an OP pesticide (methamidophos) (Annex 5, Table 6.B.3). There was no decrease in serum cholinesterase in tobacco farmers who applied methamidophos. No significant differences in auditory P300 latency and visual evoked P100 latency were reported for the group of 65 tobacco workers compared to controls (Annex 5, Table 7.B.3). Auditory EPs and visual EPs were not reported specifically for tobacco farmers who used methamidophos. Study in pesticide applicators spraying fenthion (India) 13. Auditory EPs were recorded in 32 pesticide applicators spraying fenthion in a cross-sectional study (Misra et al 1994). A group of 25 hospital employees matched for age, sex, educational status, socio-economic status who were not directly exposed to pesticides served as a control group (Annex 5, Table 6.B.3). The mean duration of exposure 10.5 y (range 1-14 y). Applicators sprayed aqueous fenthion (0.1%) by hand operated sprayer for 5-6 h/day, six days per week without using any protective clothing. It is not clear from the paper whether this was daily exposure over a season or throughout the whole year. All workers had worked till previous day before testing. Serum AChE was significantly reduced in pesticide applicators. A significant prolongation of P300 latency, but not amplitude was reported. The effect on P300 latency was only clinically significant in one exposed worker. There was no correlation between serum AChE and P300 latency (Annex 5, Table 7.B.3). Conclusion Evoked Potentials 14. There was evidence for an increase in auditory P300 latency approximately six months following acute OP poisoning and in Tokyo subway incident victims. There was also some evidence for an increase in auditory


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P300 latency in Sri Lankan vegetable farmers and pesticide applicators from India. Overall, there is some consistency between the studies of acute exposure and occupational exposure to OPs. 15. It is difficult to draw definite conclusions regarding these data. The generation of auditory P300 wave is considered to reflect attention and memory processes (Kenemans and Kähkönen, 2011; Polich and Kok, 1995). The P300 latency is affected by many factors including circadian-related factors (such as body temperature and heart rate), exercise, fatigue, alcohol and caffeine intake and smoking (Polich and Kok, 1995). The P300 wave is also altered in disease conditions such as schizophrenia, autism, depression, dementia and multiple sclerosis (Picton, 1992). 16. Given the complexity of factors which may affect the P300 response in auditory EPs, the studies conducted by Dassanayake and colleagues (2008 and 2009) included controlling for some factors which affect cognitive processing speed (age, and depression). These two studies provided evidence for increased P300 latency in patients with acute OP poisoning and in vegetable farmers applying OP pesticides. Prolonged P300 latency reflects altered attentional processing and updating of working memory. It reflects the speed of allocation of neural resources. P300 latency reflects the time required to detect and evaluate target stimuli. These data support the findings of impaired attention in neuropsychology testing.


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Table 6 NEUROPHYSIOLOGY STUDY DESIGN DETAILS First Author

Date Country Study population

Reference population

Health assessment method

Health outcome variables

Measure of Exposure

Risk factors/ bias/ confounders

Statistical analysis

A. Acute Exposure with Chronic Effects

6.A.1. Cohort studies - none

6.A.2. Case-control studies Dassanayake 2008 Sri Lanka 32 patients with

acute OP poisoning underwent examination (aged 14-56 y). 21 of these subjects underwent the initial and six month follow-up investigations.

32 age (5y) sex matched healthy volunteers from the community (age 15-52 years) . A hospitalised control group of paracetamol overdose patients (n=9, age 14-27 years) were also studied 21 healthy controls underwent a six month follow-up

Auditory P300 ERPs were recorded after clinical recovery from the acute cholinergic phase. usually on day of discharge from hospital. Healthy controls were tested on an appointment basis. Paracetamol controls were tested on day of discharge from ward. Subjects abstained from alcohol, tobacco and coffee in 24 h prior to test and were advised to have at least 6 h sleep the night before testing. ERPS were recorded over the Cz position.

N100, P200, N200 and P300 components quantified

OP formulations taken by the 32 patients included chlorpyrifos (14), dimethoate (4), phenthoate (4), fenthion (2), malathion (2), coumaphos (1), oxydemeton methyl (1) and profenfos (1). The type of OP was not known in 3 instances. 31 had ingested OPs intentionally.

Patients and controls who had chronic exposure to OPs, hearing impairment, pre-existing illnesses or medication that may predispose to cognitive dysfunction or who consumed alcohol (>21 units/week for men, >14 units/week for women) were excluded. Acute OP poisoning in the past was an exclusion criterion for controls.

Data assessed using Student’s non-paired t-test. The ERP data were initially compared suing ANOVA. The outcome measure of OP poisoned group where ERP data were available for two occasions were assessed using paired t-test

Murata (identified from references cited by Dassanayake 2008)

Japan 1997 18 subjects exposed to sarin in the Tokyo subway attack.* (16 clerks, plasterer and maid). At time of testing (September-November 1995**)

18 sex and age (within 2years) matched controls. (19-59 y) (students, medical assistants, clerks)

Auditory event related potentials (ERPs) Subjects were told to count the target tone mentally. ERPs measured using disk electrodes placed at the vertex, mastoids and forehead. The P300 component was first

P300, P100 amplitude/latency, N100, BAEP I, III and V peaks) latencies CVRR parameters CVRR ratio of standard deviation

No direct measure of exposure in this publication. When sarin cases first arrived at hospital they exhibited signs and symptoms of cholinergic poisoning. 15/18

No confounding disorders in controls. No significant differences in age, alcohol or tobacco use between sarin cases and

Analysis using paired-sample t-test and Mann-Whitney test. The relation of neurophysiological data to ChE, PTSD score, age and gender


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First Author





Measure of Exposure



none had obvious opthalmological, cardiovascular, neurological or other confounding disorders. Serum ChE levels were in normal range for 13/18 cases. *Approximately 150 viictims **Attack took place in March 1995.

maximal positive wave between 250-500 ms, the N100 component by non-target tone was recorded to assess whether the change in P300 is independent of early auditory responses. Pattern reversal Visual EP, P100 measured. Brainstem Auditory Evoke Potential (BAEP) was measured with subject lying using click signals presented to the right ear. The responses were averaged 1,024 times after amplification and filtration. Three hundred R-R intervals on ECG were measured. Cardiovascular parameters (CVRR) reported. Self rated questionnaire used to assess PTSD.

of R-R interval to the mean (CVRR %), Low Frequency component (LF) (0.04-0.15Hz) and High Frequency component (HF) (0.15-0.4Hz). Coefficient of variation for each component (C-CVLF and C-CVHF % (ratio of square root of each component power spectral density (PSDLF and PSDHF) The LF/HF ratio

had serum ChE below the normal range

controls. tested by multiple regression analysis.

6.A.3. Cross-sectional studies Araki 2005 Japan 47 subjects (24

male, 23 female mean age 42.1 (SD13.2) recruited from victims of Tokyo sarin attack. 10 were diagnosed with unequivocal PTSD related to attack. 2 of these subjects with psychiatric co-morbidity were excluded. Final group of 8 (2 with current PTSD and 6 with history of PTSD)

18 victims from the group of 47 who had never had unequivocal or partial PTSD. 5 excluded on basis of having received hypnotics (for insomnia), and unsuccessful ERP measurements due to artifacts or technical problems.

Subjects performed an oddball (auditory) task to elicit P300 event related potentials (ERPs) which were measured using scalp EEG. 5-6 years after the incident.

Subject characteristics included Impact of Event-Scale (revised) (IES) , symptom clusters of PTSD in CAPS (reexperiencing of the event, avoidance/numbing and hypervigilance, and STAI trait and state anxiety scores. Concentrations of serum cholinesterase were measured.

No measures of exposure reported. ERP measurements undertaken 5-6 years after the incident.

Confounding effects of IES-R scores STAI (anxiety) scores and serum cholinesterase which differed between the groups was evaluated

Group differences in P300 amplitude and latency evaluated uisnf ANOVA. Speraman’s rho calculated for correlations between P300 amplitudes (Cz,Pz) and clinical measures present and lifetime scores in CAPS. A Bonferroni correction was used (P value


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First Author





Measure of Exposure



The Mini-International neuropsychiatric Interview was administered on day of ERP recordings

set at 0.008) Spearman’s correlations calculated between P300 amplitudes (Cz/Pz) and IES-R scores, STAI scores and concentrations of cholinesterase.

Spencer 2001 USA Subjects recruited by telephone interview. Initially 3219 veterans contacted (923 in Khamisiyah, 927 non-Khamisiyah, 1369 non-deployed. Of 211 attempts to recruit for neurophysiological testing, 176 contacts were made. 23 were ineligible, 38 refused, 115 agreed to attend. Of these subjects 98 underwent neurophysiological testing. 43 subjects were classified as exposed to sarin at Khamisiyah (data from 42 subjects available)

26/98 subjects non-Khamisiyah, 29 non-deployed

Testing was undertaken between Jan 26, 1999 to June 14, 2000 (approximately 8-9 years after the Khamisiyah incident).

Participants had an EEG recorded while awake with eyes closed at rest or while performing a simple auditory perception task. Trans cranial evoked potential with muscle recordings from abductor pollicis brevis, adductor digiti minimi, tibalis anterior and soleus. Onset latency and average peak amplitudes recorded. (Data from this investigation not reported)

Computer-assisted telephone interview used to assess exposure.

Deployed subjects were more likely to have a high school education or less, were slightly younger or more likely to be of a minority race. These factors were considered when comparing health status. However it is not clear whether such factors were specifically considered for the neurophysiological testing.

Multivariate analysis of variance was used to assess differences among deployment groups with respect to. relative activity of EEG data; p-values are reported

6.A.4. Case series studies - none


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First Author





Measure of Exposure



6.A.5. Case reports - none

B. Chronic Low-Level Exposure

6.B.1. Cohort studies - none

6.B.2. Case-control studies - none

6.B.3. Cross-sectional studies Browne 2006 Israel Volunteers were

taken from a rural agricultural community were OPs were regularly used. Living in community for an average of 26y, resident 25-150m from fields. 291 subjects (149 males, 142 females aged 4-90y (36.5y ±21.7y) 65 were agricultural workers. 28 were excluded (history of CNS-related diseases) 60 gave written consent. EEG data were available for 19 exposed subjects. Biochemical assessment (AChE, BChE and PON) for 30 exposed subjects (10 of whom were agricultural workers)

Controls taken from urban areas with no history to anti-cholinergic agents, and with no known neurological or psychiatric diagnoses. There were 91 controls for biochemical analyses (or each biochemical analysis Ache, BChE and PON) and 9 for EEG analyses (selection of control subjects not reported)

EEG was recorded from 25 surface electrodes placed according to 10-20 system with additional electrodes to detect eye movements and electrocardiaram activity. Discrete frequency bands were measured (spectral power) (delta 1.5-6HZ, theta 6.5-8 Hz, alpha 1 8.5-10 Hz, alpha 2 105-12 Hz, beta 1 12.5-18Hz, beta 2 18.5-21 Hz, beta 3 21.5-30 Hz. Population averaged power of each electrode was calculated. Low resolution brain electromagnetic tomography (LORETA) was used for localisation of presumptive cortical sources of EEG. LORETA estimates the distribution of absolute current density for brain electrical activity and displays it on a dense grid of 2,394 voxels. . Calculation of LORETA is limited to cortical gray matter and hippocampi and has a spatial resolution of 1-2 cm. Average current

EEG and LORETA visualisation of activated regions. Neuropsychology testing (see separate summary).

No specific details provided. Authors indicate that OPs used included fenitrothion, chlorpyrifos, monocrotophos, ethion and azinphos-methyl. Qualitative exposure to OPs (AChE impregnated discs located at distances 5 x 20 m from field) Reduced serum cholinesterase (paper unclear whether plasma or serum cholinesterase measured) reported in exposed (41% of control P<0.001, Student’s t-test)

[Authors report control subjects had age and gender distribution similar to exposed (exposed mean age 46y, control 42y; exposed 58% male, control 56% male). It is note stated but this information may relate to the 19 exposed and 9 un-exposed with EEG data]

Student’s t-test used to analyse differences in the area under the curve for each discrete band of the EEG. Statistically significant differences between LORETA values were tested by using a non-parametric t test on a voxel-by-voxel basis, with correction for repeated measure. (Thresholds of P<0.01, P<0.05 and P<0.1 were used)


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First Author





Measure of Exposure



source densities for 23 distinct brain regions were calculated. Plasma paraoxonase activity determined by microtitre plate adaption of assay described by Furlong (Anal Biochem, 180, 242-247, 1989 at pH 8.5) Plasma arylesterase and plasma cholinesterase were measured

Dassanayake 2009 Sri Lanka Vegetable farmers (males) applying OPs for more than 5 years. 52 interviewed of which 14 excluded (9 with alcohol consumption >21 units (1 unit =10ml alcohol), 4 with diabetes/hyperglycaemia (blood glucose >140 mg/dl), and 1 with psychiatric illness). ERP data for 35 used ( mean age 50.1±7.6 y, age 35-64 y, duration of pesticide use 26.0±10.4 y) (Exclusion criteria also included history of acute OP poisoning, hearing impairment)

Male hospital labourers identified by open recruitment. 42 contacted the researchers, 40 attended interview. One excluded (alcohol consumption .21 units/week) and a further subject excluded (blood glucose >140 mg/dl. Data for 38 controls obtained. (mean age 49.0±7.2 y, range 25-58 y).

A complete clinical evaluation was undertaken to exclude clinically evident but previously undiagnosed neurological conditions. Subjects abstained from alcohol, tobacco and coffee in 24 h prior to test and had at least 6h sleep the night before testing. All subjects abstained from applying/handling agrochemicals for 4 days prior to test. An auditory oddball paradigm was applied to elicit ERPs (conforming to International Federation of Clinical Neurophysiologists) guidelines, counting target frequencies. ERPs recorded at Cz and Pz positions (reference over mastoids, and ground at Fp). ERPs recorded (eyes closed) resulting from 25o stimuli (200 standard tone (75 dbb, 1000 Hz) and 50 target tone (75 db, 2000 Hz)

ERPs recorded and averaged. N1 (60-160 ms), P2 (120-250 ms), N2 (170-340 ms) P300 (250-600 ms) and P300 amplitude.

No specific exposure data obtained. Farmers had sprayed at least 3 times in previous 6 months. OP formulations used contained dimethoate (30 farmers), chlorpyrifos (13), quinalphos (6), profenfos (2\), phenoate (2), fenitrothion (1), fenthion (1) Details of PPE worn not provided.

Authors accounted for effect of age on ERP measurements. .

All components were readily distinguished at the Cz site. ERP component latencies were initially compared using a mixed model ANOVA (Grenhouse-Geisser corrected degrees of freedom) Subsequent differences compared using Student’s t-test with Bonferroni correction. Analysis of covariance performed on P2, N2, P300 components separately taking into account the preceding component as a covariate. Amplitude data


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First Author





Measure of Exposure



for P300 at the Cz and Pz was analysed using mixed modle ANOVA (within subjects Cz and Pz and between subject groups)

Kimura 2005 Malaysia 76 male tobacco farmers (average age 45.1 years, SD 7.2) randomly selected from National Tobacco Board (NTB) register. Main pesticides used in the farms were either pyrethroids, dinitroaniline, organochlorines, carbamates, or the organophosphate methamidophos (trade name Tamaron). The authors note that 26 farmers used Tamaron (although most farmers used two or more pesticides in combination) (methamidophos) years working on tobacco farms 1-5 (18 subjects), 6-10 (23), 11-15 (12), 16-20 (17), >20 (14)

38 male officers of the NTB (average age 45.1 years, SD 7.1) who did not handle pesticides or wet tobacco leaves Auditory ERPS for 32 controls

Auditory evoked potential was measured (using150 tones of 1000 Hz and 20% had a frequency of 2000 Hz) P300 component was first maximal positive wave between 250-500ms latency. N100 component by non target tone was recorded. Auditory ERPs for 65 exposed subjects. Visual evoked potential (VEP) was measured in a darkened room. The pattern on the screen consisted of white black squares, reversing at a rate of 2 Hz. One positive peak (P100) was recorded using standard EEG electrodes (occipital cortex, forehead and left mastoid. Responses were averaged for128 times

Auditory and Visual EPs

Information on pesticide use on tobacco farms in Kelantan reported. No specific exposure estimate for OP use provided Information on serum cholinesterase obtained. Most of 76 farmers applied pesticides using knapsack sprayers. Practice of using PPE varied (mask 68%, rubber gloves 47%, long boots 59%. All washed hands and face after spraying and changed clothes (75/76).

Not reported Student’s t-test was used.

Misra (identified from references cited in

1994 India 32 pesticide workers engaged in spraying fenthion.

25 hospital employees matched for age, sex, educational

Auditory ERPs measured in subjects resting in a couch, avoiding blinking. Platinum needles fixed at CZ and FZ

N1 max negativity 80-120 msec. P2 max positivity 100-250 msec.

Exposure duration and type of exposure recorded. Reported subjects

53% exposed subjects took alcohol (mean 550.5 ml/week

Student’s t test, cut off for normalcy + 2SD. Linear


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First Author





Measure of Exposure



Dassanayake 2008)

status, SES, not exposed directly to pesticides.

referred to mastoids, ground at FpZ. Additional surface electrodes measured electrooculogram.

N2 most prominent negative after 200 msec following P2, preceding P3. P3 maxi positivity 270-600 msec. ERP data concentrated on CZ. Serum AChE measured.

had mild symptoms post spraying but not clinically overt cholinergic toxicity.

(range 50-2100), 53 % smoked (average 23 cigarettes/day (range 1-40), caloric intake 2432 Kcals/day (range 1900-4200). In controls 20% took alcohol (660 ml/wk (range 50-1500), caloric intake 2635 Kcals/day (range 1600-4000) smoking not reported.

correlation between AChE and electrophysiology .

6.B.4. Case series studies - none

6.B.5. Case reports - none


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Table 7. NEUROPHYSIOLOGY RESULTS AND CONCLUSIONS

First Author

Date Published Major Findings Strengths /

Limitations Conclusions


7.A.2. Case-control studies Dassanayake 2008 SIX MONTH FOLLOW UP AUDITORY ERP TEST OF ACUTELY POSIONED SUBJECTS

EXPOSURE. Dose levels not reported. HEALTH OUTCOMES 25 poisonings were impulsive acts of self-harm. Six patients were treated for depression (fluoxetine) at the time of neurophysiology testing. Patients were subject to neurophysiological testing at two time points a) at 5-45 days (median 12 days) (1-42 days after last dose of atropine (median 6 days) and 1-42 days after last dose of pralidoxime (median 7 days) b) from 124-402 days after poisoning (median 182 days) 8/9 paracetamol poisonings were categorised as impulsive acts of self-harm. None developed liver failure or hepatic encephalopathy. Neurophysiological tests 2-6 days (median 3 days) after poisoning. A significant prolongation of P300 latency (ms) was reported at the first test ; OP poisoned (n=9) 385.3 (34.7) Matched controls with paracetamol poisoning (n=9) 341.0 (22.4) (P=0.016) Matched healthy controls (n=9) 344.9 (25.4) (P=0.008) (post hoc Bonferroni test) No effect on N100 latency, P200 latency, N200 latency or P300 amplitude were reported at the firs test. Comparison of initial and follow-up P300 data

Measure First assessment (n=21)

Follow-up (n=21)

Significance

Strengths Subjects with documented acute poisoning with OPs (None developed clinically detectable seizures). Age/sex matched controls (including paracetamol overdose controls) Limitations Small study. No control data for follow-up testing. Significance values for post-hoc Bonferroni test was different in text (P=0.003)and tables (P=0.008) Authors suggest longer term follow-up with repeated ERPs should be undertaken.

Authors considered that neurophysiological tests were not affected by medications used or by the occurrence of depression in some patients. The finding of prolonged P300 latency in this study replicated finding published for Tokyo subway victims (Murata K et al J Neurol, 244, 601-6, 1997) Prolonged P300 latency reflects altered attentional processing and updating of working memory . It reflects the speed of allocation of neural resources. P300 latency reflects the time


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First Author



N100 latency (ms)

108.6 (12.2) 106.2 (13.3) 0.29

P200 latency (ms)

176.2 (27.5) 174.3 (21.6) 0.70

N200 latency (ms)

230.0 (36.3) 225.5 (28.6) 0.60

P300 latency (ms)

366.1 (37.1) 364 (34.9) 0.79

P300 amplitude (uV)

12.60 (7.48) 13.23 (7.47) 0.73

The increased P300 latency was preserved at the follow-up test.

required to detect and evaluate target stimuli. Noted that study of Misra UK et al,Nurophysiol, 34, 197-203, 1994 in fenthion sprayers not identified in 1999 COT review. Noted that study of Murata K, J Neurol, 244, 601-6, 1997 of Tokyo subway victims not previously reviewed.

Murata K 1997 SIX TO EIGHT MONTH FOLLOW UP OF TOKYO SARIN ATTACK VICTIMS (AUDITORY ERPs) EXPOSURE Not reported. But subjects had signs/symptoms of cholinergic poisoning and reduced serum cholinesterase on admission. HEALTH OUTCOMES The (auditory) P300 and (visual) P100 latencies in cases were significantly prolonged. (P<0.001 and P<0.05 respectively. Data presented in graphical form only. PTSD score was significantly higher in sarin cases compared to unexposed controls (analysis of covariance P<0.05). All the N100, BAEP and CVRR parameters were comparable between groups In sarin cases ChE was significantly related to CVRR, C-CVLF and C-CVHF

Strengths Neurophysiological testing of subjects with acute exposure to sarin who had recovered from acute cholinergic phase of toxicity. Limitations Sample/selection bias, only 18 out of potential 150 sarin cases investigated.

No definite conclusions can be reached from this study. The P300 data were considered consistent with abnormal performance of attention and/or memory function

7.A.3. Cross-sectional studies Araki 2005 STUDY OF AUDITORY ERP MEASUREMENTS IN TOKYO SUBWAY VICTIMS WITH PTSD

EXPOSURE No details provided

Strengths Subjects with well documented exposure to sarin.

No conclusions regarding OP exposure and effects on event –


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First Author



HEALTH OUTCOMES Statistically significant group differences in characteristics (No significant differences in age, gender, education (years) SES and Parental SES)

Variable PTSD victims (8)

Victims without PTSD (13)

Group comparison P value (student’s t-test)

IES-R 27.2(15.0) 10.9 (9.8) 0.017 CAPS total * lifetime

64.1 (18.6) 7.7 (6.8) <0.001

CAPS total* present

27.5 (13.1) 4.9 (5.5) <0.001

STAI trait anxiety

48.0 (10.3) 40.2 (7.0) 0.057

STAI state anxiety

44.0 (7.0) 36.7 (7.3) 0.036

Serum cholinesterase

110.8 (29.2) 150.3 (20.5) 0.020

Neither Reaction Time or % response accuracy differed between the two groups. * Californian assessment of PTSD. Groups differed significantly in P300 amplitude (main effect of group F1,19=4.80 P=0.041 while group-by-electrode interaction was not significant. For P300 latency there was no significant main effect of group.

Variable Victims with PTSD (8)

Victims without PTSD (13)

Reaction time (ms)

391 (87) 367 (64)

Response accuracy %

99.4 (1.6) 100.0

P3 amplitude (Pz) (uV)

9.2 (5.1) 13.6 (5.8)

P3 latency at PZ (ms)

319 (41) 332 (41)

Limitations Study doesn’t specifically investigate effects of OP exposure on event related potentials. No unexposed control group studied

related potentials can be derived from this study.


16

First Author



In the PTSD group there was a significantly negative correlation between present score of the cluster C of the CAPS and P300 amplitude (uncorrected P=0.007, corrected P=0.042 two-tailed) There were no significant correlations between P300 amplitudes and IES-R scores or between STAI scores and concentrations of cholinesterase for either group.

Spencer 2001 KHAYMISIYAH INCIDENT VETERANS (8-9 YEARS POST INCIDENT) EEG DATA

EXPSOURE Subjects position in relation to demolition of Bunker known. Modelled exposure reported in other publications

HEALTH OUTCOMES

Analysis of the EEG data (alpha= 8-13 Hz, beta= 14-30 Hz and theta= 4-9Hz was reported for occipital-central, mid-temporal, parietal-occipital under the different conditions tested.

No significant differences among deployment groups were found in any of the brain regions under any experimental condition (p=0.08-0.73)

Comparisons were also made between non-deployed sub group and a deployed subgroup formed from pooling Khamisiyah and non-Khamisiyah. No significant differences were found (p= 0.1-0.90)

A final comparison was made between Khamisiyah veterans who took part in, or observed, the demolition of Bunker 73 (n=7) and those who did not (n=25) showed no difference (p=0.21-0.93)

Results of trans cranial evoked potentials not reported.

Strengths Objective assessment of CNS function (EEG). Limitations; knowledge of positioning of subjects during the Khamisiyah incident used as proxy for sarin exposure. Only small number of subjects identified underwent neurophysiological testing.

No evidence for effects on EEG

B. Chronic Low Level Exposure

7.B.3. Cross-sectional studies Browne 2006 AGRICULTURAL COMMUNITY EEG DATA

EXPOSURE. Qualitative assessment of OPs (AChE impregnated discs responded to 6 ppm

Strengths Detailed EEG analysis supported by

The authors conclude exposure leads to


17

First Author



OP) reported positive exposures at distances up to 100 m from a sprayed field (discs placed prior to spraying field with tractor drawn sprayers. Assessment carried out 12 h after spraying) . HEALTH OUTCOMES Averaged spectral power revealed generally lower power in the exposed group which reached statistical significance only in the theta band (ANOVA P=0.03). When individual power was normalised, significantly lower power was found in the theta band and increased power in the beta 3 band (P=0.04 and P=0.03 respectively. Topographically, the decrease of theta power was most prominent over the central region whereas the increase in beta 3 power showed a more frontal distribution. Statistical nonparametric mapping of LORETA revealed significant differences. Decreased theta intensity bilaterally in the amygdala, the hippocampus, the subcallosal area, the parahippocampal gyrus and the anterior and posterior cingulate gyrus in exposed subjects. Areas with decreased intensity in the beta 3 band included the hippocampus and parahippocamal gyrus bilaterally. Increased sources (beta3) were found in the inferior, middle and superior frontal gyri. (all values P<0.05) Increased delta in exposed was found in the bilateral prefrontal areas and alpha1 and alpha2 bands localised to the occipital region. The authors investigated whether changes in current sources in mesial temporal lobes structures is associated with poor memory performance, Mean current density in both right and left temporal lobe (median part) for exposed individuals with low and high performance in memory tests. Exposed individuals who performed poorly on memory tests (13.3% and 16% for immediate and delayed recall) also showed 10 times lower beta activity and 50% increase in theta compared to exposed individuals who showed better performance (32% and 38% for immediate and delayed recall). However due to small sample size (n=6) these changes did not reach statistical significance. The authors also reported that there was significantly increased beta 3 activity in the frontal cortical regions of exposed individuals with PON1 Q192R allele. LORETA values revealed that exposed individuals with PON1 Q192R had increased frontal and decreased temporal activity (P<0.03)

LORETA to visualise regions with changed electrical activity. Limitations Exposure qualitatively measured for one field. Small selected group of exposed and controls underwent neurophysiological testing. Reduced serum cholinesterase but unclear whether this related to subjects who underwent neurophysiological testing. The role of potential confounding factors not considered.

changes in the distribution of sources of brain activity in specific regions (mainly limbic structures and frontal cortices. These changes are associated with deficits in visual memory It is not possible to associate the changes reported with OP exposure. The neurophysiology and neuropsychology tests appear to have been undertaken in different subjects taken from the exposed and control groups.

Dassanayake 2009 VEGETABLE FARMERS AUDITORY ERP DATA EXPOSURE No specific exposure data reported. Information on OP formulations used

Strengths ERP measurements in chronic exposed

Authors concluded there was a progressive


18

First Author



available. HEALTH OUTCOMES Age distributions of two groups were similar (farmers 50.1 ±7.6 years, controls 49.0 ±7.2 years p=0.511) There was a positive correlation between age and P300 latency (in controls r=0.328, p=0.045, farmers r=0.380, p=0.032) The N2 latency was correlated with age in farmers (r=0.347, p=0.048) The farmers made significantly more counting errors in the oddball tasks (4.8 (3.6), control 3.2 (2.7) p=0.044) In the analysis of averaged waveforms the analyses using mixed ANOVA revealed a significant effect of group [F (1,66=9.07, p=0.003] and component [F (1.934,127.6)=2115.0 p<0.001] and a component x group interaction [F(.1.934,127.6)=5.739, p=0.005]. There was evidence for a greater intergroup differences in later ERP components. Intergroup differences for individual components was analysed using Bonferroni correction (4 groups 0.0125 (0.05/4) showed a significant delay in the P300 in farmers. No significant changes were seen in N2 latency (Bonferroni correction 0.0167, and P2/N1 latencies (Bonferroni correction of 0.025)

Peak Farmers (n=35)

Controls (n=38)

Significance

N100 latency

103.5 (16.5) 99.1 (10.5) 0.173

P200 latency

186.4 (18.0) 178.4 (21.4) 0.094

N200 latency

240.5 (24.4) 226.3 (25.4) 0.019

P300 latency

394.5 (47.0) 362.4 (27.0) 0.0006

P300 amplitude Cz

14.9 (8.6) 12.1 (5.7) 0.108

P300 amplitude Pz

12.4 (5.1) 11.9 (5.2) 0.670

Counting 4.8 (3.6) 3.2 (2.7) 0.044

farmers compared to non exposed hospital labourers. Limitations ERP recorded at two sites only. EOG not recorded and no method of artefact rejection (eye movement, subvocalisation) although subjects instructed to keep eyes closed.

increasing delay in ERP components (significant at N2 and P300). Delay at N2 represented cumulative slowing of early stages of attentional processes. However P300 delay evident even after accounting for delay in preceding component. P300 latency reflects speed of allocation of neuronal resources and updating of working memory. The authors concluded effects were similar to those reported in Tokyo subway survivors and corroborated neurobehavioural test results of OP exposure.


19

First Author



errors When the delay in the preceding N2 component was taken into account, the delay in P300 latency was still significant [F (1,66)=7.107, p=0.010]. neither N2 or P2 group differences were significant after adjusting for the preceding component latency. ANOVA of P300 amplitudes at Cz and Pz sites did not show a significant group main effect [F(1,68)=1.426, p>0.237] but there was a significant site x group interaction [F (1,68) = 5.113, p=0.009] Testing of simple group effects did not show any effects (see table above). However farmers had a significantly larger P300 at Cz than at Pz (t=2.631, p=0.013). Such an effect was not seen in the control group.

Kimura 2005 TOBACCO FARMERS: BRAIN EVOKED POTENTIALS EXPOSURE Subject recall of pesticides used. 26/76 reported methamidophos use. Spraying per month (all pesticides) 1-2 (17 subjects), 3-4 (42), 5-6 (5), 7-8 (8), >8 (3) (not sure 1 subject). Spraying per week 1 (54 subjects), 2 (9), 3 (5), 4 (0), 5 (0) HEALTH OUTCOMES No reduction in serum cholinesterase reported. No effects on Visual Evoked Potential. VEP P100 (latency m sec) exposed 92.4 (SD 5.7) (n=64), compared to control 93.1 (SD 5.4) (n=34) No effects on Auditory ERPs. Auditory ERP P300 3.4.1 (SD 37.3) n=65 exposed , compared to 301.8 (SD 27.6) n=32 control. (latency msec) 0-1Hz postural sway in the anterior-posterior direction with eyes open was significantly increased which the authors suggest indicated a vestibulo-crebellar effect of Methamidophos exposure. Authors report that similar effects were reported in Tokyo subway victims.

Strengths Study of tobacco workers with documented use of OPs Limitations. No specific OP results presented (data refers to a range of pesticides used by tobacco farmers). No consideration of confounding factors reported

No definite conclusions can be reached from this study.

Misra 1994 PESTICIDE SPRAYERS: AUDITORY ERPs EXPOSURE: Mean age of workers (32.1 y (range 19-55 y)) and mean duration of exposure 10.5 y (range 1-14 y). They sprayed aqueous fenthion (0.1%) by hand operated sprayer for 5-6 h/day, six days per week without using any protective clothing. It is not clear from the paper

Strengths Objective measure of ERPs in subjects with OP exposure.

Authors conclude a subtle changes in auditory ERP P3 latency


20

First Author



whether this was daily exposure over a season or throughout the whole year. All workers had worked till previous day before testing. HEALTH OUTCOMES Pesticide sprayers reported clinical symptoms, headache (56%), giddiness (44%), eye irritation (20%), anorexia and paresthesia ((11%), depressed mood (95), insomnia/somatic concern (3% each) P3 component was elicited in 28 pesticide sprayers (27 controls). P3 latency was increased.

Parameter Control (n=27) mean ±SD

Exposed (n=28) mean ±SD

Latency CZ N1 92.2±9.2 90.6±10.1 CZ P2 165.3±23.3 170.1±14.3 CZ N2 227.3±35.8 230.8±42.7 CZ P3 331.7±32.0 351.2±42.0 FZ P3 306.3±44.3 343.3±46.7 Amplitude CZ N1 8.8±3.5 9.9±10.1 CZ P2 10.6±4.6 11.5±3.5 CZ N2 7.6±3.8 7.8±3.5 CZ P3 13.4±6.4 13.8±7.1 FZ P3 11.5±5.8 14.6±8.3

In concurrent neuropsychological tests Benton Visual Retention test performance was significantly inferior (preservation, memory quotient, visual reproduction, logical memory and associated learning were impaired. The linear correlation of P3 with BVRT was not significant. Serum AChE in exposed was 2093.0 (672 U/l) and 3015.7 (1284.7 U/l) in controls (P<0.01) There was no linear correlation between AChE and P3 (or BVRT).

Limitations Only one subject had a clinically significant increased P3 latency. No direct measure of exposure was reported. However serum cholinesterase levels were significantly reduced.


21

Table 8. EXPOSURE SCENARIO AND NEUROPHYSIOLOGICAL EFFECTS A. ACUTE EXPOSURE WITH CHRONIC EFFECTS Table 8.A.2. Case control studies

Test Name

First Author and Date Dassanayake 2008 Acutely poisoned cases. 6-month follow-up.

Murata 1997 Tokyo subway vicitms 6-8 months follow-up

Auditory Evoked-Related Potentials

N100 amplitude N100 latency - - P200 latency - N200 latency - P300 amplitude - P300 latency + + Visual Evoked Potential P100 latency + Test 1 Test 2

Table 8.A.3. Cross-sectional studies

Test Name / Diagnosis

First Author and Date Araki 2005

Tokoyo subway victims with PTSD, 5-6 years post incident.

Spencer 2001 Khaymisiyah incident, 8-9 years post

incident


P300 amplitude + P300 latency - EEG -


22

B. CHRONIC LOW-LEVEL EXPOSURE Table 8.B.3. Cross-sectional studies

Test Name

First Author and Date Browne

2006 Agricutural community,

Israel

Dassanayake 2009

Vegetable farmers,

Sri- Lanka

Kimura 2005

Tobacco farmers Malaysia

Misra, 1994

Pesticide workers

India EEG beta activity (prefontal cortex) + theta activity (hippocampus, parahippocampal regions, cingulate cortex)

+


N100 latency - - N100 amplitude - P200 latency - - P200 amplitude - N200 latency - - N200 amplitude - P300 latency + - + P300 amplitude Cz - - P300 amplitude Pz - - Visual evoked potential -


1

TOX/2012/26 Annex 6 COMMITTEE ON TOXICITY OF CHEMICALS IN FOOD, CONSUMER PRODUCTS AND THE ENVIRONMENT EVALUATION OF PSYCHIATRIC ILLNESS OVERVIEW OF COT 1999 REPORT 1. The COT evaluation of psychiatric outcomes can be found in Chapter 7, paragraphs 7.61 to 7.70 of the 1999 report (Annex 16). 2. The COT 1999 document reported that the Working Group considered a small number of studies providing evidence of increased psychiatric morbidity following acute poisoning by OPs, and also some studies with no past history of acute OP poisoning where chronic low-level exposure might be presumed. The COT report also considered a group of symptoms which together have been called Chronic Organophosphate Induced Neuropsychiatric Disorder (COPIND) by Davies et al 1999, and reported on the findings of a few studies addressing the relation of OP pesticide exposure to the risk of suicide in farmers. Studies in Individuals with a History of Acute OP Poisoning 3. The COT considered a small number of studies providing evidence of increased psychiatric morbidity following acute poisoning by OPs: Reidy et al 1992 found higher levels of anxiety and depression in farm workers acutely exposed to mevinphos in combination with a carbamate and dithiocarbamates; Steenland et al 1994 reported increased tension and confusion on follow-up of reported cases of poisoning in California; Savage et al1988 reported an excess of depression in cases of poisoning in Colorado and Texas. However the COT concluded that, overall, in relation to psychiatric morbidity following acute poisoning, the available published information at the time suggested that the abnormalities of mood recorded in the studies were not of sufficient severity to require treatment. The report stated that ‘The limited evidence available does not allow any firm conclusions to be drawn regarding the risk of developing psychiatric illness in the long term as a consequence of acute poisoning by OPs’. Studies in Individuals with No Past History of Acute OP Poisoning 4. A few cross-sectional surveys of people who had worked with OPs were reviewed for evidence of psychiatric symptoms following chronic exposure with no past history of acute OP poisoning. The findings of 3 studies are positive: Stephens et al 1995 reported more psychiatric disorders in sheep farmers exposed to OPs than in controls who worked as quarrymen; London et al 1998 found an excess of dizziness and sleepiness in South African pesticide applicators exposed to OPs; Amr et al 1997 found an increased prevalence of depression, irritability and erectile dysfunction in Egyptian pesticide applicators. Conversely, 2 studies reported


2

negative findings: Ames et al 1995, describing agricultural pesticide applicators, and Fiedler et al 1997, considering disordered emotion or personality in a sample of fruit farmers. 5. Two studies considered a possible association between pesticide exposure and the risk of suicide in farmers. A Canadian study, Pickett et al 1998, and a UK study, Hawton et al 1998, found no evidence that OP exposure was a major factor in the excess of suicide among Canadian or British farmers. 6. The COT report stated that ‘in general, the evidence relating psychiatric illness to OPs is insufficient to allow useful conclusions’. It suggested that further studies are required to investigate whether low-level exposure to OPs causes psychiatric disease in small sub-groups of exposed persons. It further suggested that people with disease and not currently employed in an occupation giving rise to OP exposure should also be considered. OVERVIEW OF LITERATURE PUBLISHED AFTER COT 1999 REPORT 7. In this review, the search was expanded to include not only exposure to OP pesticides, but also evidence from exposure to the OP sarin in two incidents in Japan, the Tokyo subway sarin attack of 1995 and the release of sarin in a residential area of Matsumoto in 1994, and evidence from literature relating to the Khamisiyah incident in the Gulf War in 1991, which involved the destruction of a munitions dump containing sarin and cyclosarin. Twenty-nine of the papers reported psychiatric outcomes. Of these, 19 papers reported on effects of exposure to OP pesticides; 7 papers reported on the sarin attacks in Japan (6 on the Tokyo subway incident and 1 on the Matsumoto sarin release), and 3 papers reported on exposure to sarin after the Khamisiyah incident in the 1991 Gulf War. ACUTE EXPOSURE WITH CHRONIC EFFECTS 8. Four studies reported on acute exposure to OPs and examine chronic effects. Seven studies report on the effects of sarin release in Japan, one on the Matsumoto incident in 1994, and six on the Tokyo subway sarin attack of 1995. Three studies reported on psychiatric outcomes of exposure to sarin and/or cyclosarin during the 1991 Gulf War, as a result of the destruction of a munitions dump at Khamisiyah in Iraq which was known to contain these two chemicals. Acute Exposure to OP Pesticides Study of occupationally exposed subjects in Nicaragua 9. Delgado et al 2004 is a small cohort study which followed 53 persons admitted to hospital after acute exposure to OP pesticides at hospital discharge, 7 weeks after hospital discharge, and again after 2 years. The mean Q16 neuropsychiatric symptom score showed an increase over 2 years for all exposed persons (Mean 1.58 at discharge to 2.19 at 2 years’ follow-up), but also a smaller


3

increase among the unexposed persons (Mean 1.72 at discharge to 1.84 at 2 years’ follow-up, Annex 6, Table 7.A.1, Delgado 2004, ‘Mean and standard deviation of Q-16 score’). The study had adjustments for a number of relevant confounders, including other neurotoxic exposures, but it also had several limitations. The study lost 11 workers to follow-up after 2 years, leaving only 28 subjects. The controls used worked in fishing and cattle farming, so some selection bias might have been introduced by comparing these workers to poisoned workers in agriculture. The results were further limited by the fact that the Q16 metric includes physical as well as psychiatric symptoms, so is not a clear indicator of purely psychiatric effects, and no individual symptoms are reported, only an overall score. Furthermore, the Q16 was developed to monitor central nervous system effects on workers exposed to solvents (Lundberg et al 1997), and may not have the same validity for monitoring workers exposed to OPs. US cross-sectional study 10. Stallones et al 2002b is a US cross-sectional study that questioned 761 respondents about their use of OPs and whether they had suffered pesticide-related illness. The evidence relating specifically to OPs is indirect. The study reported increased odds of having a pesticide-related illness if respondents applied crop OPs (Annex 6, Table 7.A.3, Stallones 2002b, ‘Odds of experiencing pesticide-related illness and type of pesticide used’), and that using an OP rather than any other pesticide was significantly associated with having had a pesticide-related illness (OR 2.34, 95% CI 1.17-4.66, Annex 6, Table 7.A.3, Stallones 2002b, ‘Several characteristics and Odds of experiencing pesticide-related illness’). The strength of the study is its size, and an association was suggested between exposure and persisting irritability and symptoms of depression. However, there remains a potential for measurement bias, since exposure was self-reported, and the psychiatric symptoms investigated are for an association with pesticide-related illness, not specifically acute exposure to OPs. Furthermore, the questionnaire used in the study was developed by the study authors and is not a validated instrument. Studies of banana plantation workers in Costa Rica 11. The Wesseling et al 2002 and 2010 cross-sectional studies were performed in Costa Rica, and reported on men working on banana plantations who had had an acute exposure to OP pesticides and received treatment at a health centre, but whose symptoms were not sufficiently severe to require hospitalisation. The same population is analysed in both studies, with 81 subjects in the 2002 study, and 54 OP-poisoned subjects in the 2010 study. 12. Wesseling et al 2002 reported total scores for the Q16 questionnaire and the Brief Symptom Inventory (BSI) test (Annex 6, Table 7.A.3, Wesseling 2002, ‘Q-16 and BSI scores for OP-poisoned workers’). The Q16 score showed a significant increase in neuropsychiatric symptoms at the p<0.01 level for the crude difference of means between poisoned and non-poisoned subjects, and the BSI grand total has a Regression Coefficient of 15.7 and a 95% CI of 5.5-25.8, which suggests significance. However, although both questionnaires are validated instruments, the paper reports only overall scores, not individual symptoms, so it is difficult to assess the true detrimental effects of the OP poisoning without further detail.


4

13. Wesseling et al 2010 reported in more detail on the same set of workers and the dimensions of the BSI, but only 54 OP-poisoned workers were included in this study. Median values for all nine dimensions of somatisation, obsessive-compulsiveness, interpersonal sensitivity, depression, anxiety, hostility, phobia, paranoia and psychoticism, were higher, and interquartile ranges wider, for the 54 OP-poisoned workers compared to 130 referent workers (Annex 6, Table 7.A.3, Wesseling 2010, ‘Scores for BSI dimensions among banana workers with history of OP poisoning’). The ORs for a T score ≥63, which is considered to be clinically relevant, are elevated for all nine dimensions, and 95% CIs indicate significance in all areas except paranoia (Annex 6, Table 7.A.3, Wesseling 2010, ‘Prevalence of abnormal scores among banana workers with history of OP poisoning’). This study suggested an overall excess of symptoms of psychological distress in OP-poisoned workers at least 1 year after the poisoning episode. However, it must be considered that workers were very likely to have been co-exposed to other pesticides, especially carbamates, so the association with OPs is not clear-cut. Furthermore, the 2010 study re-visits data that was collected some 15 years previously in 1994. Only 57% of the original group of poisoned workers could be located in 2010, so there is a potential for selection bias as it is unknown how similar the non-located workers were to those who could be followed up. Exposure to Sarin in Incidents in Japan Sarin release in Matsumoto in 1994 14. Nakajima et al 1999 is the one study reporting on the release of sarin in a residential district of Matsumoto in 1994. The study focused on just two symptoms, bad dreams and insomnia, using questionnaires devised by the authors at 1 year and 3 years after the attack. It was setup as a cohort study, with the first survey conducted 3 weeks after the attack, involving 2,052 residents, then a 1-year survey involving 1,237 respondents out of the original 2,052, and a further survey at 3 years after the incident, in which there were 836 respondents out of 2000 people living in the same area who were contacted. The questionnaires used were unvalidated instruments. At 1 year after exposure, there were no significant differences in the prevalence of bad dreams and insomnia between three different groupings of patients, those who were hospitalised, those who consulted doctors but did not need hospitalisation, and those who had symptoms but did not consult a doctor (Annex 6, Table 7.A.1, Nakajima 1999, ‘Prevalence of symptoms 1 year after sarin exposure in Matsumoto incident’). At 3 years after the incident, the Odds of having bad dreams remain non-significant, although the OR for insomnia is 2.48 with a 95% CI suggesting borderline significance, 1.07-5.78 (Annex 6, Table 7.A.1, Nakajima 1999, ‘Symptoms of sleep disturbance 3 years after sarin exposure in Matsumoto incident’). Tokyo subway sarin attack in 1995 15. Six studies related to the subway attack of 1995. Four of these considered specifically Post Traumatic Stress Disorder (PTSD), a potentially debilitating anxiety disorder that can occur after a traumatic experience. Some of the symptoms of


5

PTSD, such as anxiety, depression, sleep disturbances, have been considered as potential long-term effects of OP exposure but are also core psychological symptoms that would be expected in a proportion of people experiencing a traumatic event such as the subway sarin release. It is therefore difficult to separate the possible effects of the OP exposure from the psychological effects of the event. Nevertheless, PTSD was considered in this section because of the possibility that some of the core symptoms could be associated with the physical exposure to sarin. PTSD in victims of sarin exposure 16. Kawana et al 2001 followed up 283 patients from among those given emergency treatment at St Luke’s International Hospital, Tokyo, on the day of the attack. The group of patients was followed up 2, 3 and 5 years after the attack, with the number of patients still involved dropping to 206 after 3 years and 191 after 5 years. The study used a questionnaire developed by St. Luke’s Hospital, so it is not a validated instrument. The symptoms examined were in 3 categories associated with PTSD, those of Avoidance, Hyper-arousal, and Re-experience, and the results were positive, indicating that a number of victims continued to experience symptoms associated with PTSD up to 5 years after the attack(Annex 6, Table 7.A.1, Kawana 2001, ‘Frequency of psychiatric symptoms in victims of Tokyo subway attack’). However, only the frequency of symptoms and the percentages of people affected were reported, and no further statistical analysis was performed. 17. Ohtani et al 2004 also made use of the St. Luke’s Hospital questionnaire, but it was a small cross-sectional study that considered only 34 victims questioned at 5 years after the attack. It used several validated tests for the evaluation of PTSD, all of which gave positive results: the Impact of Event Scale – Revised (IES-R), which gave a mean total score of 16.4, suggesting that victims presented several symptoms of PTSD; the Clinician-Administered PTSD Scale (CAPS), which identified 11 PTSD sufferers out of the 34 victims; the Mini International Neuropsychiatric Interview (MINI), which indicated that 6 subjects with PTSD also had other mental disorders; and the State-Trait Anxiety Inventory (STAI), which gave mean scores of both state and trait anxiety that were significantly higher in the victims with PTSD than in the non-PTSD group (Annex 6, Table 7.A.3, Ohtani 2004, ‘Scores obtained on tests’). Overall, the findings were that both PTSD and other non-specific mental symptoms persisted in some of the victims of the sarin attack. The study found that 32.4% of the subjects developed PTSD during the 5 years after the attack. 18. Two studies by Tochigi et al (2002 and 2005) also considered a group of 34 victims, although it is not clear whether this was the identical group to the Ohtani study. The Tochigi studies included a reference population of 34 age-matched volunteers, whereas there was no reference population in the Ohtani study. Tochigi et al 2002, like Ohtani, reported significant positive results for the IES-R and development of PTSD due to the attack, and significantly increased anxiety on the STAI (Annex 6, Table 7.A.1, Tochigi 2002, ‘Diagnosis of PTSD: IES-R and STAI scores in victims of Tokyo subway attack’). However, Tochigi identified 8 victims who developed PTSD, in contrast to the 11 sufferers in the Ohtani study. The MINI indicated that 7 out of the 8 PTSD victims also suffered from other mental disorders (Annex 6, Table 7.A.1, Tochigi 2002, ‘Results of the MINI’). The Tochigi et al 2002


6

study was also able to report serum cholinesterase levels, which showed some correlation with the development of PTSD (Annex 6, Table 7.A.1, Tochigi 2002, ‘Cholinesterase levels in victims 5 years after Tokyo subway attack’, and ‘Cholinesterase levels in 25 victims immediately after, and 5 years following, the Tokyo subway attack’). All 34 victims had measures of serum cholinesterase levels taken 5 years after the attack, and levels were seen to be significantly reduced in victims who had developed PTSD compared to controls; cholinesterase levels were available for 25 victims immediately after the attack, and reduced levels were seen to be correlated significantly with scores on the present IES-R score, suggesting co-morbidity in the victims with PTSD. 19. The Tochigi et al 2005 study examined results for the group of 34 victims on the CAPS scale, and considered specific factors of the CAPS scale in greater detail. A significant positive correlation was reported between reduced cholinesterase levels 1-3 days after the subway attack and the total CAPS scores, as well as for the specific factor of re-experiencing (re-living the traumatic event) in the Current PTSD score, and the factors of re-experiencing and avoidance (of places or things associated with the event) in the Lifetime PTSD score (Annex 6, Table 7.A.1, Tochigi 2005, ‘Cholesterol, uric acid and cholinesterase levels in victims of Tokyo subway attack’). However, the overall number of subjects included in the study was small (8 who developed PTSD out of 34), and it is therefore difficult to draw firm conclusions. Other psychiatric outcomes of sarin exposure in the Tokyo attack 20. Two studies reported on psychiatric outcomes other than PTSD which they link to the sarin subway attack. Kawada et al 2005 reported on sleep disturbances, specifically in women aged 30 to 69 years, and compared to control female subjects who lived along a busy road, Route 17, in Maebashi City (Annex 6, Table 7.A.3, Kawada 2005, ‘Prevalence of insomnia in female victims of Tokyo subway attack compared to controls’). The questionnaire used was developed by the study authors, and therefore not a validated instrument, and the findings are not conclusive that the aspects of insomnia experienced by victims are related to their sarin exposure. 21. Nishiwaki et al 2001 reported overall scores on the Impact of Events Scale (IES), which measures psychological stress after a traumatic event, and the General Health Questionnaire (GHQ), which evaluates general mental health (Annex 6, Table 7.A.3, Nishiwaki 2001, ‘IES and GHQ scores in victims of Tokyo subway attack’). No individual symptoms were reported, but the 54 subjects, who were exposed in the Tokyo subway sarin incident either because they were rescue workers or police officers, are divided into a high-exposed group of 25 subjects who were hospitalised immediately after the attack, and 29 low-exposed subjects who attended hospitals as outpatients. There was some evidence that the IES and GHQ scores increase in a dose-dependent manner, from the lowest score for non-exposed referent subjects, to a higher score for the low-exposed group, and the highest score for the high-exposed group. However, the p values were reported not to indicate any significant differences between referents and exposed individuals.


7

Exposure to sarin and cyclosarin in the Khamisiyah incident in the Gulf War 22. Three cross-sectional studies reported on psychiatric outcomes of exposure to sarin and/or cyclosarin during the 1991 Gulf War, when a munitions dump at Khamisiyah, known to contain these two chemicals, was destroyed. 23. McCauley et al 2001 used a Gulf War survey instrument developed in the US, and gave ORs and 95% CIs for psychiatric symptoms reported by subjects approximately 9 years after the detonation of the munitions dump at Khamisiyah (Annex 6, Table 7.A.3, McCauley 2001, ‘Psychiatric symptoms reported by Gulf War veterans approximately 9 years after Khamisiyah incident’). There appeared to be significant ORs for personnel deployed to the Gulf (n=1,263), and located both within and outside a 50km radius of Khamisiyah at the time of the detonation, compared to non-deployed personnel (n=516) who had been on active duty at the time, but were not deployed to the Gulf. The symptoms investigated are changes in memory, difficulty sleeping, depression, unusual irritability or anger, mood swings, having a choking sensation, problems following directions or instructions, difficulty concentrating, and effects from confined places, all of which are found to be significant. However, when the study considered only subjects deployed to the Gulf, and divided them into Khamisiyah and non-Khamisiyah groups, none of the ORs remained significant. When the Khamisiyah personnel were further divided into ‘Khamisiyah witness’ (persons involved in or watching the detonation) and a ‘non-Khamisiyah witness’ (persons not directly involved) groups, symptoms of depression, changes in memory and difficulty sleeping appeared to be significant again. The apparent difference in significance might be due to problems with establishing exposure and the degree of exposure. Misclassification is possible, as it was found that 50 subjects considered to be in the Khamisiyah-deployed group reported they had never been in Iraq. Recall bias is likely, since data collection took place 8 to 9 years after the event, and selection bias also is a possibility because the sample was limited to people who could be contacted easily in the US because their telephone numbers were readily available. Overall, although there seemed to be significant psychiatric effects on subjects deployed to the Gulf compared to those deployed elsewhere, the results do not provide conclusive evidence for an association between exposure to sarin and psychiatric symptoms. 24. The McCauley et al 2002 study used data from the same subjects as the 2001 study, and considers several severe psychiatric symptoms: PTSD, major depression with hospitalisation, obsessive-compulsive disorder, and other psychiatric disease or psychosis (Annex 6, Table 7.A.3, McCauley 2002, ‘Frequency of diagnosed medical conditions reported by Gulf War veterans approximately 9 years after Khamisiyah incident’). As in the 2001 study, associations are significant when subjects deployed to the Gulf were compared with subjects deployed elsewhere, but were not found to be significant when Khamisiyah and non-Khamisiyah groups were compared. Again, the limitations in the two studies regarding exposure misclassification and bias might be responsible for the non-significant results of the Khamisiyah group potentially exposed to sarin, but the findings do not provide evidence of an association between sarin exposure and psychiatric effects. 25. Proctor et al 2006 is another cross-sectional study performed in the US, focusing on 140 subjects from the Devens Cohort Study of 1991 Gulf War Veterans.


8

The subjects were divided into a high, moderate and low/no exposure group, based on plume modelling of estimated release and dispersion of sarin and cyclosarin around the Khamisiyah site, and the presumed location of subjects in the area. The degree of exposure of individuals to sarin was not established. The study used two validated tests, the Brief Symptom Inventory (BSI) and the Mississippi PTSD Scale, but scores were found to be greater for the low/no exposure group than the high exposure group (Annex 6, Table 7.A.3, Proctor 2006, ‘Mississippi PTSD and BSI scores in Gulf War veterans 4 to 5 years after the Khamisiyah incident’). The percentage of subjects with a Current PTSD diagnosis and major depression diagnosis was also higher for the low/no exposure group, and the Profile of Mood States (POMS) did not indicate that higher exposure is significantly related to such symptoms as fatigue, tension, depression, anger or confusion (Annex 6, Table 7.A.3, Proctor 2006, ‘Current PTSD and Major Depression diagnosis in Gulf War veterans 4 to 5 years after the Khamisiyah incident’, and ‘POMS test results’). Thus, the findings of this study were negative, but the high to low exposure comparisons were inconsistent and it was difficult to establish the level of exposure of individuals with any certainty. Conclusions on Acute Exposure with Chronic Effects 26. The four studies reporting on possible longer-term psychiatric effects of acute exposure to OP pesticides all have their own specific limitations; furthermore, Delgado et al 2004 and Wesseling et al 2002 and 2010 reported on small groups of subjects, and Stallones et al 2002b, which reports on a larger group, reported an association with pesticide-related illness rather than a specific association with OPs. There was insufficient evidence to draw conclusions on psychiatric symptoms being associated with acute exposure to OP pesticides. 27. The six studies relating to the effects of the Tokyo subway sarin attack all suggested that PTSD persists in some victims. In cases of PTSD, however, it is difficult to separate possible physiological and mental effects of exposure to sarin from the possible psychological response to trauma: individuals who have experienced a traumatic event could potentially develop PTSD symptoms as a response to the psychological trauma, and it is difficult to separate the possible biological impact of sarin from a possible psychological response to the incident. The Tochigi et al 2002 and 2005 studies reported reduced cholinesterase levels in some victims 5 years after the event, which may suggest a link between physiological response to sarin and PTSD symptoms, but the study comprised only 34 subjects, and the low levels were not seen in all the subjects with PTSD. Overall, the psychological response to a traumatic event should be taken into account when considering the possible effects of sarin. 28. Evidence relating to psychiatric effects and the Khamisiyah incident in the Gulf War was limited. The two McCauley studies appeared to find associations between experiencing psychiatric symptoms and being deployed to the Gulf, but not with presence in the vicinity of Khamisiyah and potential exposure to sarin. The Proctor study findings were difficult to interpret as they found significant associations between symptoms and low or no exposure to sarin, and no significant associations with high exposure. There appeared to be uncertainties in the estimation and


9

modelling of exposure in this incident, which caused difficulties in the interpretation of the findings. CHRONIC LOW-LEVEL EXPOSURE 29. Fifteen studies reported on chronic low-level exposure. US Studies from the Agricultural Health Study 30. Three studies were taken from the Agricultural Health Study, a large and ongoing project in which a cohort of farmers and pesticide applicators and their families was enrolled between 1993 and 1997. The aim of the project is to gather information about the health of the participants and to observe changes over a period of time. 31. Beseler et al 2008 is a cross-sectional survey of 534 private pesticide applicators who self-reported a physician diagnosed depression, with a large reference population of 17,051 controls who reported never having been diagnosed with depression or feeling depressed more than once a week in the previous year. Controls were drawn from the same population as cases, which reduced the possibilities of confounding. The results of logistic regression models gave an adjusted OR of 1.78, 95% CI 1.27-2.50, for ‘Ever used OPs’ and diagnosed depression (Annex 6, Table 7.B.3, Beseler et al 2008, ‘Association of use of OPs with diagnosed depression’). However, the questionnaire used was devised by the study authors and is not a validated instrument, and exposure was self-reported, so there remains a possibility of some measurement bias. 32. Kamel et al 2005 is a cross-sectional study considering the number of neurological symptoms experienced by 18,782 licensed pesticide applicators participating in the Agricultural Health Study. The applicators were grouped into ‘cases’, those who had experienced 10 or more neurological symptoms in the year before enrollment, and ‘controls’, who had experienced less than 10 neurological symptoms: 20% of the applicators were ‘cases’, and 80% were ‘controls’. The specific psychiatric symptoms considered were fatigue, tension, insomnia, irritability, depression, absent-mindedness, and difficulty concentrating, using a questionnaire which was based on the Q16, and therefore not a validated instrument (Annex 6, Table 7.B.3, Kamel 2005, ‘Association of psychiatric symptoms with exposure to OPs’). For the highest category of lifetime days of use, which was more than 500 days, the ORs for experiencing the psychiatric symptoms with high frequency compared to low frequency were significantly greater than 1 for all seven, and the 95% CIs for all seven symptoms were greater than 1. The authors found dose-related associations of symptom count to cumulative exposure to OPs, suggesting that chronic, non-acute, exposure may be associated with an increased risk of adverse psychiatric effects. 33. Beard et al 2011 used mortality data obtained by linking the Agricultural Health cohort to state mortality files and the National Death Index, to investigate


10

suicides in the cohort. A total of 110 suicides were identified from among 81,998 cohort members, using International Classification of Diseases codes to identify suicides. The strengths of the study were the large size of the whole cohort, the fact that information on pesticide use was collected before suicides occurred, and the adjustment of Hazard Ratios for a large number of potential confounders. The findings of this study were negative, and did not suggest an association between suicide and use of OPs, or of some specific OPs, by pesticide applicators (Annex 6, Table 7.B.1, Beard 2011, ‘Association between suicide and OP use among applicators and spouses’). US Cohort Study of Chlorpyrifos Production Workers 34. Albers et al 2004b is a small cohort study conducted in the USA by the Dow Chemical Company in Midland, Michigan. The study evaluated 2 groups of chemical workers at the Company on 2 occasions, one at baseline and the other 1 year later. 53 employees who were engaged in the manufacture of chlorpyrifos took part at baseline and 1 year follow-up; 58 employees engaged in manufacture of Saran plastic film, with no known exposure to neurotoxicants, were used as a reference group, and 60 participated at baseline, with 58 also participating at 1 year follow-up. Although the cohort was small, the study included measurement of urinary excretion of OP metabolites as a measure of exposure (Annex 6, Table 7.B.1, Albers 2004b, ‘Exposure’), and estimates of air levels used to establish estimates of historic cumulative chlorpyrifos exposure were based on personal air sampling data. The study reported on only one possible psychiatric effect, that of anxiety, as measured by the Brief Symptom Inventory (Annex 6, Table 7.B.1, Albers 2004b, ‘BSI score for anxiety in chlorpyrifos production workers’), and found that there did not appear to be a significant association between exposure to chlorpyrifos and anxiety (P-value of 0.93). US Studies Reporting on Indoor Spraying of OPs 35. Three American studies reported on indoor spraying of OPs. Rehner et al 2000 is a cross-sectional study of 115 households whose homes were sprayed with methyl parathion, and where heads of households had applied to the Mississippi State Department of Health for help. The Environmental Protection Agency tested levels of methyl parathion in the homes by wipe tests, and defined four levels of exposure from the results. Thus, exposure was well documented. The study considered depression as the health outcome of interest, using the validated Center for Epidemiological Studies - Depression Scale (CES-D) as the instrument (Annex 6, Table 7.B.3, Rehner 2000, ‘Mean CES-D scores for subjects exposed to methyl parathion sprayed indoors’). Probable clinical depression is defined as a score exceeding 16, and 55% of participants had scores over 16. Comparing CES-D mean scores across the four contamination levels was not significant (p=0.942); however, a t test examining depression by gender indicated that women’s mean CES-D scores were significantly higher than men’s (p=0.009), and ANOVA examining depression by income groups indicated that people with lower incomes had significantly more depressive symptoms than people with higher incomes (p=0.004). The number of respondents’ depressive symptoms was highest among those who had been exposed the longest to methyl parathion (p<0.001), and a hierarchical regression supported the view that length of exposure was the only


11

variable predictive of depression (p=0.04). Thus, this study suggested that length of exposure to the OP methyl parathion sprayed indoors is more critical than level of exposure when considering depressive symptoms; furthermore, the symptoms may be a psychological response to the situation rather than a physical response to the chemical. 36. The Cox et al 2005 study was published 5 years after Rehner et al 2000, and also related to the spraying of methyl parathion (MP) in homes in Mississippi. Cox et al 2005 focused on data for 121 subjects with levels of MP >150μg/100cm³ in their home, determined by wipe samples taken from the home. It appeared to be a separate study to Rehner, with no indication of overlap between the subjects studied. Cox et al 2005 reports AChE levels, with no difference seen in average AChE levels between the high exposure group and controls. The psychiatric symptoms assessed in the most highly exposed subjects (n=60, levels of MP >260μg/100cm³) were insomnia and depression (Annex 6, Table 7.B.3, Cox 2005, ‘Frequency of insomnia and depression in subjects whose homes contained high levels of methyl parathion’). No validated instrument was used to assess these symptoms; the report of increased insomnia and depression was based on self-reported symptoms by subjects themselves, in an interview with a medical physician. 37. The third study which considered indoor spraying is Kilburn (1999), a cross-sectional study of 22 chlorpyrifos-exposed subjects seen in a neurotoxicology clinic. The health outcome of interest was again depression. Concentrations to which individuals were exposed are known for 6 patients whose offices were sprayed, and 4 patients who had personal clothing or rugs analysed for chlorpyrifos. The remaining 12 self-reported their exposure. The controls for this study were 264 individuals recruited at random from voter registration rolls in two towns, and who had not been exposed to neurotoxic chemicals. POMS mean scores were elevated for the exposed subjects compared to randomly selected controls (p=0.0001), as were mean depression scores (p=0.0002) (Annex 6, Table 7.B.3, Kilburn 1999, ‘POMS scores for subjects exposed to chlorpyrifos sprayed indoors’). A significantly increased frequency of 7 psychiatric symptoms – somnolence, insomnia, waking frequently, sleeping only a few hours, irritability, mood swings, loss of libido – with P value of 0.0001 for all seven, was also observed in exposed subjects (Annex 6, Table 7.B.3, Kilburn 1999, ‘Frequency of psychiatric symptoms for subjects exposed to chlorpyrifos sprayed indoors’). The POMS scores were suggestive of a possible association between indoor exposure to chlorpyrifos and depressive symptoms, but the number of exposed subjects considered was very small, and only 10 had a measure of exposure. UK Studies of Effects of Chronic Low-Level Exposure 38. The Solomon et al 2007 cross-sectional study investigated postulated COPIND (chronic organophosphate-induced neuropsychiatric disorder) symptoms and the psychiatric symptoms of anxiety and depression. COPIND is discussed in the ‘Other Neurological Effects’ section (Annex 11). In the analysis of anxiety and depression, the study used the validated Hospital Anxiety and Depression Scale, as well as responses of subjects to the Brief Symptom Inventory to derive a ‘somatising tendency’ score, that is, a tendency to experience unexplained physical complaints. The strength of the study was a large number of subjects, 9,844 exposed to sheep


12

dip, other insecticides or other pesticides, and a reference population of 6,109 subjects who had never worked with any type of pesticide. The exposed population were divided into a group who had worked with sheep dip, a group who had worked with other insecticides but never with sheep dip, and a third group who had worked with other pesticides, but never sheep dip or insecticides (Annex 6, Table 7.B.3, Solomon 2007, ‘Association of anxiety and depression with occupational exposure to pesticides and somatising tendency’). Adjustments were made for a number of potential confounders. The results of the study showed no clear association of working with any category of pesticide, including sheep dip or insecticides, and current anxiety or depression (for example, adjusted Prevalence Ratio of anxiety for men who had ever worked with sheep dip is 1.0, 95% CI 0.9-1.2, and for depression 1.1, 95% CI 0.9-1.3, where the Prevalence Ratios are on the border-line of significance and the Confidence Intervals also). Nor was there an association between anxiety or depression and handling sheep dip concentrate (Annex 6, Table 7.B.3, Solomon 2007, ‘Association of anxiety and depression with use of sheep dip’). However, the results for ‘somatising tendency’ suggested a significant association between increased anxiety and depression with greater somatising tendency, and the study authors suggested that there may be a psychological component to the reported symptoms. 39. Mackenzie Ross et al 2010 is a cross-sectional study of 127 UK sheep farmers, of whom 67 were working and 60 retired. The study specifically aimed to include subjects who had retired on ill-health grounds. It used a validated test, the Hospital Anxiety and Depression Scale, and reported that 46.9% of farmers had scores above clinical cut-offs for depression compared to only 6.5% of controls, giving a p value <0.001 for significance (Annex 6, Table 7.B.3, Mackenzie Ross 2010, ‘Hospital Anxiety and Depression Scale results’). With regard to anxiety, 41.5% of farmers scored in the clinical range, compared to 22.1% of controls, giving a p value <0.01 for significance. However, there were a number of limitations to the study, such as the recruitment methods used, which could have resulted in selection bias, and the fact that the control group were rural police officers and not farmers, which limited the strength of the presented evidence. 40. Jamal et al 2002a is a case-control study of 72 sheep farmers in the UK, drawn from a larger cross-sectional study of 685 subjects. The 72 farmers are classified as having ‘No’, ‘Possible’, and ‘Probable or definite’ neuropathy, with 15, 34 and 23 subjects in the respective categories. The scales used were the Hospital Anxiety and Depression Scale, and the General Health Questionnaire, which are both validated instruments, but the information reported in the paper was only a brief summary of possible psychiatric effects (Annex 6, Table 7.B.2, Jamal 2002a). The authors say that significant differences were observed among the 3 groups, with the ‘Probable or definite’ neuropathy group scoring consistently significantly higher than the other two, but no scores or comparisons were reported, and there was insufficient evidence presented to allow conclusions to be drawn. The study authors stated that the psychiatric results will be described in a separate paper. 41. The fourth UK study, Stephens et al 2004, focused mainly on neuropsychological test results and outcomes, and reported little evidence on psychiatric outcomes. The General Health Questionnaire (GHQ), a validated instrument, was used to gain information on psychiatric effects of spraying in 37


13

orchard sprayers, compared to 36 pig farm workers and 31 construction workers (Annex 6, Table 7.B.3, Stephens 2004, ‘GHQ score for vulnerability to psychiatric disorder in orchard sprayers compared to controls’). A P value of 0.070 was obtained for vulnerability of sprayers to psychiatric disorder as assessed by the GHQ, which does not suggest that a significant number of OP-exposed workers were above the psychiatric case threshold; in fact, there were greater numbers of psychiatric cases among pig farm workers and construction workers than among the orchard sprayers. However, only limited evidence for an association with psychiatric effects was available in the paper, and there was the potential for selection bias through the choice of workers in other occupations being compared with the OP sprayers. Studies Analysing the Effects of One Spraying Season 42. Four cross-sectional studies examined the psychiatric effects of a spraying season of OPs. Salvi et al 2003, performed in Brazil, described workers involved in family agriculture of tobacco, and had a number of strengths that would support the import of its findings: it uses the MINI, which is a validated measuring instrument; diagnoses of AXIS-1 psychiatric disorders were made by trained psychiatrists; and AChE levels were measured in participants as an indication of their exposure. The results did not indicate a significant difference in scores during OP exposure and after 3 months of OP washout (Annex 6, Table 7.B.3, Salvi 2003, ‘Mini-mental scores for tobacco workers exposed to OPs’). However, the sample size was small (37 workers in Evaluation 1 during the season of use of OPs, and only 25 who returned for Evaluation 2 after 3 months of no use), and study authors reported co-use by subjects of other chemical compounds besides OPs. 43. Bazylewicz-Walczak (1999) is a study of 26 female greenhouse workers in Poland using OPs, in which the Profile of Mood States (POMS) and Subjective Symptoms Questionnaire were used to assess the psychiatric effect of one season of spraying by comparing the results of a pre-season and post-season examination. The results did not suggest any adverse psychiatric effects of the spraying season on the workers. However, when the greenhouse workers were compared to 25 controls employed in the same gardening enterprises but in jobs not involving spraying, some significant effects were seen on the POMS results and on the Subjective Symptoms Questionnaire (Annex P, Table 10.B.3, Bazylewicz-Walczak 1999, ‘Results for Profile of Mood States and Subjective Symptoms Questionnaire’). Strengths of the study are that validated questionnaires were used, and that measures of exposure were available as results of air sampling in the greenhouses, and concentrations of pesticides on clothing and on skin of exposed workers. Limitations are that only one season was studied, and that the number of subjects was small. It was difficult to come to firm conclusions on the evidence available. 44. Bayrami et al 2012 is a study of 40 horticulture farmers in Iran exposed to OPs and monitored through the course of one spraying season. AChE levels were measured, and the Symptom Checklist-90-Revised test was used to assess levels of potential psychological distress in the workers (Annex 6, Table 7.B.3, Bayrami 2012,’Scores for dimensions in the Symptom Checklist-90-Revised’). Of the 9 symptom dimensions in the test, only the dimension of somatisation was found to be significant in farmers compared to controls not employed in agriculture (p 0.03).


14

However the study was small, and details of exposure were self-reported, although the authors reported some evidence of lower AChE activity in farmers compared to controls. 45. Keifer et al 2000 is a US study of 137 orchard thinners that investigated the effects of a season of thinning in OP-sprayed orchards. Only a very brief comment is made on psychiatric outcomes, but no report is made of scores on the BSI or Q-16, so it is difficult to evaluate the results (Annex 6, Table 7.B.3, Keifer 2000). Conclusions on Chronic Low-Level Exposure 46. Overall, the evidence for an association between chronic low-level exposure to OPs and psychiatric symptoms was limited, and sometimes inconsistent. Studies which reported overall scores on a variety of general tests provided inconclusive evidence, and each study had limitations. Positive results were reported for anger/irritability, in particular by Kamel et al 2005, a large study of applicators enrolled in the Agricultural Health Study, which provided some evidence suggestive of an effect. The association with anxiety, however, was inconclusive, with both positive and negative results reported; in fact the well-designed Albers et al 2004b and Solomon et al 2007 studies suggested no association. There were also conflicting results for the outcome of depression, with two large US studies, Beseler et al 2008 and Kamel et al 2005, reporting positive findings, while the large UK study, Solomon et al 2007, did not find an association between several different exposure scenarios and depression. Another US study, Rehner et al 2000, examined different causes not related to OP exposure, and concluded in relation to OPs that only the length of exposure was significant, not the level. No association was found with suicide by the Beard et al 2011 study using data from the Agricultural Health cohort. 47. More limited evidence was presented for an association between exposure and confusion, fatigue or tension, sleep disturbances, and absent-mindedness, largely by the Kamel et al 2005 study but also by the smaller Bazylewicz-Walczak et al 1999 cross-sectional study. Two studies found evidence of somatisation, Solomon et al 2007 and also Bayrami et al 2012; Solomon et al 2007 explores the association of somatisation with anxiety and depression apart from association with OP exposure, and suggested that further investigation may be needed as to whether people exposed to a perceived environmental hazard may be prone to develop symptoms which have no toxic cause. SUMMARY 48. The table below provides a summary of the main psychiatric outcomes and findings described in this section:


15

Psychiatric Outcome

Exposure Positive findings reported by study authors

Negative findings reported by study authors

Overall Conclusion

A. Acute Exposure with Chronic Effects General Increased symptomatology, including psychiatric symptoms; increase in Q16 score

Poisoning/acute exposure to OP pesticides

Delgado 2004, Wesseling 2002

Insufficient evidence to draw conclusions

Increase in BSI score As above Wesseling 2002, Wesseling 2010

Increased MINI mental scores, IES or GHQ

Sarin in Tokyo subway incident

Ohtani 2004, Tochigi 2002 and 2005

Nishiwaki 2001 Some evidence of symptoms of psychological distress in victims, but response to emotional trauma has to be considered

Anger/Irritability Poisoning/acute exposure to OP pesticides

Stallones 2002b Association with OP exposure limited

Sarin/cyclosarin in Gulf War incident

McCauley 2001, Proctor 2006

Limitations in the studies do not allow firm conclusions to be drawn

Anxiety Sarin in Tokyo subway incident

Ohtani 2004, Tochigi 2002

Tochigi 2005 Inconclusive evidence

Depression Sarin/cyclosarin in Gulf War incident

McCauley 2001, Proctor 2006

Proctor 2006, McCauley 2002

Inconclusive evidence


Stallones 2002b Association with OP exposure limited

Confusion/Fatigue/ Tension


Proctor 2006 Insufficient evidence

Mood Swings Sarin/cyclosarin in Gulf War incident

McCauley 2001 Insufficient evidence

PTSD Sarin/cyclosarin in Gulf War incident

Proctor 2006, McCauley 2002

Limitations in the studies do not allow firm conclusions to be drawn


Ohtanii 2004; Kawana 2001; Tochigi 2002, Tochigi 2005

Overall, studies suggest PTSD persists in some victims of Tokyo sarin incident; psychological response to trauma must be taken into account

Sleep Disturbances

Matsumoto sarin release

Nakajima 1999 Inconsistent findings, quality and consistency of evidence variable


Kawada 2005


McCauley 2001


Stallones 2002b

B. Chronic Low-Level Exposure General Increased MINI mental, GHQ, POMS, Subjective Symptoms Questionnaire, DSM-IV AXIS-I diagnoses (clinical disorders)

OP pesticides, chronic

Kilburn 1999, Salvi 2003 (DSM-IV diagnoses)

Salvi 2003 (GHQ), Stephens 2004, Bazylewicz-Walczak 1999

Inconsistent results, limited evidence and limitations in the studies


16

Psychiatric Outcome

Exposure Positive findings reported by study authors

Negative findings reported by study authors

Overall Conclusion

Anger/Irritability OP pesticides, chronic

Bazylewicz-Walczak 1999, Kilburn 1999, Kamel 2005

Some evidence suggestive of effects

Anxiety OP pesticides, chronic

Bazylewicz-Walczak 1999, MacKenzie Ross 2010

Albers 2004b, Solomon 2007

Inconsistent results; the more robust studies suggest no association

Depression OP pesticides, chronic

Bazylewicz-Walczak 1999, Beseler et al 2008, Kamel 2005, Mackenzie Ross 2010

Solomon 2007 Conflicting results regarding an association with chronic occupational exposure to OPs; some evidence suggestive of effects

Indoor spraying of OP pesticides, chronic

Rehner 2000, Cox 2005, Kilburn 1999

Causes of depression other than exposure to OP pesticides need to be considered

Suicide OP pesticides, chronic

Beard 2011 Data from large cohort study does not suggest an association

Confusion/Fatigue/ Tension


Bazylewicz-Walczak 1999, Kamel 2005

Bazylewicz-Walczak 1999

Some evidence suggestive of an association

Sleep Disturbances


Kamel 2005, Kilburn 1999



Absent-Mindedness


Bazylewicz-Walczak 1999, Kamel 2005


Somatisation OP pesticides, chronic

Solomon 2007, Bayrami 2012

Associated with anxiety and depression rather than specific exposure to OPs


17

Table 6: PSYCHIATRIC STUDY DESIGN DETAILS First Author





Measure of Exposure




6.A.1. Cohort studies Delgado 2004 Nicaragua Individuals

hospitalised in Leon and Chinandega between 1 July 1992 and 15 December 1996 for acute OP poisoning. Follow-up I was at time of discharge, follow-up II at 7 weeks after discharge, and follow-up III at 2 years after discharge. Data for 62 persons available at examinations I and II, and 53 persons at examination III. 31 were classed as moderate occupational exposure, 15 as severe occupational exposure, and 7 as severe non-occupational exposure

At examinations I and II, 39 non-poisoned men from fishing and cattle cooperatives located around Leon, Nicaragua. At examination III there were 28 workers (11 lost to follow-up)

Modified Spanish version of the Q-16 to assess neuro-psychiatric symptoms, validated in Nicaragua. It is a sum score of 16 symptoms; each symptom was coded 0=no or 1=yes

16 symptoms concerning problems with memory and concentration, irritability and sadness, tiredness, palpitations, chest tightness, headaches, dizziness, numbness and tingling, difficulty buttoning clothing, loss of sensitivity, and sleeping problems

Specific type of OP identified, and lifetime days of exposure to OPs quantified; assessment based on severity of the symptoms, and circumstances of the poisoning (occupational, accidental, suicide attempt)

Age, education, lifestyle, general health and other previous neurotoxic exposures, alcohol consumption, loss of consciousness for over 30 minutes and diagnosed by a physician, occupational exposure to vibrating machinery, history of chronic diseases

Two-way analyses of variance (time/exposure), for 3 levels of exposure – low, moderate, severe; linear regression analysis, mean values and SD reported; for the Q-16 symptom score by exposure status, means and SDs are reported with P-values for the main effect of exposure and for the interaction between time and exposure; mean changes between each exposure group and the unexposed at 7 weeks and 2 years are presented

Kawana 2001 Japan 582 sarin patients given emergency treatment at St Luke’s

655 victims of the Tokyo sarin attack studied by a non-governmental

Symptoms reported on the St. Luke’s Questionnaire, which was developed by the investigators and had

Depressed mood, avoidance of subject of the incident,

Release of sarin in the subway; the quantity that individuals were

Not considered symptom frequencies and percentages for the years 1997,


18

First Author





Measure of Exposure



International Hospital, Tokyo, on the day of the sarin subway attack (20 March, 1995)

organisation; 88 victims of the 1994 Matsumoto sarin attack; 87 Matsumoto controls

33 five-choice Likert scale items; 11 questions concerned psychological symptoms

diminished interest (numbing), irritability, difficulty falling or staying asleep, hypervigilance, flashbacks, distressing dreams or nightmares, fear in the subway or at the incident

exposed to is not known

1998 and 2000 are reported for the sarin patients, and comparisons are made using Fisher’s exact test; p values of <0.05 are indicated; symptom frequencies are also compared between 1997 and 1998, and 1998 and 2000, using Fisher’s exact test, and p values of <0.1 are indicated; a comparison of symptom incidence is made between the St Luke’s patients and the three reference populations (percentages reported)

Nakajima 1999 Japan First survey conducted 3 weeks after the attack, and involved 2052 residents in an area 1050 metres north to south and 850 metres east to west where about 12 litres of sarin were released on the night of June 27, 1994,in

919 out of the 1237 respondents to the 1 year survey who were classified as non-victims of the sarin attack, because they did not have a diagnosis of sarin toxicity and did not experience muscarinic and/or nicotinic symptoms immediately after

Questionnaires at 1 year and 3 years after the attack. Questions asked respondents whether they were given a diagnosis of sarin toxicity or had experienced muscarinic and/or nicotinic symptoms, and also asked about hospitalisations and consultations with doctors after the sarin attack; the 1-year survey contained

For psychiatric outcomes, the symptoms investigated were bad dreams and insomnia

It is known that about 12 litres of sarin were released in the specific area of Matsumoto City, but individual exposure, or proximity of individuals to the release, were not measured; 93 persons had cholinesterase activity measured 4

Not considered The significance of prevalence of symptoms subjectively described 1 year after the sarin exposure was tested by the χ2 test; the relationship between cholinesterase activity 4 weeks after sarin


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Matsumoto City, with the sarin release site in the centre; results of first survey reported in a previous paper; Survey conducted 1 year after the incident, with 1237 respondents out of the original 2052; 318 of the 1237 were sarin victims, defined as such either because they were given a diagnosis of sarin toxicity, or experienced mucsarinic and/or nicotinic symptoms immediately after the attack; for the 3 year survey, 2000 persons living in the same area about 3 years after the incident were approached; there were 836 respondents, of whom 167 were classed as victims

the sarin incident; for the 3 year survey, 669 people out of the 836 respondents were classed as non-victims

13 questions regarding specific symptoms, and whether those had been experienced within the preceding month; the same 13 symptoms were asked about in the 3-year survey

weeks after the sarin exposure and again for the 1 year survey

exposure and symptoms 1 year after the event was tested by One-way ANOVA, p values reported; differences in prevalence of symptoms between victim and non-victim groups in the 3 year survey evaluated using χ2 test or Fisher’s exact test if number in any one group too small; ORs and 95% CIs for association between sarin exposure and symptoms 3 years after the sarin incident reported

Tochigi 2002 Japan 34 victims (20 males, 14 females) of the Tokyo subway sarin attack who were treated in emergency for

34 age-matched and mostly sex-matched volunteers (18 males, 16 females),mainly recruited from staff

Clinician Administered Scale for PTSD (CAPS); IES-R (Impact of Event scale-Revised) questionnaire, with questions regarding current PTSD symptoms;

PTSD, anxiety Release of sarin in the subway; serum cholinesterase level within 1-3 days of the attack was available in 25

Current and previous status of other mental disorders was evaluated using the Mini-International

One-way analysis of variance (ANOVA), Student’s t-test and Wilcoxon test to compare


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Measure of Exposure



acute sarin intoxication and followed up at a general hospital in Tokyo

of the Tokyo University Hospital

MINI (Mini International Neuropsychiatric Interview); STAI (State-Trait Anxiety Inventory); control subjects completed the Revised NEO Personality Inventory (NEO-PI-R); serum levels of total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, uric acid and cholinesterase investigated in all subjects

victims (13 males, 12 females)

Neuropsychiatric Interview; no subjects had a history of PTSD due to other events

among or between subject groups; correlation between IES-R and serological studies analysed using Pearson correlation analysis; P values reported

Tochigi 2005 Japan 34 victims (20 males, 14 females) of the Tokyo subway sarin attack who were treated in emergency for acute sarin intoxication and followed up at a general hospital in Tokyo

34 age-matched and mostly sex-matched volunteers (18 males, 16 females),mainly recruited from staff of the Tokyo University Hospital

Clinician Administered Scale for PTSD (CAPS); MINI (Mini International Neuropsychiatric Interview); STAI (State-Trait Anxiety Inventory); serum levels of total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, uric acid and cholinesterase investigated in all subjects

Correlation between serum levels of cholesterol, uric acid and cholinesterase and severity of PTSD symptoms

Release of sarin in the subway; serum cholinesterase level within 1-3 days of the attack was available in 25 victims (13 males, 12 females)

Current and previous status of other mental disorders was evaluated using the MINI, and co-morbid DSM-IV disorders - panic disorder, major depression and agoraphobia - were confirmed among some of the 8 victims who developed PTSD due to the sarin attack

Pearson’s correlation coefficient calculated for correlation between the CAPS scores and serological studies in the victims, and for the STAI scores and serological studies in the victims; some r values are reported, and correlations with p values of <0.05 and <0.01 are indicated

6.A.2. Case-control studies - none

6.A.3. Cross-sectional studies Kawada 2005 Japan 161 subjects who

were victims of the 1995 Tokyo

Control female subjects lived along Route 17 of

Self-administered questionnaire; study authors referred to

Insomnia, defined as: poor sleep in the preceding

Subjects had been victims of the release of sarin in

No other risk factors evaluated;

Chi square test carried out to compare the


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Measure of Exposure



subway sarin attack and who attended the annual health examination related to sarin exposure in 2003 ; out of these, 75 women aged 30 to 69 years (n=61 aged 30 to 49, and n=14 aged 50-69) were chosen for this analysis because control data was available for female subjects only

Maebachi City, Gunma Prefecture, Japan: n=186 controls aged 30 to 49, and n=145 controls aged 50-69

criteria of ICD-10 (International Statistical Classification of Diseases and Related Health Problems, 10th version, WHO 1992) and DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, American Psychiatric Association 1994) for the definition of insomnia

month, and the presence of one or more of the following symptoms: difficulty falling asleep, intermittent awakening, early morning awakening, feeling of light overnight sleep, occurring one or more times a week

the Tokyo subway

limitations due to subjective evaluation of sleep, and lack of a standardised definition of insomnia; questionnaire was not validated by performance of a multiple sleep latency test or overnight sleep monitoring

distribution of frequencies of insomnia or insomnia-related factors between exposed and control subjects; p values reported

McCauley 2001 Study undertaken in the USA, exposure thought to have occurred at Khamisiyah, Iraq, where a munitions dump was destroyed

653 Khamisiyah veterans who were operating within a 50 km radius of the Khamisiyah Ammunition Storage Point where the munitions were destroyed; veterans had to be resident in one of the 5 states of Oregon, Washington, California, North Carolina or Georgia

610 Non-Khamisiyah military personnel who were deployed in the Gulf during the Gulf War, but were outside the 50 km radius and had no known exposure to nerve agents; 516 Non-Deployed personnel who had been on active duty at the time of the Gulf War, but not deployed to the Gulf; participants were also residents of one of the 5 states of Oregon, Washington, California, North Carolina or Georgia

Self-reported symptoms on a questionnaire; Computer Assisted Telephone Interviews conducted

Changes in memory, difficulty sleeping, depression, unusual irritability/anger, mood swings, choking sensation, problems following directions or instructions, difficulty concentrating, effects from confined places

No direct measure; exposure was assumed for troops within a 50 km radius of the detonated chemical warfare agents. Self-reported information on personnel movement in the Khamisiyah area included

ORs and 95% CIs adjusted for potential confounders of age, gender, and region of residence; selection bias possible because sample limited to persons whose telephone numbers could be tracked down by common search mechanisms; the Khamisiyah population of veterans was not evenly distributed throughout the US, so there may be bias due

Logistic regression, reporting ORs and 95% CIs; categorical data analysed using χ2 tests, and the continuous variable of age analysed using t-tests


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to location; also possible misclassification, because 50 subjects considered by the Department of Defense to be in the Khamisiyah-deployed group reported they had never been in Iraq

McCauley 2002 As above As above As above Self-reported conditions diagnosed by a physician since the Gulf War; Computer Assisted Telephone Interviews conducted

PTSD, major depression with hospitalisation, obsessive compulsive disorder, report of other psychiatric disease or psychosis

As above As above Frequency of diagnosed medical conditions in Gulf War veterans reported; ORs and 95% Cis calculated for the likelihood of reporting medical conditions diagnosed by a physician since the Gulf War

Nishiwaki 2001 Japan 56 male exposed subjects in the Tokyo subway sarin attack of 1995, consisting of rescue team staff members of the Tokyo Fire Department, and police officers in the Metropolitan Police Department; subjects were also divided into a high-

52 non-exposed referent subjects matched for age and occupation, including rescue team staff members and police officers from the same departments as the exposed subjects

Impact of Events Scale (IES) and 30-item General Health Questionnaire (GHQ); validated Japanese-language versions of both tests were used

Psychological stress after a traumatic event measured by the IES, and general mental health evaluated by the GHQ

No precise measure of exposure to sarin during the attack, so high-exposed persons were deemed to be those hospitalised immediately after poisoning, and low-exposed were those who attended hospitals as outpatients

Age, height, weight, alcohol consumption, smoking status, educational level; psychometric variable scores (IES and GHQ) were used as possible confounders of the stabilometry, vibration

Means and Standard Deviation calculated, and Student’s t-test or Welch’s method used to compare exposed with referents; p values calculated


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exposed group of 25 who had been hospitalised immediately after the attack, and a low-exposed group of 29 who attended hospitals as outpatients

perception threshold, and neurobehavioural tests

Ohtani 2004 Japan 34 victims (20 males, 14 females, 21-69y, mean age male 50.2±11.9 y, female 39.6 ±10.4) of the Tokyo subway sarin attack who were treated at emergency wards for acute sarin intoxication at St. Luke’s International Hospital in Tokyo, and who agreed to undertake the survey and undergo psychological and psychiatric investigations; this study was conducted at Tokyo University

None for the 34 victims studied; for evaluating PTSD, 11 of the victims who had been diagnosed with current or lifetime PTSD were defined as the PTSD group, and the remaining 23 subjects were used as a non-PTSD group for comparison

Self-rating questionnaire evaluating 34 subjective somatic and mental symptoms, developed by staff at St. Luke’s International Hospital; IES-R (Impact of Event scale-Revised) questionnaire, concerning symptoms associated with PTSD; STAI (State-Trait Anxiety Inventory), concerning patients’ current and general anxiety; MINI (Mini International Neuropsychiatric Interview); CAPS (Clinician-Administered Post-Traumatic Stress Disorder Scale)

PTSD: re-experiencing, avoidance and numbing, hyperarousal; tension; nightmares; insomnia

Release of sarin in the subway; the quantity that individuals were exposed to is not known

Not considered Frequency of symptoms, percentages reported; mean scores of both state and trait anxiety on the STAI, reporting SD, degrees of freedom, t value, and P value for significance; in correlational analysis, Pearson product moment coefficients were calculated among the indices of the IES-R, CAPS, and WMS-R; concerning the CAPS score, the sum of the scores of frequency and intensity was calculated for both current and lifetime diagnosis, respectively


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Measure of Exposure



Proctor 2006 Study undertaken in the USA, exposure thought to have occurred at Khamisiyah, Iraq, where a munitions dump was destroyed

Stratified, random subset of 1991 Gulf War veterans from the Devens Cohort Study, n=140; persons in units with exposure levels greater than 0.072 mg min/m³ were defined as the high exposure group (n=23); persons in units with exposure levels greater than General Population Limit but no more than 0.072 mg min/m³ were defined as the moderate exposure group (n=47)

Persons in troop units for which no exposure level was estimated because they were not in locations within the modelled plume areas (n=70); the majority were located in Saudi Arabia coastal cities during the time period

Medical and occupational history questionnaire; several scales for assessing psychological symptomatology: Brief Symptom Interview (BSI); Mississippi Scale for Post Traumatic Stress Disorder (PTSD); Clinician Administered Scale for PTSD (CAPS); Structured Clinical Interview for DSM-IIIR Mental Disorders; mood states at time of testing using the Profile of Mood States (POMS)

Diagnosis and scores for symptoms of PTSD; diagnosis of depression; mood states of fatigue, tension, depression, anger, or confusion at time of testing

Plume modelling of estimated release and dispersion of sarin and cyclosarin around the Khamisyah site; persons were considered exposed if they were located in an area with an exposure estimate greater than the General Population Limit of 0.01296 mg min/m³, defined by the US Army and Centers for Disease Control and Prevention. Exposure estimates ranged from 0.035 to 0.144 mg min/m³

Unit group, age, gender, general intelligence using WAIS-R (Wechsler Adult Intelligence Scale – Revised) Information score, PTSD symptomatology using the Mississippi PTSD scale, history of head injury; co-exposure to cyclosarin as well as sarin may affect results

Analyses of variance (ANOVAs) and Student’s t tests for continuous variables, χ² or Fisher’s exact tests for categorical variables, for comparison of demographic and descriptive characteristics across the 3 exposure groups; analyses conducted at group level, not individual level, so analyses of covariance methods were used, with adjustments for various factors; Cohen’s effect size is reported; p values are reported throughout

Spencer 2001 Study undertaken in the USA, exposure thought to have occurred at Khamisiyah, Iraq, where a

3219 Gulf War veterans identified (923 Khamisiyah, 927 non-Khamisiyah, 1369 non-deployed). 1833 interviews taken, and data reported for 1779 subjects. 653 were deployed to Khamisiyah

Number of veterans outside the Khamisiyah region (610) or not deployed (516)

Level 1 neurobehavioural tests completed on 624 participants; these tests included several psychological tests: Penn PTSD, Mississippi PTSD, multiple scales of the SF-36 (health survey giving physical and mental health summary measures), and Beck Depression Scale

Performance on psychological tests, but no specific psychiatric or psychological health outcomes reported

Not reported Not reported Not reported


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munitions dump was destroyed

Stallones 2002b USA 761 repsondents to the questionnaire concerning neurological symptoms and exposure to pesticides, including several specific OPs; 69 respondents reported a pesticide-related illness

692 respondents who did not report a pesticide-related illness

Self-reported pesticide poisoning and neurological symptoms on questionnaire

Feeling irritable, depressed, sleeping more than usual, having difficulty falling asleep

Self-reported use of pesticides, including specific OPs

Regression analyses adjusted for age and gender; other co-variates considered were years of schooling, alcohol consumption and farm income; the analyses relate to pesticide-related illness, not specifically OP-related illness, although the use of crop OPs and some specific OPs is a significant factor in assessing pesticide-related illness; co-exposure in respondents to pesticides other than OPs is likely

Univariate and multivariate conditional regression; stepwise logistic regression; ORs and 95% CIs reported; Mantel-Haenszel χ2 test for trend for neurological symptoms significantly associated with acute pesticide-related illness, P values reported

Wesseling 2002 Costa Rica 81 workers, men aged 15-55 years at time of testing, from banana plantations; subjects had had mild poisoning caused by cholinesterase-inhibiting pesticides at least

130 non-poisoned workers, who included field workers and employees in packing plants who had never had any contact with cholinesterase-inhibiting pesticides, but also

Red blood cell RBC) and plasma cholinesterase levels determined at time of testing; review of medical file and report to National Insurance Institute of poisonings; for neuropsychiatric symptoms, use of Q-16 questionnaire which had been validated in

Total scores on the Questionnaire-16 (Q-16) and Brief Symptom Inventory (BSI) – no report of individual symptoms

Self-reported exposure to cholinesterase-inhibiting pesticides as well as to other pesticides; self-reported number of days exposure per year and number of years exposed;

Adjustments made for age, education, long-term exposure to cholinesterase inhibitors, alcohol intake, solvent exposure, loss of consciousness, examiner, time of

Multiple linear regression analyses to assess potential associations between poisoning and test scores; confounding assessed by determining


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Measure of Exposure



1 year prior to testing, and had received treatment at a health centre but had not been hospitalised

included some field workers who had worked with cholinesterase-inhibiting pesticides in varying degrees

Nicaragua, and Brief Symptom Inventory (BSI)

fraction of the day exposed was estimated from job title; report of use of protective equipment; RBC and plasma cholinesterase levels determined at time of testing

day of testing, plasma cholinesterase levels

whether the crude difference of means for each outcome changed meaningfully after addition of potential confounders to a univariate model; unstandardised and standardised coefficients and 95% CIs estimated for subjects, and p values reported

Wesseling 2010 Costa Rica 78 male banana workers who had worked on banana plantations for at least 1 year, and who were aged 15-55 years of age; 54 workers had had mild poisoning caused by OP pesticides, and were treated at a health centre, but were not hospitalised; the remaining 24 were carbamate poisoned workers

130 non-poisoned workers randomly selected from company payrolls

Brief Symptom Inventory (BSI), a standardised questionnaire with a Spanish version which has been used in different cultural settings to measure psychological distress

The nine dimensions of the BSI: somatisation, obsessive-compulsiveness, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety, paranoid ideation, psychoticism; also suicidal ideation

Information about the pesticide responsible for poisoning from medical record and compensation claim to National Insurance Institute, otherwise from self-report; No detail of severity of poisoning in outpatient reports, but presumed to be mild because none of subjects hospitalised; plasma cholinesterase levels measured as indication of recent exposure to cholinesterase inhibiting pesticides

Adjustments made in calculating the prevalence of abnormal scores on the BSI for age, education, recent and cumulative exposure, alcohol, head injury, time of day of examination, examiner

Multivariate logistic regression to compare the odds for presenting abnormal symptom scores among OP-poisoned workers against the odds among the non-poisoned subjects: ORs and 95%CIs reported; adjusted ORs also computed for suicidal ideation


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Measure of Exposure



6.A.4. Case series studies - none

6.A.5. Case reports - none


6.B.1. Cohort studies Albers 2004b USA All Dow employees

engaged in the manufacture of chlorpyrifos (aged 18-65y) who were employed on 1 Sept 1999. Of 66 potentially eligible, 53 took part (80%). All these subjects took part in 1 year follow up

Dow employees engaged in manufacture of Saran (plastic film) with no known exposure to neurotoxicants. Out of 74 workers asked, 60 (81%) participated, with 58 also participating at 1 year follow-up

Brief Symptom Inventory (BSI)

Anxiety Mean score on the BSI

Chlorpyrifos exposed and referents were matched for age, height, weight, BMI, reading scale, smoking pack year and anxiety

Mean and SD for anxiety score on the BSI reported; P-value given

Beard 2011 USA 110 suicides identified through state mortality files and the National Death Index occurring in Agricultural Health Study (AHS) participants between enrolment in the AHS (from 1993 to 1997) and 31 May 2009

81,998 cohort members

Mortality data including date and cause of death obtained by linking Agricultural Health cohort to state mortality files and the National Death Index; International Classification of Diseases codes used to identify suicides

Suicide Self-reported information in questionnaire on pesticide use, including years of use and average days of use per year. Information obtained on 10 pesticide categories, including OPs, and on ever use of 50 specific pesticides

Age, sex, state of residence, race/ethnicity, education level, marital status, number of children in family as a measure of social connection, size of farm worked last year, alcohol consumption during past 12 months, smoking status, ever diagnosed with heart disease or diabetes as measures of chronic disease

Cox proportional hazards regression models to estimate hazard ratios (HR) and 95% CIs for the association of suicide with each measure of pesticide use


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Measure of Exposure



6.B.2. Case-control studies Jamal 2002a UK In this nested

case-control study there were 72 subjects, all sheep farmers, who were classified as having NO, POSSIBLE, PROBABLE OR DEFINITE neuropathy from results in a larger cross-sectional study

15 subjects classified as having NO neuropathy could be considered controls

Mood and affect were assessed by the General Health Questionnaire, which evaluates psychiatric morbidity, and the Hospital Anxiety and Depression scale

Anxiety and depression

Self-reported on questionnaire

Authors state that confounders which were NOT allowed for in the study, and which might need to be considered, were stress,genetic differences, difference in ability to detoxify compounds, and possible combinations of exposure with synergistic effects.

Number of subjects and percentage experiencing neurological symptoms is reported

6.B.3. Cross-sectional studies Bayrami 2012 Iran 40 male

horticulture farmers, age range 15-80, from the village of Hafte in Arak, Iran, exposed to OPs through working with OP pesticides, monitored through the course of a spraying season

40 workers from the same village who were not engaged in any agricultural work, and with no history of job-related exposure to OPs

Psychological symptoms and distress measured by the SCL-90-R (Symptom Checklist-90-Revised); comprehensive clinical examination also performed, and blood samples collected through the course of a spraying season to measure AChE activity; subjects also requested to have EEG test

As a measure of psychological distress, the nine primary symptom dimensions of the SCL-90-R were evaluated: somatisation, obsessive-compulsiveness, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety, paranoid ideation and psychoticism

Self-reported information on working history and OP exposure through questionnaire

Control group of 40 workers were matched on age, sex and education level

Two sample t test used for statistical comparisons, Pearson correlation coefficient for association between variables; all data presented as mean ±SD, with p values of less than 0.05 considered statistically significant

Bazylewicz-Walczak

1999 Poland 26 women working as greenhouse workers,

25 women considered unexposed as the

2 symptom questionnaires that form part of the Neurobehavioural Core

From the Profile of Mood States: tension, anger,

Air sampling carried out at 5 measuring sites in the

Participants were matched with respect to sex,

Two-way ANOVA performed to


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Measure of Exposure



performing standard gardening jobs, employed at 3 large gardening enterprises

control group; these women were employed in canteens, kitchens and administrative jobs in the same 3 gardening enterprises

Test Battery (NCTB) recommended by the WHO, in a Polish adaptation – the Profile of Mood States, and Finnish Subjective Symptoms Questionnaire

fatigue, depression and confusion; from the Subjective Symptoms Questionnaire: fatigue, sleep disturbances, absent-mindedness, depression, lability

greenhouses; concentrations of pesticides on clothing determined from pads placed on different parts of garments, and level of pesticides on the skin determined in washes from the skin

age, level of education and place of habitation; co-exposure of greenhouse workers to other pesticides – carbamates, synthetic pyrethroids and dithiocarbamates

assess differences between exposed and control groups, p values reported

Beseler et al 2008 USA 534 private pesticide applicators, enrolled in the Agricultural Health Study between 1993 and 1997 in Iowa and North Carolina, who self-reported a physician diagnosed depression

17,051 controls who reported never having been diagnosed with depression and who did not feel depressed more than once a week in the past year

Self-reported physician-diagnosed depression reported in questionnaire

Depression Self-reported information on lifetime use of pesticides reported in questionnaire

The OP specific information Is adjusted by state of residence, marital status, age, solvent exposure, alcohol, smoking, education, working a job off the farm, and having no crops or animals

Logistic regression; univariate and multivariable models; results are reported as ORs with 95%CIs

Cox 2005 USA 121 subjects from 49 homes in the state of Mississippi with high levels of methyl parathion (MP), >150μg/100cm²

Control group of 170 subjects from 56 homes in state of Mississippi with non-detectable or very low levels of MP, <15μg/100cm²

Medical evaluation by physician blinded to level of MP in the home; medical history and physical examination included; children of 12 and under had growth evaluations performed; AChE activity measured

Most frequent psychiatric symptoms reported were insomnia and depression

10cm-square wipe samples taken of flat surfaces in different rooms in each house, and results averaged; samples analysed for MP using gas chromatography and mass spectrometry

Evaluating physician judged whether signs or symptoms could be explained by other disease states, tobacco use, second hand tobacco exposure, or other situations

ANOVA and t tests to compare continuous data; Bonferonni test to determine significance in multiple groups; logistic regression to compare findings for groups, p values reported for frequency of symptoms between groups, and OR of


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Measure of Exposure



increased frequency of symptoms in homes with highest levels of MP

Kamel 2005 USA 18,782 white male licensed private pesticide applicators; these were grouped into ‘Cases’ and ‘Controls’ in an evaluation of the association of summary measures of neurologic symptom prevalence with pesticide use and exposure; ‘Cases’ were the 20% of applicators who experienced ≥ 10 symptoms in the year before enrollment

‘Controls’ were the 80% of applicators who experienced < 10 symptoms in the year before enrollment, in an evaluation of the association of summary measures of neurologic symptom prevalence with pesticide use and exposure

Self-reported disease states reported in questionnaire ; symptom questions were based on the Q-16

Fatigue, tension, insomnia, irritability, depression, absentmindedness, difficulty concentrating

Self-reported exposure

Adjustments made for age, state, education, smoking and alcohol use

Linear regression for number of symptoms and logistic regression for dichotomous outcomes; ORs for having ≥ 10 symptoms in the year before enrollment, and ORs for experiencing a specific symptom with high frequency compared with low frequency and OP exposure

Keifer 2000 USA 137 farm workers of both sexes recruited from several orchards in the Wenatchee area, Washington State; 90 workers were tested a second time in year 2

For each worker recruited from an orchard, a worker was recruited from a non-agricultural workplace, including garment manufacturing, hotels and restaurants; non-agricultural workers were matched on age, education to within 3 years and

The depression scale from the Brief Symptom Inventory; the Q-16 neurological symptom questionnaire in Spanish

Depression Self-reported work history, and reported home pesticide exposure, in questionnaire; blood taken for testing AChE levels; testing also undertaken for paraoxonase, chlorpyrifos oxonase and diazinon oxonase on most participants

Model included the confounders of gender, age haemoglobin, Peabody Picture Vocabulary score, thinning hours in 1994 and seasons of thinning pre-1994; many ‘unexposed’ controls reported some work

Multiple linear regression and logistic regression performed; coefficients for effect reported and delta score for difference between Year 2 and Year 1 scores, with p value for the model


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gender exposure in agriculture in the cherry harvest, although OPs are not used on cherries and the exposure was not considered to be significant in terms of pesticide exposure; no objective means of validating workers’ reported previous work experience, and total seasons of farm work may have been underestimated, biasing results towards the no effect direction

Kilburn 1999 USA 22 chlorpyrifos-exposed subjects, aged 34-77 years, seen in a neurotoxicology clinic between 1991 and 1997; they represent 6% of 384 patients evaluated for possible neurobehavioural effects of chemicals

264 men and women aged 18-83 years, unexposed to neurotoxic chemicals, recruited at random from voter registration rolls in Wickenburg, AZ and Smithfield, LA.

Profile of Mood States (POMS)

Depression in particular

Schedules of pesticide application were found for offices of 6 patients, so concentrations of applied chlorpyrifos were known; 4 patients had personal clothing or rugs analysed for chlorpyrifos; for other subjects, self-reported exposure on questionnaire

Age, education, gender, height, family income, hours of general anaesthesia, weight; pre-existing nervous system disease, head trauma; POMS score as possible confounder on neurobehavioural test scores

POMS test scores and standard deviations reported for exposed and unexposed; also symptom frequencies, as means with standard deviations; both compared by analysis of variance, and p values reported

Mackenzie 2010 UK 127 UK sheep 78 controls who For psychiatric symptoms, Anxiety and Self-reported Possible χ2 test


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Measure of Exposure



Ross farmers exposed to OPs (67 working, 60 retired) from the North and South West of the UK; recruitment through writing to farm owners listed on relevant databases, and a more random selection through telephoning every fith person on lists held by the Wool Marketing Board; some farmers were recruited through advertising or replied to articles in the media

were rural police workers and who had never worked in the farming industry (38 working, 40 retired); recruited by advertising through local constabularies, the National Association of Retired Police Officers and the Police Press

The Hospital Anxiety and Depression Scale was used; self-reported medical history, including history of psychiatric, neurological or other serious medical problems ; sample of venous blood taken for analysis of PON1 polymorphisms and activity level

depression exposure in interview through current work, exposure history (number of days or years spent working with OPs), and report of years since last performed sheep dipping

selection bias through different recruitment methods used, and because of differences between working and retired cohorts

performed to compare proportions of subjects in the clinical range for depression and anxiety, p values reported

Rehner 2000 USA 115 heads of households whose homes had been sprayed with methyl parathion; households had been tested and classified by the Environmental Protection Agency (EPA), if a head of household had submitted an application for assistance to the Mississippi State Department of Health

None The Center for Epidemiological Studies- Depression Scale (CES-D), which defines a probable clinical depression as scores exceeding 16; the higher the score, the greater the number of depressive symptoms

Depression The EPA tested methyl parathion levels in the homes by ‘wipe tests’ in the building, and defined 4 levels of exposure, with Level I being the highest and Level IV the lowest

Gender, race, income and length of exposure to methyl parathion

ANOVA used to compare the CES-D mean scores across the four contamination levels, p value reported; hierarchical regression performed to examine the relationship between depression and gender, ethnicity, income , and length of exposure to MP, and a value for


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Measure of Exposure



R² is reported

Salvi 2003 Brazil 37 workers involved in family agriculture of tobacco in December 2001, when they had been working with OPs for 3 months; second evaluation in March 2002 conducted in 25 workers from the original sample who returned for the follow-up, when they had been off OP exposure for 3 months

No unexposed group is included

MINI (Mini International Neuropsychiatric Interview); number of patients with AXIS-I Psychatric Diagnosis (DSM-IV)

Psychiatric AXIS-I diagnosis; score on MINI-mental scale

plasma AchE levels

time and type of OP exposure, use of protective clothing, activities with OPs, history of acute toxicity, alcohol and tobacco use were noted as characteristics of the 25 subjects who completed both evaluations; possible bias because affected subjects might have enrolled more readily in the study, and there is no unexposed comparison group

Comparisons between the two evaluations performed with ANOVA; statistical significance defined as p<0.05

Solomon 2007 UK 9844 men born between 1933 and 1977 resident in 3 rural areas of England/ Wales, of whom 1913 had at some time worked with sheep dip, 832 had worked with other insecticides but never with sheep dip,and 990 had worked with other pesticides but never with sheep dip or insecticides

6109 men born between 1933 and 1977, resident in 3 rural areas of England/ Wales, who had never worked with any type of pesticide

Hospital Anxiety and Depression Scale for psychiatric outcomes; Brief Symptom Inventory (BSI) for questions about somatic symptoms, which were used to derive a ‘somatising tendency’ score, partitioned to 4 levels; questions regarding symptoms of anxiety and depression, and somatic symptoms, related to past 7 days

Psychiatric symptoms associated with COPIND (chronic organophosphate-induced neuropsychiatric disorder), in particular anxiety and depression

Self-reported on questionnaire, lifetime history of work with the following five categories of pesticides: sheep dip, other insecticides, herbicides, fungicides and wood preservatives

Age, area, and a supplementary analysis was undertaken excluding 2320 men who might have worked with pesticides in the past year to investigated possible confounding by acute effects of recent exposure to pesticides

Associations of psychiatric symptoms with exposure to pesticides and other possible risk factors were examined by modified Cox regression, and results summarised as prevalence ratios (PRs) with associated 95% CIs


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Measure of Exposure



Stephens 2004 UK 37 male orchard sprayers from 53 orchard businesses in Hereford, Worcester, East Anglia, Kent and Sussex

26 pig farm workers from same geographical areas as the participating orchards; 31 construction workers from employees of a South Yorkshire construction company

General Health Questionnaire (GHQ)

Vulnerability to psychiatric disorder

Self-reported exposure to pesticides; orchard sprayers took part in an occupational hygiene survey the previous year, in which they were found to have used chlorpyrifos on average for 5 days per year for 14 years; OP metabolites DEP and DETP were measured in urine the morning after spraying in this previous survey, and compared to a sample taken in the present study at time of neuro-psychological testing

Age, weekly alcohol consumption and educational level recorded

GHQ scores were analysed using χ2 test , a p value is reported

6.B.4. Case series studies - none

6.B.5. Case reports - none Table 7. PSYCHIATRIC ILLNESS RESULTS AND CONCLUSIONS

First Author




7.A.1. Cohort studies Delgado 2004 NICARAGUAN STUDY OF 53 SUBJECTS EXPOSED TO OP POISONING

EXPOSURE Low exposure in the study was considered to be moderate occupational poisoning.

Strengths: Prospective cohort design with persons

The results for psychiatric outcomes after


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31 subjects were considered to have had this type of exposure, resulting in symptoms of weakness, dizziness, headache or blurred vision, in combination with at least one symptom out of: vomiting, sialorrhoea (excessive salivation), fasciculations (muscle twitches), abdominal pain, diarrhoea, miosis (contraction of the pupil), wheezing or crepitus (crackling sounds). The medium exposed group were 15 persons who had had severe occupational poisoning, including the above symptoms and either diminished consciousness or cyanosis. The high exposed group consisted of 7 subjects with severe poisoning via the oral route; these were non-occupational poisonings, six of which were suicide attempts and one was a massive accidental ingestion. Types of OP that had been used by subjects were: methamidophos (20 subjects), chlorpyrifos (17), edifenphos(6), methy parathion (4), terbufos (3), phorate (1), malathion (1), fenthion (1) HEALTH EFFECTS The adjusted Q-16 neuropsychiatric symptom score increased over 2 years in all the groups who had been exposed by poisoning with OP pesticides in this Nicaraguan study, the low-exposed, medium-exposed and high-exposed groups, including also a smaller change among the unexposed persons. The greatest increase in the number of symptoms was observed in the medium-exposure group, which increased from 3.9 to 10.8 symptoms (after exponentiation) from hospital discharge to 2 years. The table below shows the unadjusted means and standard deviations of the log of the Q-16 symptom score by exposure status, with P-values from the two-way analyses of variance (unadjusted) and analyses of covariance (adjusted for age, education, and alcohol consumption), contrasting each exposed group with the unexposed group: Mean and standard deviation of Q-16 score

Unexposed (N=28)

All exposed (N=53)

Low exposed (N=31)

Medium exposed (N=15)

High exposed (N=7)

Mean SD Mean SD Mean SD Mean SD Mean SD At discharge 1.72 0.60 1.58 0.90 1.52 0.96 1.58 0.74 1.83 0.97

7 weeks’ follow-up 1.72 0.49 1.46 0.97 1.43 0.97 1.27 1.03 2.03 0.70 2 years’ follow-up 1.84 0.68 2.19 0.73 2.10 0.78 2.38 0.62 2.15 0.76

P value for main effect (exposure) Unadjusted - 0.90 0.65 0.92 0.25 Adjusted - 0.85 0.60 0.99 0.35

P value for interaction between time and exposure (days of use of OPs)

followed up after 2 years Limitations: 28% of referent group lost to follow-up No direct measure of exposure Small study group size The Q-16 symptom score includes symptoms relating to the peripheral nervous system as well as psychiatric symptoms, so is not a clear measure of psychiatric effects; the Q-16 was developed to monitor central nervous system effects on workers exposed to solvents, and may not have the same validity in workers exposed to neurotoxic chemicals

acute exposure to OPs suggest an increase in symptoms in the exposed group overall; however, the Q-16 gives an overall score, not a report on individual symptoms, and includes physical as well as psychiatric symptoms


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Unadjusted - 0.007 0.020 0.001 0.80

Adjusted - 0.008 0.025 0.002 0.93

Thus, the adjusted P-values for increase in number of symptoms with time were P= 0.025 for the low exposed group, P=0.002 for the medium exposed group, and P=0.93 for the high exposed group. For all exposed, the adjusted P-value was 0.008. In summary, neuropsychiatric symptoms were in excess 2 years after hospital discharge in the low- and medium-exposure groups and all the groups combined; results were imprecise for the small high-exposure group

Kawana

2001 STUDY FOLLOWING UP VICTIMS OF THE TOKYO SUBWAY SARIN ATTACK TREATED AT ST LUKE’S HOSPITAL IN TOKYO, UP TO 5 YEARS AFTER THE INCIDENT EXPOSURE Unknown quantity of sarin released in the Tokyo subway; symptoms self-reported on questionnaire HEALTH EFFECTS The table below indicates the frequency of psychiatric symptoms included in the St. Luke’s Hospital questionnaire in victims of the Tokyo subway sarin attack: Frequency of psychiatric symptoms in victims of Tokyo subway attack

Frequency and percentage of symptoms in St. Luke’s Hospital sarin patients Symptom 1997(No. and %

of 283 patients) 1998 (No. and % of 206 patients)

2000 (No. and % of 191 patients)

Depressed mood 42* (14.8) 20 (9.7) 25 (13.1)

Avoidance of the subject of the incident

42 (14.8) 29 (14.1) 30 (15.7)

Diminished interest, numbing

18 (6.4) 9 (4.4) 11 (5.8)

Irritability 23 (8.1) 16 (7.8) 14 (7.3)

Difficulty falling or staying asleep

21 (7.4) 18 (8.7) 15 (7.9)

Hypervigilance 7 (2.5) 8 (3.9) 3 (1.6) Flashbacks 41 (14.5) 28 (13.6) 25 (13.1) Distressing dreams, nightmares

26 (9.2) 19 (9.2) 11 (5.8)

Fear in the subway or at the incident

25 (8.8) 18 (8.7) 21 (11.0)

Strengths The study follows up a group of patients for 2, 3 and 5 years after the sarin attack Limitations The study is largely descriptive, with limited statistical analysis of the results The questionnaire used was developed by St. Luke’s Hospital, and is thus an unpublished, non peer-reviewed instrument No possible confounding factors are examined

The study suggests that some victims of the sarin attack who were treated at St. Luke’s Hospital continued to have psychological symptoms up to 5 years after the incident. The number of victims diagnosed with PTSD depends upon the diagnostic criteria used


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In the table above, the psychological symptoms had no significant decrease in incidence, apart from ‘Depressed mood’, which decreased significantly (*P <0.1) in 1998. The symptoms fall into 3 categories associated with Post Traumatic Stress Disorder (PTSD), those of Avoidance, Hyperarousal, and Re-experience. The frequencies and percentages show that a number of victims continued to experience these symptoms up to 5 years after the attack. In a comparison of symptom incidence (%) of the St. Luke’s Hospital group with the 3 reference groups (655 victims of the Tokyo sarin attack studied by a non-governmental organisation (NGO), 88 victims of the 1994 Matsumoto sarin attack, and 87 Matsumoto controls), the incidence of all psychological symptoms was reported to be higher by the NGO. The symptom incidence rate was lowest in the Matsumoto control group, and the rates of both the St. Luke’s and Matsumoto victims groups were in the middle. The incidence of diagnosed PTSD according to different PTSD diagnostic criteria was also compared in the St. Luke’s group and the NGO cohort. The incidence reported for the NGO group was consistently higher, and at least double the St. Luke’s incidence. Adding physical symptoms to the diagnostic criteria (PTSD-Nakano) made diagnosis of PTSD much more frequent in the St. Luke’s group, with an incidence of 9.7% for 1998 rising to 14.1% in 2000

Nakajima 1999 STUDY OF VICTIMS OF THE INCIDENT OF SARIN RELEASE IN A RESIDENTIAL DISTRICT OF MATSUMOTO IN JAPAN IN 1994 EXPOSURE 12 litres of sarin were released in a residential area of Matsumoto City, but individual exposure, or proximity of individuals to the release, were not measured or estimated; 93 persons had cholinesterase activity measured 4 weeks after the sarin exposure and again for the 1 year survey The relationship between cholinesterase activity four weeks after sarin exposure and symptoms 1 year after the sarin incident in 93 subjects is given in the table below: Cholinesterase activity 4 weeks after sarin exposure and symptoms 1 year after the event in 93 subjects

Symptoms Number of subjects

Erythrocyte-Anticholinesterase (IU/L)

Serum- Anticholinesterase (IU/L)

Strengths 93 of the subjects had cholinesterase activity records from 4 weeks after the incident which could be considered for any association with symptoms at 1 year after the event The study follows some subjects for up to 3 years after the event Limitations The symptoms of bad dreams and

There is insufficient evidence from the study to suggest that the symptoms of bad dreams and insomnia were significant health outcomes for victims of the Matsumoto sarin attack: evidence is limited in scope, no validated tests are used , and the number of victims reporting the symptoms is very small


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Symptoms (any type of symptoms)

Yes 20 No 73

1.38±0.42● 1.56±0.29

5.57±0.77 5.48±0.93

Bad dreams Yes 3 No 90

1.6,1.4,1.5 1.52±0.33

6.57±0.85● 5.46±0.88

Insomnia Yes 4 No 89

1.53±0.10 0.52±0.33

6.15±1.08 5.47±0.88

Notes: subjects are divided into those who experienced symptoms and those who did not; the numbers given represent the mean ±SD, or the values when the numbers were 3 or less; ● significant differences were noted between subjects who had and did not have symptoms at p<0.05 From the table it can be seen that, of the 93 subjects with AChE measurements, those who had any type of symptoms 1 year after the event had had lower erythrocyte AChE activity 4 weeks after the event; also, those who had bad dreams 1 year after the event had had significantly lower activity of serum AChE, but the mean values for erythrocyte AChE are not significantly different in subjects reporting bad dreams, and the number of subjects reporting the symptom was very small (3). HEALTH EFFECTS The two psychiatric symptoms reported on throughout the surveys are bad dreams and insomnia; the prevalence of these symptoms described subjectively 1 year after sarin exposure in the Matsumoto incident is given in the table below: Prevalence of symptoms 1 year after sarin exposure in Matsumoto incident

Symptoms Admitted patients

Out patients Non- patients

Total

Bad dreams 3 (27.3) 3 (11.5) 0 (0) 6 (11.1)

Insomnia 2 (18.2) 4 (15.4) 2 (11.8) 8 (14.8)

Notes: Admitted patients are victims who required hospitalisation; Out patients were victims who consulted doctors but did not require hospitalisation;

insomnia are self-reported and self-assessed There is limited statistical analysis of the results


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Non-patients were those who had symptoms but did not consult a doctor Numbers of subjects are reported in the table, with percentages in brackets Thus, at 1 year after exposure, there were no significant differences in symptom prevalence between the three groupings of patients. The table below shows the relationship between sarin exposure and the psychiatric symptoms 3 years after the sarin incident: Symptoms of sleep disturbance 3 years after sarin exposure in Matsumoto incident

Symptoms Non-victims 669

All victims 167

Odds Ratio (95% CI)

Victims A 167

Odds Ratio (95% CI)

Bad dreams 7 (1.0) 5 (3.0) 2.92 (0.92-9.32)

2 (1.2) 1.15 (0.24—5.56)

Insomnia 15 (2.2) 9 (5.4)● 2.48 (1.07-5.78)

2 (1.2) 0.53 (0.12-2.28)

Notes: Non-victims are subjects who did not have one or more symptoms immediately after the sarin incident; All victims are subjects who had one or more symptoms immediately after the sarin incident; Victims A are subjects who had one or more symptoms both 1 and 3 years after the sarin incident ● significant differences noted between victims and non-victims at p<0.05 A significant difference between victims and non-victims is noted for insomnia, but with a small number of subjects reporting the symptom (9)

Tochigi 2002 STUDY OF PTSD IN VICTIMS OF THE TOKYO SUBWAY SARIN INCIDENT IN 1995 EXPOSURE Release of sarin in the Tokyo subway; serum cholinesterase level within 1-3 days of the attack was available in 25 victims (13 males, 12 females) out of the total 34. Serum levels of total cholesterols and of uric acid were measured in all 34 victims. The authors give the reason for investigating cholesterol and uric acid levels that these may change under emotionally stressful conditions and in mental disturbances, including anxiety disorders; however, only the AChE levels are directly relevant to the biological effect of sarin and therefore to this review. There were no significant differences in serum levels of cholesterols and uric acid between victims and controls 5 years after the attack; however, serum cholinesterase level was significantly reduced 5 years after the attack in victims with the development of PTSD compared to controls, as shown in

Strengths The study contains biomonitoring data for all subjects and controls (cholinesterase levels) at 5 years after the attack, and also has a record of cholinesterase levels in approximately two-

Although the study results indicate reduced cholinesterase levels 5 years after the sarin attack in victims who developed PTSD, the suggested association between the exposure and


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the following table: Cholinesterase levels in victims 5 years after Tokyo subway attack

Number of subjects Serum cholinesterase, Ch-E (IU/l), 5 years after attack

Victims 34 313±77 Development of PTSD (-) 26 (+) 8

324±81 276±47*

Controls 34 347±76 Note: * P<0.02 (t= -2.52, P<0.02, t-test) Serum cholinesterase levels immediately after the exposure to sarin, as well as 5 years after the attack, were available in 25 victims and are shown in the table below: Cholinesterase levels in 25 victims immediately after, and 5 years following, the Tokyo subway attack

Development of PTSD

Number of subjects

Within 1-3 days of the attack (IU/l)

5 years after the attack (IU/l)

(-) 18 127±42 319±58 (+) 7 101±31 276±48

The difference in levels between victims with and without development of PTSD, immediately after the attack and 5 years later, did not reach statistical significance; however, the serum cholinesterase level within 1-3 days of the attack had a significant correlation with scores on the present IES-R score, which are reported below (r= -0.515, P=0.008) HEALTH EFFECTS The results for diagnosis of PTSD and scores of IES-R (Impact of Event Scale – Revised) and STAI (State trait Anxiety Inventory) in 34 victims of the Tokyo subway sarin incident are given below:

thirds of subjects immediately after the attack for comparison Use of the MINI (Mini-International Neuropsychiatric Interview) for the subjects is useful in indicating the current and previous status of other mental disorders, which could potentially affect development of PTSD The study uses validated measuring instruments Limitations Small sample size Co-morbidity as a confounding or complicating factor - many of the subjects who developed PTSD were evaluated as also suffering from other mental disturbances, which could suggest that subjects who are susceptible to

development of PTSD has to be treated with caution because of the small number of subjects, and the effect of co-morbidity: the possibility that individuals who already suffer from a mental disorder may be more susceptible to develop PTSD


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Diagnosis of PTSD: IES-R and STAI scores in victims of Tokyo subway attack Notes: * P<0.02 **P<0.02, comparison between victims with the development of PTSD and controls (t-test) ***P<0.05, compared with the victims without the development of PTSD (t-test) 8 victims developed PTSD due to the sarin attack; out of those, 2 were currently diagnosed with PTSD. No subjects had a history of PTSD due to other events; the IES-R score was significantly higher in the 8 victims with the development of PTSD than those without the development (P<0.02, t-test); STAI scores were elevated in victims with development of PTSD: State Anxiety of the STAI was increased in victims with development of PTSD, compared to those without development (P<0.01, t-test) and to controls (P<0.05, t-test); Trait Anxiety of the STAI was also elevated in victims with the development of PTSD compared to those without development (P<0.05, t-test); there was a trend for a weak correlation between the IES-R score and the STAI scores, but the correlations were not statistically significant; Results of the MINI According to the MINI, high co-morbidity of other mental illnesses was observed in victims who developed PTSD: 2 currently suffering from agoraphobia, 2 from panic disorder (one with

Number of subjects (male: female)

Age (mean ±SD)

IES-R (mean ±SD)

State Anxiety (mean ±SD)

Trait Anxiety (mean ±SD)

Victims 34 (20:14)

43.8±13.4 16.1±12.3 39.8±8.3 41.5±9.7

Development of PTSD due to the attack (-) 26 (15:11) (+) 8 (5:3)

43.3±12.5 45.5±16.8

13.4±11.0 25.0±12.5*

37.7±7.7 46.4±6.5**

39.6±7.4 47.5±13.7***

Currently diagnosed with PTSD 2 (2:0)

59.0±14.1

41.0±12.7

43.5±5.0

58.0±5.7

Controls 34 (18:16)

43.7±12.5 Not determined

38.4±8.4 40.9±8.3

other mental disorders may be more likely to develop PTSD, and Trait Anxiety score of STAI is significantly higher in victims who developed PTSD than in victims who did not


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agoraphobia and the other without), 1 from major depression, 1 from major depression with agoraphobia, 1 from risk of suicide; most of the victims without development of PTSD were not suffering from other mental illnesses, except one currently suffering from alcoholism

Tochigi 2005 ANALYSIS OF SUBCLUSTERS OF PTSD SYMPTOMS IN VICTIMS OF THE TOKYO SUBWAY SARIN INCIDENT DESCRIBED IN PREVIOUS (TOCHIGI 2002) PAPER EXPOSURE Release of sarin in the Tokyo subway; serum cholinesterase level within 1-3 days of the attack was available in 25 victims (13 males, 12 females) out of the total 34. Serum levels of total cholesterols and of uric acid were measured in all 34 victims. HEALTH EFFECTS 8 out of 34 victims developed PTSD due to the Tokyo subway sarin attack; among these 8, the following co-morbid DSM-IV disorders were confirmed using the Mini-International Neuropsychiatric Interview: panic disorder n=2, major depression n=2, agoraphobia n=2. Current and Lifetime scores of the PTSD symptoms were evaluated in all 34 victims using the CAPS scale, and Pearson’s correlation coefficients between the CAPS factors and serum cholesterols, cholinesterase or uric acid are shown in the table below: Cholesterol, uric acid and cholinesterase levels in victims of Tokyo subway attack

Diagnosis of PTSD

Factors T-cho HDL-C LDL-C UA Ch-E Ch-E within 1-3 days of the attack

Current Re-experiencing

-0.069 0.226 -0.202 -0.351* -0.267 -0.499*

Avoidance -0.169 0.182 -0.299 -0.086 -0.182 -0.335 Increased

arousal -0.052 0.126 -0.167 -0.196 -0.071 -0.333

Total -0.113 0.208 -0.263 -0.248 -0.196 -0.452* Lifetime Re-

experiencing -0.098 0.278 -0.279 -0.256 -0.337 -0.528**

Avoidance -0.102 0.373* -0.260 -0.247 -0.399* -0.400* Increased

arousal 0.108 0.200 -0.043 -0.180 -0.217 -0.358

Total -0.031 0.313 -0.211 -0.250 -0.350* -0.465* Notes: T-cho: total cholesterol HDL-C: high-density lipoprotein cholesterol

Strengths cholinesterase levels for all subjects and controls at 5 years after the attack are used from the 2002 study, and measurements from the previous study are also available for approximately two-thirds of subjects immediately after the attack The CAPS and STAI are validated measuring instruments Limitations These results are published as a Letter to the Editor, and may not have been peer reviewed Small number of victims with PTSD

The study presents some results that show significant correlations of aspects of PTSD with cholinesterase levels immediately after the attack; however, the number of subjects in the study is too small to draw firm conclusions about the effect of exposure to sarin on cholinesterase levels and subsequent development of PTSD. There may also be a confounding effect of co-morbidity on the development of PTSD


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LDL-C: low-density lipoprotein cholesterol UA: uric acid Ch-E: cholinesterase * p<0.05 ** p<0.01 The results show that the majority of significant correlations of aspects of PTSD are with cholinesterase levels immediately after the attack. Current and lifetime scores of re-experiencing, and the lifetime score of avoidance, as well as the overall current and lifetime PTSD score, are correlated with serum cholinesterase level after the attack. Pearson’s correlation coefficients between scores for STAI and serum cholesterols, cholinesterase or uric acid in the 34 victims and 34 matched controls are shown in the table below: Correlation coefficients between STAI scores and cholesterol, uric acid and cholinesterase levels in victims of Tokyo subway attack

Subjects Factors T-cho HDL-C LDL-C UA Ch-E Ch-E within 1-3 days of the attack

Victims State anxiety -0.082 0.358* -0.319 -0.243 -0.059 -0.297

Trait anxiety 0.100 0.234 -0.110 -0.051 0.039 0.000

Controls State anxiety -0.094 0.069 -0.152 -0.176 -0.066 N.D. Trait anxiety -0.270 0.049 -0.334 -0.206 -0.153 N.D.

Notes: T-cho: total cholesterol HDL-C: high-density lipoprotein cholesterol LDL-C: low-density lipoprotein cholesterol UA: uric acid Ch-E: cholinesterase N.D.: not determined * p<0.05 The correlations between State and Trait Anxiety with cholinesterase levels and cholinesterase levels 1-3 days after the attack in the victims do not indicate significance

7.A.3. Cross-sectional studies Kawada 2005 STUDY OF INSOMNIA IN FEMALE VICTIMS OF THE TOKYO SUBWAY SARIN ATTACK

Strengths Subjects and

In view of the limitations, the


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EXPOSURE Quantity of sarin to which individuals were exposed is not known HEALTH EFFECTS The results from the questionnaire relating to insomnia as an outcome of the Tokyo subway sarin attack are given in the table below: Prevalence of insomnia in female victims of Tokyo subway attack compared to controls

Prevalence of insomnia (%) evaluated by self-administered questionnaires to Japanese women ages 30 to 69 years Age (yr) Poor Sleep S1 S2 S3 S4 Insomnia Case 30-49 39.3** 31.1** 16.4** 16.4** 23.0** 31.1* n=61 (24) (19) (10) (10) (14) (19) Control 30-49 16.7 11.8 6.5 2.2 5.4 17.2 n=186 (31) (22) (12) (4) (10) (32) Case 50-69 50.0* 28.6 28.6 21.4* 28.6* 35.7 n=14 (7) (4) (4) (3) (4) (5) Control 50-69 11.0 19.3 11.7 6.2 9.0 24.8 n=145 (16) (28) (17) (9) (13) (36)

Notes: S1=difficulty falling asleep S2=intermittent awakening S3=early morning awakening S4=a feeling of light over-night sleep Numbers of subjects given in brackets * p<0.05 ** p<0.001 Control female subjects lived along Route 17 of Maebashi city In summary, for the younger exposed group age 30-49, percentages of poor sleep, difficulty falling asleep, intermittent awakening, early morning awakening, feeling of light overnight sleep and insomnia were significantly higher than those for the control group; for the older exposed group age 50-69, there was a significantly higher prevalence of poor sleep, feeling of light overnight sleep and early morning awakening compared to the control group. When the prevalence of insomnia was compared among the victims by the chi square test, there was a significant difference in the distribution of participants, with more participants being under 50 years of age

controls are precisely grouped by sex and age Limitations Evaluation of sleep is subjective, and accuracy of questionnaire not verified by performance of multiple sleep latency test or overnight sleep monitoring No standardised definition of insomnia is used The criteria used to define insomnia are taken partly from DSM-IV, which is criticised by some psychologists as lacking validity and reliability Small number of victims in the age 50 to 69 group (n=14) Authors report that the data obtained are from victims who attended the health check in 2003, and not chosen through a precise sampling

results of this study do not provide conclusive evidence that the insomnia experienced by victims is related to their sarin exposure


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procedure from the entire number of subjects exposed to sarin

McCauley 2001 STUDY OF GULF WAR VETERANS POSSIBLY EXPOSED TO SARIN AT KHAMISIYAH, IRAQ EXPOSURE Exposure to sarin was assumed for troops within a 50 km radius of the detonated chemical warfare agents at Khamisiyah in Iraq HEALTH EFFECTS Odds Ratios and 95% CIs for psychiatric symptoms reported by subjects approximately 9 years after the detonation of the munitions dump at Khamisiyah in Iraq are shown in the table below: Psychiatric symptoms reported by Gulf War veterans approximately 9 years after Khamisiyah incident

Deployed (n=1,263) vs. Non-deployed (n=516) OR (95% CI)

Khamisiyah (n=653) vs. Non-Khamisiyah (n=610) OR (95% CI)

Khamisiyah Witness (n=162) vs. Non-Khamisiyah Witness (n=405) OR (95% CI)

Changes in memory 5.6 (4.3-7.4) 1.3 (1.0-1.7) 1.7 (1.2-2.4)

Difficulty sleeping 4.1 (3.1-5.5) 1.0 (0.8-1.4) 2.0 (1.2-3.5) Depression 3.7 (2.7-5.1) 0.9 (0.6-1.2) 1.6 (1.1-2.4)

Unusual irritability/anger 4.1 (3.1-5.6) 1.0 (0.6-1.5) 1.3 (0.9-1.9) Mood swings 4.8 (3.6-6.7) 1.1 (0.8-1.5) 1.4 (1.0-2.1)

Choking sensation 2.1 (1.3-3.7) 0.9 (0.5-1.7) 1.6 (0.9-3.1) Problems following directions or instructions

4.3 (2.6-7.6) 1.0 (0.6-1.5) 1.4 (0.8-2.4)

Difficulty concentrating 4.3 (3.1-6.2) 1.0 (0.8-1.4) 1.3 (0.9-1.9) Effects from confined places

3.6 (2.3-6.0) 0.6 (0.4-0.9) 1.6 (0.9-2.7)

Notes: ‘Deployed’ were personnel deployed within a 50km radius of Khamisiyah, as well as personnel deployed in the Gulf but outside the 50km radius; these results are adjusted for age, gender, and region of residence ‘Non-deployed’ were personnel who had been on active duty at the time of the Gulf War, but not deployed to the Gulf

Strengths Relatively large number of subjects took part in the study Limitations No direct measures of exposure Several possible sources of bias: selection bias possible because sample limited to persons whose telephone numbers could be tracked down by common search mechanisms; the Khamisiyah population of veterans was not evenly distributed throughout the US, the majority having North Carolina or Georgia addresses, so there may be bias due to location; also possible misclassification,

Due to the limitations of the study, in particular the difficulty of establishing precise location of personnel, the evidence provided is not conclusive that subjects directly involved in the detonation of chemical warfare agents containing sarin and cyclosarin, or located close to the site of the detonation, were more significantly affected than other personnel deployed in the Gulf area; significant associations relate more clearly to Gulf War veterans compared to veterans not deployed to the Gulf


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The ‘Khamisiyah vs. Non-Khamisiyah’ results are adjusted for age and gender; ‘Khamisiyah’ were personnel thought to be operating within a 50km radius of the munitions dump; The Khamisiyah personnel are further divided into ‘Khamisiyah Witness’, a subgroup who had been involved in or watched the Khamisiyah detonation (n=162), and ‘Non-Khamisiyah Witness’ (n=405) who had not been involved; the remaining 86 individuals were unsure which subgroup they belonged to The results appear to indicate significant Odds Ratios for subjects Deployed to the Gulf versus Non-Deployed subjects; however, in the Khamisiyah versus Non-Khamisiyah analysis, there do not appear to be any significant associations. When the Khamisiyah subjects are subdivided into Witness and Non-Witness, there appear to be some significant associations again, suggesting that those who were directly involved in the detonation or may have been closest to the site may have suffered more symptoms. However, the precise location of personnel is in some doubt: 86 subjects among the Khamisiyah personnel were unsure whether they were ‘Khamisiyah witness’ or ‘Non-Khamisiyah witness’. Furthermore, the precise location of personnel generally is in some doubt, as the authors report that 50 subjects thought to be by the Department of Defense in the Khamisiyah-deployed group reported that they had never been in Iraq. Due to the difficulty of precisely establishing exposure of individuals, the results cannot be seen as conclusive evidence of an association between presumed exposure and symptoms in the subjects. Co-exposure to other chemicals was also possible

because 50 subjects considered by the Department of Defense to be in the Khamisiyah-deployed group reported they had never been in Iraq recall bias because the data collection took place 8-9 years after the events

McCauley 2002 STUDY OF GULF WAR VETERANS POSSIBLY EXPOSED TO SARIN AT KHAMISIYAH, IRAQ EXPOSURE Exposure to sarin was assumed for troops within a 50 km radius of the detonated chemical warfare agents at Khamisiyah in Iraq HEALTH EFFECTS Frequency of diagnosed medical conditions reported by Gulf War Veterans and 95% likelihood-based CIs for several psychiatric symptoms are shown in the table below: Frequency of diagnosed medical conditions reported by Gulf War veterans approximately 9 years after Khamisiyah incident

Symptom

Frequency (%) ND KHAM NKHAM n=516 n=653 n=610

OR (95% CI) Deployed KHAM vs ND vs NKHAM

PTSD 0.8 7.0 8.7 14.9(5.6-60.9) 1.0(0.6-1.7) Major depression with hospitalisation

0.6 1.7 2.0 5.1(1.5-32.1) 1.1(0.4-3.3)

Obsessive compulsive disorder

1.0 1.8 1.3 1.7(0.7-5.1) 1.6(0.5-5.4)

Strengths and Limitations as above for McCauley 2001 study

The findings of the study do not appear to provide evidence of psychiatric effects associated with exposure to the detonation of sarin/cyclosarin; any significant associations relate to Gulf War veterans when compared to veterans who were not deployed to the Gulf


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Other psychiatric disease or psychosis

2.3 3.1 5.2 1.7(0.9-3.4) 0.6(0.3-1.2)

Notes: KHAM – Khamisiyah veteran; NKHAM – non-Khamisiyah veteran; ND – veteran not deployed to the Gulf The results suggest that veterans potentially exposed to low levels of sarin/cyclosarin in the vicinity of Khamisiyah did not differ from other veterans in the Gulf not deployed to Khamisiyah on self-reported psychiatric diagnoses (KHAM vs. NKHAM). Some significant differences were observed in the comparison of outcomes among veterans deployed to the Gulf and other veterans who were not in the Gulf War (Deployed vs. ND)

Nishiwaki 2001 EFFECTS OF SARIN ON RESCUE WORKERS AND POLICE OFFICERS INVOLVED IN THE TOKYO SUBWAY SARIN ATTACK EXPOSURE No measure of quantity of sarin to which rescue workers were exposed, so high-exposed persons were deemed to be those hospitalised immediately after poisoning, and low-exposed were those who attended hospitals as outpatients HEALTH EFFECTS Scores for the two tests of psychiatric outcomes reported in the study, at 3 years after the Tokyo subway sarin attack, are given in the table below: IES and GHQ scores in victims of Tokyo subway attack

EXPOSED High (n=25) Low (n=29) Mean SD Mean SD

REFERENTS (n=52) Mean SD

Psychometric test

IES score 13.3 14.2 12.0 11.9 8.0 12.5

GHQ score 4.4 5.6 3.3 2.1 2.8 3.6

The p values are reported not to indicate any significant differences, but both the IES and GHQ scores can be seen to increase in a dose-dependent manner

Strengths Exposed and referent subjects are taken from the same groups of workers, and matched by age Use of two validated tests Limitations Small sample size Individual psychiatric symptoms not reported, only an overall score The main focus of the study were tests of neurobehavioural function, and the IES and GHQ scores were used as possible confounding variables in the analysis

The test scores reported in the study do not suggest significant differences between exposed and unexposed workers in the Tokyo subway sarin incident in outcomes of psychological stress and general mental health; there is some suggestion that scores increase in a dose-dependent manner, but evidence is not substantial

Ohtani 2004 STUDY OF PTSD SYMPTOMS IN VICTIMS OF THE TOKYO SUBWAY SARIN ATTACK 5 YEARS Strengths The study


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AFTER THE INCIDENT EXPOSURE Quantity of sarin not known; subjects had been treated in emergency wards after the attack HEALTH EFFECTS Using the self-rating questionnaire developed by staff at St. Luke’s International Hospital, the following mental symptoms were found most frequently in subjects: recollections of the event in 65.5% of subjects; avoidance of places that trigger recollections of the trauma in 48.3% of subjects; tension in 43.3% of subjects; forgetfulness in 42.9% of subjects. SCORES OBTAINED ON TESTS IES-R results The IES-R (Impact of Event Scale - Revised) is a self-rating questionnaire associated with PTSD symptoms, and contained 22 questions. Subjects had to answer each question by choosing one of 5 levels, from extremely (rated as 4 points) to not at all (0 points). The mean total score was 16.4 (SD 12.1), and a score between 12 – 32 suggests a person presents several symptoms of PTSD. The most frequent symptom was feeling watchful and on guard; CAPS results On the CAPS evaluation (Clinician-Administered Post-Traumatic Stress Disorder Scale), 11 victims were evaluated as having lifetime PTSD, and 5 of those had current PTSD. The most frequent symptoms were difficulty in falling or staying asleep in the current diagnosis, and psychological distress at exposure to cues in the lifetime diagnosis. MINI results The 11 patients diagnosed with lifetime or current PTSD were defined as the PTSD group, and the 23 subjects not diagnosed with PTSD as the non-PTSD group. According to MINI (Mini International Neuropsychiatric Interview), 6 subjects in the PTSD group had other mental disorders such as agoraphobia, panic disorder with or without agoraphobia, major depression, or depression with agoraphobia; there were no mental disorders in the non-PTSD group. STAI results On the STAI (State-Trait Anxiety Inventory), the mean scores of both state and trait anxiety were significantly higher in the PTSD group than in the non-PTSD group: state anxiety in PTSD group, mean 46.0, SD 6.3, in non-PTSD group, mean 36.8, SD 7.4, d.f.=32, t=2.50, P<0.01; trait anxiety in PTSD group, mean 46.6, SD 12.2, in non-PTSD group, mean 39.0, SD 7.2, d.f.=32, t=2.50, P<0.05 Correlational analysis

Study uses several tests for the evaluation of PTSD; apart from one, the St. Luke’s Questionnaire, the tests used are standard, validated tests Limitations Small group of subjects High prevalence of PTSD found in this study may be at least partly caused by the bias of subject selection

suggests that both PTSD and non-specific mental symptoms persist in some victims of the Tokyo subway sarin release 5 years after the attack


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the total IES-R score was correlated significantly with both total current and total lifetime CAPS scores: current CAPS score, d.f.=32, r=0.521, P<0.01; lifetime CAPS score, d.f.=32, r=0.418, P<0.05 Overall result for PTSD 32.4% of the subjects developed PTSD during the 5 years after the attack

Proctor 2006 STUDY OF GULF WAR VETERANS CONSIDERED TO HAVE BEEN EXPOSED TO SARIN AFTER DETONATION OF THE MUNITIONS DUMP AT KHAMISIYAH, IRAQ EXPOSURE Plume modelling of estimated release and dispersion of sarin and cyclosarin around the Khamisyah site was performed; persons were considered exposed if they were located in an area with an exposure estimate greater than the General Population Limit of 0.01296 mg min/m³, defined by the US Army and Centers for Disease Control and Prevention. Exposure estimates ranged from 0.035 to 0.144 mg min/m³ HEALTH EFFECTS The table below shows results for some psychiatric outcomes in Gulf War veterans thought to have been exposed to sarin and cyclosarin when the munitions dump at Khamisiyah was blown up in 1991: Mississippi PTSD and BSI scores in Gulf War veterans 4 to 5 years after the Khamisiyah incident

Reported above are the mean scores for the group, with 3 Standard Deviation extreme scores (top or bottom) recorded in brackets; both the Mississippi PTSD score and BSI index of severity are greater for the low-no-exposure group than the high exposure group, which is contrary to what might be expected, and the scores appear to increase with decreasing exposure. The p values do not suggest significance.

High exposure group (n=23) (exposure range: >0.072-0.144 mg min/m³)

Moderate exposure group (n=47) (exposure range: 0.01296-0.072 mg min/m³)

Low-no exposure group (n=70) (exposure range: <0.01296 mg min/m³)

p-value

Mississippi PTSD scale score

66.7(16.8) 73.0(18.4) 74.1(25.3) 0.36

BSI – general severity index for psychological symptoms

0.55(0.55) 0.65(0.61) 0.75(0.82) 0.45

Strengths Modelled estimate of exposure is available Data collected before the Khamisiyah incident was publicly disclosed, so less scope for recall bias The study uses validated measuring instruments Limitations Small sample sizes Results for psychiatric outcomes appear to show a trend in the opposite direction of what might be expected – the high exposure group had the lowest symptom and severity scores of the 3 exposure groups

The results for psychiatric outcomes in Gulf War veterans exposed to sarin and cyclosarin in the Khamisiyah incident do not provide conclusive evidence of effects of exposure


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Current PTSD and Major Depression diagnosis in Gulf War veterans 4 to 5 years after the Khamisiyah incident

High exposure group (n=23) (exposure range: >0.072-0.144 mg min/m³)

Moderate exposure group (n=47) (exposure range: 0.01296-0.072 mg min/m³)

Low-no exposure group (n=70) (exposure range: <0.01296 mg min/m³)

p-value

Current PTSD diagnosis (%)

0 6.4 5.8 0.48

Current MDD (major depression) diagnosis (%)

0 2.1 12.9 0.03

The percentage of PTSD and MDD diagnosis is higher for the low-no-exposure group, and the diagnosis of major depression appears to be significant for the low-exposure group POMS test results higher exposure was not significantly related to mood state on the POMS test when examined by exposure category, or in linear trend models; in linear trend models, the adjusted parameter estimates were: -1.2 (95% CI (-3.8, 1.2), p=0.32) for fatigue; 0.11 (95% CI (-2.0, 2.2), p=0.92) for tension; -1.8 (95% CI (-4.2, 0.46), p=0.12) for depression; -1.2 (95% CI (-3.9, 1.5), p=0.39) for anger; -1.3 (95% CI (-3.2, 0.57), p=0.17) for confusion

Spencer 2001 STUDY REPORTING ON GULF WAR VETERANS PRESUMED TO BE EXPOSED TO SARIN/CYCLOSARIN WHEN THE MUNITIONS DUMP AT KHAMISIYAH WAS DETONATED, AND COMPARISON MADE WITH NON-KHAMISIYAH AND NON-DEPLOYED CONTROLS EXPOSURE No direct measure of exposure. Khamisiyah veterans were present within a 50km radius of the munitions dump when it was detonated HEALTH EFFECTS Authors state that significant differences were found between Khamisiyah and non-Khamisiyah veterans in psychological tests (Penn PTSD, Mississippi PTSD, multiple scales of SF-36 - a health survey with 36 questions giving physical and mental health summary measures, and Beck Depression scale), but no details or scores are given. Authors report that statistical differences were also found between veterans from different cities and different ethnic groups, and that it was not possible to extract information independent of regional differences

Insufficient details are given to allow assessment of strength of results

Insufficient evidence of psychiatric effects presented to allow conclusions to be drawn

Stallones 2002b US STUDY OF DEPRESSIVE SYMPTOMS IN FARM RESIDENTS WITH AGRICULTURAL Strengths This is a large


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EXPOSURE TO PESTICIDES, INCLUDING OPs EXPOSURE Self-reported exposure to pesticides on questionnaire; pesticides used on the farm included the OPs terbufos, chlorpyrifos, phosmet, dichlorvos (and other classes of pesticides) HEALTH EFFECTS The table below indicates that if a respondent applied crop OPs, there were increased odds of having a pesticide-related illness: Odds of experiencing pesticide-related illness and type of pesticide used

Type of pesticide % (n) Odds Ratio 95% CI

Total crop organophosphates 30.6(232) 1.76 1.06-2.92 Livestock organophosphates 35.1(266) 0.99 0.59-1.66

Crop terbufos (Counter) 20.8(159) 1.96 1.14-3.36

Crop chlorpyrifos 8.0(61) 0.90 0.35-2.34 Other crop organophosphates 10.5(80) 2.45 1.29-4.64

The table below shows the results of a further logistic regression analysis, showing the estimated ORs and 95% CIs of having had a pesticide-related illness and the characteristics indicated: Several characteristics and Odds of experiencing pesticide-related illness

Characteristic Odds Ratio 95% CI Gender 0.27 0.14-0.52

Depressed 2.39 1.36-4.20 Sleeping too much 3.09 1.62-5.89

Use OP 2.34 1.17-4.66

Using an OP rather than another pesticide was significantly associated with having had a pesticide-related illness. The table below shows the psychiatric symptoms that were investigated for association with having had a pesticide-related illness; for the 3 out of 4 with 95% CIs suggesting significance, a χ2 for trend and P value are reported: Psychiatric symptoms associated with having had a pesticide-related illness

Neurological Odds 95% CI χ2 for trend P value

Relatively large study population of 761 respondents Limitations The questionnaire used is not a validated instrument The regression analyses relate to pesticide-related illness, not specifically OP-related illness, although the use of crop OPs is a significant factor in assessing pesticide-related illness; co-exposure to pesticides other than OPs is likely The characteristics of ‘Depressed’ and ‘Sleeping too much’ are seen to be significantly associated with pesticide-related illness, independently of whether subjects were also exposed to OPs or not

study which suggests an association between use of OPs and symptoms of irritability and depression. Its main limitation is that associations are with pesticide-related illness, and only indirectly with OPs


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symptom Ratio

Felt irritable? 1.84 1.08-3.12 4.89 0.0270

Felt depressed? 2.82 1.65-4.81 14.77 0.0001

Been sleeping more than is usual for you?

3.58 1.95-6.58 14.42 0.0001

Had difficulty falling asleep? 1.45 0.84-2.53 not given not given

Thus, out of 4 psychiatric symptoms, 3 were significantly associated with reporting a pesticide-related illness, and OPs applied to crops were found to be associated with reporting a pesticide-related illness in this study population

Wesseling 2002 STUDY OF EFFECTS OF MILD POISONING ON BANANA WORKERS IN COSTA RICA EXPOSURE Poisoning was verified from worker’s medical file, report of the National Insurance Institute, information provided by the company about nematocide used, and worker’s report HEALTH EFFECTS The table below indicates the scores on the Q-16 and BSI tests of banana plantation workers previously poisoned with a cholinesterase-inhibiting pesticide, compared with scores for non-poisoned workers: Q-16 and BSI scores for OP-poisoned workers

Note: ** p<0.01 A significant increase in neuropsychiatric symptoms was observed in the OP-poisoned workers. OP-poisoned workers had a worse performance of -0.2 (standardised regression coefficient, 95% CI -0.38 to -0.02) on the Q-16, and a worse performance of -0.28 (standardised regression coefficient, 95% CI -0.41 to -0.11) on the BSI, compared to non-poisoned workers. As the table indicates, there is a significant difference at the p<0.01level for the crude difference of means and the regression coefficient. However, the Q16 95% CI includes 1.0

Test

Poisoned Mean (SD) n=81

Non-poisoned Mean (SD) n=130

Crude Difference of Means

Regression Coefficient

95% Confidence Interval

Questionnaire -16 (no.yes answers)

9.1 (4.2)

6.5 (3.8)

2.6**

1.7**

0.5-3.0

Brief Symptom Inventory (grand total)

45.8(25.3)

26.8(40.7)

19.0**

15.7**

5.5-25.8

Strengths Use of validated questionnaires for psychiatric symptoms Limitations Only overall scores are reported for the questionnaires, no individual symptoms are reported No clear measure of ‘poisoned’; some of the ‘non-poisoned workers’ group had also reported experiencing mild symptoms after exposure 42% of all the identified cases of poisoning from medical records could not be located, so there may also be

It is difficult to evaluate the detrimental psychiatric effects of mild OP poisoning based on the results of this study, as no individual symptoms are reported


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selection bias Wesseling 2010 STUDY OF SYMPTOMS OF PSYCHOLOGICAL DISTRESS AND SUICIDAL IDEATION AMONG

BANANA WORKERS WITH A HISTORY OF OP POISONING IN COSTA RICA EXPOSURE Poisoning was verified from worker’s medical file, report of the National Insurance Institute, information provided by the company about nematocide used, and worker’s report HEALTH EFFECTS Descriptive parameters of the Brief Symptom Inventory (BSI) dimensions among banana workers are given in the table below: Scores for BSI dimensions among banana workers with history of OP poisoning

Referents, N=130 Organophosphate poisoned, N=54

BSI dimensions Median Min-Max Interquartile range

Median Min-Max Interquartile range

Somatisation 0.29 0-3.29 0.86 1.00 0-3.29 2.03

Obsessive-compulsiveness

0.50 0-3.50 0.83 1.00 0-3.83 1.92

Interpersonal sensitivity

0.25 0-3.25 0.75 0.75 0-3.75 1.56

Depression 0.17 0-2.67 0.67 0.59 0-3.67 1.27

Anxiety 0.33 0-2.33 0.83 0.75 0-3.67 1.21

Hostility 0.20 0-3.00 0.65 0.69 0-3.40 0.85

Phobia 0.20 0-2.60 0.60 0.40 0-4.00 0.80

Paranoia 0.20 0-3.00 0.99 0.60 0-3.80 1.05

Psychoticism 0.25 0-2.75 0.75 0.50 0-3.25 1.06

General severity index

0.37 0-2.31 0.61 0.86 0-3.31 1.09

The table indicates that median values for the 9 BSI dimensions were higher, and interquartile ranges wider, for the OP-poisoned workers compared to referent workers. The table below shows the prevalence of abnormal scores (T scores ≥63) for psychological distress assessed with the BSI, for OP-poisoned workers compared to never-poisoned referents: Prevalence of abnormal BSI scores among banana workers with history of OP poisoning

Strengths Use of validated instrument to assess symptoms of psychological distress Sampling of the referent workers was random, from the company payrolls of national companies and one large multinational company; workers commonly move from one company to another, so the sample is thought to be representative of the total population of banana workers Limitations Workers may have been exposed to both OPs and carbamates during their work, besides the documented poisoning episodes with one or other pesticide, which may have an effect on symptoms Potential for selection bias,

The results of this study suggest a possible association between mild OP poisoning and some symptoms of psychological distress, including depression and anxiety, but the study re-visits results collected many years previously, with a potential for selection bias because only 57% of the poisoned workers could be located. The severity of the poisoning cannot be assessed in the non-located workers


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Notes: all scores were dichotomised according to the corresponding T scores in the BSI manual for male non-patients, with a cut-off T score of ≥63, which is considered a clinically relevant level; ORs were adjusted for age, education, recent and cumulative exposure, alcohol, head injury, time of day of examination, and examiner The above table indicates that logistic regression models gave a significantly higher probability for abnormal BSI scores among OP-poisoned workers, compared to referents, in the symptom dimensions of somatisation, obsessive-compulsiveness, interpersonal sensitivity, depression, anxiety, hostility, phobia, psychoticism and the general severity index. The table below shows the prevalence of suicidal ideation in OP-poisoned workers compared to referents: Prevalence of suicidal ideation among banana workers with history of OP poisoning

Poisoned/non- N Suicidal Prevalence Adjusted 95% CI

Referents, N=130 Organophosphate poisoned, N=54 Prevalence T score ≥63 Prevalence T score ≥63 n (%) OR n (%) OR 95%CI

BSI dimensions

Somatisation 46 (35.4) 1.00 34 (63.0) 3.10 1.61-6.00

Obsessive-compulsiveness

38 (29.2) 1.00 29 (53.7) 3.18 1.61-6.24

Interpersonal sensitivity 28 (21.5) 1.00 24 (44.4) 2.91 1.48-5.76

Depression 38 (29.2) 1.00 27 (50.0) 2.42 1.26-4.66

Anxiety 36 (27.7) 1.00 27 (50.0) 2.24 1.14-4.41

Hostility 25 (19.2) 1.00 19 (35.2) 2.28 1.12-4.63

Phobia 54 (41.5) 1.00 31 (57.4) 1.90 1.00-3.61

Paranoid ideation 33 (25.4) 1.00 18 (33.3) 1.44 0.71-2.92

Psychoticism 56 (43.1) 1.00 32 (59.3) 2.19 1.12-4.30

General severity index 42 (32.3) 1.00 34 (63.0) 3.56 1.84-6.92

because only 57% of the subjects with reported work-related poisoning could be located The presence of suicidal thoughts is based on the answer to a single question, and other potential sources of stress were not addressed


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poisoned individuals

ideation % OR

Referents 130 8 6.2 1.00

Organophosphate-poisoned

54 12 22.2 3.72 1.41-9.81

Note: ORs adjusted for age, education, time of day of examination and head injury It can be seen that OP-poisoned workers were over three times more likely to have had suicidal thoughts (in the previous month) compared to the reference population. To put these scores in the context of other investigations conducted by the study authors, it was found that OP-poisoned workers had a clear overall excess of symptoms of psychological distress compared to carbamate-poisoned workers, who only had increased scores for somatisation. The highest prevalence of suicidal ideation was among workers with ≥2 poisoning episodes, for both OP- and carbamate-poisoned workers


7.B.1. Cohort studies Albers 2004b STUDY OF EFFECTS OF EXPOSURE TO CHLORPYRIFOS IN CHLORPYRIFOS PRODUCTION

WORKERS EXPOSURE Urinary excretion of OP metabolites was measured: excretion of 3,5,6 trichloro-2-pyridinol (TCP) was significantly higher in chlorpyrifos-exposed subjects (192.2 µg/g creatinine vs 6.2 µg/g creatinine in referents) (P<0.0001). Plasma butyrylcholinesterase (BuChE activity) was measured, and was significantly lower in chlorpyrifos-exposed subjects (7155 vs 8183 mu/ml; P<0.01). The ratio of lowest BuChE during the follow-up period to baseline was significantly lower in chlorpyrifos exposed subjects. The daily excretion of TCP equated to systemic daily doses of 576-627 µg/day. HEALTH EFFECTS The Brief Symptom Inventory (BSI) was used to measure anxiety: BSI score for anxiety in chlorpyrifos production workers

Variable Chlorpyrifos subjects (n=53) MEAN (SD)

Referents (n=60) MEAN (SD)

P-value

Anxiety 48.0 (11.8) 47.8 (13.8) 0.93

Strengths Biomonitoring measures are available for subjects and referents in the study Limitations The focus of the analysis in the study are the neurological and neuropsychological effects of exposure, and there is only limited information on psychiatric effects

Very limited information on psychiatric effects is reported in this study; anxiety does not appear to be a significant effect of exposure


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The P-value is for the t-test for the difference in means, and is not significant at the P<0.05 level

Beard 2011 STUDY OF SUICIDE AND PESTICIDE USE, INCLUDING OPs AND SPECIFIC PESTICIDES, AMONG PESTICIDE APPLICATORS AND SPOUSES IN THE AGRICULTURAL HEALTH STUDY EXPOSURE Cumulative lifetime days of use of OPs evaluated for applicators and spouses. For applicators only, suicide and dose response was calculated for the specific OPs chlorpyrifos, fonofos and terbufos HEALTH EFFECTS The results for an association between suicide and OP use among applicators and spouses are shown in the table below: Association between suicide and OP use among applicators and spouses

Variable Cases n PY*

Total n PY*

Adjusted HR** (95% CI)

Ever personally mixed or applied OP insecticides NO YES

26 84

170 532

28,870 52,979

377,315 713,702

Reference 0.82(0.50-1.33)

Ever personally mixed or applied specific pesticide:

Chlorpyrifos 40 263 22,784 308,881 0.99(0.66-1.48) DDVP*** 8 58 5,571 75,408 1.05(0.51-2.18) Diazinon 34 208 18,850 254,163 1.16(0.76-1.76) Fonofos 15 120 10,957 147,693 0.85(0.49-1.49) Malathion 62 388 40,702 548,502 0.93(0.61-1.42) Parathion 14 86 7,815 105,287 0.94(0.53-1.67) Terbufos 24 176 19,527 263,680 0.67(0.42-1.07)

Notes: *PY – person-years **HR – Hazard Ratios adjusted for age at enrolment, sex, number of children in family, frequency of alcohol consumption during past 12 months, smoking status ***DDVP – 2,2-dichlorovinyl dimethyl phosphate These results do not suggest an association between OP use, or the use of several specific OPs, and suicide among pesticide applicators and their spouses

Strengths Prospective cohort study with large number of subjects Information on pesticide use was collected before suicides occurred Limitations Small number of suicides (n=110) meant low power for estimating HRs for individual pesticides Most participants were farmer owners or operators, or commercial applicators, and not farm workers, so results may not be generalisable to farm workers, potentially more highly exposed to pesticides than AHS farmers Other information potentially useful to suicide analysis, such as information on financial situations, divorce,

The findings of the study do not suggest that OP use, or the use of some specific OPs, is associated with increased risk of suicide in pesticide applicators and their spouses in the AHS


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or death of family members, was not available from the AHS questionnaires; neither was information on personality or access to mental health care

7.B.2 Case-control studies Jamal 2002a UK STUDY OF SHEEP FARMERS AND DIPPERS EXPOSED TO OP PESTICIDES

EXPOSURE Self-reported exposure history on questionnaire HEALTH EFFECTS The authors provide only a brief summary of possible psychiatric effects in this paper: they state that psychological symptom rating scale variables showed significant differences among the three groups, with the probable or definite neuropathy group consistently significantly higher than the other two, inferring greater anxiety and depression. The scales used were the Hospital Anxiety and Depression Scale and the General Health Questionnaire. Increasing severity of neuropathy, as based on symptoms and sensory tests in the clinic, was associated with anxiety and depression as measured in the neuropsychological tests.

Strengths A nested case-control study of subjects with abnormal indices of peripheral neuropathy identified from a larger cross-sectional survey, which had 685 subjects; 72 subjects are included in this case-control study Limitations No evidence, such as test scores, of the statements made regarding anxiety and depression is presented Possible co-exposure to different pesticides

Insufficient evidence is presented in this study to allow any conclusions regarding exposure to OPs and the symptoms of anxiety or depression


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7.B.3 Cross-sectional studies Bayrami 2012 STUDY OF 40 HORTICULTURE FARMERS EXPOSED TO OPs IN IRAN

EXPOSURE Measurement of AChE activity indicated that levels in farmers were lower than in controls : AChE activity in farmers exposed to OPs

Group AChE levels (kU/L) (mean±SD)

Controls 35.73±4.89

Farmers 31.24±7.11

p value 0.001

HEALTH EFFECTS Results for the Symptom Checklist-90-Revised (SCL-90-R), as shown in the tables below, do not appear to indicate significant levels of psychological distress except for the dimension of somatisation: Scores for Dimensions in the Symptom Checklist-90-Revised

Group SOM OBS INT DEP ANX HOS PHOB PAR PSY Controls 0.48

±0.4 0.71 ±0.64

0.66 ±0.61

0.73 ±0.66

0.44 ±0.52

0.52 ±0.52

0.29 ±0.36

0.83 ±0.61

0.46 ±0.44

Farmers 0.75 ±0.53

0.71 ±0.57

0.67 ±0.62

0.62 ±0.47

0.54 ±0.56

0.48 ±0.52

0.35 ±0.42

0.86 ±0.77

0.42 ±0.41

p value 0.03 0.98 0.93 0.43 0.46 0.75 0.54 0.86 0.65 Notes: SOM – somatisation; OBS – obsessive-compulsive; INT - interpersonal sensitivity;

Group GSI PST PSDI Controls 0.59

±0.43 29.19 ±19.32

0.16 ±0.006

Farmers 0.62 ±0.42

33.33 ±22.02

0.142 ±0.007

p value 0.73 0.43 0.35

Strengths AChE levels were measured in participants, as well as a number of biochemical and haematological parameters Limitations Small study size No details of exposure are given It is not clear if the SCL-90-R is an appropriate instrument to measure psychological distress in this study population

Apart from somatisation, the results of the study do not suggest significant levels of psychological distress in the study population of 40 Iranian farmers during one spraying season


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DEP – depression; ANX – anxiety; HOS – hostility; PHOB – phobic anxiety; PAR – paranoid ideation; PSY – psychoticism; GSI – global severity index; PSDI – positive symptom distress index; PST – positive symptom total The authors comment that, although there appears to be evidence of lower AChE activity in farmers compared to controls, the incidence of neuropsychological disorders is a complex multivariate phenomenon, and does not clearly follow AChE depression

Bazylewicz-Walczak

1999 STUDY OF BEHAVIOURAL EFFECTS OF OCCUPATIONAL EXPOSURE TO OPs IN POLISH FEMALE GREENHOUSE WORKERS EXPOSURE Data for 26 Polish women working in greenhouses was reported as total OPs (combined dermal/respiratory exposure) on days 1-6 after the restriction period, a period of 1-3 days after spraying of pesticides when workers may not enter the sprayed area. Most frequently used compounds were dichlorvos, methamidophos, methidathion and pirimiphos-methyl. Workers had additional exposures to carbamates, synthetic pyrethroids and dithiocarbamates Exposure was seasonal, each worker being in contact with OPs for approximately 7-46 days. There were cycles of 4-7 days of spraying with an interval of approximately 3 days between spray applications. The daily cumulative exposure (combined inhalation/dermal) on 6 consecutive days after the restriction period was between 0.001-0.009 % of the toxic dose (based on dermal LD50s). The duration of spraying varied between 1-24 years (mean 11.9y SD 6.8y). Concentrations of OP pesticides in the air in greenhouses did not exceed acceptable limits; exposure level of workers measured from levels on clothing and skin was found to be low, below 0.010% of the toxic dose based on acute dermal LD50. HEALTH EFFECTS Mean values and standard deviations are reported for Profile of Mood States variables and Subjective Symptoms Questionnaire variables for a pre-season examination (January- February), before intensive spraying in the greenhouses began, and for a second post-season examination (June), conducted in the period following major spraying operations. The Profile of Mood States results after one season of exposure did not indicate negative changes in the emotional state of the subjects. On the Subjective Symptoms Questionnaire, the only adverse psychiatric effect was an increase in fatigue, but it was not statistically significant. Two-way ANOVA was also performed, and statistically significant results are shown in the table below. The significance level α was taken to be 0.10 for these results: Results for Profile of Mood States and Subjective Symptoms Questionnaire

Name of test variable

Factor ‘season’ F p*

Factor ‘group’ F p*

Interaction F p*

Strengths Monitoring - air levels of OPs recorded in greenhouses, and exposure of workers measured by analysis of levels on clothing and skin Limitations Small sample size The results are analyses undertaken in a single year, not observations over a longer span of time

The results of the study suggest that greenhouse workers seasonally exposed to non-toxic levels of OPs did not suffer detrimental psychiatric effects after one season of spraying, but comparison with non-exposed controls suggest some significant increases in effects (fatigue, anger and depression) after longer-term exposure. The small number of subjects studied is a limitation, as well as the fact that the results relate to a single year


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Profile�of Mood States Te�s�on – anxiety 4.92 � .03 4.49 .04 NS NS Anger – hostility NS NS 2.95 (.09) NS NS Fatigue – inertia NS NS 5.57 .02 NS NS Depression – dejection 3.91 .05 5.44 .02 NS NS Confusion – bewilderment 4.37 .04 NS NS NS NS Subjective Symptoms Questionnaire

Fatigue NS NS NS NS 2.73 (.10) Sleep disturbances 4.70 .03 NS NS NS NS Absent-mindedness 3.62 (.06) 2.72 (.10) NS NS Depression 11.91 .001 NS NS NS NS Lability 5.40 .02 NS NS NS NS Neurovegetative symptoms 3.90 .05 NS NS NS NS

Notes: NS=not significant Factor ‘season’= single spraying season effects Factor ‘group’ = long-term effects of exposure Interaction= interaction between short- and long-term effects of exposure * figures in brackets indicate a significance level of .05<p≤.10 In the category ‘Factor ‘season’’, results obtained in the pre-season and post-season examination were compared for exposed workers. The significant p values are for improvement in mood and general feeling; thus there were no detrimental effects observed after one season of spraying. In ‘Factor ‘group’’, the exposed and control groups were compared for possible long-term effects of exposure. The exposed group reported a higher degree of tension, anger, fatigue-inertia and depression, and also absent-mindedness. No significant interaction effects were seen between short- and long-term effects of exposure at the p<0.05 level

Beseler et al 2008 DEPRESSION AND PESTICIDE EXPOSURE AMONG U.S. PRIVATE PESTICIDE APPLICATORS IN THE AGRICULTURAL HEALTH STUDY EXPOSURE Self-reported information on lifetime use of pesticides reported in questionnaire. Low exposure was <226 days (median 225 days is reference group); Intermediate exposure was 226-752 days; High exposure was >752 days, above the 90th centile. Acute high-intensity pesticide exposures (i.e. High Pesticide Exposure Event (HPEE), or physician-diagnosed pesticide poisoning) were also reported.

Strengths Large number of cases and controls available, because a cross-sectional analysis is made of data from a large cohort study (Agricultural Health

The findings of this study, which has a large number of cases and controls, suggest a possible association between OP exposure and


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HEALTH EFFECTS The results for logistic regression models of diagnosed depression with the specific class of OP pesticides are shown in the table below: Association of use of OPs with diagnosed depression

Cases Controls Unadjusted Adjusted Pesticide class [no.(%)] [no.(%)] OR(95% CI) OR(95% CI) Ever used OPs

No 38 (7.1) 1,969 (11.5) Reference Reference

Yes 496 (92.9) 15,082 (88.5) 1.70(1.22-2.38) 1.78(1.27-2.50)

These results suggest an association between use of OPs and a diagnosis of depression. For this analysis, the whole sample of 534 cases and 17,051 controls is used, which includes chronic cumulative exposure, although some cases will also have experienced an acute high-intensity pesticide exposure, or physician-diagnosed pesticide poisoning. The study as a whole suggests that acute high-intensity pesticide exposure, and cumulative exposure to pesticides, may contribute to depression in pesticide applicators.

Study) Controls are drawn from same population as cases, which minimises potential confounding Limitations Most of the results in the study analyse an association between depression and pesticide exposure in general, and only the one result given refers specifically to exposure to OPs. Information on diagnosed depression and pesticide exposure is self-reported

reported diagnosed depression

Cox 2005 EVALUATION OF POTENTIAL HEALTH EFFECTS ON RESIDENTS OF SPRAYING OF METHYL PARATHION IN THE HOME EXPOSURE Authors report no difference in average cholinesterase levels between low- and high-exposure groups HEALTH EFFECTS Authors report no significant differences in any of the health outcome measurements between the exposure group (121 homes containing >150μg/100cm²) and the control group (homes containing <15 μg/100cm²). However, some differences in health outcome measures were seen at the highest exposure levels, that is, where average levels of methyl parathion in the home were >260μg/100cm²; the table below indicates the psychiatric symptoms that were reported with increased frequency by subjects whose home contained >260μg/100cm² of methyl parathion: Frequency of insomnia and depression in subjects whose homes contained high levels of

Strengths Measurement of levels of methyl parathion in homes Medical evaluations performed by physicians Limitations Methyl parathion levels were averaged over several rooms in the house

Individuals living in homes with high levels of methyl parathion may have an increased incidence of insomnia and depression; however, average AChE levels were not lower in the high-exposed group than in the low-exposed group, so the


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methyl parathion

Symptom Frequency* <260μg/100cm²

Frequency >260μg/100cm²

P OR(95% CI)

n 231 60

Insomnia 17% 29% 0.03 2.1(1.1-4.3)

Depression 11% 22% 0.03 2.2(1.1-4.9)

* the percentage of residents who reported the symptoms

Many high samples occurred under kitchen sink and in cabinets, areas to which not all family members would be exposed No attempt made to measure potential exposures to methyl parathion or other OPs outside the home Subjects not blinded to the methyl parathion in their homes, so possible recall bias

symptoms do not appear to be related to AChE depression

Kamel 2005 STUDY OF NEUROLOGIC SYMPTOMS IN U.S. PRIVATE PESTICIDE APPLICATORS IN THE AGRICULTURAL HEALTH STUDY EXPOSURE Self-reported exposure; cumulative use of OPs (lifetime days, without or with use in past year) was calculated. Cumulative use also calculated for other chemical classes, including organochlorines, carbamates, pyrethoids HEALTH EFFECTS In models including cumulative use, applicators in the highest category of OP use (>500 lifetime days) were 1.6-1.7 times more likely to experience ≥10 symptoms in the year before enrollment, and averaged 1.0-1.1 additional symptoms, compared with the lowest quartile. Associations of specific psychiatric symptoms with OP exposure among licensed pesticide applicators enrolled in the Agricultural Health Study 1993-1997 (n=18,782) are shown in the table below: Association of psychiatric symptoms with exposure to OPs

Strengths Large study size, because a cross-sectional analysis is made of data from a large cohort study (Agricultural Health Study) Limitations Questionnaire used is not a validated instrument, only based on the Q-16 Only 44% of private applicators eligible to participate completed the

The study results suggest a possible association of some psychiatric symptoms with cumulative lifetime exposure to OPs, although the questionnaire used is not validated, and co-exposure to other pesticides besides OPs may be a confounding factor


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Notes: estimates are for the highest category of lifetime days of use, >500 days * estimates for which the 95% Confidence Interval excludes 1.00 Overall, the authors found dose-related associations of symptom count to cumulative exposure to OPs, whether or not individuals with history of pesticide poisoning or a high-exposure event were included, suggesting that moderate (non-acute) exposure itself is associated with increased risk

Symptom Exposure to Organophosphates ( OR for experiencing symptom with high frequency compared to low frequency )

Fatigue 2.34* Tension 1.82* Insomnia 1.70* Irritability 1.93* Depression 2.09* Absentmindedness 2.13* Difficulty concentrating 2.07*

relevant questionnaire, so potential bias is a concern Symptoms and exposure are self-reported, raising concern of potential recall or reporting bias Likely co-exposure of participants to other pesticides

Keifer 2000 U.S. STUDY OF NEUROLOGICAL EFFECTS OF OPs ON FARM WORKERS ENGAGED IN ORCHARD THINNING EXPOSURE Self-reported work history, and reported home pesticide exposure, in questionnaire; blood taken for testing AChE levels; testing also undertaken for paraoxonase, chlorpyrifos oxonase and diazinon oxonase on most participants HEALTH EFFECTS Scores from the BSI (Brief Symptom Inventory) depression scale or answers to the Q-16 test are not reported; the only comment made on psychiatric symptoms is that the ordinal scores from the depression scale from the BSI, and the dichotomous answers to the neurological symptoms from the Q-16, were examined for their relationship to exposure, and none of the symptom questions asked showed a significant relationionship

Strengths 90 of the original 137 subjects were followed up for a second year and tested again Limitations There is very little report of psychiatric symptoms from this study

The study authors state that there is no significant relationship between exposure and depression, but no details of scores or answers to questions are reported

Kilburn 1999 U.S. STUDY OF EFFECTS OF CHLORPYRIFOS SPRAYED INDOORS ON 22 SUBJECTS EXPOSURE Schedules of pesticide application were found for offices of 6 patients, so concentrations of applied chlorpyrifos were known; 4 patients had personal clothing or rugs

Strengths Objective measures of exposure were available for 10 of

The Profile Of Mood States scores in this study are suggestive of


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analysed for chlorpyrifos; for other subjects, self-reported exposure on questionnaire HEALTH EFFECTS The POMS scores for exposed and unexposed subjects are shown in the table below: POMS scores for subjects exposed to chlorpyrifos sprayed indoors

Exposed (22) [mean±SD]

Unexposed (264) [mean±SD]

p value

Profile of Mood States

Mean score 89.7±36.6 21.0±31.6 0.0001

Depression mean score

15.3±3.4 12.9±2.3 0.0002

POMS and depression scores are elevated in the exposed group. The table below indicates the frequency of psychiatric symptoms that were included in the questionnaire used in the study: Frequency of psychiatric symptoms for subjects exposed to chlorpyrifos sprayed indoors

Exposed (mean±SD)

Unexposed (mean±SD)

p

Somnolence 5.8±3.0 2.5±2.1 0.0001

Insomnia, cannot fall asleep

6.6±3.3 3.0±2.5 0.0001

Wake frequently 6.7±3.3 2.8±2.3 0.0001

Sleep few hours 7.1±3.5 2.9±2.6 0.0001 Irritability 7.2±2.4 3.5±2.3 0.0001

Mood swings 5.6±3.1 2.6±2.4 0.0001 Loss of libido 6.5±3.4 3.2±2.7 0.0001

There appears to be a significantly increased frequency of all 7 of these symptoms in exposed subjects compared to unexposed. The authors further report that there was no significant increase in psychiatric illnesses, but that 63% of the exposed group had been prescribed tranquilisers after the exposure and onset of

the exposed participants The POMS is a validated instrument Limitations Small study size The questionnaire relating to symptom frequencies was devised by the group and is not a validated instrument

a possible effect on exposed subjects, but the small size of the study, and the fact that exposure measures were available for only 10 out of 22 of the exposed subjects, have to be considered


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symptoms, which was a significant difference from the unexposed group Mackenzie Ross

2010 STUDY COMPARES SHEEP FARMERS AND CONTROL GROUP OF RURAL POLICE OFFICERS IN THE UK, INCLUDING SOME SUBJECTS WHO HAD RETIRED FROM ILL HEALTH EXPOSURE Self-reported work and exposure history; various exposure indices are reported in the table below: Exposure indices for sheep farmers

Exposure indices

Mean SD Range

No of years spent working with OPs

Working group

23.05 10.72 8-49

Retired group 26.11 15.17 5-66 No of days spent working with OPs

Working group

3.77 6.90 0.5-30

Retired group 2.84 3.54 1-21 Years since last dip

Working Group

9.48 8.78 0-37

Retired group 11.26 7.65 0-42 HEALTH EFFECTS To assess mood disorder, the Hospital Anxiety and Depression Scale was used. Hospital Anxiety and Depression Scale results 46.9% of farmers had scores above clinical cut-offs for depression, compared to only 6.5% of controls: χ2 test for depression = 33.97, p<.001; 41.5% of farmers scored in the clinical range for anxiety compared to only 22.1% of controls; χ2 test for anxiety = 7.25, p<.01. Thus the χ2 test showed that a significantly higher proportion of farmers scored in the clinical range for both anxiety and depression.

Strengths The study included participants who had retired on ill health grounds PON1 levels were analysed in all study participants Limitations Exposure is estimated by self-report, and so is medical history. The control group were not farmers, but authors claim it is impossible to find a group of farmers without history of exposure to OPs. A number of different recruitment methods were used in the study, which could result in selection bias, and therefore differences between working and retired groups. Small sample sizes. Study reports only percentages of subjects affected by anxiety and depression, not scores

The study suggests an association between OP exposure and depression and anxiety in farmers, but the limitations detract from the strength of the presented evidence; the PON1 results show no evidence of poor metabolisers in the study


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The PON1 results showed that there were similar numbers of participants in each phenotype group, with similar levels of arylesterase, and the arylesterase levels suggested that there were no poor metabolisers in the study

Rehner 2000 STUDY OF DEPRESSION IN VICTIMS OF THE SOUTH MISSISSIPPI METHYL PARATHION DISASTER EXPOSURE 115 heads of households whose homes had been sprayed with methyl parathion applied for assistance to the Mississippi State Department of Health, and these homes were tested and classified by the Environmental Protection Agency (EPA). The EPA tested methyl parathion levels in the homes by ‘wipe tests’ in the building, and defined 4 levels of exposure, Level I being the highest and Level IV the lowest. HEALTH EFFECTS The mean Center for Epidemiological Studies – Depression Scale (CES-D) scores, and the Standard Deviation, for the methyl parathion victims in Mississippi is given in the table below: Mean CES-D scores for subjects exposed to methyl parathion sprayed indoors Variable M SD p

Level of contamination

Level I 19.97 12.00 0.942

Level II 20.67 9.47

Level III 20.56 12.31

Level IV 18.81 11.51

Gender

Male 15.41 11.18 0.009

Female 21.65 10.89

Ethnicity

Black 22.41 11.15 0.047

Latino (n=1) 15.00

White 17.29 10.91

Income groups

<$19,999 23.20 12.19 0.004

Strengths Exposure was quantified by measurement of methyl parathion levels in homes from wipe samples The CES-D is a validated instrument Other factors influencing the possibility of having depressive symptoms, such as low income, are included in the analysis Limitations The findings in this study relate to an OP pesticide applied indoors, and may be less applicable to situations where OPs are applied outdoors in the open air

The findings of this study suggest an association between length of exposure to an OP pesticide, applied indoors, and depressive symptoms in exposed subjects


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55% of all participants had CES-D scores greater than the clinical cut-off score of 16. The table indicates that comparing the CES-D mean scores across the four contamination levels was not significant (p=0.942); however, the t test of depression by gender indicates that women’s mean CES-D score was significantly higher (p=0.009) than men; the t test of depression by race indicates marginal significance (p=0.047) that black Americans had more depressive symptoms than white people; the ANOVA of depression by income groups was significant (p=0.004) and indicates that people with lower incomes had more depressive symptoms than people with higher incomes. The number of respondents’ depressive symptoms was highest among those who had been exposed to methyl parathion for the longest time: r=0.248, p<0.001. A hierarchical regression indicated that length of exposure was the only variable predictive of

$20,000-29,999 23.09 12.21

$30,000-39,999 17.24 8.50

>$40,000 14.50 8.10


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depression, giving a significant R2 change of 0.034 (p=0.04) Thus, only length of exposure, regardless of contamination level, was predictive of depression among people exposed

Salvi 2003 STUDY OF TOBACCO WORKERS CHRONICALLY EXPOSED TO OPs IN BRAZIL EXPOSURE OP exposure history obtained via questionnaire (chlorpyriphos and acephates most predominant pesticides used). Plasma AChE levels of all subjects were within the normal range (3.2-9.0 U/l), and were not different between on- and off-exposure periods (4.7±0.9 and 4.5 ±1.1 U/l respectively). First evaluation was performed when the workers were exposed to OPs; the second evaluation was conducted 3 months later, when the workers had been off exposure for 3 months. HEALTH EFFECTS The number of current AXIS-I psychiatric diagnoses was 36, among the 37 subjects in Evaluation1 and during exposure to OP; diagnoses made by 2 trained psychiatrists; 18 of the 37 subjects (48%) had a current psychiatric diagnosis in the first interview; 13 (35%) had a current diagnosis of generalized anxiety disorder, and 8 (21%) had a current episode of major depression. In Evaluation 2, for the 25 individuals who completed both evaluations and after 3 months without OP exposure, the number of individuals affected with any psychiatric diagnosis dropped from 11 to 7. The table below shows the Mini-mental scores recorded in both evaluations: Mini-mental scores for tobacco workers exposed to OPs

Parameter ON exposure OFF exposure

Statistical analysis (p)

(n=37) (n=25) (n=25)

Mini-mental score

24.86±3.77

24.56±3.71

25.16±2.97

0.33

Notes: data presented as mean±SD statistical analysis refers only to the 25 subjects evaluated twice ON exposure= during exposure OFF exposure= after exposure The Mini-mental scores are within the expected range, and the statistical analysis further suggests that there is no significant difference in scores during OP exposure, and after 3 months of OP washout.

Strengths Study uses the MINI, a validated measuring instrument, and diagnoses of AXIS-I psychiatric diagnoses were made by trained psychiatrists; AChE levels available as measure of exposure Limitations Small sample size, and 12 of the original 37 were lost to follow-up. No unexposed group for comparison. Authors report frequent co-use by subjects in the study of at least one other chemical compound besides OPs. Limited psychiatric analysis, as the study reports only one set of scores, the MINI mental scores. The results of the

The study results do not provide evidence of ongoing mental or psychiatric symptoms as a result of exposure to OPs


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two measures used do not support each other: AXIS-I diagnosis suggests a large number of psychiatric diagnoses, while the MINI mental scores fall within the expected range for the population, and do not indicate influence by exposure to OPs

Solomon 2007 UK STUDY OF NEUROPSYCHIATRIC SYMPTOMS IN PAST USERS OF SHEEP DIP AND OTHER PESTICIDES EXPOSURE Self-reported on questionnaire, lifetime history of work with the following five categories of pesticides: sheep dip, other insecticides, herbicides, fungicides and wood preservatives HEALTH EFFECTS The aim of the study was to investigate postulated COPIND (chronic organophosphate-induced neuropsychiatric disorder) symptoms, and other psychiatric symptoms. COPIND symptoms are discussed in the ‘Other – Neurological’ section and tables. The psychiatric symptoms investigated were anxiety and depression, which are discussed here. In the study sample as a whole, 1984 (20.2%) men were classed as having symptoms of anxiety and 1161 (11.8%) as having symptoms of depression. Associations of psychiatric symptoms with occupational exposure to pesticides and somatising tendency are shown in the table below: Association of anxiety and depression with occupational exposure to pesticides and somatising tendency

Risk Factor

All subjects n

Anxiety*+ n PR (Prevalence PR Ratio) (95% CI)∞ (95% CI)§

Depression*+ n PR PR (95% CI)∞ (95% CI)§

Occupational exposure to

Strengths Large number of subjects, despite the fact that the response rate was only 31% A number of adjustments are made for possible confounding factors Limitations There may be selection bias, given the low response rate, with an over-representation of subjects with symptoms in the study sample The ‘somatising tendency’ score is a measure created by

The study results do not suggest an association of symptoms of anxiety or depression specifically with use of sheep dip or insecticides. The suggestion of the authors that psychological mechanisms may be responsible for symptoms (persons exposed to a perceived environmental hazard may be more prone to develop symptoms which may not have a toxic cause) would need to be verified in


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pesticides Never worked with pesticides

6109 1170 1.0 1.0 701 1.0 1.0

Ever worked with sheep dip

1913 424 1.1(1.0-1.2) 1.0(0.9-1.2) 259 1.2(1.0-1.4) 1.1(0.9-1.3)

Ever worked with insecticides, never with sheep dip

832 174 1.0(0.8-1.2) 1.0(0.8-1.1) 95 0.9(0.7-1.1) 0.9(0.7-1.1)

Ever worked with other pesticides, never with sheep dip or insecticides

990 216 1.1(0.9-1.2) 1.0(0.9-1.2) 106 0.9(0.8-1.1) 0.9(0.7-1.0)

Somatising tendency score**

0 5263 572 1.0 1.0 310 1.0 1.0 0.1-0.5 2705 591 2.0(1.8-2.3) 2.0(1.8-2.3) 303 1.9(1.6-2.2) 1.9(1.6-2.2)

0.6-1.0 1214 471 3.6(3.2-4.1) 3.6(3.2-4.1) 290 4.1(3.5-4.8) 4.1(3.5-4.8) >1.0 581 341 5.6(4.9-6.4) 5.6(4.9-6.4) 254 7.4(6.3-8.7) 7.4(6.3-8.7)

Notes: * for each outcome, the comparator for risk estimates was all subjects who did not have the outcome + possible or probable ∞ adjusted for age (in 8 bands) and area § adjusted for age and area, and also mutually adjusted ** ‘somatisation’ means experiencing medically unexplained symptoms. The authors used responses to the Brief Symptom Inventory (BSI) to derive a ‘somatising tendency’ score, which they divided into the 4 levels shown above ( 81 of the 9844 subjects were excluded because somatising tendency score was missing). The prevalence of anxiety and depression increased with somatising tendency. However, the prevalence of anxiety and depression showed no clear relation to work with any category of pesticide, and no association was seen with working with sheep dip, or with insecticides The following table shows associations with psychiatric symptoms among men who had, at some time, worked with sheep dip:

the study authors and is not a validated instrument

further studies


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Association of anxiety and depression with use of sheep dip

Risk Factor

All past users of sheep dip n+

Anxiety* n PR PR (95% CI)∞ (95% CI)§

Depression* n PR PR (95% CI)∞ (95% CI)§

Lifetime use of sheep dip (days)

<10 416 90 1.0 1.0 48 1.0 1.0

10-49 700 150 1.0(0.8-1.3) 1.0(0.8-1.3) 84 1.1(0.7-1.5) 1.1(0.8-1.5) 50+ 693 160 1.2(0.9-1.5) 1.0(0.8-1.4) 110 1.4(1.0-2.0) 1.2(0.9-1.8)

Handled sheep dip concentrate

No 544 129 1.0 1.0 78 1.0 1.0

Yes 1369 295 0.9(0.7-1.1) 0.9(0.7-1.1) 181 0.9(0.7-1.2) 0.9(0.7-1.2) Ever experienced symptoms within 48h of work with sheep dip

Never 1355 258 1.0 1.0 155 1.0 1.0 Fever or chills¥ 101 33 1.7(1.2-2.5) 1.1(0.8-1.6) 26 2.3(1.5-3.5) 1.5(1.0-2.3)

≥4 symptoms¥ 156 58 2.0(1.5-2.6) 1.2(0.9-1.6) 47 2.6(1.9-3.7) 1.6(1.1-2.2)

Notes: * for each outcome, the comparator for risk estimates was all men who did not have the outcome ∞ adjusted for age (in 8 bands) and area § adjusted for age and area, and somatising tendency + information on risk factors was missing for some subjects ¥ these risk factors overlapped; each was analysed separately relative to never having experienced acute symptoms The above table indicates that anxiety and depression were more common among those who reported having experienced multiple symptoms or fever/chills on at least one occasion within 48h


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of using sheep dip. However, anxiety and depression were not associated with handling sheep dip concentrate. The table below shows age-standardised prevalence rates for consulting a GP for 2 types of psychiatric symptoms: Prevalence rates for consulting a GP for certain types of psychiatric symptoms

Reason for consulting GP

Never worked with pesticides n Prevalence (%)*

Ever worked with sheep dip n Prevalence(%)*

Ever worked with insecticides, never with sheep dip n Prevalence (%)*

Ever worked with other pesticides, never with sheep dip or insecticides n Prevalence (%)*

Change in personality (e.g. tendency to depression or irritability)

501 8.2

168 9.0

60 7.2

84 8.4

Thoughts about self-harm or committing suicide

149 2.5

55 2.9

28 3.3

37 3.9

Note: * prevalence rates are directly standardised to the age distribution of the total study sample The table indicates that the lifetime prevalence for consulting a GP for the 2 categories of psychiatric symptoms described was marginally higher among men who worked with sheep dip than in those who had never worked with pesticides. However, consultations because of thoughts of self-harm or suicide were marginally more common among men who had worked with other pesticides but not sheep dip. Overall, past use of sheep dip or insecticides was not associated with current anxiety or depression. There was also no relation to handling the concentrate, which is a known determinant of higher exposure. However, the authors suggest that there may be a psychological component to the reported symptoms, especially because there is a strong association with somatising tendency.

Stephens 2004 UK STUDY OF EFFECTS OF CHRONIC LOW-LEVEL EXPOSURE TO OPs IN ORCHARD SPRAYERS

Strengths The GHQ is a

It is not possible to draw conclusions


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EXPOSURE Urine DEP/DEPT metabolites were similar in each group (p=0.207), 0-35 nmol/mmol creatinine in orchard sprayers, 0-12.19 nmol/mmol creatinine in pig farm workers and 0-11.48 nmol/mmol creatinine in construction workers. Orchard sprayers regularly used chlorpyrifos and had participated in an HSE survey. They were found to have used chlorpyrifos for a median of 5d/year (range 0.5-13 d/yr) for a median of 14 years (range 2-25 yrs) at an intensity sufficient to produce median summed DEP and DETP metabolites of 10.20 nmol/mmol creatinine (range 0-65.0 nmol/mmol creatinine). The predominant pesticide sprayed at the time of the study was chlorpyrifos. The majority (73%) of orchard sprayers reported previously using pesticides in applications outside of spraying top fruit in orchards: spraying soft fruit 18, non- fruit crops 12, dipping sheep 7, treating other farm animals 9, spraying to control insects 6, spraying amenity land 2, commercially spraying in households 2. The majority (73%) of pig farmers reported previously using pesticides: spraying soft fruit 2, non- fruit crops 10, dipping sheep 4, treating other farm animals including pigs 17, spraying to control insects 3, spraying amenity land 4, commercially spraying in households 2. Two construction workers reported using pesticides (control of insects 1 and commercially spraying households 1) HEALTH EFFECTS Most of the results in the paper concern neuropsychological test results and outcomes. There is one measure using the General Health Questionnaire which is reported, and is given in the table below: GHQ score for vulnerablilty to psychiatric disorder in orchard sprayers compared to controls

Orchard sprayers (n=37)

Pig farm workers (n=36)

Construction workers (n=31)

Uncorrected P

Vulnerability to psychiatric disorder on GHQ (n above psychiatric case threshold)

3

7

9

0.070

The P value does not suggest that a significant number of OP-exposed workers were above the

validated instrument Limitations Individual symptoms are not reported. It is difficult to assess the possible psychiatric effects of exposure from the limited information given. Some of the pig farm workers and even the construction workers may have also been exposed to OPs, so the reference groups are not truly unexposed individuals

about psychiatric effects on the limited evidence available; the focus of the paper is neuropsychological effects


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psychiatric case threshold


75

Table 8. EXPOSURE SCENARIO AND PSYCHIATRIC EFFECTS A. ACUTE EXPOSURE WITH CHRONIC EFFECTS Table 8.A.1. Cohort Studies

Test

First Author and Date Delgado

2004 2 year

follow up All

exposed

Delgado 2004 2 year follow

up Low exposed


up Medium exposed


up High exposed

Kawana 2001 2 year follow

up


up


up

Nakajima 1999 1 year

follow-up


follow-up

Tochigi 2002

Tochigi 2005

General Q-16 - increase in number of symptoms

+ + + -

Anxiety State-Trait Anxiety Inventory (STAI)

+ -

PTSD Symptomatology St. Luke's Hospital Questionnaire Symptoms associated with PTSD

+ + +

Impact of Event Scale-Revised (IES-R) for PTSD symptoms +

Clinician-Administered Post-Traumatic Stress Disorder Scale (CAPS) and correlation with reduced cholinesterase 1-3 days after sarin subway attack

+

Current PTSD re-experiencing + Current PTSD avoidance - Current PTSD increased -


76

Test

First Author and Date Delgado

2004 2 year

follow up All

exposed


up Low exposed


up Medium exposed


up High exposed


up


up


up


follow-up


follow-up

Tochigi 2002

Tochigi 2005

arousal Current PTSD total score + Lifetime PTSD re-experiencing + Lifetime PTSD avoidance + Lifetime PTSD increased arousal -

Lifetime PTSD total score + Sleep Disturbances Nakajima questionnaire, bad dreams

- -

Nakajima questionnaire, insomnia - +

Table 8.A.3. Cross-Sectional Studies

Test First Author and Date

Kawada 2005

McCauley 2001

McCauley 2002

Nishiwaki 2001

Ohtani 2004

Proctor 2006

Stallones 2002b

Wesseling 2002

Wesseling 2010

General Brief Symptom Inventory (BSI) + + BSI T score ≥63, clinically relevant level +

General Health Questionnaire (GHQ) - Impact of Event Scale (IES) -


77


Kawada 2005

McCauley 2001

McCauley 2002

Nishiwaki 2001

Ohtani 2004

Proctor 2006

Stallones 2002b

Wesseling 2002

Wesseling 2010

Mini International Neuropsychiatric Interview (MINI)

+

Q-16 + Anger/Irritability Gulf War survey, anger - Profile of Mood States (POMS), anger - Stallones questionnaire, irritability + Anxiety State-Trait Anxiety Inventory (STAI) anxiety in PTSD sufferers

+

Depression BSI suicidal ideation + Gulf War survey, depression + Gulf War survey, major depression with hospitalisation from sarin exposure

-

POMS, depression - Stallones questionnaire, depression + Structured Clinical Interview for DSM-IIIR Mental Disorders (SCID), Major Depression Diagnosis (MDD)

+

Confusion/Fatigue/Tension POMS, confusion - POMS, fatigue - POMS, tension - Mood swings Gulf War survey, mood swings + PTSD Clinician-Administered Post-Traumatic Stress Disorder Scale (CAPS) -


78


Kawada 2005

McCauley 2001

McCauley 2002

Nishiwaki 2001

Ohtani 2004

Proctor 2006

Stallones 2002b

Wesseling 2002

Wesseling 2010

Gulf War survey, PTSD from sarin exposure -

Impact of Event Scale-Revised (IES-R) + Mississippi Scale for PTSD - St. Luke's Hospital Questionnaire + Sleep disturbances Nakajima questionnaire, bad dreams Nakajima questionnaire, insomnia Gulf War survey, difficulty sleeping + Kawada questionnaire, insomnia in younger age group

+

Kawada questionnaire, insomnia in older age group

-

Stallones questionnaire, sleeping more than usual

+

Other Gulf War survey, obsessive compulsive disorder from sarin exposure

-

Gulf War survey, other psychiatric disease or psychosis

-

B. CHRONIC LOW-LEVEL EXPOSURE Table 8.B.1. Cohort Studies



79

Albers 2004b

Beard 2011

Anxiety Brief Symptom Inventory (BSI) Anxiety

-

Suicide Beard study, mortality data and exposure questionnaire -

Table 8.B.3. Cross-Sectional Studies

Test

First Author and Date Bayra

mi 2012

Bazylewicz Walczak

1999 one spray

season

Bazylewicz Walczak

1999 longer term

effects

Beseler et al 2008

Cox 2005

Kamel 2005

Kilburn 1999

Mac-kenzie Ross 2010

Rehner 2000

Salvi 2003 1st

evaluation Dec 2001

Salvi 2003 2nd

evaluation

Mch 2002

Solomon 2007

Stephens 2004

General DSM-IV AXIS-I Psychiatric Diagnosis + -

General Health Questionnaire (GHQ) -

Mini International Neuropsychiatric Interview (MINI)- mental scores

- -

Profile of Mood States (POMS) mean score +

Subjective Symptoms Questionnaire -

Davies questionnaire, frequency of symptoms

Kamel Q-16-based questionnaire +

Anger


80

Test


mi 2012

Bazylewicz Walczak

1999 one spray

season

Bazylewicz Walczak

1999 longer term

effects

Beseler et al 2008

Cox 2005

Kamel 2005

Kilburn 1999


Rehner 2000

Salvi 2003 1st

evaluation Dec 2001

Salvi 2003 2nd

evaluation

Mch 2002

Solomon 2007

Stephens 2004

POMS: anger + Kamel questionnaire + Kilburn questionnaire + Anxiety Hospital Anxiety and Depression Scale: anxiety

+ -

POMS: anxiety + Depression Center for Epidemiological Studies - Depression Scale (CES-D)

+

Hospital Anxiety and Depression Scale: depression

+ -

Kamel questionnaire + POMS: depression + + Subjective Symptoms Questionnaire: depression

-

Subjective Symptoms Questionnaire: neurovegetative symptoms associated with depression, e.g. appetite disturbance, difficulty concentrating

-

Beseler questionnaire: +


81

Test


mi 2012

Bazylewicz Walczak

1999 one spray

season

Bazylewicz Walczak

1999 longer term

effects

Beseler et al 2008

Cox 2005

Kamel 2005

Kilburn 1999


Rehner 2000

Salvi 2003 1st

evaluation Dec 2001

Salvi 2003 2nd

evaluation

Mch 2002

Solomon 2007

Stephens 2004

depression Cox evaluation, high exposure group: depression

+

Confusion/Fatigue/Tension

POMS: confusion - POMS: fatigue- inertia + Subjective Symptoms Questionnaire: fatigue -

POMS: tension + Kamel questionnaire- fatigue +

Kamel questionnaire- tension +

Sleep Disturbances Subjective Symptoms Questionnaire: sleep disturbances

-

Cox evaluation, high exposure group: insomnia +

Kamel questionnaire + Kilburn questionnaire + Other Outcomes Kamel questionnaire: absent-mindedness

+

Subjective Symptoms Questionnaire: absent- +


82

Test


mi 2012

Bazylewicz Walczak

1999 one spray

season

Bazylewicz Walczak

1999 longer term

effects

Beseler et al 2008

Cox 2005

Kamel 2005

Kilburn 1999


Rehner 2000

Salvi 2003 1st

evaluation Dec 2001

Salvi 2003 2nd

evaluation

Mch 2002

Solomon 2007

Stephens 2004

mindedness

Subjective Symptoms Questionnaire: lability (emotional changeability)

-

Kamel questionnaire: difficulty concentrating

+

Somatisation + + Table 9. OVERVIEW OF PSYCHIATRIC TESTS The grouping of tests below on the basis of predominant symptoms is based on the information given in the papers which have been summarised. It was noted that many tests examine multiple outcomes, so the citation of a particular test might be relevant to more than one outcome. The information is summarised here to aid members in interpreting the psychiatric studies. General Test Description Brief Symptom Inventory (BSI) The BSI instrument provides patient-reported data to help support clinical decision-making.

Psychologists, psychiatrists, physicians, nurses, and other healthcare professionals can use the BSI instrument to help: assess patients at intake for psychological problems; objectively support care management decisions; measure patient progress during and after treatment to monitor change; provide outcomes measurement for treatment programs and providers through aggregated patient information. Key features: the BSI test is brief and requires only 8-10 minutes to complete, making it well-suited for


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Test Description repeated administrations over time to evaluate patient progress; the BSI instrument provides an overview of a patient's symptoms and their intensity at a specific point in time; the Global Severity Index (GSI) is designed to help quantify a patient's severity-of-illness and provides a single composite score for measuring the outcome of a treatment program based on reducing symptom severity; the reliability, validity, and utility of the BSI instrument have been tested in more than 400 research studies

DSM-IV AXIS-I psychiatric diagnosis

The Diagnostic and Statistical Manual of Mental Disorders (DSM) is the standard classification of mental disorders used by mental health professionals in the United States. It is not only used for patient diagnosis and treatment, but is also important for collecting and communicating accurate public health statistics. The DSM consists of three major components: the diagnostic classification, the diagnostic criteria sets, and the descriptive text. The DSM uses a ‘multiaxial’ system for assessment. This assessment model is designed to provide a comprehensive diagnosis that includes a complete picture of not just acute symptoms but of the entire scope of factors that account for a patient's mental health. There are five axes in the DSM diagnostic system, each relating to a different aspect of a mental disorder: Axis I: This is the top-level diagnosis that usually represents the acute symptoms that need treatment; Axis 1 diagnoses are the most familiar and widely recognized (e.g., major depressive episode, schizophrenic episode, panic attack). Axis II: Axis II, is for personality disorders and developmental disorders such as mental retardation. Axis II disorders, if present, are likely to influence Axis I problems. For example, a student with a learning disability may become extremely stressed by school and suffer a panic attack (an Axis I diagnosis). Axis III: Axis III is for medical or neurological conditions that may influence a psychiatric problem. For example, diabetes might cause extreme fatigue which may lead to a depressive episode.


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Test Description Axis IV: Axis IV identifies recent psychosocial stressors such as a death of a loved one, divorce, losing a job, etc., psychosocial and environmental problems Axis V: Axis V identifies the patient's level of function on a scale of 0-100, (100 is top-level functioning). This is known as the Global Assessment of Functioning (GAF) Scale

General Health Questionnaire (GHQ)

The General Health Questionnaire is a screening device for identifying minor psychiatric disorder. It is suitable for all ages from adolescent upwards – not children. It can be used with the general population or with patients in any sort of non-psychiatric clinical or primary care settings. It is used by researchers and clinicians, and was created by David Goldberg (Goldberg and Hillier, 1979). It is a self-administered questionnaire which focuses on two major areas – the inability to carry out normal functions and the appearance of new and distressing psychological phenomena. The GHQ screens for non-psychotic disorders and is available in the following versions: GHQ-60: the fully detailed 60-item questionnaire GHQ-30: a short form without items relating to physical illness GHQ-28: a 28 item scaled version – assesses somatic symptoms, anxiety and insomnia, social dysfunction and severe depression GHQ-12: a short form – may be used for research studies

Impact of Event Scale (IES) The IES was developed by Mardi Horowitz, Nancy Wilner, and William Alvarez to measure current subjective distress related to a specific event (Horowitz et al 1979). Horowitz observed that the most commonly reported responses to traumatic stressors fell into 2 major response sets: intrusion and avoidance. The IES is considered one of the earliest self-report measures of post-traumatic disturbance. The IES is a broadly applicable self-report measure designed to assess current subjective distress for any specific life event. It is an instrument that can be used for repeated measurement over a period of time. Its sensitivity to change makes it useful for monitoring the client's progress in therapy. The scale consists of 15 items, 7 of which measure intrusive symptoms (intrusive thoughts, nightmares, intrusive feelings and imagery), 8 measure avoidance symptoms (numbing of responsiveness, avoidance of feelings, situations, ideas), and combined they


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Test Description provide a total subjective stress score. All items of the IES are anchored to a specific stressor. Respondents are asked to rate the items on a 4-point scale according to how often each has occurred in the past 7 days. The 4 points on the scale are: 0 (not at all), 1 (rarely), 3 (sometimes), and 4 (often)

Mini International Neuropsychiatric Interview (MINI)

The Mini-International Neuropsychiatric Interview (M.I.N.I.) is a short structured diagnostic interview, developed jointly by psychiatrists and clinicians in the United States and Europe, for DSM-IV and ICD-10 (International Classification of Diseases, latest version, by World Health Organisation) psychiatric disorders. With an administration time of approximately 15 minutes, it was designed to meet the need for a short but accurate structured psychiatric interview for multi-centre clinical trials and epidemiology studies and to be used as a first step in outcome tracking in non-research clinical settings. In their paper (Sheehan et al. 1998), the authors describe the development of the M.I.N.I. and its family of interviews: the M.I.N.I.-Screen, the M.I.N.I.-Plus, and the M.I.N.I.-Kid. They report on validation of the M.I.N.I. in relation to the Structured Clinical Interview for DSM-III-R, Patient Version, the Composite International Diagnostic Interview, and expert professional opinion, and they comment on potential applications for this interview

Profile of Mood States (POMS) Released in 1971, the POMS assessment is a measure of affective mood state fluctuation in a variety of populations, including psychiatric outpatients, medical patients, and in sports psychology. The POMS identifies and assesses transient, fluctuating affective mood states. The POMS Standard & Brief measure six identifiable mood or affective states: 1) Tension-Anxiety 2) Vigour-Activity 3) Depression-Dejection 4) Fatigue-Inertia 5) Anger-Hostility 6) Confusion-Bewilderment. The POMS can be re-administered on a weekly basis, which is long enough to detect the respondent’s mood responses to his or her current life situation, but short enough to assess acute treatment effects. Different waiting periods may be more appropriate for certain research studies, so the POMS items may be interpreted as referring to last week, last month or last year

Q-16 The Q16 questionnaire (Lundberg et al 1997) was developed in Sweden to monitor effects on the central nervous system among workers exposed to solvents but it has also been used on workers exposed to other neurotoxic agents. It contains 16 short questions with yes or no response alternatives on symptoms commonly described by workers exposed to solvents. The understanding of the questions has been investigated by physicians, psychologists, and workers. The reliability was studied by test-retest procedures.


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Test Description It is common practice in Sweden to monitor groups exposed to solvents for possible over-exposure by noting their symptom rate in the Q16 compared with reference groups - for example, the questionnaire has been used to evaluate preventive programmes. The Q16 has also been used to screen exposed people for chronic toxic encephalopathy due to exposure to organic solvents, which is a recognised occupational disease in Sweden. It has been recommended that people with more than six symptoms in the Q16 should be referred for further medical and psychiatric examination. This questionnaire has become commonly used in research on neurotoxic effects of exposure to organic solvents, but it is unknown how positive responses to Q16 questions relate to other outcomes intended to measure similar effects

Revised NEO Personality Inventory (NEO-PI-R)

The Revised NEO Personality Inventory is a psychological personality inventory containing 240 items, and measuring the personality traits of extraversion, agreeableness, conscientiousness, neuroticism and openness to experience. It was developed by Paul Costa Jr and Robert McCrae for use with adults aged over 17 years without overt psychopathology.

Symptom Checklist-90-Revised (SCL-90-R)

The Symptom Checklist-90-Revised (SCL-90-R) is a 90-item self-report symptom inventory developed by Leonard R. Derogatis in the mid-1970s (Derogatis et al 1973) to measure psychological symptoms and psychological distress. It is designed for use with individuals from the community, as well as individuals with either medical or psychiatric conditions. The SCL-90-R assesses psychological distress in terms of nine primary symptom dimensions and three summary scores called global scores. The main symptom dimensions are labelled Somatization (SOM), Obsessive-Compulsive (OBS), Interpersonal Sensitivity (INT), Depression (DEP), Anxiety (ANX), Hostility (HOS), Phobic Anxiety (PHOB), Paranoid Ideation (PAR), and Psychoticism (PSY). The global measures are referred to as the Global Severity Index (GSI), the Positive Symptom Distress Index (PSDI), and the Positive Symptom Total (PST)

Subjective Symptoms Questionnaire

A questionnaire developed by Bazylewicz-Walczak et al 1999 that is a Polish adaptation of the Neurobehavioural Core Test Battery recommended by the WHO (Johnson ed., 1990). The questions relating to subjective symptoms are based on the POMS and on a Finnish Subjective Symptoms Questionnaire

Kamel Q-16 based questionnaire

Questionnaire developed by Kamel et al 2005, based on the Q-16


87

Anger/Irritability Test Description Gulf War survey Survey instrument used in the McCauley et al 2001 study, in which the authors developed their

own questionnaire concerning current health symptoms, including psychiatric symptoms. The instrument is based on previous surveys of unexplained symptoms in Gulf War veterans (McCauley 1999, 2 studies)

Kamel (2005), Kilburn (1999), Stallones (2002b) questionnaires

Questionnaires developed by the respective study authors including questions relating to anger and irritability

POMS, anger Questions relating to Anger-Hostility in the POMS questionnaire described previously Anxiety Test Description BSI: Anxiety The Anxiety sub-scale of the Brief Symptom Inventory Hospital Anxiety and Depression Scale (HADS)

The scale is commonly used by doctors to determine the levels of anxiety and depression that a patient is experiencing. It is a 10 point scale, and if a patient scores the lowest possible value of 1 they are considered to possibly need clinical psychiatric treatment. If the patient scores the maximum points (10) they will be considered clinically stable. Questions are asked by a clinician to determine the number of points. Some questions are geared towards anxiety and others towards depression. The patient is asked about his or her feelings over the past week, and is asked to answer what his/her immediate reaction is, to more accurately reflect their feelings

State-Trait Anxiety Inventory (STAI)

The STAI was developed to provide reliable, relatively brief, self-report scales for assessing state and trait anxiety in research and clinical practice (Spielberger,1983). It consists of two 20-item scales for measuring the intensity of anxiety as an emotional state (S-Anxiety) and individual differences in anxiety proneness as a personality trait (T-Anxiety). In responding to the S-Anxiety items, subjects report the intensity of their feelings of anxiety ‘right now, at this moment’ by rating themselves on the following 4-point scale: (1) Not at all, (2) Somewhat, (3) Moderately so, (4) Very much so. Responses to the T-Anxiety items require subjects to indicate how they generally


88

Test Description feel by reporting how often they have experienced anxiety-related feelings and cognitions on a 4-point scale: (1) Almost never, (2) Sometimes, (3) Often, and (4) Almost always. Items were selected for the original STAI (Form X) on the basis of highly significant correlations with the most widely used measures of anxiety at the time the inventory was developed by Spielberger in 1972 (Auerbach and Spielberger, 1972). In subsequent research, strong correlations of the STAI scales with measures of depression contributed to recognising that the content of several items adapted from other anxiety measures were more closely related to depression than anxiety (e.g., ‘I cry easily’; ‘I feel blue’). In developing the revised STAI (Form Y), the conceptual definitions of state and trait anxiety were further clarified, and items with depressive content were replaced (Spielberger,1983)

POMS: anxiety Tension-anxiety is one of the six sub-scales of the self-rated POMS, described above. The POMS as a whole includes 65 items, rated on a scale from 0, ‘not at all,’ to 4, ‘very much so’, and the scores on the six subscales are summed to give a total mood disturbance score

Depression Test Description BSI suicidal ideation The Brief Symptom Inventory, described above, consists of 53 questions on symptoms occurring

within a 1-month time frame. Forty-nine of the questions address nine dimensions: somatisation, obsessive-compulsiveness, interpersonal sensitivity, depression, anxiety, hostility,phobic anxiety, paranoid ideation and psychoticism. There is one question that specifically addresses suicidal ideation: (during the last month) ‘Have you had thoughts of ending your life?’ ; this is the question used in the Wesseling et al 2010 study

Center for Epidemiological Studies - Depression Scale (CES-D)

One of the most common screening tests for helping an individual to determine his or her depression quotient, the Center for Epidemiologic Studies Depression Scale (CES-D) was originally developed by Lenore Radloff of Utah State University. The quick self-test measures depressive feelings and behaviours during the past week.

Gulf War survey: depression Survey instrument used in the McCauley et al 2001 study described in the ‘Anger/irritability’ section above


89

Test Description Hospital Anxiety and Depression Scale (HADS)

Questions concerning depression on the HADS, described above

Beseler et al (2008), Kamel (2005), Stallones (2002b) questionnaires

Questionnaires developed by the respective study authors including questions relating to depression

POMS: depression One of the six sub-scales on the POMS scale described above: 3) Depression-Dejection Subjective Symptoms Questionnaire: depression; neurovegetative symptoms associated with depression

Questionnaire developed by Bazylewicz-Walczak et al 1999, described above, questions relating to depression. With regard to neurovegetative symptoms, clinical depression is characterised by a persistent sad mood as well as by changes in physical and mental functioning. These symptoms are called ‘neurovegetative’ because they are caused by the changes in the nervous system that occur during depression. Doctors look for the neurovegetative signs of depression to determine whether a patient is clinically depressed or simply experiencing normal grief or sadness. Neurovegetative symptoms affect functions such as sleep, energy levels and ability to concentrate

Structured Clinical Interview for DSM-IIIR Mental Disorders (SCID)

The SCID is a semi-structured interview for making the major Axis I DSM-III-R diagnoses. Axis 1 diagnoses are the most familiar and widely recognized (e.g., major depressive episode, schizophrenic episode, panic attack). The SCID is administered by a clinician and includes an introductory overview followed by nine modules, seven of which represent the major axis I diagnostic classes. Because of its modular construction, it can be adapted for use in studies in which particular diagnoses are not of interest. Using a decision tree approach, the SCID guides the clinician in testing diagnostic hypotheses as the interview is conducted. The output of the SCID is a record of the presence or absence of each of the disorders being considered, for current episode (past month) and for lifetime occurrence.

Confusion/Fatigue/Tension Test Description POMS: confusion, fatigue, tension

Three of the six sub-scales of the Profile of Mood States described above. The three sub-scales are: Tension-Anxiety, Fatigue-Inertia, and Confusion-Bewilderment


90

Kamel (2005) questionnaire Questionnaire developed by the study authors, including questions relating to fatigue and tension Subjective Symptoms Questionnaire

Questionnaire developed by Bazylewicz-Walczak et al 1999, described above, questions relating to fatigue

Mood swings Test Description Gulf War survey: mood swings Survey instrument used in the McCauley et al 2001 study described previously, questions

concerning mood swings PTSD symptomatology Test Description Clinician-Administered Post-Traumatic Stress Disorder Scale (CAPS)

The CAPS has been called the gold standard in PTSD assessment. It is a 30-item structured interview that corresponds to the DSM-IV criteria for PTSD (Diagnostic and Statistical Manual of Mental Disorders, the standard classification of mental disorders used by mental health professionals in the United States, described above). The CAPS can be used to make a current (past month) or lifetime diagnosis of PTSD or to assess symptoms over the past week. In addition to assessing the 17 PTSD symptoms, questions target the impact of symptoms on social and occupational functioning, improvement in symptoms since a previous CAPS administration, overall response validity, overall PTSD severity, and frequency and intensity of five associated symptoms (guilt over acts, survivor guilt, gaps in awareness, depersonalisation, and derealisation). For each item, standardised questions and probes are provided. As part of the trauma assessment (Criterion A), the Life Events Checklist (LEC) is used to identify traumatic stressors experienced. CAPS items are asked in reference to up to three traumatic stressors. The CAPS was designed to be administered by clinicians and clinical researchers who have a working knowledge of PTSD, but can also be administered by appropriately trained para-professionals. The full interview takes 45-60 minutes to administer, but it is not necessary to administer all parts (e.g., associated symptoms). The most frequently used scoring rule is to count a symptom as present if it has a frequency of 1


91

Test Description or more and an intensity of 2 or more. Severity scores can also be calculated by summing the frequency and intensity ratings for each symptom

Impact of Event Scale – Revised (IES-R)

The IES-R is a 22-item self-report measure that assesses subjective distress caused by traumatic events. It is a revised version of the older version, the 15-item IES (Horowitz et al 1979). The IES-R contains 7 additional items related to the hyperarousal symptoms of PTSD, which were not included in the original IES. Items correspond directly to 14 of the 17 DSM-IV symptoms of PTSD. Respondents are asked to identify a specific stressful life event and then indicate how much they were distressed or bothered during the past seven days by each ‘difficulty’ listed. Items are rated on a 5-point scale ranging from 0 (‘not at all’) to 4 (‘extremely’). The IES-R yields a total score (ranging from 0 to 88) and subscale scores can also be calculated for the Intrusion, Avoidance, and Hyperarousal subscales

Mississippi Scale for PTSD The Mississippi Scale for Post-Traumatic Stress Disorder is a 35-item self-report scale derived from the Diagnostic and Statistical Manual of Mental Disorders (DSM-III) criteria for the disorder, and relates specifically to combat-related PTSD

St Luke’s Hospital Questionnaire

Questionnaire used in the Kawana et al 2001 and Ohtani et al 2004 studies; the questionnaire survey evaluated 34 subjective somatic and mental symptoms. It was developed by the staff of St. Luke’s International Hospital in Tokyo, where a number of victims of the Tokyo subway sarin attack in 1995 were treated. A three-point scale was used to assess the severity of the symptoms (severe was rated 2 points, mild 1, and none 0). It is a self-rating questionnaire, with the mental symptoms divided into two groups, PTSD symptoms and non-specific mental symptoms. The latter were defined by the authors as post-sarin poisoning mental (PSPM) symptoms

Sleep disturbances Test Description Kawada (2005) questionnaire Questionnaire developed by the authors, questions concerning insomnia in younger and older

age groups Kamel (2005), Kilburn (1999) Questionnaires developed by the respective study authors including questions relating to sleep


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questionnaires Nakajima (1999) questionnaire Questionnaire developed by the authors, questions concerning bad dreams and insomnia Gulf War survey Survey instrument used in the McCauley et al 2001 study described previously, questions

concerning difficulty sleeping Stallones (2002b) questionnaire Questionnaire developed by the authors, questions concerning sleeping more than usual Subjective Symptoms Questionnaire

Questionnaire developed by Bazylewicz-Walczak et al (1999) described above, questions relating to sleep disturbances

Other outcomes Test Description Kamel (2005) questionnaire Questionnaire developed by the study authors, includes questions regarding absent-mindedness

and difficulty concentrating Subjective Symptoms Questionnaire

Questionnaire developed by Bazylewicz-Walczak et al 1999, described above, questions relating to absent-mindedness and emotional changeability

Somatisation, somatising tendency

A measure devised by the authors of the Solomon et al 2007 study. Authors used questions from the Brief Symptom Inventory about somatic symptoms, and the responses were used to derive a ‘‘somatising tendency’’ score, which was partitioned to four levels


1

TOX/2012/26 Annex 7 COMMITTEE ON TOXICITY OF CHEMICALS IN FOOD, CONSUMER PRODUCTS AND THE ENVIRONMENT EVALUATION OF PERIPHERAL NEUROPATHY AND NEUROMUSCULAR DYSFUNCTION OVERVIEW OF COT 1999 REPORT Studies in Individuals with a History of Acute Poisoning 1. In relation to the long-term sequelae of acute poisoning, the COT concluded that: “Peripheral neuropathy (PN), as one feature of organophosphate-induced delayed polyneuropathy (OPIDPN), is a well-established complication of OP poisoning by OPs that inhibit the enzyme neuropathy target esterase (NTE). The neuropathy is predominantly motor but possibly also sensory. Compounds that produce more than 70% inhibition of NTE give positive results in the hen test Compounds evaluated as giving a positive result in the hen test are not used in the United Kingdom and have not been approved or licensed by regulatory agencies (i.e. the Veterinary Medicines Directorate or the Pesticides Safety Directorate). The balance of evidence indicates that acute poisoning by other OPs, which do not inhibit NTE, can also lead to persistent PN detectable by neurophysiological tests. If this occurs, most cases are not at a level that would give rise to symptoms.” 2. For doses lower than that which causes frank toxicity, the COT concluded that: “No studies have examined the long-term effects of a single exposure to OPs insufficient to cause toxicity. However, the findings in individuals with prolonged and repeated low-dose exposures, and in those who have suffered recognised acute poisoning, together indicate that any risk of serious health effects from such limited exposure must be small.”

Studies in Individuals with No Past History of Acute Poisoning

3. The COT concluded that: “The balance of evidence indicates that low-level exposure to OPs does not cause PN. If effects on peripheral nerve function sufficient to cause severe disability do occur, they must be rare." OVERVIEW OF LITERATURE PUBLISHED AFTER COT 1999 REPORT Definition of Peripheral Neuropathy 4. The COT 1999 report defines peripheral neuropathy (PN) as a disorder that affects nerves outside the brain or spinal cord. This is in accordance with definitions provided in the published literature, which consider PN in general terms as any disorder of the peripheral nervous system (PNS) where damaged nerves are unable


2

to send messages from the brain and spinal cord to the muscles, skin and other parts of the body. It should be noted that this definition also encompasses effects on the autonomic nervous system (ANS). However, the COT 1999 report analysed ANS abnormalities separately from other PN effects. The report also considered cranial nerve effects within the PN/NMD section. Therefore, for consistency, it was decided that the review would adopt a similar approach and exclude ANS abnormalities from the PN/NMD health outcome category, and focus on motor and sensory functions of both types of peripheral nerves i.e. the spinal nerves that go from the spinal cord to the skin and muscle of the limbs, hands and feet, and the cranial nerves that go from the brain to the eyes, mouth, ears and other parts of the head (that are responsible for visual acuity, visual fields, papillary reactions, extra-ocular movements, corneal reflexes, smell, hearing, balance, taste, swallowing, gag reflexes, and facial movements and sensations). ANS abnormalities are analysed under a separate health outcome category (Annex 8). 5. Motor function examinations often include assessment of gait, station i.e. posture and equilibrium (balance). ‘Dizziness’ is a non-specific symptom, but can have cranial nerve aetiologies (i.e. it could be due to vertigo or disequilibrium/imbalance associated with cranial nerve VII dysfunction). Therefore, this section also includes studies that evaluated dizziness when appropriate tests for vertigo and imbalance were conducted. NB. Dizziness or ‘presyncope’ can also be caused by autonomic nerve abnormalities (e.g. orthostatic hypotension) and is discussed in the ANS section where appropriate. 6. Reflexes provide information on the integrity of the CNS and PNS. Decreased reflexes tend to indicate a peripheral problem while lively or exaggerated reflexes indicate a problem with the CNS. Somatic reflexes that involve the spinal nerves known as ‘stretch’ or ‘deep tendon reflexes’ (e.g. biceps-, brachioradialis-, extensor digitorum-, triceps-, patellar-, ankle jerk-, and plantar- or babinski- reflexes) and those that involve the cranial nerves (e.g. corneal and gag reflexes) are analysed under the PN/NMD health outcome category where appropriate. 7. Members should note that although PN can impact on neuropsychological function and affect fine motor skills and co-ordination (as evidenced for example by finger-tapping, pegboard and manual dexterity tests), this section primarily reports on effects on motor and sensory systems in relation to the PNS, and not the higher centres of the CNS. 8. Detailed information of the studies summarised in this section can be found in Annex 7, which presents information on the study design used (Table 6), and the results, strengths/weaknesses and conclusions (Table 7).


3

Acute Exposure with Chronic Peripheral Neuropathy and Neuromuscular Dysfunction 9. For the purpose of this review, acute exposure is defined as a single incident where the exposure is continuous and may last up to a number of days, e.g. four days. 10. Fourteen epidemiological studies evaluated the chronic effects of acute exposure to OPs in relation to the development of peripheral neuropathy (PN) and neuromuscular dysfunction (NMD). These investigations comprised five cohort studies (Kawana et al 2001, Miranda et al 2002ab, 2004, Nakajima et al 1999), six cross-sectional studies (McCauley et al 2001, Nishiwaki et al 2001, Spencer, 2001, Stallones and Beseler, 2002b, Miyaki et al 2005, Jalali et al 2011) one case series (Dahlgren et al 2004) and two case reports (Loh et al 2010, Soummer et al 2011). There were no case-control studies evaluating persistent PN/NMD following acute OP exposure. 11. Acute exposure to OPs arose largely from poisoning episodes. Two studies examined the effects of acute exposure to OPs insufficient to cause acute toxicity (McCauley et al 2001, Spencer, 2001). NB. Keifer et al examined the acute effects of low-dose exposure to OPs but the period of exposure was over a season i.e. sub-chronic (See para. 112 in the chronic exposure section) (Keifer et al 2000). 12. Poisoning (or attempted poisoning) incidents were either:

a. accidental i.e. following occupational exposure (Miranda et al 2002ab, 2004, Stallones and Beseler, 2002b) or residential exposure (Dahlgren et al 2004); or

b. intentional i.e. the result of suicide attempts (Miranda et al 2002ab, 2004; Jalali et al 2011., Soummer 2011), or deliberate chemical attacks during warfare (McCauley et al 2001, Spencer, 2001, Loh et al 2010) or terrorism (Nakajima et al 1999, Kawana et al 2001, Nishiwaki et al 2001, Miyaki et al 2005).

13. Members should note that the 1994/1995 sarin terrorist attacks in Japan and the 1991 Gulf War Khamisiyah incident are included in the current evaluation (despite not being evaluated in the COT 1999 report) because they represent defined exposure incidents where a large number of individuals received definite acute exposure to specific OP chemical agents, which are not pesticides and have a different toxicological profile from approved OPs. 14. The main causes of PN include systemic disease (e.g. diabetes mellitus, cancer), specific hereditary conditions, trauma, infection (e.g. HIV, Hep B), vitamin deficiency, exposure to neurotoxic chemicals, alcohol abuse, smoking and age (due to an increased prevalence in individuals aged over 50 years) (Rubin, 2008). Unless otherwise stated, studies accounted for some or all of the possible confounding effects of age, gender, education, occupation, socioeconomic status, lifestyle, existing medical conditions, and exposure to other neurotoxic agents.


4

15. Paragraphs 17 to 66 provide detailed summaries of these studies. NB. For each summary, the first paragraph briefly describes the study design, with the main findings and conclusions outlined in subsequent paragraphs. Cohort studies Nicaraguan Acute Poisoning Hospital Patients 16. Miranda et al evaluated motor neurological impairment in a cohort of 59 Nicaraguan male patients (from an original cohort of 77 patients) hospitalised for acute poisoning after accidental or intentional exposure to OP pesticides (Miranda et al 2002a). Thirty-nine cattle ranchers and fishermen (from an original cohort of 74 men) who had never experienced pesticide poisoning were used as an external comparison group (although the authors noted that several had been exposed to pesticides). Comparison of demographic and lifestyle characteristics of the referent and exposed group showed they had similar education levels. The authors explain that the reduced cohorts arose from subjects either dying before the first evaluation or data going missing, patients not completing the second evaluation (due to death, or for reasons not reported), concerns over possible exposure misclassification, patients relocating, or patients refusing to participate/continue (that comprised one and three subjects in the exposed and unexposed groups respectively). No further analyses were conducted to evaluate the characteristics of the four non-participants/drop-outs. The authors divided the pesticides the patients were exposed to into either neuropathic OPs (which can cause PN i.e. methamidophos, chlorpyrifos, and fenthion) and non-neuropathic OPs (i.e. not known to cause PN e.g. methyl parathion, malathion, terbufos, phorate, ediphenfos, and metaphox). Exposure assessment relied on information collected from case histories and field visits to confirm the reports, and a biological assessment of exposure that was performed in 35 patients. The severity of poisoning was also used as an indication of the level of exposure, and was categorised as being either mild (n=0), moderate (n=37) or severe (n=22) depending on the extent and severity of symptoms. Patients with intentional poisonings were the most severe cases (n=6). Poisoning severity was verified by the finding that the level of erythrocyte cholinesterase activity in one severely poisoned patient (measured at the time of hospital admittance) was 85% below the lower limit of the normal level in the control group. Subjects were followed up twice: (i) at hospital discharge i.e. up to 24 days after poisoning, and (ii) approximately seven weeks after poisoning. The authors did not specify whether the hospital-based examinations were conducted by experts. However, a standardised procedure was used to assess hand-grip and pinch strength (kg) in the subject’s dominant hand (via dynamometer and pinch gauge respectively). It is not reported whether investigators were blinded to the subject’s exposure or health status. Potential confounders were considered, which included days between exposure and each examination, and OP cumulative exposure. Only age was adjusted for in subsequent analyses. 17. Compared to controls, grip and pinch strength was significantly diminished in patients who were moderately and severely poisoned with known neuropathic pesticides seven weeks after poisoning (p<0.05) (Miranda 2002a, Table 7.A.1,


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Annex 7). Individuals poisoned with non-neuropathic OPs produced non-significant impairments. The authors also observed possible dose-dependent decreases in grip strength in patients poisoned with neuropathic OPs, which was notably worse in the severely poisoned subgroup (n=16) compared to the moderately poisoned subgroup (n=25), although the significance of this was not reported. Further analysis of subjects severely poisoned with neuropathic OPs (n=16) revealed more marked grip and pinch impairments for poisonings that were intentional (n=6) compared to those resulting from occupational accidents (n=10) (p<0.05). These impairments in the intentional subgroup worsened at the second examination, and was confirmed to be statistically significant in subsequent analyses of mean changes in grip and pinch strength between both examinations (p<0.05). 18. The findings of this study provide evidence of subsequent dose-related motor impairments in individuals acutely poisoned with neuropathic OPs. Given the short follow-up period it was suggested that these effects represented symptoms of the intermediate syndrome or OPIDPN. However, the study was not without its limitations. The authors used only seven controls in the second examination, as most men migrated and were unreachable. The authors did not adequately control for potential selection bias. In addition, the authors failed to account for the effect of muscle training as a potential confounder, which can influence a subject’s muscular load on hands/grip and pinch strength. 19. In a separate paper, Miranda et al report the findings of their evaluation of sensory fibre impairment in the same cohort of Nicaraguan patients (Miranda et al 2002b) (Table 6.A.1, Annex 7). A fewer number of subjects were used (56 patients and 38 controls who had similar education and socioeconomic backgrounds). Erythrocyte cholinesterase activity was reported for 21 patients, which revealed a dose-dependent decrease in activity with poisoning severity (although the authors did not indicate whether this was significant). As in the previous study, subjects were followed up at hospital discharge and approximately seven weeks after poisoning. Large diameter myelinated sensory fibre impairment was evaluated by measuring the quantitative tactile vibration thresholds (log mic) in subject’s dominant index finger and great toe using standardised procedures. Multiple linear regression was used to identify potential associations; a regression coefficient with a p-value of less than 0.05 was considered statistically significant. The authors accounted for several confounders (as in the previous study) including occupational exposure to vibrational machinery, and the effect of four different examiners. Again, only age was found to be a significant confounder and adjusted for in subsequent analyses. 20. No significant group and intra-individual differences were observed for patients poisoned with non-neuropathic OPs (n=16) (Miranda 2002b, Table 7.A.1, Annex 7). For men poisoned with neuropathic OPs (n=40) the results for their index fingers were not significantly different from controls or between examinations. However, significantly increased toe vibrotactile thresholds were observed in the second examination for patients with severe intentional poisonings compared to controls. As in the previous study, patients who were severely poisoned intentionally (n=5) with known neuropathic OPs had a significantly higher mean change difference in toe thresholds (0.52 log mic; SD=0.6; RC=0.60[0.24 – 0.97])) compared to controls (-0.09 log mic; SD=0.34 (n=39)).


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21. This study therefore provides evidence of subsequent sensory impairment in subjects acutely poisoned with known neuropathic OPs. This appears to be dependent on the severity of poisoning and type of agent; the largest impairments arising in those who intentionally poisoned themselves with neuropathic OPs. However, as with the previous study, the loss of subjects to follow-up in both exposed and unexposed groups represents a possible source of selection bias, although the authors noted that in the first examination there was no difference in vibration thresholds between individuals in the comparison group who did and did not participate in the second evaluation. Nonetheless, the short duration of the follow-up period should be considered when interpreting the study’s significance. 22. The authors undertook a further two year follow-up study in the same cohort of patients and addressed some of the above limitations of their earlier studies (e.g. non-participation of subjects) (Miranda et al 2004) (Table 6.A.1, Annex 7). Abnormalities of both motor and sensory function (i.e. hand grip/pinch strength, and vibrotactile thresholds of big toe/ index finger respectively) were evaluated in 48 male patients (from an original group of 71) and 28 of 74 controls (due to a further 11 patients and 11 controls not examined a third time). 23. Sensory and motor impairments were still apparent two years after subjects were severely poisoned with neuropathic OP pesticides. These patients (n=14) remained significantly weaker than controls in all examinations (particular with regards to grip strength) although part of their strength recovered in examination III (evidenced by a higher mean value for grip strength) (Miranda 2002a, Table 7.A.1, Annex 7). Motor effects resolved in men poisoned with non-neuropathic OPs, and those moderately poisoned with neuropathic OPs. The vibrometry findings of the current study contrasted with the vibrometry findings observed in earlier studies (Miranda et al 2002b). Subjects that were severely poisoned with neuropathic OP pesticides and previously had increased toe vibrotactile thresholds (but no impairment of index finger in the second examination), displayed significantly increased index finger vibrotactile thresholds at the third examination (n=14), but no significant increased thresholds for the big toe (Miranda et al 2004). The authors also investigated whether the loss of subjects to follow-up could have biased the results by comparing the test scores of subjects who participated only in examination I with those who participated in both the first and second examination, and also comparing the scores of those who participated in only I and II with those who participated in all three. The reason for non-participation was not found to be due to health, and loss to follow-up did not seem to have altered the results for most of the comparisons. However, the authors noted that two subjects with severe intentional poisoning with neuropathic OPs dropped out at examination II and both had considerably higher vibration thresholds at the time of examination II than those who remained in the study. 24. Based on the reduced hand strength results, the authors concluded that their study showed that patients severely poisoned with neuropathic OP pesticides displayed mainly motor (and less sensory) abnormalities up to two years after the incident. The resolution of some of these symptoms was found to be consistent with the pattern of OP-induced delayed polyneuropathy (OPIDPN), which has been previously considered by the COT to be a well-established sequelae of acute OP


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intoxication (which occurs two to three weeks after poisoning and is known to resolve and/or persist for years). Victims of the Sarin Terrorist Attacks in Japan 25. Nakajima et al used an author-generated questionnaire survey to collect symptom data on 2052 residents of the 1994 Matsumoto sarin attack (Nakajima et al 1999). All victims were Matsumoto inhabitants living or staying in the area at the time of the incident (i.e. within 1050 metres north to south and 850 metres east to west of the spot where about 12 litres of sarin was released on the night of June 27). The authors used an internal comparison group i.e. non-victims defined as residents who were not diagnosed with definite muscarinic and/or nicotinic symptoms immediately after the incident. Subjects were followed up at various times after the attack, which ranged from three weeks up to three years (although the current study evaluated symptoms reported one and three years after the attack - the results of the earlier surveys were reported in an earlier paper (Nakajima et al 1998)). Erythrocyte and serum AChE activity levels were measured only in victims admitted to hospital four weeks after sarin exposure. Health examinations were also performed (albeit inconsistently i.e. for those who requested an examination). No objective neurological tests were used to validate symptoms or collect data on the background prevalence of the sarin-related symptoms, and the authors accounted for the confounding effects of age and gender only. 26. Postcode mapping revealed that the percentage of symptomatic subjects were greatest in the block where sarin was released (Table 7.A.1, Annex 7). Of the 318 victims and 919 non-victims who responded in the first year survey (60.3% participation rate), nine victims reported having asthenia (muscle weakness) which was associated with a significantly lower erythrocyte AChE activity compared to victims who did not report having this symptom (p<0.01). A total of 167 victims and 669 non-victims responded to the three-year survey, there being a lower compliance in the victim group (36.1% of the victims immediately after the incident) compared to the non-victim group (54.5%). Victims (n=14) were 5.5 times more likely to report symptoms of ‘asthenia’ (i.e. muscle weakness) compared to non-victims (n=11) (p<0.001), and 2.5 times more likely to report ‘shoulder stiffness’ (p<0.01). Possible cranial nerve involvement was indicated by victims being more likely to report having experienced ‘narrowing of visual fields’ compared to non-victims (OR=3.52[1.17-10.63]; p<0.05) n=6, 7 respectively). However, in the absence of objective verification of optic nerve damage, it would be imprudent to assume that the latter symptom represented a definite PN-related outcome as it is quite non-specific (i.e. visual field deficits could also have a non-neurological aetiology). 27. The authors concluded that there was a possible association between sarin exposure and chronic symptoms reported in victims. However, the study did not use an unexposed comparison group and did not attempt to control for potential confounding exposures. The authors did not systematically conduct objective health assessments (or present the results of those for subjects who requested them) which would otherwise help towards verifying the reported symptoms. Furthermore, the background prevalence of these symptoms was unclear and there was no attempt to control for possible selection bias from the low questionnaire compliance (especially


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in victims) or account for possible information/ measurement bias caused by over-recall of symptoms in the victim group. 28. Kawana et al, also used an author-generated questionnaire to investigate whether 582 victims of the 1995 Tokyo sarin subway attack treated locally in St Luke’s International Hospital suffered chronic symptoms after two, three and five years (Kawana et al 2001). Three referent groups were used in a comparative analysis of symptom incidence: these comprised of two sarin-exposed referents (i.e. 88 Matsumoto sarin victims, and 655 sarin victims of the Tokyo incident analysed by a non-governmental organisation), and an unexposed group of 87 subjects taken from controls used in a study of the Matsumoto 1994 attack (Nakajima et al 1998). The questionnaire response rate was low and inconsistent, which declined from 48.6% to 32.8% in subsequent follow-ups. This was not analysed further to rule out selection bias. Exposure assessment was based on subject’s admittance to the hospital as a victim. The authors did not attempt to stratify the level of exposure or conduct any form of biological exposure assessment. No objective health assessments were performed. 29. Muscle stiffness was among one of the frequently reported symptoms (>10%), although the symptom frequency rate did not change significantly from survey to survey (Kawana 2001, Table 7.A.1, Annex 7). The symptom frequency rate was highest for the NGO Tokyo victim group (31.8%), followed by victims of the current study of St Luke’s Hospital subjects at (14.7%), and was lowest in the Matsumoto control group (8%). The authors did not report whether these represented significant differences between groups. NB. It is assumed that the follow-up period used for the symptom incidence comparison was five years, although this is not clear in the report. Another frequently reported symptom was ‘dim vision’, which can arise from dysfunction in the optic nerve. However, in the absence of further testing it would be imprudent to classify ‘dim vision’ as a cranial nerve issue as it could also result from dysfunction in either the eye or brain. 30. There were several limitations associated with this study that included the lack of account of any potential confounders, questions over the appropriateness of the external comparison groups (as the authors did not elaborate on whether subjects were matched), no attempt to rule out possible recall bias, and no use of measures of association. This study, therefore, provides a descriptive account of self-reported chronic symptoms in sarin victims, which prevents any meaningful inferences from being drawn. Cohort studies summary 31. Overall, the findings from cohort studies investigating chronic peripheral neuropathy in subjects acutely exposed to NTE-inhibiting and non-NTE-inhibiting OP pesticides/agents provide weak evidence for the development of peripheral neurological abnormalities. Three of the five cohort studies were conducted by the same author (Miranda et al) which provided limited evidence of chronic neurological impairment in Nicaraguan patients acutely exposed to high levels of neuropathic OPs. The authors postulated that the impairments (which were largely motor) were most likely the result of OPIDPN. However, muscle strength tests heavily rely on


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subject co-operation and are, therefore, limited in terms of their reliability, and the authors did not corroborate these with supportive data from electrophysiological tests. Furthermore, the QST tests conducted were only partial as they did not include assessment of small nerve fibre function via thermal threshold tests. 32. The remaining two cohort studies investigated the long-term effects of acute poisoning to an OP agent that does not inhibit NTE i.e. sarin poisoning in victims of the two terrorist attacks in Japan (Nakajima et al 1999; Kawana et al 2001). Both studies reported an increased prevalence of a select few PN-related symptoms compared to controls e.g. asthenia (muscle weakness), shoulder and muscle stiffness. However, these findings relate to symptoms obtained using subjective health assessments i.e. questionnaire surveys. No data from objective and routinely applied neurological examinations or neurophysiological assessments were reported in either study to verify these symptoms. Cross-sectional studies 33. Six cross-sectional studies investigated the long-term effects of acute OP exposure. Iranian Acute Poisoning Hospital Patients 34. Jalali et al. sought to specifically assess electrophysiological peripheral nerve effects in patients hospitalised for intentional acute OP poisoning (Jalali et al 2011). Eight of the 342 patients admitted to the Poisons Treatment Unit of the Imam Reza hospital, Iran, were considered eligible for the study. These patients had recovered from the acute phase of intoxication with either chlorpyrifos (n=3), malathion (n=2), or azinphosmethyl (n=2) and displayed clinical signs and symptoms of muscle weakness or peripheral neuropathy. NB. For one patient the identity of the OP used was not determined/ recorded. The extent of the patients acute signs were used to classify the patients as being either moderately or severely poisoned (n= 3 and 5 respectively), which was largely dependent on whether the patients exhibited respiratory failure. The authors noted that three patients revealed symptoms of Intermediate Syndrome. Exposure data gathered from case histories and clinical manifestations were verified via measurement of AChE activity that was significantly negatively correlated with severity of poisoning (p<0.0001). Nerve conduction velocity (NCV) tests of the median, ulnar, peroneal and tibialis nerves, and electromyography (EMG) of various muscles of the hands, arms and legs were conducted by investigators (Jalali 2011, Table 6.A.3, Annex 7). However, the times lapsed since the onset of poisoning varied from 10 days (i.e. during hospitalisation) to 210 days (i.e. 6 months after discharge): three patients were tested during hospitalisation or <1 week after discharge; four patients within 6 weeks after OP exposure and one patient 6 months after discharge. The authors accounted for most potential confounders and excluded patients if they co-ingested alcohol or any other chemicals/pharmaceuticals. Instead of a referent group the authors used normal values for NCV test comparisons. 35. All eight patients exhibited sensory dysfunction with motor dysfunction arising in four patients (it is unclear whether this summary result refers solely to


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electrophysiological tests or to the clinical evaluations conducted prior to them). The authors briefly reported on the EMG findings and noted identification of a sensory-motor peripheral polyneuropathy with a predominantly distal sensory deficit. No further information was provided. NCV results for the upper and lower limbs were inconsistent (Jalali 2011, Table 7.A.3, Annex 7). The NCV in the right and left tibial nerves were significantly lower than normal values (e.g. 44.6 cf. 55.9 m/s in the left tibial nerve vs. normal values (p=0.023), while, the NCV of the left ulnar nerve was significantly higher than the normal value (i.e. 65.6 vs. 62.7; p=0.034). This is unsurprising given that subjects had variable testing times since exposure. However, more consistent results were observed for other nerve conduction parameters measured. Sensory latencies (ms) in each nerve were significantly higher in all eight patients compared to normal values (highest p value = 0.017). Furthermore, the authors also found that sensory latency was negatively correlated to NCV (r=-0.558, r2=0.312, p<0.0001). Additional impairments were also demonstrated by the significantly lower motor and sensory wave amplitudes (mV) compared to normal (p<0.0001). The authors report that these impairments were significantly worse in the lower limbs compared to the upper limbs as indicated by the lower and higher average values of the motor NCV and sensory latencies respectively (p<0.0001). However, the authors noted that these electrophysiological changes were not significantly correlated with clinical severity or AChE activity. 36. The findings of this study suggest individuals acutely poisoned with OP pesticides bear electrophysiological deficits (particularly sensory) in their peripheral nerves up to six months post exposure. However, it is unclear whether these results merely reflect changes associated with the development of OPIDPN as the electrophysiological evaluations combine subjects poisoned with pesticides known to inhibit NTE (i.e. chlorpyrifos) and those that do not (i.e. malathion and azinphosmethyl). In addition, the strength of these positive findings is further compromised by the study’s very small size, its lack of a control group, the absence of dose-response relationship, and the fact the study tested different subjects at different times since being exposed (with the longest duration being six months in one patient). Police Officers/Rescue Teams at 1995 Tokyo Subway Sarin Attack 37. Vibration perception threshold tests and stabilometry were used to evaluate post-traumatic stress-related vibratory sensations and disequilibrium respectively in 54 (from an original 57) sarin-exposed male rescue team staff members and police officers of the 1995 Toyko subway attack (Nishiwaki et al 2001). Controls comprised of 52 age-matched male office members who were not exposed. Subjects were followed-up between 34-45 months after the incident and were divided into two groups according to their level of exposure based on the severity of poisoning i.e. whether subjects were hospitalised immediately after poisoning (high exposed group) or attended hospital as outpatients (low-exposed group). No biological measures of exposure were performed to verify these categories. Comparison of the demographics between the different groups showed significant differences for height and weight (p<0.05). Vibration perception threshold determinations in the index or middle fingers and stabilometry using a strain-gauge-type force platform, were conducted by members of the author’s research group who had no knowledge of


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subject’s exposure status (Nishiwaki 2001, Table 6.A.3, Annex 7). Various potential confounding variables were included in the regression analysis, which included height and weight but not previous use of vibrational machinery. Subjects were excluded if they were routinely occupationally exposed to other chemicals such as solvents and reagents. For the stabilometry tests, two subjects were excluded for having low back pain or a history of auditory ossicles fracture. 38. The authors reported that none of the parameters for vibration perception thresholds or stabilometry had any relation to exposure. Although two body-sway parameters in the low-exposed group were significantly larger than those in the referent group this was not dose-related (Nishiwaki 2001, Table 7.A.3, Annex 7). 39. This study did not provide any convincing evidence of chronic PN-related neurological effects in subjects exposed to a non-NTE inhibitory OP. However, the study is limited by severity of exposure being based solely on hospitalisation admission status obtained from a questionnaire (there being no objective exposure assessment conducted and thus a possible source of misclassification bias). Furthermore, the sole use of QST tests as a measure of sensory impairment is limited by its subjective nature. Subway Workers at 1995 Tokyo Subway Sarin Attack 40. The same research group sought to determine dose-dependent nervous system effects in another cohort exposed during the attack (Miyaki et al 2005). Twenty-three male subway workers (representing a 65% participation rate) were subdivided into high and low exposure groups based on their responses to a self-administered questionnaire, which asked subjects whether they were hospitalised immediately after poisoning (high-exposure) or attended hospital as an outpatient (low-exposure). For comparisons, 13 referent subway workers who were not exposed to sarin also participated. Investigators, who were blinded to the subject’s exposure status performed various standardised computerised neurobehavioural tests, which included stabilometric tests that used a strain-gauge type force platform with subjects asked to keep their eyes open or closed (Miyaki 2005, Table 6.A.3, Annex 7). The authors used Student’s t-test to compare mean values between the exposed group and referents, and ANOVA among the high vs. low exposed groups and referent group. Most potential confounders were accounted for. 41. No significant exposure-related changes were observed for any of the body-sway parameters evaluated (Miyaki 2005, Table 7.A.3, Annex 7). Possible selection bias cannot be ruled out as the authors did not evaluate background characteristics of non-participating subjects. The crude exposure assessment may have introduced measurement bias and thus weakened the study’s ability to observe dose-dependent changes. US Farm Residents 42. Stallones & Beseler based their evaluation of neurological symptoms in a study population of 761 US farm residents (operators and their spouses) on data


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collected through a non-standardised questionnaire interview (Stallones and Beseler, 2002b) (Table 6.A.3, Annex 7). Subjects were selected from a sample of farms (479) in an eight county area of North Eastern Colorado. This represented a 57% response rate (835 eligible farms were initially contacted). Subsequent comparisons of farming practices between farms that refused and those that participated were found to be similar. Exposure assessment was based on data collected via a questionnaire in relation to farm characteristics (provided from operators only), use of pesticide equipment, working hours and number of pesticide applications conducted in the previous year. Subjects reported using mainly terbufos (used by 20.8% of subjects) and other OPs (e.g. chlorpyrifos, phosmet, and dichlorvos)/carbamates (used by 37.1% of subjects) to treat crops and livestock respectively. NB. Other non-OP pesticides/herbicides were also used albeit to a lesser extent. Neurological assessment comprised of questions relating to symptoms experienced in the past month (ranked according to occurrence), and having any physician-diagnosed pesticide related illnesses. Sixty-nine subjects reported having ever experienced a physician-diagnosed pesticide illness and were defined as cases. The remaining 692 respondents (who answered ‘No’) were used as internal controls. Age and gender were found to be significant variables and were adjusted for in subsequent analyses. 43. Subjects with previous pesticide poisoning had a two-fold increased likelihood of reporting ‘difficulty moving fingers and grasping things’ compared to controls (95% CI[1.06-4.09]). Other PN-related symptoms failed to reach statistical significance (Stallones & Beseler 2002b, Table 7.A.3, Annex 7). Chi-squared test for trend revealed that having a pesticide-related illness was significantly associated with ‘loss of muscle strength in legs or feet’ and ‘difficulty moving fingers and grasping things’ (P=0.0365 and 0.0007 respectively). 44. These findings are limited by the fact that subject’s had co-exposure to other non-OP pesticides, and the physician-verified pesticide poisoning was based solely on self-reports; there being no objective tests to verify the symptoms or exposures reported. Furthermore, the authors noted that the time-sequence of exposure and outcome was flawed. US Desert Storm Veterans Deployed in Khamisiyah, Iraq 45. McCauley et al, and Spencer report different findings of the same study of 653 US troops believed to have been within a 50 km radius of the Khamisiyah Ammunition Storage Point. These troops are believed to have sustained low-level exposure to sarin/cyclosrin following ground detonation of chemical munitions during the first two weeks of March 1991 (McCauley et al 2001, Spencer, 2001). 46. McCauley et al used a telephone computer-assisted survey to ascertain neurological and neurophysiological symptoms experienced during the first two weeks of the Ground War in Khamisiyah, Iraq (March 1991), and at the time the study was conducted (approximately eight years later) (Table 6.A.3, Annex 7). The authors were able to contact 2918 (90.6%) of 3219 veterans in their sampling pool. Contactable veterans were found to be significantly more likely to have college or advanced degrees, be married, Caucasian and male. Only 2388 (81.8%) of the


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subjects reached by phone were subsequently eligible and of these, 555 (19.0%) refused to participate resulting in 1833 completed telephone interviews (i.e. a 77% participation rate). Participants compared to non-participants/ non-responders were significantly more likely to be Caucasian and on active rather than reserve duty. The final study participant population comprised of 1779 interviews (due to 54 being excluded for providing incorrect deployment information). The 653 Khamisiyah-deployed subjects were subdivided according to whether they witnessed the explosions/operations or not (i.e. 162 KHAM-witness and 405 KHAM non-witness respectively) – 86 subjects were unsure. Two groups were used for comparisons: 610 military personnel who were deployed elsewhere and assumed to have no known exposure to nerve agents (i.e. non-Khamisiyah aka. N-KHAM); and 516 non-deployed military personnel (ND). Comparative analysis revealed significant differences between groups in relation to current region of residence, gender, age, race, and military status (i.e. whether regularly active or activated reserve). Data on potential confounding exposures during the duty were also collected. No objective assessments of exposure and health outcomes were conducted. The authors categorised the level of sarin exposure within the 50km radius as ‘low’. However, this does not account for the fact that the troops closest to the chemical munitions would have received greater exposures than those further away. The authors used trained interviewers and a standardised interview technique. 47. After adjusting for confounding effects of age, gender and region of residence, the authors found that during the first two weeks after the Ground War the KHAM-witness subgroup were significantly twice as likely to report having experienced acute symptoms of ‘muscle twitching’, ‘cramping’, and ‘weakness and tingling of the hands/feet’ than the KHAM non-witness group (95% CIs were greater than the value one; p-values not reported). Significant cranial nerve symptoms reported included ‘vertigo’ and a ‘hoarse voice’ (although in the absence of further tests it would be imprudent to classify ‘a hoarse voice’ as having a cranial nerve aetiology as it is a non-specific symptom). NB. All these symptoms were considered to be mild acute health effects of acute exposure to low-levels of OPs and not demonstrative of episodes of acute illness consistent with high exposure to AChE inhibiting agents. However, eight years later, the reported symptoms in the KHAM group as a whole were not significantly different from the N-KHAM. Although, KHAM group differences were apparent: the KHAM-witness subgroup being almost twice as likely to report symptoms of ‘tingling’, ‘burning’, and ‘sensation of pins and needles’ compared to KHAM-non witness subgroup (OR=1.7[1.1-2.8]) (McCauley 2001, Table 7.A.3, Annex 7). Moreover, all deployed veterans (KHAM and N-KHAM) were significantly more likely to also report experiencing these symptoms, and several other PN-related symptoms e.g. ‘numbness', ‘loss of muscle strength’ and ‘balance/coordination’, and ‘cramping, pains or muscle stiffness’ compared to non-deployed veterans (ND) (ORs ranged from 2.2 – 2.8). The previously reported symptoms suggestive of cranial nerve dysfunction were not among the current health symptoms reported. 48. The findings from this study should be considered with caution in view of the fact that some of the positive results were not specific to sarin-exposed Khamisiyah-based troops. This non-specific result is unsurprising given that the author’s question the status of some of the KHAM and N-KHAM subjects (these groups are reported to have been categorised by the US Department of Health): about 8% of


14

those classified as KHAM subjects reported never being in Iraq, and about 9% of the N-KHAM subjects claimed to have been within the 50km radius of Khamisiyah, with a third of these being involved or watching the detonations. In addition to possible misclassification bias, the KHAM subjects had been notified of their potential exposure to chemical warfare agents and were also informed on what the likely symptoms were (which thereby potentially introduced information and recall bias). Other limitations include possible selection bias arising from the sampling process, as it was limited to individuals with easily accessible telephone numbers. However, this study is noted for its assessment of the long term effects of an acute low-level exposure to an OP. 49. In a related study, Spencer 2001 extends the above findings of McCauley et al (2001) to report on a two-tiered clinical assessment of subjects who completed the telephone interview, and who were subsequently recruited for neurobehavioural testing (Level I) and a focused clinical neuromuscular and neurophysiological examination (Level II) (Spencer, 2001) (Table 6.A.3, Annex 7). In this study, subjects are classified as either Khamisiyah (KHAM), Non-Khamisiyah (N-KHAM) or Non-deployed (ND). The authors noted that the telephone interview sought information on hospitalisations and conditions that had been diagnosed by a physician. Furthermore, an adapted survey instrument was used to gather detailed information on the nature of confounding exposures, and telephone numbers were tracked using more extensive tracking measures to reduce possible selection bias. The authors also conducted upper and lower limb somatosensory evoked potential testing (recording relevant peak latency and amplitudes) on a total of 42 KHAM subjects who completed the Level II testing, and 26 N-KHAM subjects and 28 ND subjects. 50. Interview survey results were reported in an attached manuscript entitled “McCauley et al under review”, which was subsequently published in McCauley et al (2002). The findings revealed no differences between subjects in the KHAM group and N-KHAM group during the time period of the Khamisiyah detonation (although an unreported sub-analysis revealed that mild immediate responses to OPs were evident in a small number of KHAM subjects who witnessed the detonations compared to those who did not). Nine years after the incident, KHAM subjects did not differ from other deployed troops on reports of any medical conditions or hospitalisations (Spencer 2001/McCauley 2002, Table 7.A.3, Annex 7). However, deployed troops were significantly 1.5 times more likely to report a diagnosis of a ‘slipped disk or pinched nerve’ compared to non-deployed troops (95%CI[1.1-2.0]), although this is not specific to sarin-exposed Khamisiyah based troops. No group differences were observed from the neurological and neurophysiological examinations. Cross-sectional studies summary 51. None of the cross-sectional studies separately examined the chronic effects of acute exposure to OPs that inhibit/age NTE. The findings in subjects acutely exposed to non-NTE inhibiting OP pesticides/agents provide weak evidence for the development of persistent peripheral neurological abnormalities. This is because the positive findings observed were either: (i) non-specific for OP-exposed subjects and


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solely based on self-reported data, or (ii) the authors combined both NTE-inhibiting and non-NTE-inhibiting OPs together in their evaluations. Furthermore, the results of objective health assessments were largely negative. Nishiwaki et al conducted stabilometric and quantitative vibration threshold tests and did not observe any significant differences between sarin-exposed and unexposed subjects of the 1995 Tokyo subway terrorist attack (Nishiwaki et al 2001). Stabilometric assessment of a second sarin-exposed cohort of the same terrorist incident also yielded no significant differences from non-exposed groups (Miyaki et al 2005). Significant electrophysiological nerve deficits were reported in a study of symptomatic patients who attempted suicide by ingesting an OP pesticide either capable of inhibiting NTE, or one without the ability (Jalali et al 2011). However, because the study combined all the patients for the electrophysiological evaluations it was not possible to evaluate the development of persistent subclinical effects in those subjects who poisoned themselves with the non-NTE inhibiting OPs. Two studies examined the chronic effects of acute exposure in subjects thought to be exposed to levels insufficient to cause acute toxicity (McCauley et al 2001, Spencer, 2001). Increased odds of reporting sensory symptoms were apparent (but not specific) to the sarin exposed group, however there was no objective evidence to substantiate these symptoms clinically or subclinically. Case series/reports US Family Acutely Poisoned from Residential Diazinon Exposure 52. One case series and two case reports investigated the chronic neurological effects of acute OP exposure. The case series study involved a US family accidentally exposed to diazinon following a pesticide company’s mistaken application to the family house interior (Dahlgren et al 2004). A battery of health tests were conducted three years after the initial exposure, which included assessment of muscle grip strength and other neurological parameters (Table 6.A.4, Annex 7). A comprehensive assessment of external exposure levels were performed via wipe, air and bulk sampling. The authors also tested urine samples for the diazinon parent compound, which was found to be negative. The authors did not test for diazinon metabolites or cholinesterase levels. 53. In addition to acute effects experienced soon after exposure, the chronic effects observed three years later included ‘weak tone of oral musculature’ in a two-year-old child exposed in-utero at five months gestation. No chronic peripheral neuropathy-related symptoms arose in adults (Dahlgren 2004, Table 7.A.4, Annex 7). The authors also did not report the results of the grip strength tests. The study concluded on the susceptibility of children to the central neurotoxic effects of OP exposure and provides no evidence of lasting peripheral nerve effects in adults acutely exposed.


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Deliberate Subcutaneous Injection of Chlorpyrifos in a 24-Year Old Male Patient 54. Soummer et al report on the long-term neurological effects of acute OP poisoning in a 24 year-old man of North African descent who injected chlorpyrifos subcutaneously into his forearms, and also ingested two psychoactive drugs (alprazolam (5mg) and olanzapine (100mg)) in a suicide attempt (Soummer et al 2011) (Table 6.A.5, Annex 7). The patient was conscious on arrival but subsequently deteriorated and went into a coma for approximately six weeks. Biological exposure assessment included evaluation of plasma butyrylcholinesterase and RBC AChE activities, and plasma and urine levels of chlorpyrifos and its metabolites: 3,5,6-trichloro-2-pyridinol (TCP), diethylphosphate (DEP), and diethylthiophosphate (DETP). The authors noted that the patients butyrylcholinesterase and AChE activity remained low until day 60 (i.e. <500 IU/L, normal:8000-18,000) which was suggestive of prolonged poisoning. This was further supported by the presence of chlorpyrifos in the subjects serum 20 days post admission, and in the forearm muscle on day 50 (44 µg/kg); the urinary metabolite levels were equivalent to occupational exposure levels. The subject underwent manual and electrophysiological testing (via repetitive nerve stimulation) of his proximal and distal muscles at 50 days, 120 days (at hospital discharge) and one year post-admission. 55. The authors did not present any quantitative data for electrophysiological component of the assessment. Prior to hospital discharge, the results were suggestive of Intermediate Syndrome and OPIDPN. On hospital discharge, the patient’s signs and electrophysiological data suggested improvement of previous proximal muscle abnormalities of upper limbs, while limb weakness and deficits in the distal muscles of lower limbs prevailed (Soummer 2011, Table 7.A.5, Annex 7). These latter symptoms were reported as being present and severe at the one-year follow-up, which the authors suggested was demonstrative of irreversible OP-related peripheral neuropathy. It is further suggested that the detection of chlorpyrifos in the body fat and forearm muscles acted as reservoirs causing the patient to receive prolonged and continuous exposure as opposed to a single bolus dose. 56. This study provides evidence of a potentially irreversible and severe peripheral nerve deficit in an individual one year after being subcutaneously exposed to a high dose of chlorpyrifos, and later accompanied by a more sub-acute continuous phase. The patient also ingested psychoactive agents at toxic levels and this should also be considered, as this may have impacted on the more subjective (manual) assessments. Furthermore, the authors provide very brief information on the testing procedures, which makes a critical assessment of the methods used more difficult. US Army Sergeant Deployed in Iraq 57. Loh et al. conducted neurological and neurophysiological tests on a senior male Army explosive ordinance disposal (EOD) sergeant acutely exposed to sarin from an improvised explosive device (IED) in May 2004 (Loh et al 2010) (Table 6.A.5, Annex 7). The sergeant was exposed while transporting an exploded IED he had placed inside his enclosed vehicle, and minutes later developed acute symptoms typical of OP exposure. The authors did not specify whether this


17

represented a poisoning episode but noted that his erythrocyte cholinesterase activity was depressed by 39%, which returned to normal weeks later as did his state of health. No external measures of exposure were performed at the time of exposure (although the authors postulated that the sergeant may have been exposed to levels insufficient to cause acute toxicity). The subject began to experience chronic symptoms two months following his return to duty. Further tests were performed approximately eight months later. The authors briefly noted that the neurological examination comprised of cranial nerve, motor, sensory, reflex and gait tests. The neurophysiological examination included both EMG and NCS. No further descriptions are provided. 58. The subject complained of episodic dyscoordination and imbalance; however the findings of the neurological and electrophysiological tests were unremarkable (Loh 2010, Table 7.A.5, Annex 7). 59. This study reported an absence of subclinical or clinical effects which did not corroborate the symptoms reported by an individual with potentially low-level acute exposure to sarin. Case series/reports summary 60. The data collected from the single case series and two case reports of subjects acutely exposed to OP pesticides provide no evidence for the development of chronic peripheral neurological abnormalities (in adults) by agents that do not inhibit NTE. However, severe chronic effects thought to be consistent with the development of OPIDPN were reported in a study, where the subject was acutely poisoned with the NTE-inhibiting OP pesticide chlorpyrifos (Soummer et al 2011). Another study examined the chronic effects of acute exposure in a subject thought to be exposed to levels insufficient to cause acute toxicity and found no significant clinical or subclincal effects (Loh et al 2010). Chronic Effects Following Acute Exposure Discussion Summary 61. Regardless of the type of OP used, most of the evidence for the development of persistent peripheral nerve effects in acutely exposed individuals came from cohort and to lesser extents cross-sectional studies and one case report study. No case-control studies examined the long-term effects of acute exposure to OPs. The evidence from cohort studies was based on either self-reported data collected from questionnaires (Nakajima et al 1999, Kawana et al 2001) or more objective data derived from specific neurological (albeit slightly psychophysical) tests (Miranda et al 2002a, Miranda et al 2002b, Miranda et al 2004). The cross-sectional studies on balance, were largely inconclusive: there being limited evidence from studies using self-reported data, whose effects were not specific to exposed individuals (McCauley et al 2001, Stallones and Beseler, 2002b), and limited evidence for electrophysiological effects from a single study of questionable design (Jalali et al 2011). Evidence for neurophysiological effects provided by the case report study was compromised by the patient’s intoxication with other neuroactive agents


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(Soummer et al 2011). Most of these studies had limitations that affected the reliability of their findings. 62. The following paragraphs respond specifically to the issues raised in the COT 1999 Report’s conclusions on PN & NMD with regards to: (i) the paucity of objective and symptomatic evidence of chronic PN effects in individuals acutely poisoned by OPs that do not inhibit the neuropathy target esterase (NTE); (ii) the lack of studies examining the long-term effects of a single exposure to OPs insufficient to cause acute toxicity (Table (i)). 63. None of the studies exploring chronic effects of acute exposure to OPs that inhibit/age NTE (and known to cause OPIDPN) involved subjects exposed in the UK (Table (i) below). Subjects were either from Nicaragua (Miranda et al 2002a, Miranda et al 2002b, Miranda et al 2004), the US (Stallones and Beseler, 2002b), Iraq (Jalali et al 2011), or France (Soummer et al 2011). The Nicaraguan cohort subjects were exposed to methamidophos and chlorpyrifos (including other OPs that do not inhibit NTE) and the authors discriminate between the effects of the neuropathic and non-neuropathic OPs. The results of the grip and pinch strength dynamometry and vibrometry tests were not significant for non-neuropathic OPs. However, subjects exposed to neuropathic OPs demonstrated motor impairments and possibly sensory impairments. The authors suggested that this was possibly related to development of OPIDPN. Subjects in the US cross-sectional study were mainly exposed to terbufos (21% subjects), which does not inhibit NTE (although they were also exposed to other OPs that inhibit NTE (i.e. chlorpyrifos and dichlorvos), and other non-OP pesticides) (Stallones and Beseler, 2002b). These authors did not discriminate the results according to the type of OP used. Therefore, while they observed an increased prevalence of reporting motor dysfunctions of the hand, it is not possible to determine the nature of the OPs that this outcome refers to. A similar drawback arises in the cross-sectional study of eight Iranian patients poisoned with either chlorpyrifos, malathion or azinphosmethyl (Jalali et al 2011); this study does provide limited evidence for electrophysiological effects of nerve impairment in symptomatic patients but because the outcomes were evaluated irrespective of the OP agent used, it is not possible to determine which OP exposures these effects relate to. Finally, evidence for neurological and electrophysiological effects of severe peripheral nerve deficits described in a 24-year old male poisoned with chlorpyrifos supports current knowledge on the long-term effects of acute poisoning with OPs that inhibit and age NTE (Soummer et al 2011). Therefore, the findings of these studies accord with the conclusions of the COT 1999 report. 64. Studies investigating the chronic effects of acute exposure to OPs that do not inhibit NTE do provide evidence of symptomatic PN effects in exposed subjects. However, these relate to increased symptom frequencies that are not corroborated by any objective evidence for clinical or subclinicaleffects. The two cohort studies reporting on the 1994 and 1995 sarin attacks in Japan (that observed largely symptomatic motor effects in exposed subjects), were based on self-reports, and the authors did not corroborate these symptoms with more objective tests (Nakajima et al 1999, Kawana et al 2001). In contrast, Nishiwaki et al and Miyaki et al both incorporated objective health assessments (i.e. sensory thresholds and disequilibrium tests) into their cross-sectional study of sarin victims of the 1995


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subway attack, and did not observe any increased prevalence of possible PN effects (Nishiwaki et al 2001, Miyaki et al 2005). These findings, therefore, do not accord with the previous COT conclusions, as there is a lack of evidence of subclinical effects of long term PN effects in individuals acutely poisoned with non-NTE inhibiting OPs. 65. McCauley et al (2001), Spencer (2001) and Loh et al (2010) examined the development of chronic health effects in individuals assumed to be exposed to a single dose of OPs insufficient to cause acute toxicity. The first was a cross-sectional study of US troops exposed to sarin/cyclosarin following ground detonations of chemical munitions during the 1991 Gulf War in Iraq (McCauley et al 2001). The study provided very weak evidence of peripheral nerve (sensory and motor) effects as these were based solely on self-reported symptoms that were not specific to troops deployed where the detonation took place in Khamisiyah. Subjective and objective tests conducted in a related study by Spencer (2001) found no significant effects in the same group of exposed subjects. Similarly, a case study by Loh et al (2010) found no neurological and electrophysiological PN-related effects in a symptomatic acutely sarin-exposed army sergeant assessed approximately ten months after exposure. The findings of these three studies update the previous COT conclusions as they provide examples of studies examining the long term effects of a single exposure to OPs insufficient to cause acute toxicity. The observed health effects in exposed individuals were non-specific to these groups, and were based solely on subjective assessments; there being no supportive evidence for clinical or subclinical PN-related effects. Table (i). Breakdown of evidence for PN according to OP mechanism of action and country

Author Country OP pesticide exposure Evidence of chronic PN symptoms/ signs?

NTE* inhibitory Non-NTE inhibitory** No Yes Symptomatic Subclinical

Nakajima 1999 Japan

Sarin poisoning x

Kawana 2001 Japan Sarin poisoning x Nishiwaki 2001 Japan Sarin poisoning x Miyaki 2005 Japan Sarin poisoning x Miranda 2002a Nicaragua Methamidophos,

chlorpyrifos, fenthion poisoning

x

Methyl parathion, malathion, terbufos, phorate, ediphenfos and metofox poisoning

x

Miranda 2002b Nicaragua As above As above x Miranda 2004 Nicaragua As above As above x Stallones & Beseler 2002b

US Chlorpyrifos, dichlorvos poisoning

Terbufos, phosmet poisoning

x

Dahlgren 2004 US Diazinon poisoning x McCauley 2001 US/Iraq

Sarin/cyclosarin exposure x

Spencer 2001 US/Iraq Sarin/cyclosarin exposure x Loh 2010 US/Iraq Sarin exposure x Jalali 2011 Iraq Chlorpyrifos poisoning Malathion, azinphosmethyl

poisoning x x

Soummer 2011 France Chlopyrifos poisoning x x


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*NTE is a neuronal enzyme whose phosphorylation and ageing leads to the development of OPIDPN. Certain OPs are able to covalently bind and phosphorylate NTE and produce an aged enzyme. However, not all OPs that inhibit NTE activity result in OPIDPN. Only those OPs that both inhibit NTE by more than 70% and modify its structure to produce a negatively charged (aged) enzyme subsequently result in OPIDN. These OPs produce a positive result in the Hen Test and include chlorpyrifos, coumaphos, cyanofenphos, DEF (S,S,S,-tributylphosphorotrithioate), dioxabenzophos, EPN, dichlorvos, haloxon, isofenphos, leptophos, merphos, methamidophos, mipafox and trichlorfon (COT, 1999, Lotti & Moretto, 2005). NB. OPs that inhibit NTE without ageing the enzyme have protective effects against the development of OPIDPN. **Organophosphates that do not inhibit NTE (e.g. primiphos-methyl and sarin, etc) are reported to produce peripheral nerve effects through other mechanisms such as via phosphorylation of proteases, esterases or proteins involved in cell signalling, interaction with cytoskeletal proteins, excessive calcium influx in cells at nerve endings, prolonged receptor stimulation at nerve endings leading to muscle fasiciculation and necrosis, hypoxic brain damage and psychological stress from an acute episode producing PTSD (COT, 1999).


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Chronic Exposure with Chronic Effects 66. For the purpose of this review, chronic exposure is defined as a multiple repeat incident with a variable length of exposure (e.g. lasting for one season up to a lifetime). 67. Twenty-three epidemiological studies evaluated the chronic effects of prolonged low-level exposure to OPs in relation to the development of peripheral neuropathy (PN) and neuromuscular dysfunction (NMD). These comprised of four cohort studies (Albers et al 2004a, Albers et al 2004b, Albers et al 2007, Starks et al 2012b), three case-control studies (Jamal et al 2001, Jamal et al 2002a, Crawford et al 2008) and 16 cross-sectional studies (Bazylewicz-Walczak et al 1999, Horowitz et al 1999, Kilburn, 1999, Konieczny et al 1999, Keifer et al 2000, Srivastava et al 2000, Steenland et al 2000, Pilkington et al 2001, Peiris-John et al 2002, Farahat et al 2003, Albers et al 2004c, Kamel et al 2005, Kimura et al 2005, Kamel et al 2007b, Abdel Rasoul et al 2008, Hoshino et al 2008). No case series or case reports evaluated chronic effects of low-level long-term exposure to OPs. 68. The exposure to OPs was largely occupational, in which subjects either worked as:

a. chemical plant workers manufacturing either chlorpyrifos (Albers et al 2004a, Albers et al 2004b, Albers et al 2004c, Albers et al 2007), chlorfenvinphos (Konieczny et al 1999), or quinalphos (Srivastava et al 2000);

b. farmers/farm workers involved in either sheep dipping (Jamal et al 2001, Pilkington et al 2001, Jamal et al 2002a), tobacco farming (Kimura et al 2005), or working as pesticide applicators in farms/orchards (Horowitz et al 1999, Keifer et al 2000, Peiris-John et al 2002, Kamel et al 2005, Kamel et al 2007b, Crawford et al 2008, Hoshino et al 2008, Starks et al 2012b), cotton-fields (Farahat et al 2003, Abdel Rasoul et al 2008), or lawns and houses (Steenland et al 2000);

c. greenhouse workers (Bazylewicz-Walczak et al 1999). 69. A mixed cohort of individuals exposed either occupationally (i.e. via job as agricultural worker or pesticide applicator), or residentially (i.e. at offices or at home) was used by (Kilburn, 1999). 70. All studies accounted for some or all possible confounding effects of age, gender, education, occupation, socioeconomic status (SES), lifestyle, existing medical conditions, and exposure to other neurotoxic agents unless otherwise stated. 71. Paragraphs 73 to 147 provide key detailed summaries of these studies. NB. For each summary, the first paragraph briefly describes the study design, with the main findings and conclusions outlined in subsequent paragraphs.


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Cohort studies Chlorpyrifos Manufacturing Workers 72. Albers et al investigated the chronic CNS effects of approximately 10 years low-level occupational exposure to chlorpyrifos in 53 of 66 eligible manufacturing workers of the Dow Chemical Company (US) (Albers et al 2004b). Subjects had a historic cumulative exposure of 64.16 mg/m3 x days chlorpyrifos. These low levels of exposure were verified via use of biological exposure indicators i.e. red blood cell cholinesterase (AChE) activity, which were not significantly different from the 60 of 74 eligible saran-manufacturing workers (controls) selected from the same company. Controls had similar demographic characteristics to the chlorpyrifos workers and had no occupational exposure to chlorpyrifos or known/suspected neurotoxicants. As expected chlorpyrifos workers had significantly higher urinary excretion of the chlorpyrifos metabolite 3,5,6 trichloro-2-pyridinol (TCP), expressed as TCP/creatinine (Cr)) (P<0.0001), and lower plasma butyrylcholinesterase (BuChE) activity (P<0.01) compared to controls. Subjects were followed-up one year after baseline examinations, which comprised of: a standardised questionnaire designed to collect information on demographics and potential confounding exposures/ medical conditions and possible abnormal neurological symptoms; and a neurological examination that comprised of sensory and motor tests conducted solely to develop specific ratings for subsequent analyses of CNS dysfunction. The neurological assessment included evaluation of the presence of selected cranial nerve function (e.g. extraocular movements), abnormal movements (tremor), station (Romberg), gait, strength, sensation e.g. pin-pain, vibration), and muscle stretch and primitive/pathological reflexes. The authors attempted to control for possible measurement bias by using blinded trained interviewers and expert neurologists to verify symptoms elicited in the questionnaire. Factors such as age, sex, height, BMI, etc, were evaluated as potential confounders including screening for medical conditions that could potentially produce nervous system abnormalities. 73. At baseline, two subjects (one from each group) reported intermittent symmetric sensory symptoms, although these disappeared at the second evaluation (Albers 2004b), Table 7.B.1, Annex 7). No significant neurological results were observed at clinical examination, which may be related to small sample size. Despite this, the study was well designed in view of its high subject participation rate (80% and 98% at baseline and follow-up respectively), use of a comparable referent group that controlled for the potential effects of employment in the chemical industry, and exposure measurements based on reliable sources that were further verified. 74. Albers et al do specifically set out to evaluate the effects of chronic low-level chlorpyrifos exposure on the peripheral nervous system (PNS) for the same study group as reported in a separate publication (Albers et al 2004a). In addition to a standardised questionnaire, the authors conducted a range of objective tests comprising of a clinical neurological examination of the PNS with known specificity and sensitivity, and extensive sensory and motor nerve conduction studies (NCS) performed on the dominant side of the median ulnar, sural and peroneal nerves (Albers 2004a, Table 6.B.1, Annex 7). The NCS were well established in terms of reliability and precision. Subjects were also subdivided into four neuropathy groups


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(i.e. confirmed, probable, possible, and subclinical) depending on their symptoms, signs and NCS results. 75. None of the findings from any of the assessments supported an association (Albers 2004a, Table 7.B.1, Annex 7). No subject reported persistent sensory or motor symptoms suggestive of neuropathy, and neither the neurological examination nor the NCS yielded significant group differences. Furthermore, the number of subjects fulfilling any of the four diagnostic categories of neuropathy were not significantly different between groups at either evaluation. It was suggested that the one-year follow-up was too early to detect additional clinical and possibly subclinical peripheral nerve health effects. 76. Consequently, Albers et al. conducted a dose-effect analyses of the same cohort to identify subclinical adverse effects that would be indicative of PN (Albers et al 2007). The authors also noted that the subject selection process included potentially vulnerable individuals to control for potential selection bias caused by healthy worker effects. As in the preceding two Albers studies (Albers et al 2004ab), subject’s chlorpyrifos exposure was significantly higher than controls as demonstrated by their interim chlorpyrifos exposure (i.e. period between baseline and second examination) and historic chlorpyrifos exposure estimates, and urinary excretion of TCP. Interim TCP excretion among chlorpyrifos workers suggested an estimated daily chlorpyrifos exposure of 576-627 µg/day and indicated levels approximately 30% of the internal dose received by a typical subject exposed during a working day at the threshold limit value of 200 µg/m3. These low exposure levels were verified by their similar AChE activities to controls. Subjects were also subdivided into workers with historic chlorpyrifos exposure either below or exceeding 20 mg/m3 x days for the dose-response analysis. Both sensory and motor nerve conduction studies were performed as in the previous study (Albers et al 2004c) (Albers 2007, Table 6.B.1, Annex 7). 77. The NCS findings for interim chlorpyrifos exposures were conflicting: with increasing exposure (units: interim TCP/Cr levels; µg/g Cr) the authors observed both deteriorating and enhanced electrophysiological function i.e. decreased median sensory terminal conduction velocity (m/s) (p=0.05), and increased median motor amplitude (mV) (p=0.08) respectively, although the latter was not significant (Albers 2007, Table 7.B.1, Annex 7). For historic exposures, three of the 20 NCS outcomes (i.e. motor conduction velocity (m/s), F-wave latency (ms), and summary Z score sensory conduction) showed a significant association with increasing exposure (< 20 mg/m3 x days) (p<0.05). However, the authors did not observe any significant dose-effect relationship among the 32 subjects with historic exposure exceeding 20 mg/m3 per day. 78. The authors concluded that the study was not suggestive of a causal association between long term chlorpyrifos exposure and subclinical neuropathy in chlorpyrifos manufacturing workers. Although positive findings of subclinical peripheral sensory and motor nerve dysfunction were observed, this should be considered with caution in light of the partial dose-dependent increases.


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Pesticide Applicators 79. Starks and colleagues used a range of clinical and electrophysiological tests to assess the peripheral neurological function of 678 US male private pesticide applicators enrolled in the Agricultural Health Study (AHS) between 1997 and 1998 (Starks et al 2012b). The study group came from 1807 eligible participants (represents a 39% participation rate) from Iowa and North Carolina who had (i) completed all four AHS questionnaires (of which two were self-administered and completed in Phase 1, and two were telephone-based and completed in a 5 year follow-up in Phases 2 and 3), and (ii) also reported having higher lifetime use of 10 of 16 OP pesticides. The authors noted that the study participants were similar to the eligible non-participants on several important characteristics, including age and total lifetime days of pesticide use. No comparison groups (non-applicators) were used as the authors suggested this would preclude the ability to determine the role of specific OPs in PNS dysfunction. Exposure data was collected from the four completed questionnaires and a fifth questionnaire that related to exposures experienced in the previous year and was conducted on the day of neurological testing. The data was used to create the following exposure variables: (i) ever use and (ii) cumulative lifetime days of use of any or specific OP and carbamate pesticides (used by at least 50 study participants). The authors did not provide information on the duration or level of pesticide use, and instead report participant’s ever-use of specific OPs: malathion was the most frequently reported (77%), followed by dimethoate, tebupirimfos and tetrachlorvinphos (<10%). Other OP pesticides assessed included acephate, chlorpyrifos, coumaphos, diazinon, dichlorvos, disulfoton, ethoprop, fonofos, parathion, phorate, phosmet, and terbufos. The health assessments were conducted between November 2006 to March 2008. The clinical examination was performed by a blinded physician who used standard protocols to assess vibration perception, proprioreception of the great toe, Achilles reflex, Romberg test for sway, tandem gait and postural tremor (Starks 2012b, Table 6.B.1, Annex 7). Results were dichotomised as being either normal or abnormal. Logistic regression was used to estimate odd ratios of associations between pesticide use and dichotomised test results (p values were not reported). Other specific tests PNS function conducted included hand strength tests (via dynamometer), stabilometry, and vibration threshold testing. The electrophysiological component was performed under standard procedures by one examiner, who measured several nerve conduction parameters for the dominant peroneal motor nerve (no sensory nerve conduction parameters were measured). For the above continuous outcomes, the authors used linear regression to examine any associations with pesticide use. Exposure-response modelling was also performed and Chi-squared test for trend was used to determine whether potential relationships existed for particular pesticides (p values were not reported). Most potential confounder factors were accounted for including the effect of multiple pesticides, and subjects were excluded if they reported ever being physician-diagnosed with pesticide poisoning at the time of AHS enrolment. 80. Significant associations were observed between ever-use of 13 of 16 OPs (except malathion, phorate and terbufos) and six of the seven clinical examination outcomes (except the Romberg test). Applicators had an approximate two- to three-fold increased odds of having an abnormal result (Starks 2012b, Table 7.B.1, Annex 7). For example, the odds of an applicator who used fonfos having a toe


25

proprioception abnormality was 3.06 [95% CI: 1.79-5.25]. Significant exposure-response relationships were also observed for several pesticides and the outcomes of the clinical examination, particularly with regards to abnormal toe proprioception and use of chlorpyrifos, fonofos and phosmet (which all yielded significant trends and monotonic increases in ORs). However, not all of these significant associations were positive: ever use of acephate, diazinon, and (ethoprop or parathion) were inversely associated with having an abnormal result for tandem gait, postural tremor and toe proprioception respectively. Inverse associations (i.e. better performance) with pesticide use were also observed for the PNS function tests that provide quantitative data. Phosmet use was associated with greater hand strength, while lower sway speeds with eyes open were associated with parathion and tebupirimfos use (the authors did not report the numerical data or indicate whether they were significant). This pattern of reduced risk estimates was also evident from the electrophysiological test results. For example, distal motor amplitude (mV) and ever use or log10 lifetime use of phorate and tebupirimfos were significantly associated with regression coefficients lower than unity. Similarly, ever-use of acephate or phorate, and lifetime days use of phorate or all OPs (as a summary variable) showed shorter F-wave latency (ms) (i.e. lower risk). None of the nerve conduction velocity data were significant. An increased risk of nerve abnormalities were observed with lifetime days (and ever use) of diazinon that showed greater distal motor latency (ms) (β=-0.07[-0.15 – 0.00]). The authors found that the risk increased with increasing use. 81. This study reported findings that suggest long term exposure to particular OPs may be associated with some clinical abnormalities in pesticide applicators. However, these findings do not appear to be supported by the evidence provided from more quantitative measures. Indeed, in some instances the electrophysiological data suggest an association where there is a reduced risk of nerve dysfunction with the use of some OP pesticides. The inconsistencies observed in this study could be due to the various limitations of the study, which include: lack of referent group, the large number of comparisons made that may have led to some chance findings, and the study’s reliance on self-reported exposure data (although the authors stipulate that methodological studies have shown that AHS participants provide accurate exposure information). Moreover, the effects were not specific to OPs, as some significant associations were also observed with carbamate use. The study also had data gaps (e.g. with regards to exposure levels and duration) and provided only descriptive data for both the dose response findings and the quantitative neurological test results, which does limit the extent to which the study can be evaluated. Cohort studies summary 82. Overall, the findings from these four cohort studies investigating chronic peripheral neuropathy in subjects chronically exposed to low doses of OP pesticides provide no convincing evidence of an association (or at most an inconsistent one). The three Albers studies were conducted in the same cohort of individuals, which suggests that these are in fact subsets of the results of one study that have been reported in three different papers. Regardless of this, these studies were very carefully and reliably performed in terms of the neurological and neurophysiological


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assessment of the peripheral nerve activity, and controlling for potential confounding exposures and bias. However, the first Albers study is limited by the fact that it examined PN effects in relation to CNS abnormalities i.e. it conducted motor and sensory tests to develop specific ratings to evaluate CNS and not PNS dysfunction (Albers et al 2004b). However, the second study examined PNS abnormalities via a neurological examination and an electrophysiological test and found no group differences between exposed subjects and referents for any of the NCS results (Albers et al 2004a). The positive findings of deteriorating NCS results in this cohort came from analyses of subclinical effects in the third Albers study, which observed effects at low exposure levels but no further effects as the exposure increased over a 10-fold range (20-220 mg/m3) (Albers et al 2007). The fourth study by Stark et al (2012) observed significant positive and negative (inverse) associations between clinical abnormalities and OP use. However, further quantitative neurological tests tended to both support the negative associations and refute any positive correlations. The reduced risk of peripheral nerve dysfunction was also reflected in the findings of the electrophysiological tests, except for one NCV parameter (distal motor latency) which was positively associated with diazinon, indicating an increased risk of nerve impairment with increased use. However, this study was comparatively the weakest of the cohort studies due to various study limitations and data gaps. None of the four investigations included QST or EMG measurements as part of their neurophysiological testing battery. Case-control studies 83. This comprised the smallest group of studies investigating the development of peripheral neuropathy and neuromuscular dysfunction in individuals chronically exposed to low-levels of OPs. Two of the three case-control studies were conducted by the same author (Jamal et al) and incorporated extensive objective health assessments into their design (i.e. clinical neurological examinations, motor and sensory nerve conduction studies, quantitative sensory tests and electromyography). The third study used a telephone interview questionnaire to collect information on self-reports of hearing loss (Crawford et al 2008). One study included assessment of the neuromuscular junction (Jamal et al 2002a). Sheep Farmers 84. Jamal et al. investigated peripheral and CNS abnormalities in two groups of 16 UK sheep farmers who had more than four years exposure to diazinon, propetamphos or chlorfenvinphos (Jamal et al 2001). The first group of subjects (Group 1) comprised of 16 farmers who were randomly selected from the Organophosphate Information Network (OPIN) of 200 farmers, and diagnosed with a chronic long-term illness, and reported having previous acute OP poisoning. Group 2 comprised of 16 asymptomatic farmers with no history of acute symptoms of OP poisoning, randomly selected from an area in the west of Scotland. NB. 129 calls were made and 39 farmers were found to be eligible. However, 13 subjects did not participate (due to either refusal or the date being inconvenient) producing a 67% participation rate. The authors did not further evaluate possible background differences between participants and non-participants. Both farmer groups were comparable in terms of age, lifestyle and medical history and potential for exposure


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to OPs, and were compared to 16 healthy age and sex-matched external controls who had office-based occupations. Low OP exposure in farmers were confirmed by the lack of difference in enzyme activities of RBC AChE and serum cholinesterase from controls. A range of tests were performed that included collecting data on clinical symptoms via a clinical evaluation of reflexes, muscle power (in the right upper and lower limbs), and sensation of pin, prick, vibration, touch and position. These were all scored according to severity using modified previously established criteria. Several neurophysiological tests were also performed i.e. motor and sensory nerve conduction studies (NCS), needle electromyography (EMG), and quantitative sensory tests (QSTs) of thermal and vibration thresholds using standard techniques that were conducted by blinded investigators (Jamal 2001, Table 6.B.2, Annex 7). The authors considered most potential confounders for PN except smoking. Possible confounding due to occupation was addressed by using test sites that did not have calluses (which would impact on sensory tests). Also for vibration threshold measurements, the authors used a method that is not influenced by thickness of skin, although they did not account for exposure to vibrational machinery. 85. Both farmer groups showed evidence of peripheral nerve damage (i.e. distal axonopathy) as demonstrated by the significantly higher clinical scores (greater abnormality) compared to controls for symptoms, reflexes and sensation, vibration and cold perception thresholds (p values ranged from 0.000 to 0.023 for symptoms in Group 2) (Jamal 2001, Table 7.B.2, Annex 7). NB. Farmers in Group 1 received higher clinical scores compared to Group 2. Furthermore, heat perception threshold (degrees Celsuis) was significantly increased in Group 1 only (p=0.0). Significant motor conduction abnormalities were also observed in both farmer groups (p=0.000; n=14 and n=12 for Group 1 and 2 respectively) via presence of polyphasic units without spontaneous activity for EMG testing, which were more pronounced in Group 1. NCS results supported impaired motor and sensory conduction in both farmer groups as evidenced by the significantly increased motor, F-wave and sensory latencies (ms) and reduced compound action potential amplitudes (µV, mV) and nerve conduction velocities (m/s) compared to controls (p values ranged from 0.000 to 0.007). 86. This study provides objective evidence of peripheral nerve impairment in sheep farmers chronically exposed to low levels of OPs regardless of whether they experienced an acute poisoning episode. However, given the multiple comparisons of mean values it is possible that some of these significant findings could have arisen by chance. Furthermore, the use of an external control group who had a different potential for exposure to OPs (due to their occupation) may question the validity of some of the comparisons made with the farmer groups. 87. Jamal and colleagues sought to clinically interpret the neuropathic outcomes of 72 sheep farmers/dippers nested within a previous cross-sectional field study (that had invited 685 subjects to participate) (Jamal et al 2002a). The farmer’s previous neuropathy status was determined from the outcome of neuropathy scores derived from responses to questionnaire and sensory tests conducted in the field study. The current study repeated the neurological symptom assessment and QST measurements in the clinic to recategorise subjects into neuropathy groups and verify these assignments with more specific neurophysiological tests. Ninety-five of


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the 201 eligible subjects responded to the standardised questionnaire (47% participation rate), but only 79 also attended the clinic (39% attendance rate). Reasons for non-participation included travelling distance, and for non-attendance included lack of work cover, and delayed clinical study time. Despite these low rates, the authors reported that that there was no evidence to suggest that the non-attendees differed in any significant way than those attending. Information on chronic neurological symptoms occurring in the upper and lower limbs were collected via a neurologist-administered and previously established (yet modified) Mayo Clinic neuropathy/neurological symptom questionnaire. Questions on cranial nerve involvement were excluded. This was followed with a hospital-based clinical assessment of neurological signs (i.e. reflexes, sensation and muscle power), which was scored according to severity of symptoms, where a higher score signified increasing abnormality. The final study participant group comprised of 72 subjects (due to exclusions). Twenty-three subjects were subsequently categorised as having “definite or probable” neuropathy, with 34 subjects as “possible” and 15 as “no” neuropathy (the latter used as the control referent group). Group comparative analysis revealed that age was lower in the “no” group. Exposure assessment involved calculating a mean cumulative exposure metric based on a crude exposure index “OPEXP” that used exposure history and occupational hygiene data, as well as urinary OP metabolite concentration data. OPEXP also represented the weighted sum of cumulative exposure to concentrate and dilute dip splash. The “possible” group had the highest mean cumulative exposure followed by the “definite or probable” group, and then the controls. NB. The authors did not report the level of significance and attributed the high value in the “possible” group to the presence of a few very highly exposed people. The current study also conducted identical neurophysiological tests as those performed in Jamal et al (2001), and included an assessment of neuromuscular dysfunction via the single fibre EMG (SFEMG) test on the forearm (Jamal 2002a, Table 6.B.2, Annex 7). The authors defined sensory abnormalities as abnormal sural conduction and one or more abnormal QST values; motor abnormalities were defined as abnormal motor nerve conduction; small fibre abnormalities defined as abnormal hot or cold sensation threshold; and large fibre abnormalities defined as abnormal vibration threshold or sural nerve conduction. A diagnosis of ‘clinical neuropathy’ was based on subjects having both neurological signs and nerve conduction abnormalities. Comprehensive measures were used to minimise investigator/observer bias. Data for QST scores were based on age-dependent thresholds and the authors excluded subjects to control for confounding effects of various medical conditions/neurophysiological abnormalities. 88. The results did not show that cumulative exposure to OPs correlated with the presence of neuropathy, although the authors reiterate that the study was not designed to investigate this. However, the incidence of abnormalities increased from the “no” to “probable/definite” neuropathy group for all clinical and neurophysiological measures (except SFEMG) (Jamal 2002a, Table 7.B.2, Annex 7). This was particularly evident in what the authors describe as the most important outcome measure: subjects having clinically diagnosed neuropathy, which increased from 7% in the “no” neuropathy group to 52% in the “probable/definite” category. Sensory abnormalities were found more often than motor deficits, both symptomatically (via questionnaire) and neurophysiologically. For example, 18% of 72 subjects had sensory abnormalities compared to just 3% with abnormal motor nerve conduction. There were low numbers for SFEMG results (8% of 72 subjects), which did not


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increase with increased severity of neuropathy indicating that there was little abnormality in the neuromuscular transmission. Small fibre populations appeared to be more affected than large fibre populations, with the former being three times more common (e.g. 65% vs 21% of 72 subjects respectively). 89. The findings led the authors to conclude that the neuropathy in farmers exposed to OPs was predominantly sensory with small fibres being affected than large ones. It is further suggested that the results corroborate the usefulness of the modified Mayo Clinic methods in detecting suspected toxic neuropathy. However, the fact that the study was designed to validate previous neuropathic outcomes in farmers exposed to OPs limited its ability to demonstrate an association between OP exposure and chronic PN. Pesticide Applicators 90. Crawford et al. evaluated self-reported hearing loss and pesticide exposure in licensed private pesticide applicators enrolled in the Agricultural Health Study in 1993-1997 in Iowa and North Carolina (Crawford et al 2008). From 52,393 eligible applicators, a total of 14,229 applicators completed two standardised questionnaires at enrolment and participated in a 5-year follow-up interview. NB. Seventy-three percent of the 22,915 applicators that completed the questionnaires also completed the telephone interview. Reasons for non-participation in the telephone interview were evaluated and subject characteristics of the non-participants were compared to those who interviewed, with differences included in subsequent logistic regression models. Cases (4926) were defined as those who answered ‘Yes’ to the question: “Do you have trouble hearing in one or both ears (without a hearing aid)?” Applicators who answered ‘No’ were assigned as internal controls (9303). The authors screened for potential confounders of hearing loss i.e. loss attributable to a congenital condition, infection or injury (although use of medications such as antibiotics or antineoplastics was not included). All subjects had personally used pesticides, which the authors subdivided according to function (e.g. insecticide, herbicide, fungicide, and fumigant), class (of which OPs comprised one of four insecticides evaluated) and individual agents (e.g. for OPs subjects were exposed to chlorpyrifos, coumaphos, diazinon, DDVP, fonofos, malathion, parathion, phorate, terbufos, and trichlorfon). Data on the frequency and duration of pesticide use was collected via the questionnaires. Cumulative lifetime days of use for each pesticide class were calculated and expressed in tertiles with zero exposure used as the referent category (Crawford 2008, Table 6.B.2, Annex 7). The authors did not specify whether the telephone interview was blinded. Logistic regression was used to calculate the odds of a subject being a case at different exposure intensities, and was adjusted for age, state, noise (i.e. from farming practices and tractor use, although there was no account for firearm use), solvent and metal exposures. Estimates for trend for cumulative days of pesticide use were also calculated. NB. Due to lack of information the authors used an indirect measure of lifetime noise exposure that was based on the applicators current use of noise equipment. 91. OPs had the strongest association with hearing loss of any insecticide type, and this was largest at the highest exposure quartile (i.e. (OR=1.17[95% CI: 1.03-1.31]; n=1393/2568 for cases and controls respectively at > 129.5 cumulative lifetime


30

days use) (Crawford 2008, Table 7.B.2, Annex 7). However, this was not significant for trend with increasing cumulative exposure (p=0.08). Specific pesticide analysis showed significantly elevated associations with hearing loss for all OPs (except coumaphos) for at least one category of lifetime days of use, which was particularly pronounced for malathion, fonofos, diazinon, phorate and parathion (OR>1.2). Significant trends (p<0.05) were noted for diazinon, fonofos, phorate and terbufos. 92. Despite providing some evidence of a weak association between OP use and self-reported hearing loss (suggestive of dysfunction in the eighth cranial nerve), this study is limited by the lack of objective exposure and health assessments (and thus the potential for recall bias). Use of a potentially inaccurate variable to adjust for the confounding effects of noise from farm and non-farm equipment presents another drawback of the study. Furthermore, the absence of a positive trend with cumulative lifetime OP exposure does weaken the strength of the conclusions made. Case-control studies summary 93. Overall, the findings from the three case-control studies investigating chronic peripheral neuropathy in subjects chronically exposed to low doses of OP pesticides provide some limited evidence of a possible association. The evidence appears to be consistent regardless of whether subjective or objective neurophysiological tests were performed, of which the latter were comprehensive. However, two of the studies were conducted by the same author (Jamal et al), and the ability of the second Jamal study to demonstrate an association with increasing OP exposure was compromised by its study objective. The third study by Crawford et al (2008) used large sample sizes and reported OPs having the strongest association for ‘hearing loss’ compared to other pesticides. However, this is based on self-reported information, and the authors used a questionable variable to control for the confounding effect of lifetime noise exposure. Cross-sectional studies 94. This comprised the largest group of studies to investigate the development of peripheral neuropathy and neuromuscular dysfunction in individuals chronically exposed to low-levels of OPs. NB. In one of these studies, the duration of exposure was subchronic i.e. over a season (Keifer et al (2000). Approximately half of the 16 cross-sectional studies incorporated objective health assessments into their design (i.e. mainly clinical neurological examinations and nerve conduction studies). Chlorpyrifos Manufacturers 95. Albers et al adopted a cross-sectional design to investigate the prevelance of subclinical or clinically evident peripheral neuropathy in 53 of 66 eligible US Dow Chemical Company employees involved in the manufacture of the OP insectide chlorpyrifos (Albers et al 2004c). Subjects had approximately 10 years of chlorpyrifos exposure and a historical cumulative chlorpyrifos exposure of 64.16mg/m3 per day (as determined by calculation of geometric mean exposure levels). The authors estimated that their average daily chlorpyrifos exposure was


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approximately 17% of that received by a typical subject exposed during a working day at the permissible chlorpyrifos exposure level of 200µg/m3 (based on urinary excretion of the chlorpyrifos metabolite TCP). These levels were significantly higher than the 60 of the 74 eligible controls randomly selected from the same company but involved in saran manufacturing (P<0.0001). Both groups were similar with respect to age, gender, anthropmetric features, alcohol use, smoking history, mediation use and systemic illness. Significantly lower average plasma BChE levels in chlorpyrifos subjects compared to controls verified their higher exposure to OPs (P<0.003). An expert neurologist conducted a clinical interview to identify symptoms of neuropathy and also performed a neurological exam to identify signs of neuropathy. This was followed by sensory and motor nerve conduction studies in the median, ulnar, sural and peroneal nerves (with measurements made for the conduction velocity, latency, amplitude, F-wave latency and H-reflex) (Albers 2004c, Table 6.B.3, Annex 7). The results of these tests were used to further categorise subjects into one of three clincally-evident neuropathy or subclinical neuropathy groups (i.e. probable, possible, confirmed and subclinical) using a combination of the abnormalities from the symptoms, signs and nerve conduction testing. These were consistent with standard clinical practice. 96. The authors found no significant differences in the frequency of either clinical symptoms or signs of PN in subjects and controls (Albers 2004c, Table 7.B.3, Annex 7). There were also no significant group differences for any of the nerve conduction measures. 97. As one of the better designed studies reviewed in this report, the lack of any subjective or objective evidence for PN effects in individuals chronically exposed to low levels of chlorpyrifos represents a key finding. Nonetheless, it is possible that the small size of the study may have limited the study’s power to detect group differences, although the authors state that there was sufficient power to detect small group differences for the quantitative measures of sensory and motor nerve function. Chlorfenvinphos Manufacturers 98. Konieczny et al examined impaired respiratory muscle function in 35 Polish chemical plant workers who produced chlorfenvinphos for a mean duration of nine years (range one to 15 years) (Konieczny et al 1999). Gas liquid chromatography was used to estimate the mean air concentration which did not reach one fifth of the maximum allowable concentration of 0.01 mg/m3. Subjects low OP exposure levels were verified by measurement of their RBC AChE activity levels, which was similar to the 33 healthy male residents of the region with no occupational exposure to chemicals. No further information provided and it appears that this group was recruited solely for making biological exposure comparisons. Various spirometric tests were performed to aid assessment of PN-induced lung dysfunction, which included intrathoracic gas volume (i.e. ITGV (%) – in which increases are associated with a weakening of muscle force), maximal static inspiratory/expiriatory mouth pressure (i.e. MIP and MEP respectively (cm H2O) – which provide a measure of respiratory muscle function), and specific airway conductance (sGaw) (Konieczny 1999, Table 6.B.3, Annex 7). These were compared to a second referent group of 22 controls who had no occupational exposure to chemicals (selected solely for


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spirometric test comparisons). Comparative analysis revealed that these subjects comprised of some smokers and individuals diagnosed with chronic bronchitis. The authors also performed the repetitive nerve stimulation test on the right ulnar nerve of the forearm to assess potential neuromuscular dysfunction (NMD). This was done by recording compound action potentials of the hypothenar muscle (fingers) and estimating contraction amplitude differences. These were compared to 30 healthy male residents of the region who had no occupational exposure to chemicals. It is not specified whether these tests were performed by experts or individuals blinded to the subjects exposure status. Mean ages of all groups evaluated varied. The authors accounted for the possible confounding effects of age and smoking only, and screened for conditions known to confound respiratory function e.g. chronic bronchitis and atopic airway disease. 99. Spirometric investigations showed an elevated ITGV and decreased air conductance expressed as a percentage of mean value for 24 and 11 workers workers with abnormally high indexes respectively. The authors did not indicate whether this was significantly different to the values obtained in the 22 spirometric referents. However, chlorfenvinphos workers had significantly lower MIPs (n=3) and MEPs (n=20) compared to controls (p<0.01) (Konieczny 1999, Table 7.B.3). A subanalysis of worker’s years of exposure revealed a possible dose-related effect whereby workers with more than 10 years of chlorfenvinphos exposure (n=17) had a significantly lower MIP compared to workers with less than 10 years work exposure (n=18) (p<0.05). Findings relating to neuromuscular transmission were not clearly or fully reported; it was noted that some disturbances were found in three workers via electrostimulation myasthenic testing but no disturbances were observed in neuromuscular transmission. 100. The findings from this study suggest possible impairment of respiratory muscle function in men chronically exposed to low-levels of chlorfenvinphos. However, this assertion is based on spirometric tests alone, as there was no evidence of electrophysiologial dysfunction of the respiratory muscle (which itself was inferred from tests conducted on the hypothenar muscle of the finger). The possibility that respiratory muscle weakness could result from pathological muscle changes and overload-induced fatigue could not be ruled out. The reliability of these findings are further questioned by the study’s lack of quantitative data for the AChE activities, and electrophysiological testing of NMD, as well as there being very limited information to determine the appropriateness and validity of using three different referent groups for separate parts of the clinical and exposure assessments. Also, the fact that some of the results were reported onlyin the abstract and not in the main paper confounded data interpretation. It is worth noting that the use of spirometric tests to aid assessment of peripheral neurological dysfunction of the respiratory muscle is contraindicated by the lack of correlation between electrophysiological findings and clinical respiratory signs or spirometric abnormalities in peripheral neuropathies (Burakgazi & Hoke, 2010). Quinalphos Manufacturers 101. Srivastava et al examined various health risks (particularly CNS outcomes) in 59 Indian quinalphos (QP) manufacturing workers who had a self-reported mean


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work exposure history of approximately six years (Srivastava et al 2000) (Table 6.B.3, Annex 7). Seventeen controls were selected who were not engaged in QP manufacturing but worked for three years outside the manufacturing unit as tea vendors and roadside hawkers. The authors noted this lower number was due to only a few subjects being eligible. However, it is reported that both groups were similar with respect to age, work history, physical activity at work, sex, education, and drinking and smoking habits (p>0.05), although exposed subjects tended to be more educated, smokers and alcohol drinkers. The authors did not evaluate external/ambient levels of OP exposure but used a pretested structured interview to collect information on subjects occupational history, and also measured AChE activity. Mean blood AChE levels in QP workers were not statistically different from controls. A medical doctor conducted a general clinical evaluation on subjects after a typical days work to determine the presence of any abnormalities, which included assessment of reflexes. No neurophysiological tests were conducted. 102. QP workers showed a significantly higher prevalence of abnormal reflexes of the foot and ankle compared to controls (p<0.05) (Srivastava 2000, Table 7.B.3, Annex 7. Eighteen of the 26 QP workers with abnormal plantar reflexes had diminished or no responses, with equivocal responses observed in the remaining eight subjects. Similarly, 12 of the 20 QP workers with abnormal ankle reflexes had diminished or no responses, with equivocal responses in the remaining eight subjects. 103. The findings showed that QP-exposed subjects had a higher prevalence of abnormal reflexes of the foot and ankle. However, this finding should be interpeted with caution in view of the limited exposure assessment, lack of account of subject’s co-exposure to other chemicals e.g. potentially neurotoxic raw materials and by-products of QP manufacture, and the appropriateness of the control group, which was considerably smaller and thereforelikely to question the validity of the statistical group differences. Sheep Farmers 104. Pilkington et al examined clinically detectable abnormalities of the peripheral and autonomic nervous system in 612 UK sheep farmers/workers who had dipped sheep from 1970 (Pilkington et al 2001). Two low OP exposure groups were used for comparisons: 53 farmers without sheep-dipping experience and 107 ceramic factory workers recruited from two companies based in either England or Scotland. The response rate based on the number of eligible sheep and pig farms contacted was 88% and 62% respectively. Reasons for non-participation included being disinterested or too busy, or being excluded due to ineligibility. Participation rates for the ceramic factory workers recruited from Scotland and England were 60% and 80% respectively. Comparative group analyses showed differences in age, gender and alcohol consumption (the latter being higher in ceramic workers). The current study was an exposure-response design that utilised retrospective exposure data collected from a previous ‘first phase’ study. This earlier study used an exposure history questionnaire and an occupational hygiene study to obtain data on dipping practice in 20 farms. A dermal exposure model based on the two urinary OP metabolites DEP and DETP was derived that identified ‘handling of dip concentrate’


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and ‘splashing of dilute dip’ as principal sources of exposure among farmers. The authors calculated the following exposure variables: (i) cumulative exposure to OP dips (OPEXP) defined as the weighted sum of cumulative exposure to both concentrate and dilute dip splash; (ii) DAYS defined as the total number of dipping days; and (iii) daily intensity of exposure to both concentrate and splash (Pilkington 2001, Table 6.B.3, Annex 7). NB. No biological assessment of exposure was conducted. A technician-administered questionnaire (based on the Mayo Clinic questionnaire) was used to obtain data on sensory and motor symptoms of the upper and lower limbs that were indicative of damage to the PNS. This was performed in conjunction with a series of QST tests that assessed both thermal and vibration thresholds under standard procedures. The authors accounted for most potential confounders of PN including occupational/ recreational exposure to vibration, except smoking. For QST, the authors did not report whether subjects were monitored for boredom, fatigue or inattention (which can give rise to spurious results). 105. Sensory symptoms and muscle weakness comprised the second and third most prevelant of reported symptoms respectively within all groups (the most prevelant being autonomic symptoms) (Pilkington 2001, Table 7.B.3, Annex 7). Symptom prevalence was highest among sheep dippers (11.6%) and lowest in ceramic workers (1.9%) for both sensory and muscle symptoms. NB. Subsequent analyses of symptom prevalence was based on an overall symptom indicator that was not specific for peripheral nerve symptoms, therefore it was not possible to further evaluate the effect of low-level cumulative exposure on the odds of reporting peripheral neuropathy related symptoms. After correcting for age, group comparisons of the sensory threshold results demonstrated higher cold sensation thresholds among sheep dippers compared with non-exposed farmers (data was represented graphically). There was no evidence of differences in mean hot or vibration thresholds among these groups. After adjusting for the confounding effects of age, sex and country (i.e. England vs. Scotland) the exposure and response analyses did not show evidence of a significant association between either of the two cumulative exposure indices (OPEXP and DAYS) and the three sensory thresholds, except for a significant negative association between DAYS and hot thresholds (OR=0.94[95% CI:0.89 – 1.00]). However, vibration threshold was significantly associated with concentrate handling intensity (p=0.031) and further statistical analyses via linear regression showed that farmers who handled concentrate had a 20% higher cold threshold than those who never handled dip concentrate (p=0.05). Cold thresholds among sheep dippers who had not handled concentrate remained 41% higher than among farmers who were not sheep dippers. 106. These findings showed that sheep farmers had higher rates of self-reported sensory symptoms compared to other workers. Use of an overall symptom indicator (that included autonomic symptoms) precluded analysis of odds of reporting PN-related effects due to cumulative exposure. However the authors concluded that there was limited evidence of a weak effect of low-level cumulative exposure on reporting symptoms. Exposure to dip concentrate appeared to be an important factor as to whether farmers had abnormal cold thresholds, this effect more likely to arise in sheep dippers than other farmers. However, analyses of the effect of cumulative OP exposure did not provide consistent evidence of an association with sensory thresholds. The psychophysical nature of sensory threshold data, together


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with the absence of data from clinical examinations or electrophysiological testing, suggests any positive QST findings should be considered with caution. Tobacco Farmers 107. Kimura et al examined the effect of long-term pesticide use on the peripheral and central nervous system of 76 Malaysian tobacco farmers randomly selected from the National Tobacco Board (NTB) (Kimura et al 2005) (Table 6.B.3, Annex 7). Thirty-eight officers of the NTB who did not handle pesticides or wet tobacco were used as controls. The authors initially selected 80 farmers and 40 controls but restricted the analysis to healthy males without diabetes mellitus. Comparisons between groups showed both were similar in terms of age, height, weight, smoking and alcohol non-consumption. Interviews were performed to collect data on pesticide use and duration of work. Twenty-six farmers reported having used methamidophos (trade name Tamaron) although this was not exclusive, as the farmers used two or more pesticides in combination. Other pesticides used included pyrethroids, dinitroaniline, organochlorines and carbamates.Years of working on tobacco farms ranged from one year to more than 20 years. The authors also measured serum chloinesterase activity, which did not differ significantly between groups (data not shown). An experienced clinical technician performed nerve conduction tests on the right forearm and lower limb. This included measuring maximal motor conduction velocity (MCV) of the median and tibial nerve and the sensory conduction velocity (SCV) of the median and sural nerve. Computerised posturography was used to assess disorders in the CNS (and not the PNS). 108. Very little data on methamidophos was reported and the study did not observe any significant peripheral nerve system effects in farmers exposed to methamidophos. However, significant effects on the vestibulo-cerebullar system were observed as demonstrated by postural sway increases (Kimura 2005, Table 7.B.3, Annex 7). 109. Aside from positive data for vestibulo-cerbeullar effects of methamidophos, which were evaluated as a measure of the CNS, this study does not report evidence of chronic PN effects in tobacco farmers exposed to chronic low levels of the OP methamidophos. Limitations include small sample size (and smaller control group), as well as failure to account for co-exposure to non-OP pesticides with cholinesterase-inhibitory activity, and the possible effects of nicotine poisoning from tobacco leaves. Pesticide Applicators (Farms/Orchards) 110. Six studies investigated the effects of long term low-level exposure to OPs in subjects who routinely applied pesticides to farms/orchards. However, in one of these studies (Keifer et al 2000) the duration of exposure was subchronic. 111. Keifer et al examined the effect of a season (April to August) of low-level exposure to OPs on neurobehavioural and neurophysiological performance among a controlled group of US apple orchard thinners (Keifer et al 2000) (Table 6.B.3, Annex


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7). One-hundred and thirty-seven subjects were included in the first examination (baseline) and 90 subjects in the second examination, which was conducted six to nine months later. The authors did not attempt to control for possible selection bias due to loss of subjects to follow-up. Controls comprised of unexposed age, education and gender matched-workers recruited from a non-agricultural workplace (the authors did not specify the number used). Exposure assessment was based on self-reports of work history and recent work hours, and biochemical measures of exposure (data not presented). NB. Some controls reported exposure to farm work although the authors considered this was insignificant in terms of OP pesticide exposure. A range of neurophysiological tests were administered by trained testers who were blinded to the exposure status of the workers. This included sensory and motor nerve conduction tests, vibration threshold tests of the hand and foot, and repetitive simulation electromyography. However, the authors reported only the findings of the effects of exposure on vibration thresholds (which did not produce significant results when compared to controls). Readers are subsequently referred to previously published work by Engel et al (1998) for the remaining results, which have been reviewed in the previous COT 1999 report (where no significant differences were observed between exposed workers and controls). The authors account for some of the main confounders of PN, except for smoking. They do not control for the farmer’s fatigue during tests (as some tests were conducted after work), nor the fact that some controls had worked on farms. 112. This study did not present any quantitative data on peripheral nerve outcomes or elaborate on the neurophysiological tests conducted (Table 7.B.3, Annex 7). The study also relied on unverified self-reports of chronic pesticide exposure that could introduce potential recall bias. Together with the negative vibrometry findings, this study provides no evidence of chronic PN symptoms in acutely OP exposed subjects. 113. Horowitz et al examined peripheral nerve abnormalities in nine US fruit growers who sprayed OPs on fruit trees from mid-April to mid-September (Horowitz et al 1999) (Table 6.B.3, Annex 7). These subjects were abstracted from a previous study on the basis that they had the worst sensory nerve deficits i.e. highest vibration indices. Most applicators were farmers applying either methyl parathion, teratethyl pyrophosphate and azinphos methyl. NB. Non-OP chemicals such as fungicides were also used. Exposure assessment was based on self-reported duration of exposure, which ranged from five to more than 55 years. Given that the study sought to corroborate previous findings via the use of more extensive tests, a referent group was not used (except to establish 95% confidence limits for the electrodiganostic and quantitative sensory threshold test values). The authors screened for existing medical and neurological conditions and alcohol abuse as potential confounders for PN. 114. A physical examination revealed sensory abnormalities in four of the nine subjects i.e. reduced sensation in the lower extremeties in a stocking distribution (which included ankle jerk reflex) (Table 7.B.3, Annex 7). Computer-aided assessment of sensory thresholds revealed abnormal vibration and cold thresholds in three of the four subjects with abnormal clinical signs. The quantitative muscle strength test TQNE (i.e. Tufts Quantified Neurological Evaluation (kg)) was normal in all subjects. However, abnormalities in sensory and motor nerve conduction studies


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were apparent in subjects with or without clinical signs. These included slowed F-wave latency, H-reflex and sensory conduction velocities and reduced amplitudes of sensory evoked potentials. NB. The authors did not provide quantitative data or elaborate on the units of the parameters measured. 115. On the basis of these results the authors concluded that four of the nine fruit growers with long-term exposure to OP pesticides had clinical evidence of mild peripheral neuropathic dysfunction, which they considered was not attributable to any other cause. Furthermore, the results from the electrophysiological tests in all four subjects with clinical abnormalities and computerised sensory testing in three of the four subjects corroborated the presence of peripheral neuropathic dysfunction. However, in addition to the small sample size and lack of controls, these findings should be interpreted with caution in view of the study’s reliance on a crude exposure assessment based on self-reports, the qualitative nature of the study findings, (with no indication of the statistical significance of these findings), and the failure to account for co-exposures to other compounds used. 116. Peiris-John et al. examined neurophysiological abnormalities in 30 Sri-Lankan farmers engaged in regular spraying of OP pesticides on crop farms for approximately 14 years (Peiris-John et al 2002). The farmers resided in the Uda Walawe irrigiation scheme of Southern Sri Lanka and were also exposed to non-OP agents. No further information regarding the selection process is provided. The authors also did not specify the OP agents used, but mentioned that the farmers used AChE-inhibiting pesticides (that included carbamates) and that 80% had used anilides and pyridine (40%) in the previous month. Thirty fishermen living close by (within a 25-km radius of farm lands) but not involved in pesticide spray activities were used as controls. The authors applied restrictions to ensure only healthy men without any conditions known to confound test results were included. Group comparisons showed similarities in age, BMI and alcohol consumption. Evaluations were conducted at two time points: during a cultivation season (April-May) and between cultivation seasons or “intercultivation” (June-July). The authors noted that during the cultivation season, farmers receive an average of 20 hours of direct pesticide exposure. Mean AChE levels in farmers did not differ significantly from controls between or during cultivation seasons, although the levels in both groups were significantly reduced at the second evaluation compared to the first (p<0.01). The authors suggested that the reduced levels of AChE activity in controls may be due to drift or residential exposure. A neurological evaluation that included a clinical examination that followed a fixed protocol (no further details provided) and nerve conduction studies assessing both peripheral nerve and neuromuscular synapse function were performed by testers blinded to the subjects exposure status (2001, Table 6.B.3, Annex 7). The level of the tester’s expertise is not specified. The authors also controlled for the extent of farming activities and work with pesticides. 117. Clinical examinations revealed no abnormalities in farmers. Significantly altered sensory and motor effects were apparent between cultivation seasons as demonstrated by higher sensory conduction velocities (CVs) and lower motor CVs in farmers (n=28, 26 respectively) compared to controls (n=26) (p=0.04) (Peiris-John 2002, Table 7.B.3, Annex 7). The authors suggested that the unexpectedly higher sensory conduction velocities in farmers compared to controls during this intercultivation season may be due to the increased sensitivity of damaged


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peripheral nerves. Significantly altered effects were not apparent during the cultivation season as the sensory/motor CVs did not differ significantly between the two groups. The authors attribute this lack of difference between groups during the cultivation season to probable environmental exposure of controls. However, the different seasons appeared to have an effect on conduction velocity in both groups. Sensory CVs for farmers (n=28) and controls (n=25) were significantly reduced during the cultivation season compared to values obtained during the intercultivation (between) season (p<0.01 and p=0.04 respectively). However, the different seasons did not affect motor CVs in either group. The authors highlight how this effect parallels with the initial effect of OP poisoning which appear to first affect sensory nerves. Other neurophysiological parameters tested (i.e. sensory/motor latencies, motor amplitudes, and neuromuscular transmission amplitude) yielded no-significant differences between farmers and controls during and between cultivation seasons. 118. The findings of this study showed that farmers chronically exposed to low-level OP pesticides had limited altered sensory and motor effects. However, this should be considered with caution given the study’s failure to adjust the neurophysiological data for farmers co-exposure to non-OP agents such as carbamates, which also inhibit AChE. 119. Kamel et al used a symptoms questionnaire to evaluate neurological outcomes in US private pesticide applicators (Kamel et al 2005) (Table 6.B.3, Annex 7). Subjects (n=18, 782) were mostly farmers applying for new or renewed licences and were taken from the Agricultural Health Study (AHS) cohort of 52,400 applicators. The questionnaire was based on a previously established neurological questionnaire (Q16). Subjects provided complete information on neurological symptoms experienced during the year before enrolment. Applicators who provided incomplete information (n=2,603) differed in terms of age, education and state. The authors used the applicator’s questionnaire responses to classify subjects into two groups based on their frequency of symptoms rather than their level of exposure. Twenty-three symptoms were reported. Cases comprised of subjects with a high frequency of symptoms i.e. 10 or more symptoms (20% of applicators), while subjects who experienced less than 10 symptoms (low frequency) were used as internal controls (80% of applicators). The authors constructed several measures of pesticide exposure from the questionnaire data, which included cumulative lifetime days of pesticides, in which OPs comprised one of four subdivisions of chemical class of insecticides. The authors did not identify the specific OPs to which subjects were exposed. No biological measures of exposure were conducted. A greater proportion of control subjects had no cumulative lifetime days of OP exposure compared to cases (14% vs. 9% respectively), while the number of subjects experiencing higher levels of exposure (i.e. from 1 – 500 cumulative lifetime days) was comparable in both groups (ranged from 11% – 29%). However, slightly more cases had more than 500 cumulative lifetime days of OP use compared to controls (i.e. 6% vs 4% respectively). The authors used logistic regression to calculate the odds of experiencing a particular neurological symptom with high frequency compared with low frequency. Data was adjusted for age, state, education and smoking and alcohol consumption. 120. Several neurological symptoms were found to be associated with the highest category of lifetime days of use of OPs. These included symptoms suggestive of


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spinal nerve dysfunction i.e. ‘numbness in hands or feet’, ‘twitches or weakness in arms or legs’, ‘poor balance’ and’tremor in hands’, and symptoms suggestive of cranial nerve dysfunction i.e. ‘blurred/ double vision’, ‘changes in smell or taste’ and ‘difficulty speaking’ (Table 7.B.3, Annex 7). These all had significant odd ratios between 1.77 and 2.67 (no 95% CIs or p-values were provided except from noting that the CIs excluded 1.0), however, they were not specific to OP exposure as they were also associated with other pesticide groups. 121. Despite the use of internal comparisons of exposed individuals from the same population, the authors observed positive associations in applicators with a high frequency of reporting several peripheral neuological symptoms compared to those with a low frequency. However, the significance of this finding is limited by the subjective nature of both the exposure and health outcome assessment and the potential for recall bias. 122. Kamel et al re-analysed the above data in a separate publication by categorising the neurological symptoms a priori into groups representing several functional areas that included sensory and motor domains (Kamel et al 2007b) (Kamel 2007b, Table 6.B.3, Annex 7). Each domain (based on the previously established Q16 questionnaire) amalgamated both spinal and potential cranial peripheral nerve symptoms. The sensory domain included the following symptoms: numbness or parathesia, poor night vision, blurred or double vision, and changes in smell or taste; while the motor domain included: twitches, weakness, poor balance, tremor and difficulty speaking. The authors used the same symptoms listed in motor group, but modified the sensory group to focus on just the vision symptoms (i.e. poor night vision and blurred or double vision). 123. The authors observed a 2-fold increased risk of experiencing symptoms relating to motor function and vision among cases with the use of OPs. However, this result was presented graphically and was not specific for OPs (as the authors also evaluated the effect of other non-OP pesticide groups on the development of neurological symptoms) (Table 7.B.3, Annex 7). Furthermore, in the absence of more objective tests it would be imprudent to categorise the vision symptoms as a definite cranial nerve issue (as some can have non-peripheral nerve causes). Therefore, this reanalysis does not provide any further supportive evidence. 124. Hoshino et al used questionnaires and objective tests to assess vestibular system alterations in 18 Brazilian rural workers who complained of dizziness and had occupational exposure to pesticides (Hoshino et al 2008). Little information regarding the selection process was provided except that the subjects were located in the Corrego das Pedras and Bau regions of Rio de Janeiro. Workers were exposed to several classes of pesticides, which included exposure to OPs i.e. tamaron (methamidophos) and folidol (parathion). No control populations were used. Exposure assessment was based on self-reports of work duration and work practices. The majority of subjects had worked for more than 10 years (most having between 21 to 45 years on the job), and for more than 10 hours per day. The authors used questionnaires to obtain information on auditory and vestibular symptoms, particularly in relation to work practices. Clinical assessment included otorhinolaryngological (ear, nose and throat), audiological and vestibular examinations (which comprised of vector electronystagmography (VENG) testing).


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The audiometic assessment used previously defined hearing normality criteria to characterise normal hearing thresholds in adults, and the VENG were performed under previously defined principles. No further details are provided. To address potential confounders for vestibular disorders, subjects who smoked, drank alcohol, abused drugs or had metabolic, hormonal and neck disorders were not included in the study. The authors reported the frequency of subjects for each variable as percentages. No further statistical analyses were conducted. 125. The audiometry tests detected altered tone results in seven subjects (which included sensorineual and bilateral hearing loss) (Hoshino 2008, Table 7.B.3, Annex 7). Five of these seven subjects were aged between 46-59 years, compared to seven of the 11 subjects with normal test results who were aged between 16-35 years. Vestibular examinations revealed peripheral irritative vestibular syndrome (PIVS) in 18 subjects. For 10 subjects who claimed their dizziness worsened while working, eight had altered VENG test results. Of the 13 subjects who reported that the chemical products affected their balance, 12 were found to have altered VENG test results. Further analysis showed that age did not influence the VENG results. This contrasts with the finding that ‘number of years on the job’, ‘daily number of hours of exposure’, and ‘failure to use personal protection equipment (PPE)’ were associated with an increased number of subjects with altered vestibular examinations. 126. This study provides some limited evidence of signs of vestibular and hearing disorders suggestive of cranial nerve dysfunction in symptomatic Brazilian rural workers exposed to OP pesticides. However, the reliability of these findings is questioned in view of the small sample size, crude exposure assessment based on self-reports, lack of a comparison group and subjects co-exposures to other pesticide agents. Pesticide Applicators (Cotton Fields) 127. Two studies examined PN-related effects of long term low-level exposure to OPs in subjects who routinely applied pesticides to cotton fields. 128. Farahat et al assessed neurological sensory and motor function in 52 of 64 eligible Eygptian agricultural engineers, mixers and mechanics who worked for approximately 18 years in pesticide application departments at Berket El-Sabe district of Menoufiya Governate, Egypt (Farahat et al 2003). This represented an 81% participation rate among eligible subjects. Reasons for non-participation were due to exclusions or refusal. An external comparison group of fifty male clerks and administrators from different departments of the Ministry of Agriculture who had no occupational exposure to pesticides were used as controls. The response rate among controls invited to participate was 79%. Both groups were comparable in terms of socioeconomic class, area of residence and work. Other demographic characteristics were not significantly different between either group. A questionnaire was used to ascertain occupational history information. The engineers applied the pesticide to the cotton crops while mechanics repaired spray devices. The pesticide application was done once every 15 days with an initial treatment of insect repellent followed by application of one (or a combination of two to three) OP pesticide(s) and


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other non-OP insecticides, which included carbamates and pyrethroids. NB. Seasonal applicators were not included in the study. All cotton field workers reported having to remain in the field during the spraying seasons (June to September) and working six days per week. Mean serum AChE levels in exposed subjects was significantly lower than controls (p=0.0001), although all were within normal limits. The authors reported that serum AChE was found to be significantly associated with duration of exposure, and suggested that ageing of AChE due to long term inhibition may account for the lower levels in exposed participants. Both a questionnaire and clinical examination were used to collect neurological symptom data and signs respectively. The latter assessment included specific tests of the sensory and motor system, and reflexes (Farahat 2003, Table 6.B.3, Annex 7). No further details regarding the adminstration of these tests were provided. The authors screened for many potential confounding conditions or exposures. 129. Exposed subjects were 16 times more likely to report symptoms (and have clinical signs) of ‘numbness’ than controls (p=0.003). Other symptoms and signs although higher in exposed participants were not significant (Farahat 2003, Table 7.B.3, Annex 7). However, this finding is limited by the study’s crude measure of external exposure based on self-reports, and failure to account for the effect of multiple exposures to non-OP pesticides including carbamate, which also inhibits AChE thereby questioning the specificity of this outcome for OP exposure. The authors do, however, allude to the possibility that the observed associations were age-related due to a significant higher number of older workers reporting symptoms and signs compared to younger workers. They suggested this could be due to older workers having a longer duration of work (and thus exposure to pesticides) compared to younger workers (data not shown). 130. Consequently, the same research group investigated the prevalence of various neurological symptoms in 50 of 56 eligible Eygptian children aged between nine and 18 years who worked for approximately seven years as seasonal workers helping the agricultural engineers spray pesticides onto cotton crops (Abdel Rasoul et al 2008). The authors subdivided the children (randomly chosen from five of 50 villages within the Shebin Elkom District) into two applicator groups: a younger group comprising of 30 children aged nine to 15 years, and an older group of 20 children aged 16-18 years. Six children declined to participate yielding a response rate of 89.3%. A control group of 50 children who were friends and relatives of the applicator children and had never worked in the cotton fields were used to match on age, education and location. Group comparisons showed that both lived in the same community and attended the same schools. Both had similar demographics except for the BMI of control children being significantly higher than the applicator children (p<0.05). A questionnaire was used to ascertain their work history and pesticide exposure. Exposed children in the older group reported working for an average of approximately 21 days during the current season application. OP pesticides (i.e. chlorpyrifos) was applied in tandem with other non-OP pesticides. As in the previous study, the mean AChE activity levels were significantly lower in applicators compared to controls (p<0.05), although these were within the normal range. Members should note that the assessment of neurological symptoms (via a neurological examination) comprised an adjunctive componentof a much larger examination of neurobehavioural deficits in these children. The authors did not elaborate on the clinical neurological examinations performed by specialists, and


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account for age, education and BMI with regards to being confounders for the neurobehavioural outcomes. 131. The authors observed a significantly higher number of exposed subjects with ‘numbness’ compared to controls (i.e. 18% vs. 2% respectively, (p<0.05)) (Table 7.B.3, Annex 7). No further analyses specific to peripheral neuropathy-related symptoms were conducted. Therefore, this study provides very limited data, and is further compounded by the self-reports of external exposure, lack of account of the multiple co-exposures to other non-OP pesticides and the fact evaluation of PN effects was not a primary study aim. Pesticide Applicators (Lawns/Houses) 132. Steenland et al evaluated central and peripheral nervous system abnormalities in current and former termiticide applicators who reported using chlorpyrifos (or chlordane, which was banned in 1998) for a minimum of one year in a 12-county area of North Carolina, US (Steenland et al 2000). The source population comprised of 3605 pest-control workers, of which 3123 were screened. A total of 520 workers were found to be eligible, however only 193 (representing a 37% participation rate) were tested. Reasons for non-participation included subjects being uncontactable, unavailable, non-compliant, or not willing to participate. The authors did not evaluate potential differences between participants and non-participants. Two non-exposed groups were used for comparisons: (i) 106 age- and sex- matched friend controls and, (ii) 83 age-, sex- and race-matched blue collar workers living in the area. The authors deployed both subjective and objective methods to assess exposure. This comprised of an interview (to ascertain whether subjects were current or former applicators, with or without a history of pesticide poisoning) and biological monitoring of the chlorpyrifos metabolite, TCP, via gas chromatography to discriminate current (n=128) from former (n= 63) chlorpyrifos applicators and the non-exposed group. Subjects were also genotyped for paraoxonase polymorphisms via PCR amplification of DNA obtained from buccal swabs. A total of 18 applicants (10% of those tested) were found to have the polymorphism for increased susceptibility to chlorpyrifos effects. A similar proportion of non-exposed subjects also had this genotype. The mean duration of chlorpyrifos exposure among applicators was 2.4 years, although subjects also had years of exposure to chlordane and other pesticides. Eight subjects reported having previously been poisoned. In addition to a standardised symptom questionnaire, two neurologists trained to conduct comparable methods performed a range of tests to evaluate neurological function. This included a general clinical examination, specific neurological tests for spinal and cranial function e.g. vibrotactile test in the finger and toe, arm/hand tremor, postural sway, vision tests of visual acuity and colour, olfactory senses test (Steenland 2000, Table 6.B.3, Annex 7). Electrophysiological testing of three nerves of the dominant limbs (peroneal (motor), sural and ulnar (sensory)) was also performed. The authors accounted for most confounders (except exposure to previous vibration machinery) and pre-screened subjects for alcohol consumption via a saliva test on the morning of the health assessments. 133. The authors did not present any quantitative data and reported only on the p-values. Significant differences were observed between exposed (n=191) and non-


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exposed groups (n=189) for outcomes obtained via questionnaire, clinical exam, stabilometry tests, and nerve conduction velocity (NCV) tests (Table 7.B.3, Annex 7). No significant negative associations were observed for the tremor, smell tests or vibration sensitivity (although for the latter, increased sensitivity at 125 Hz of the toes were observed for the exposed group (p=0.03) and its subgroups i.e. the formerly exposed group (p=0.04) and the susceptible group (for fingers) (n=18; p=0.05)). In the symptom questionnaire, all exposed subjects (and previously posioned subgroup(n=8)) reported more symptoms of ‘dizziness’ and ‘loss of strength in limbs’ than the non-exposed group (p<0.05). For the clinical examination, significant impairments were observed for the previously poisoned subgroup, who had decreased wrist vibration and pin-prick sensitivity (p=0.003 and 0.002 respectively), and in the susceptible genotype group who had an increased odds of gastro-soleus reflex and Romberg test (p=0.01 and 0.04 respectively). Stabilometry findings suggested greater sway abnormalities in the exposed group compared to combined non-exposed group (p=0.04) or friend control group when assessed seperately (p=0.05). This was also apparent in applicators exposed prior to 1998. Interestingly, the abnormal Romberg test results witnessed in susceptible group was not detected in the analogous computerised sway tests. Tests for trends showed that duration of exposure in applicators was significantly correlated to length of sway for the right leg (p=0.04). Finally, the electrophysiological tests revealed significant differences between the subgroups of exposed and non-exposed groups. Compared to the NC state employees, the exposed group had significantly lower ulnar nerve amplitudes (mV) (p=0.03). This was also apparent with the currently exposed subgroup, although their sural nerve conduction was found to be significantly faster than NC state employees (p=0.05). The electrophysiological findings, however, were not correlated to duration of exposure or urinary levels of TCP metabolite. 134. This study provides some limited evidence of impaired peripheral nerve function in termiticide applicators chronically exposed to chlorpyrifos, as suggested by the positive findings of a select group of tests. However, the electrophysiological findings were limited by an inverse association for one parameter, and the lack of correlation to duration of exposure and biomarker levels. Furthermore, subjects had substantial co-exposure to other pesticide agents, and possible selection bias cannot be ruled out due to the low participation-rate. Also, the assay’s reliability for susceptibility was not particularly high as few applicators with the polymorphisms showed positive effects upon testing. Greenhouse Workers 135. Bazylewicz-Walczak et al assessed peripheral neurological symptoms as part of a much larger examination of neurobehavioural effects in 26 Polish greenhouse workers who performed standard gardening/planting jobs for approximately 12 years (range was from one to 24 years) (Bazylewicz-Walczak et al 1999). No further details of the selection process was provided. Workers were exposed to OPs on a seasonal and cyclic basis that occured in the first half of the year. Dichlorvos, methamidophos, methidathion and primiphos-methyl were the most frequently used OPs, although additional exposure to carbamates, synthetic pyrethroids and dithiocarbamates also occured. The authors conducted a comprehensive assessment of ambient levels of exposure during March to May (the most intensive


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spraying period). This involved measuring levels in air, and contamination of skin and clothes, followed by estimation of daily cumulative exposure to OPs via the Durhan & Welfe method, which takes into account both dermal and respiratory absorption routes (Bazylewicz-Walczak 1999, Table 6.B.3, Annex 7). No biological measures of exposure were performed. The daily cumulative exposure in plant workers was between 0.001-0.009% of the toxic dose, which the authors noted is classified as a low exposure level based on WHO recommendations. The authors did not appear to have performed an assessment of OP exposure in the 25 controls (matched for age, sex, education and habitation) as no exposure data are reported. Controls worked as canteen, kitchen and administrative workers at the same gardening enterprises as the greenhouse workers. An adapted subjective symptoms questionnaire was used as part of the neurobehavioural core test battery for psychological assessment, which collected information on several symptoms including paraestheisa in the fingers and the upper and lower limbs, limb weakness, tremor, vertigo, sense of taste, smell and facial numbness and other facial muscular sensations/ problems. This was performed twice: before and after the intensive spraying season. The authors did not elaborate on whether the questionnaire was blindly conducted. 136. Comparison of the first and second examinations in exposed and control subjects revealed a non-significant reduction in the number of peripheral neurological symptoms (indicated by the lower mean values of the NCTB tests i.e. 1.96 and 1.81 in exposed subjects vs 1.40 and 1.36 in controls) (Table 7.B.3, Annex 7). NB. There is no indication of what symptoms were considered as peripheral neurological symptoms. The authors conducted further analyses of the effects of a single spraying season or long-term exposure (or both) on neurobehavioural effects for significant results only. 137. In conclusion, this study does not provide any evidence of increased PN-related symptoms in greenhouse workers after the spraying season. However, it is noted that the assessment of neurological symptoms comprised an auxiliary study objective, as neurobehavioural effects was the overall health outcome evaluated. The study is further limited by its small sample size and the lack of account of co-exposure to other pesticides. 138. As with the previous study, Kilburn et al assessed neurophysiological function as a component of a much wider investigation of neurobehavioural impairments (Kilburn, 1999). Twenty-two of 384 US patients who were being evaluated in a neurotoxicology clinic for exposure to various neurotoxic chemicals were selected on the basis that they identified chlorpyrifos as the main chemical of exposure. Other major chemicals subjects were exposed to included diazonin, carbamates, pyrethroids and phenolic chemicals. Patients had different exposure scenarios i.e. either occupational or residential, although 86% of patients were exposed via the office or home. The duration of indoor exposures ranged from a single one day incident to chronic repeat events spanning two years. The authors estimated the concentration of pesticides applied in offices based on spraying schedules, and measured the residues in clothing and rugs. However, no air sampling of chlorpyrifos or other OPs were conducted in premises due to the time elapsed since exposure (which ranged from nine to 108 months). Concentrations of chlorpyrifos were assumed to be low as demonstrated by levels used in offices to make dilutions


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(ranged from 0.5% to 24% of concentrate) and the levels found in clothing and rugs (ranged from 0.21 to 0.40 µg/ft2 and 23.7 to 125 µg/ft2 respectively). Two hundred and sixty-four referents with no exposure to neurotoxic chemicals were randomly recruited from voter registration polls and used to generate predicted values for subsequent health assessment comparisons. The authors did not compare exposure levels between these two groups. All subjects were similar in most respects except for education. Other potential confounders explored included age, sex, height and other test determinants. A previously established questionnaire was used to collect frequency data on 35 common health complaints that were scaled from one to 11 depending on the extent of occurrence. Subjects were also screened for several potential confounding exposures and conditions. Objective tests were also performed that included a neurological examination (which the authors did not elaborate on) and the following ‘neurophysiological’ tests for balance, blink reflexes, grip strength, colour discrimination, visual field (which were also defined by authors as ‘physiological component of neurobehavioural testing’) (Kilburn 1999, Table 6.B.3, Annex 7). Other neurophysiological tests conducted included tests for hearing (via audiometry) and vibration sense threshold. The authors did not elaborate on the expertise of the examiners or whether these tests were blinded. 139. Comparison of symptom frequencies between exposed patients and unexposed controls showed that the most significantly elevated PN symptoms included itching, and loss of balance and decreased smell (p=0.0001) (and numb white fingers/Raynaud’s phenomena although data was not shown) (Kilburn 1999, Table 7.B.3, Annex 7). Eleven patients had peripheral neuropathy manifested by weakness and sensory changes associated with a history of painful extremities. Three of these patients had the onset of simple partial motor or temporal lobe seizures after exposure. Physical and neurological examinations showed motor weakness and abnormal reflexes in 11 patients (the authors did not indicate if this was in the same 11 patients with PN symptoms). Neurophysiological tests revealed significant impairments in mean grip strength in exposed patients compared to unexposed controls (p=0.0001); this was also evident for balance, visual fields, and colour scores (p value ranged from 0.005 to 0.0001). All subjects had elevated thresholds for detecting 128 Hz vibration (no further information was provided). Hearing was diminished in four patients. 140. This study observed increased PN-related impairments in individuals exposed to chlorpyrifos largely in the home and office. However, the significance of this finding is limited partly because the authors combined both acutely and chronically exposed subjects in their analyses, the small sample size, and the fact they measured chlorpyrifos levels in clothing and rugs without accounting for possible degradation. Furthermore, the subject’s co-exposure to other non-OP pesticides that may add or synergise with the effects of chlorpyrifos were also not accounted for. Cross-sectional studies summary 141. Overall, the findings from the cross-sectional studies provide some limited evidence of an increased prevalence of chronic peripheral neuropathy in subjects chronically exposed to low-levels of OP pesticides and chemicals. Irrespective of the study findings, nine of the 16 cross-sectional studies used symptom questionnaires


46

in their health assessment, often in conjunction with either a clinical neurological examination (Kilburn, 1999, Steenland et al 2000, Farahat et al 2003, Albers et al 2004c, Hoshino et al 2008) or a neurophysiological test (Kilburn, 1999, Steenland et al 2000, Pilkington et al 2001, Albers et al 2004c, Hoshino et al 2008); three studies used questionnaires only (Bazylewicz-Walczak et al 1999, Kamel et al 2005, Kamel et al 2007b). NB. In a few studies the assessment of neurological/ neurophysiological function comprised a minor component of a much wider investigation of neurobehaviour, which may have influenced the extent of objective tests conducted. The questionnaires gathered data generally showing increased sensory and motor symptoms in exposed subjects, except for two studies that found no increased prevalence of PN symptoms (Bazylewicz-Walczak et al 1999, Albers et al 2004c). Studies that incorporated objective tests to confirm or refute the reported symptoms found that the evidence from the clinical and neurophysiological tests were generally supportive (Kilburn, 1999, Steenland et al 2000, Pilkington et al 2001, Farahat et al 2003, Hoshino et al 2008). 142. Three studies incorporated all three strands of assessment i.e. a questionnaire, clinical examination and electrophysiological test (Kilburn, 1999, Steenland et al 2000, Albers et al 2004c). Kilburn (1999) provided consistent positive findings for questionnaire, and clinical and neurophysiological tests conducted. However, the study was limited by subjects having a variable duration of exposure, and co-exposures to other non-OP pesticides. Steenland et al (2000) also observed select positive findings in each testing strand (although these were inconsistent for the vibrometry and electrophysiological components).This study was also limited by subject’s substantial exposure to other non-OP pesticides, and the possibility that the study group was unrepresentative of the source population due to low participation rates. However, the investigation by Albers et al which is considered to be one of the few well-designed studies, found no evidence of an increased prevalence of PN in exposed subjects from their comprehensive battery of subjective and objective tests (Albers et al 2004c). Hoshino et al (2008) do not present the findings of their clinical exam but reported consistent positive data from the results of their questionnaire and specific neurophysiological tests of the vestibular system, however, the various drawbacks of the study weaken the strength of these findings. 143. Five studies did not use questionnaires to collect symptom data but conducted either a clinical neurological examination and observed positive findings (Srivastava et al 2000, Abdel Rasoul et al 2008), or performed neurophysiological tests, that on balance provided no reliable evidence of PN (Konieczny et al 1999, Keifer et al 2000, Kimura et al 2005), although the latter study reported vestibulo-cerebullar effects that were evaluated as a measure of CNS. Two studies incorporated both a clinical neurological examination and motor/sensory NCS with an additional assessment of sensory thresholds (via QSTs) (Horowitz et al 1999) or neuromuscular dysfunction (via repetitive nerve stimulations) (Peiris-John et al 2002). Study limitations aside, Horowitz et al found that four of the nine subjects had evidence for clinical effects of PN, while Peiris-John et al reported that there was no evidence for clinical effectsbut some limited evidence of electrophysiological deficits. 144. In summary, not all cross-sectional studies used objective tests to verify self-reported symptoms, but for those studies that did, the findings were generally


47

positive (from clinical neurological examinations and QSTs), or inconclusive (from NCS data). Chronic Effects Following Chronic Low-Level OP Exposure Discussion Summary 145. Positive evidence for the development of persistent peripheral nerve effects in individuals exposed to chronic low-levels of OPs came from case-control studies and to a lesser extent cross-sectional and cohort studies respectively. None of these peripheral nerve effects was reported as being severely disabling (Table (iii) below). 146. The positive data from case-control studies was based on three studies, two of which were adequately designed and conducted by the same author and incorporated extensive objective health assessments into their design (Jamal et al 2001, Jamal et al 2002a). The third study was based on self-reported data only and related to hearing loss (Crawford et al 2008).The evidence from cross-sectional studies were derived from several different investigators whose positive findings were largely based on self-reported data, or objective clinical and specific neurological tests, and QST tests; with the findings from NCS being generally inconsistent. Since both case-control and cross-sectional type studies are limited by their inability to prove causation, the lack of consistent positive evidence from the four prospective cohort studies, especially the three papers reporting on a cohort study by Albers et al (which appear to be the most reliable in terms of design), leads one to conclude that although, on balance there is some limited/weak evidence to suggest that non-severely disabling peripheral nerve effects were apparent in individuals who were exposed to low-level OPs, there is currently no strong or convincing evidence to suggest that a temporal relationship exists or that the association is a causal one. However, any inferences based on these more reliable Albers studies should be made with caution due to the fact that the first study conducted motor and sensory tests to develop specific ratings to evaluate CNS and not PNS dysfunction, and all four studies were conducted by the same author, with the same cohort of individuals, suggesting that the findings of the three Albers cohort studies are actually subsets of results of just one study. OVERALL SUMMARY 147. Tables (ii) and (iii) below summarise the main peripheral neuropathy (PN) and neuromuscular dysfunction (NMD) outcomes, and documents whether the study reported evidence of PN/NMD (i.e. significant positive findings) or observed no evidence/ no significant evidence of PN/NMD (negative findings) as described in this section. NB. The findings are categorised according to whether they were obtained from objective or subjective health assessments and are irrespective of the quality of the study.


48

Acute OP Exposure and Chronic PN/NMD 148. In conclusion, regardless of the type of OP agent used, subjects acutely exposed to OPs were more likely to report chronic symptoms of motor nerve impairment. Clinical and subclinical findings provide positive evidence of mainly motor effects in subjects poisoned with agents known to inhibit NTE. No studies provided supportive evidence for clinical/subclinical effects of chronic PN/NMD in symptomatic subjects acutely exposed to OP agents that do not inhibit NTE. In part, this was due to negative findings, but also because very few of the studies conducted all three strands of a neurological evaluation. There was no significant clinical or subclinical evidence of chronic PN/NMD in individuals acutely exposed to low-levels of OPs. In part, this was due to the paucity of studies examining these effects in symptomatic subjects. These findings do not accord with the COT 1999 conclusions on the long-term PN-related effects of acute OP exposure. Chronic OP Low-Level Exposure and Chronic PN/NMD 149. In conclusion, the weight of evidence suggests that there were increased self-reports of PN-related symptoms in subjects chronically exposed to low doses of OPs. However, there is only very limited evidence to suggest that OP exposed subjects exhibit clinical signs of motor, or sensory, impairments of the spinal and cranial nerve, or reflex impairment. Most studies found no significant clinical motor effects, and the results of QST tests appear to suggest that small rather than large sensory nerve fibres could be affected. Most of the evidence for electrophysiological motor and sensory nerve deficits was inconclusive, although two case-control studies (conducted by the same author) observed positive evidence for electrophysiological muscle deficits suggestive of muscle weakness in OP exposed sheep farmers. Very few studies examined NMD and evidence of impairment was reported in one of the four studies, which was the only study to use SFEMG. 150. The findings of the notably better designed studies (i.e. the three papers reporting on a cohort study by Albers and colleagues; and a related paper of cross-sectional design) generally observed no convincing evidence for PN effects, although inconsistent electrophysiological findings were reported in one Albers study. These findings support the COT 1999 conclusions on the long-term PN-related effects of chronic low-level OP exposure.


49

Table (ii). Crude summary of the study findings for chronic effects of acute exposure PN & NMD OUTCOME EXPOSURE

Provides data specific to use of NTE inhibiting/ ageing OPs

Provides data specific to use of Non-NTE inhibiting OPs

Does not discriminate according to type of OP

Positive1 findings reported by authors

Negative2 findings reported by authors

CONCLUSION

MOTOR IMPAIRMENT Subjective assessments

General symptoms of motor impairment

Sarin exposure

Spencer 2001

Difficulty moving fingers or grasping things

OP poisoning

Stallones & Beseler 2002b

Evidence to suggest possible motor –related symptoms regardless of type of OP

Impaired muscle strength (arms or hands)


Impaired muscle strength (legs or feet)

Stallones & Beseler 2002b,


Chlorpyrifos poisoning Soummer 2011 Muscle stiffness/cramping

Sarin poisoning

Kawana 2001, Nakajima 1999,

Sarin exposure McCauley 2001 Muscle fatigue/asthenia Sarin poisoning

Kawana 2001,

Nakajima 1999

Muscle twitching Sarin exposure McCauley 2001 Objective assessments

General clinical signs of motor impairment

Sarin exposure Loh 2010 Spencer 2001

No evidence to suggest clinical symptoms of motor impairment in subjects exposed to non-NTE inhibiting OPs.

Diazinon poisoning Dahlgren 2004

Impaired grip strength* Neuropathic OP

poisoning Miranda 2002a,

Miranda 2004 Evidence to suggest

possible muscle impairment appears to be associated with exposure to NTE-inhibiting OPs

Non-neuropathic OP poisoning

Miranda 2002a Miranda 2004


50

PN & NMD OUTCOME EXPOSURE






CONCLUSION

Impaired pinch strength*

Neuropathic OP poisoning

Miranda 2002a, Miranda 2004

Evidence to suggest possible muscle impairment/weakness appears to be associated with exposure to NTE-inhibiting OPs


Miranda 2002a Miranda 2004

Muscle weakness Chlorpyrifos exposure Soummer 2011

Motor NCS deficits Sarin exposure Loh 2010

No evidence to suggest motor NCS deficits in subjects exposed to non-NTE inhibiting OPs. These negative findings are also associated with low-level acute exposures. Inconsistent evidence of motor NCS deficits was provided from one study that does not discriminate between the type of OP

Chlorpyrifos, malathion and azinophosmethyl poisoning

Jalali 2010 Jalali 2010

Sarin exposure Spencer 2001

EMG deficits Sarin exposure

Loh 2010 No evidence to

suggest EMG deficits in subjects exposed to non-NTE inhibiting OPs. These negative findings are also associated with low-level acute exposures. Evidence of EMG deficits was provided from one study that does not discriminate

Chlorpyrifos, malathion and azinophosmethyl poisoning

Jalali 2010


51







CONCLUSION

between the type of OP

SENSORY IMPAIRMENT Subjective assessments

General symptoms of sensory impairment

Sarin exposure

Spencer 2001

No strong evidence to suggest possible sensory–related symptoms regardless of type of OP

Numbness or tingling in fingers

OP poisoning


Numbness or tingling in toes


Paresthesia Sarin exposure McCauley 2001 Objective assessments

General clinical signs of sensory impairment

Sarin exposure Loh 2010 Spencer 2001

No evidence to suggest clinical symptoms of sensory impairment in subjects exposed to non-NTE inhibiting OPs

Diazinon poisoning Dahlgren 2004

Abnormal vibrotactile threshold (toe)*


Miranda 2002b, Miranda 2004 Inconclusive evidence to suggest abnormal vibration thresholds in subjects exposed to NTE-inhibiting OPs. No evidence to suggest abnormal vibration thresholds in subjects exposed to non-NTE-inhibiting OPs


Miranda 2002b

Abnormal vibrotactile threshold (index finger)*


Miranda 2004 Miranda 2002b


Miranda 2002b

Sensory NCS deficits Chlorpyrifos,

malathion and azinophosmethyl poisoning,

Jalali 2010 Inconclusive evidence to suggest sensory NCS deficits

Sarin exposure Spencer 2001 CRANIAL NERVE IMPAIRMENT


52







CONCLUSION

Subjective assessment

Hoarse voice Sarin exposure McCauley 2001 No strong evidence to suggest cranial nerve effects in subjects exposed to non-NTE inhibiting OPs. Positive subjective evidence provided in two studies of subjects poisoned with non-NTE inhibiting OPs

Visual field deficits Sarin poisoning Nakajima 1999 Kawana 2001

Vertigo Sarin exposure McCauley 2001 Objective assessment

General cranial nerve exam

Sarin exposure Loh 2010

Diazinon poisoning Dahlgren 2004 Postural sway increases

Sarin poisoning Nishiwaki 2001 Miyaki 2005

IMPAIRED REFLEXES

Sarin exposure Loh 2010 Limited number of studies. No evidence of impaired reflexes in subject exposed to Non-NTE inhibiting OP

NMD Chlorpyrifos poisoning Soummer 2011 Limited number of

studies. Evidence of NMD in subject exposed to an NTE-inhibiting OP assessed via repetitive nerve stimulation

1 Study reported significant evidence of PN/NMD 2 Study observed no evidence or reported no significant evidence of PN/NMD *These tests are partly subjective/ psychophysical as they rely on the subject’s participation i.e. there is no way to differentiate the results of subjects who are biased (whether consciously or subconsciously) toward an abnormal study from those with organic disorders.


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Table (iii). Crude summary of the study findings for chronic effects of chronic low-level exposure PN & NMD OUTCOME

OP EXPOSURE Positive1

findings reported by authors

Negative2


CONCLUSIONS

MOTOR IMPAIRMENT Subjective assessment

General symptoms of motor impairments

Chlorpyrifos Albers 2004a Limited evidence to suggest possible motor –related symptoms. However, findings from the more well- designed studies (Albers et al) suggest that the evidence is lacking.

OPs used in sheep dip

Jamal 2002a

Muscle weakness

Chlorpyrifos Albers 2004b Albers 2004a

Dichlorvos, Methamidophos, Methidathion, Pirimiphos-Methyl


OP pesticides Kamel 2005 Kamel 2007b

Chlorpyrifos Kilburn 1999 OPs in sheep dip Pilkington

2001

Chlorpyrifos Steenland 2000

Tremor OP pesticides Kamel 2005

Kamel 2007b

Abnormal movements


Objective assessment

General clinical signs of motor impairment

Tebupirimfos, Dimethoate, Disulfoton Ethoprop, Dichlorvos, Chlorpyrifos, Coumaphos, Fonofos, Phosmet, Tetrachlorvinphos

Starks 2012b No strong evidence to suggest clinical symptoms of motor impairment.

Diazinon, Acephate, Ethoprop, Parathion,

Starks 2012b

Diazinon, Propetamphos, Chlorfenvinphos

Jamal 2001


Jamal 2002a

OP pesticides (chlorpyrifos)

Abdel-Rasoul 2008

Profenofos, Chlorpyrifos, Triaziphos and Phorate

Farahat 2003

Methyl parathion, Tetraethyl pyrophosphate and Azinphos-methyl

Horowitz 1999

OP pesticides Peiris-John 2002

Proximal muscle weakness

Chlorpyrifos Albers 2004b Kilburn 1999

Respiratory muscle weakness

Chlorfenvinphos Konieczny 1999

Increased Chlorpyrifos Albers 2004b


54

PN & NMD OUTCOME



Negative2


CONCLUSIONS

muscle tone Abnormal station or gait

Chlorpyrifos Albers 2004b Albers 2004c

Reduced hand strength

Several OPs Starks 2012b Chlorpyrifos Kilburn 1999

Abnormal movements (e.g. tremor, fasciculations )

Chlorpyrifos Albers 2004c Profenofos, Chlorpyrifos, Triaziphos and Phorate

Farahat 2003


Motor NCS deficits

Chlorpyrifos Albers 2007 Albers 2004a Albers 2004c

Inconclusive electrophysiological evidence of motor NCS deficits. Findings from the more well- designed studies (Albers et al) generally suggest that the evidence is lacking. Two studies provide evidence of deficits in the muscle.

Phorate, Tebupirimfos, Acephate

Starks 2012b

Diazinon Starks 2012b Diazinon, Propetamphos, Chlorfenvinphos

Jamal 2001


Jamal 2002a


Horowitz 1999

Azinphos-methyl Keifer 2000 Methamidophos Kimura 2005 OP pesticides Peiris-John

2002 Peiris-John 2002

EMG deficits Diazinon,

Propetamphos, Chlorfenvinphos OPs used in sheep dip

Jamal 2001 Jamal 2002a

SENSORY IMPAIRMENT Subjective assessment

General symptoms of sensory impairment


Limited evidence to suggest possible sensory symptoms. However, the findings of the more-well designed studies (Albers et al) suggest that the evidence is lacking.


Jamal 2002a

Chlorpyrifos Kilburn 1999 OPs in sheep dip Pilkington

2001

Paraesthesia Dichlorvos,

Methamidophos, Methidathion, Pirimiphos-Methyl


Numbness Profenofos,

Chlorpyrifos, Triaziphos and Phorate

Farahat 2003

OP pesticides Kamel 2005 Skin itching Chlorpyrifos Kilburn 1999


General clinical signs of sensory

Chlorpyrifos Albers 2004a Inconclusive evidence of clinical Dichlorvos, Starks 2012b


55

PN & NMD OUTCOME



Negative2


CONCLUSIONS

impairment Tetrachlorvinphos, Chlorpyrifos Coumaphos, Fonofos, Phosmet

symptoms of sensory impairment. Findings from the more well- designed studies (Albers et al) generally suggest that the evidence is lacking.

Ethoprop, Parathion

Starks 2012b


Jamal 2001


Jamal 2002a

OP pesticides (chlorpyrifos)

Abdel-Rasoul 2008


Farahat 2003


Horowitz 1999


Numbness Profenofos,

Chlorpyrifos, Triaziphos and Phorate

Farahat 2003

Increased vibration threshold

Several OPs Starks 2012b Inconclusive evidence of abnormal vibration thresholds


Jamal 2001


Jamal 2002a


Horowitz 1999

Azinphos-methyl Keifer 2000 Chlorpyrifos Kilburn 1999 OPs in sheep dip Pilkington

2001 Pilkington 2001


Increased thermal thresholds


Jamal 2001 (hot/cold)

Limited evidence of abnormal thermal thresholds (especially for cold) OPs used in sheep

dip Jamal 2002a (hot/cold)


Horowitz 1999 (cold)

OPs in sheep dip Pilkington 2001 (cold)

Pilkington 2001 (hot)

Sensory NCS deficits

Chlorpyrifos Albers 2007 Albers 2004a Albers 2004c

Inconclusive electrophysiological evidence of sensory NCS deficits. Findings from the more well- designed studies (Albers et al) generally suggest that the evidence is


Jamal 2001


Jamal 2002a

Methyl parathion, Tetraethyl pyrophosphate and

Horowitz 1999


56

PN & NMD OUTCOME



Negative2


CONCLUSIONS

Azinphos-methyl lacking. Azinphos-methyl Keifer 2000 OP pesticides Peiris-John

2002 Peiris-John 2002


Steenland 2000

CRANIAL NERVE IMPAIRMENT Subjective assessments

Hearing loss Chlorpyrifos, Diazinon, DDVP, Fonofos, Malathion, Parathion, Phorate, Terbufos, Trichlorfon

Crawford 2008

Limited evidence of cranial nerve symptom abnormalities. Negative findings based on one study that had several study limitations

Imbalance OP pesticides Kamel 2005

Kamel 2007b

Chlorpyrifos Kilburn 1999 Vertigo Dichlorvos,

Methamidophos, Methidathion, Pirimiphos-Methyl


Smell impairments



OP pesticides Kamel 2005 Chlorpyrifos Kilburn 1999

Taste impairments



OP pesticides Kamel 2005 Facial weakness/ numbness



Difficulty speaking


Double vision OP pesticides Kamel 2005

Kamel 2007b

Objective assessments

General cranial nerve signs

OPs in sheep dip Jamal 2002a Inconclusive evidence of clinical cranial nerve abnormalities. Difficult to compare as studies are testing different functions. Slightly consistent evidence for audio-vestibular impairments. However, findings of Albers study (considered to be well designed) found no evidence of abnormal sway or impaired eye movements.


Farahat 2003

Chlorpyrifos Steenland 2000 (Romberg test)

Steenland 2000 (Romberg test)

Audiometric deficits

Methamidophos and parathion

Hoshino 2008

Chlorpyrifos Kilburn 1999 Abnormal balance*

Chlorpyrifos Albers 2004b Several OPs Starks 2012b Chlorpyrifos Kilburn 1999 Methamidaphos Kimura 2005 Chlorpyrifos Steenland

2000


57

PN & NMD OUTCOME



Negative2


CONCLUSIONS

Vestibular abnormalities*

Methamidophos and parathion

Hoshino 2008

Impaired eye movements

Chlorpyrifos Albers 2004b

Abnormal nystagmus

Chlorpyrifos Albers 2004b

Visual field deficits*

Chlorpyrifos Kilburn 1999 Steenland 2000

Colour impairments*

Chlorpyrifos Kilburn 1999 Steenland 2000

Impaired smell Chlorpyrifos Steenland

2000 IMPAIRED REFLEXES

Muscle stretch reflex abnormalities


Inconclusive evidence of reflex impairments. Findings from the more well- designed studies (Albers et al) generally suggest that the evidence is lacking.



Chlorpyrifos Kilburn 1999 Ankle reflex abnormality

Phosmet, Tebupirimfos

Starks 2012b


Farahat 2003


Horowitz 1999

Quinalphos Srivastava 2000

Plantar reflex abnormality


Farahat 2003


Knee reflex abnormality


Farahat 2003


Blink reflex Chlorpyrifos Kilburn 1999 Gastroc-soleus reflex

Chlorpyrifos Steenland 2000 (susceptible genotype)

Steenland 2000

NMD OPs used in sheep

dip Jamal 2002a No strong evidence

to suggest NMD based on three cross-sectional studies that used repetitive nerve stimulation. Evidence from one

Azinphos-methyl Keifer 2000 Chlorfenvinphos Konieczny

1999 OP pesticides Peiris-John

2002


58

PN & NMD OUTCOME



Negative2


CONCLUSIONS

case-control that used SFEMG.

1 Study reported significant evidence of PN/NMD 2 Study observed no evidence or reported no significant evidence of PN/NMD * May not be reflective of a peripheral nerve disorder


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Table 6. PERIPHERAL NEUROPATHY & NEUROMUSCULAR DYSFUNCTION STUDY DESIGN DETAILS First Author





Measure of Exposure




6.A.1. Cohort studies Kawana 2001 Japan 582 male and

female sarin patients (mean age ranged from 36.6 ± SD 13.6 years to 39.7 ± 13.4 years old) treated at St Luke’s International Hospital, Tokyo, on the day of the attack (i.e. 20 March, 1995). Subjects were followed up 2, 3 and 5 years following the sarin attack (i.e. 1997, 1998 and 2000). Response rates were 48.6%, 35.3% and 32.8% (corresponding to 283 victims in 1997, 206 in 1998 and 191 in 2000 i.e. the current analysis) respectively

Three groups were used for a comparative sub-analysis of symptom incidence: two groups comprised of subjects exposed to sarin i.e. (i) 655 victims of Tokyo sarin attack studied by a non-governmental organisation (NGO); and (ii) 88 victims of the 1994 Matsumoto sarin attack); and one group of unexposed subjects (iii) 87 Matsumoto controls of the 1994 Matsumoto sarin attack

St Luke’s symptom questionnaire, containing 33 five-choice Likert scale items i.e. 14 physical symptoms (that included stiff muscles and fatigue), 8 eye-related symptoms and 11 psychological symptoms

Psychological, physical and eye related symptoms reported at follow-up. These were used in part as the diagnostic criteria for post-traumatic stress disorder

Subject’s status as a victim of exposure to the 1995 sarin attack in Tokyo’s subway system (following admittance to St Luke’s International Hospital for treatment)

None considered Symptom frequencies/ percentages were reported for St Luke’s Hospital sarin patients at each follow-up period and compared using Fisher’s exact test (i.e. 1997 vs. 1998, and 1998 vs. 2000); p values reported *p < 0.05; **p < 0.1; symptom incidence (%) compared in 3 groups exposed to sarin, and one control group

Miranda 2002a Nicaragua 59 (from originally 77) male patients, median age 25.0 years (range 14-64 years) hospitalised for acute OP

39 (from original 74) cattle ranchers and fishermen, median age 30.0 years (range 15-49 years), who had

Hand grip and pinch strength was performed according to a standardised procedure: subjects were seated with shoulder adducted and neutrally

Motor neurologic impairment. NB. The authors noted that impairment of muscle strength that diminishes

Pesticide used for poisoning was determined by patient report on hospital examination and

Age, education, lifetime OP pesticide exposure history (obtained via interview

Multiple linear regression analysis; a regression coefficient with a p value < 0.05


60

First Author





Measure of Exposure



poisoning between 1992 and 1996: (8 intentional [2 moderate and 6 severe], and 51 occupational [35 moderate and 16 severe]). Subjects were followed up at hospital discharge (1 – 18 days after the poisoning event, median 6 days, 77% during first 10 days) and approx 7 weeks (18 – 128 days, median 49 days, 84% between 24 and 90 days) after poisoning. Reasons for reduced cohorts include missing and incomplete datasets, death, inability to locate and refusal

never experienced pesticide poisoning (although some had been exposed to pesticides, n=20). Controls were followed up evenly over the whole examination period with intervals between the examinations of 28 to 281 days (median 66 days). From the group initially examined 32 men were not examined a second time due to inability to locate them (migrated)

rotated, elbow flexed at 90o, forearm in neutral position, and wrist between 0o and 30o dorsiflexion and between 0o and 15o ulnar deviation; grip and pinch strength in dominant hand (nb. 90% of participants were right handed) tested using an adjustable-handle Jamar dynamometer and B & L Engineering pinch gauge, respectively; pinch strength examined using key and palmar modalities; mean of three successive trials used as the outcome

with time is suggestive of development of neuropathy

field visits to confirm the reports were made in 35 patients. The authors categorise pesticides according to whether they were neuropathic (e.g. methamidophos, chlorpyrifos and fenthion) or non-neuropathic (methyl parathion, malathion, terbufos, phorate, ediphenfos and metofox). Exposure was categorised as being either mild (i.e. symptoms of weakness, dizziness, headache or blurred vision), moderate (i.e. mild symptoms plus ≥1 of following: sialorrhea, vomiting, abdominal pain, diarrhoea, miosis, tongue or chest fasiculations, wheezing or crepitus, without depression of consciousness) and severe (i.e. mild and moderate symptoms plus marked alteration of consciousness). Erythrocyte cholinesterase activity was also

questionnaire, with cumulative exposure to OPs [no. of years working with OPs x no. of days of application/year] divided into none, low and high), alcohol consumption, body mass index all evaluated as potential confounders; authors also considered exposures to other chemical agents, medical history, occupational exposure to vibrating machinery , time between poisonings and each of the two examinations

was considered statistically significant. Jonckheere-Terpstra test was used to evaluate trends for means for ordered categories. Paired-samples t-test was used to evaluate intra-individual mean change between first and second examinations for every hand-strength test


61

First Author





Measure of Exposure



measured in selected patients. Exposure was further categorised as either moderate and severe non-neuropathic poisonings and moderate and severe neuropathic poisonings

Miranda 2002b Nicaragua 56 (from an original 77) male patients, median age 28 years (range 14-64 years) hospitalised for acute OP poisoning between 1992 and 1996: (6 intentional [1 moderate and 5 severe], and 50 occupational [34 moderate and 16 severe]). Poisoning was categorised as mild, moderate or severe. Subjects were followed up twice: (i) at hospital discharge (1 – 24 days after the poisoning event, median 7 days, 81% during first 10 days); and (ii) approx 7 weeks (24 – 128 days, median 50 days, 77% between 24

38 (from an original 74) male members of fishing and cattle co-operatives, median age 30.0 years (range 15-49 years), who had never experienced pesticide poisoning. The authors noted that some referents had been exposed to pesticides i.e. 11 cattle farmers, with occasional pesticide exposure. Controls were followed up over the whole examination period with intervals between the examinations of 28 to 281 days (median 66 days). From the group initially examined 33 men were not examined a

Cutaneous quantitative tactile vibration thresholds were measured using Vibration II (Sensortek, Inc: Clifton, NJ) on the dominant index finger and great toe. The vibrometer is a control box that displays the reading of vibration amplitude units and two transducer boxes, each with a protruding 1.5cm vibrating post. The Method of Limits procedure was used to determine each digit threshold. Five readings (trials) were made per digit and two were readings averaged (after discarding the first, lowest and highest reading). The resulting data was converted into microns of vibration amplitude (A) and the distribution of vibrotactile thresholds were normalised into log mic. The threshold mean change was calculated as the arithmetic difference between 1st and 2nd examinations. Room

Large-diameter myelinated sensory fibre impairment (i.e. peripheral nerve dysfunction); the authors noted that these fibres are especially susceptible to axonal degeneration induced by OPs

The pesticide used for poisoning was determined by patient report taken on hospital examination; field visits to confirm the reports were made in 32 patients. The authors categorised pesticides according to whether they were neuropathic (e.g. methamidophos, chlorpyrifos and fenthion) or non-neuropathic (i.e. methyl parathion, malathion, terbufos, phorate, ediphenfos and metofox). Poisoning severity was categorised as being either mild (i.e. symptoms of weakness, dizziness, headache or blurred vision), moderate (i.e. mild symptoms plus ≥1 of following:

Callus formation (known to influence vibration threshold), age, height, education, alcohol consumption, days between poisoning and the examination, examiner, OPs cumulative exposure. NB. The authors also considered exposures to other chemical agents, medical history, occupational exposure to vibrating machinery, the effect of four different examiners, and time

Multiple linear regression; vibrotactile thresholds were used as the outcome variables as well as mean changes in index finger and toe vibrotactile thresholds (i.e. difference in vibration thresholds between the two evaluations). A regression coefficient with a p value < 0.05 was considered statistically significant


62

First Author





Measure of Exposure



and 80 days) after poisoning. NB. One severely poisoned patient refused to participate in second examination. From the group initially examined 12 poisoned men were not examined a second time due to inability to locate them (i.e. had migrated), and one because it could it be confirmed that the poisoning agent was an OP. Six with severe poisonings died before completing either the first or second evaluation

second time due to inability to locate them (i.e. had migrated), and two refused to participate in the second examination. The authors noted that vibration thresholds for these individuals not differ from those who did participate in the second examination

temperature during first and second examinations were as (29.4-32.4oC) and (28.4-33.2oC) respectively. Four different examiners performed the vibrometry tests, but subjects were tested by the same examiner on both occasions

sialorrhea (drooling), vomiting, abdominal pain, diarrhoea, miosis, tongue or chest fasiculations (muscle twitch), wheezing or crepitus (i.e. clicking or cracking sound of joints), without altered mental state) and severe (i.e. mild and moderate symptoms plus marked alteration of consciousness). Erythrocyte cholinesterase activity was also measured in patients with moderate and severe poisonings. Exposure was further categorised as either moderate and severe non-neuropathic poisonings and moderate and severe neuropathic poisonings

Miranda 2004 Nicaragua 48 male patients (from an original group of 71) hospitalised for acute OP poisoning between 1992 and 1996: (4 intentional and 44 occupational). Fifteen men were

28 healthy male cattle ranchers and fishermen, (from an original group of 74), median age 30.3 years (range 17-51), who had never experienced pesticide poisoning. NB.

Clinical examination and interview. Grip and pinch strength measurements were performed according to previously noted standardised procedure (Miranda et al 2002a). Vibration thresholds were measured as previously noted using a Vibration II

Sensory and motor abnormalities

Pesticides used were determined by patient reports on hospital examination and confirmed by field visits. Poisoning was classed as mild, moderate or severe according to

Age, education, lifetime exposure to OPs, alcohol consumption, body mass index, lead exposure, neurological side effects of medicinal drugs;

Vibration threshold distributions were normalised with log transformation; linear multiple regressions; comparisons expressed as


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poisoned with non-neuropathic OPs (median age 19.5 years, range 14-39); 19 men were moderately poisoned with neuropathic OPs (median age 22.5 years, range 15-64) and 14 men were severely poisoned with neuropathic OPs (median age 23 years, range 15-62). NB. None of the poisonings were classed as mild. Subjects were followed up at hospital discharge, approx 7 weeks and between 24 and 32 months (mean 28 months) after poisoning. From the group initially examined 12 poisoned men were not examined a second time and an additional 11 men the third time due to inability to locate them (migrated). No new poisonings occurred during this 2 year period

The authors noted that some referents had been exposed to pesticides (n=10) but none reported ever having been poisoned. From the group initially examined, 35 men were not examined a second time due to inability to locate, with an additional 11 men for the third evaluation due to similar reasons two years on

Sensor for dominant index finger and big toe (Miranda et al 2002b). Tests were performed at room temperature of 29-33oC for third examination

a predefined list of signs and symptoms. Pesticides were classed as either non-neuropathic OPs (e.g. methamidophos, chlorpyrifos and fenthion) and neuropathic OPs (e.g. edifenphos, methyl parathion, phorate, malathion and terbufos) as previously described (Miranda et al 2002ab)

authors also addressed exposures to other chemical agents, medical history, possible selection bias due to attrition (by comparing test scores of subjects who participated the various examination permutations)

mean and SD, age adjusted regression coefficient with 95% CI; a regression coefficient with p<0.05 was considered significant

Nakajima 1999 Japan

[1 year survey]: 318 male and

[1 year survey]: 919 male and

Questionnaire-related surveys were conducted 3

Various symptoms

Residence in the Matsumoto area,

Age and gender Evaluation focussed on


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female victims of the Matsumoto sarin attack (taken from 1237 respondents of an original cohort of 2052 people). Sarin victims were identified as those diagnosed with definite muscarinic and/or nicotinic symptoms immediately after the sarin incident (starting from evening of 27th to 28th June 1994). NB. The authors did not specify the mean age of victims one year after exposure but only the mean age of the victims who were symptomatic (i.e. victims who reported symptoms associated with sarin one year after exposure) and for those for which symptoms resolved (Table 10). Subjects were all inhabitants living or staying in the area of the sarin attack in Matsumoto City. [3 year survey]: 167 sarin victim

female respondents identified as non-victims i.e. were not diagnosed with definite muscarinic and/or nicotinic symptoms immediately after the sarin incident. [3 year survey]: 669 respondents identified as non-victims (mean age 54.2 years, SD ±21.5)

weeks, 4 months, 1, 2 and 3 years after the sarin incident to clarify symptoms and complaints. The questionnaire collected data on health status including: having a sarin toxicity diagnosis or experiencing muscarinic/nicotinic symptoms immediately after the sarin exposure; experiencing delayed symptoms; hospitalisations and consultations with doctors; and current symptoms. Health examinations were conducted after each survey for those requesting consultations (no further information was provided)

associated with sarin exposure were investigated. Symptoms relating to peripheral neuropathy included asthenia (muscle weakness) and shoulder stiffness

1050 metres north to south and 850 metres east to west where about 12 litres of sarin were released on the night of June 27, 1994, with the sarin release site in the centre. Erythrocyte and serum AChE activity levels were measured in victims admitted to hospital 4 weeks after sarin exposure

symptom prevalence/likelihood of reporting symptoms one and three years after the attack. Chi-square test was used in the 1-year survey to test the significance of the prevalence of symptoms reported one year after sarin exposure during medical consultations, (p values were reported). In the 3-year survey chi-squared test was used to evaluate differences in prevalence of symptoms between two groups (victim and non-victim) and that of people with or without each symptom during the surveys (p values were reported). NB. Fisher’s exact test was used for small group numbers. One-way ANOVA was used to test the difference in


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respondents (taken from 836 respondents); mean age 44.4 years, SD ±19.8). 27 of these victims had been hospitalised immediately after the incident, 109 had consulted doctors and 31 victims did not consult any doctor

erythrocyte and serum AChE activity at an early stage of intoxication between people with or without symptoms (p values were reported). ORs and 95% CIs were calculated for symptoms in victim group

6.A.2. Case-control studies - none

6.A.3. Cross-sectional studies Jalali 2011

Iran Male and female patients investigated between Mar 2005 to Mar 2006 with intentional acute moderate to severe OP poisoning who recovered from the acute phase of intoxication. Eight patients (aged 13-59 years) from 342 patients hospitalised for OP poisoning in the Poisons Treatment Unit of Imam Reza hospital, had clinical signs or symptoms of muscle weakness

None. NB. The authors used normal values for NCV test comparisons. No further information was provided

Electrophysiological tests were conducted between 10 and 210 days since onset of OP poisoning (i.e. during hospitalisation or < 1 week during discharge (n=3), within 6 weeks after OP exposure (n=4) or 6 months after discharge (n=1)). Nerve conduction velocity (NCV) tests were performed using surface electrodes to measure sensory (orthodromic) and motor NCV (stimulated proximally and distally) in the right and left median, ulnar, peroneal and tibialis. For the motor NCV the difference in time latencies was divided by the distance of the two excitation points.

Peripheral nerve effects

OP poisoning was diagnosed via case history of OP ingestion, clinical manifestations and decreased AChE activity (no further biomarker information was provided). OP used in suicide attempt was identified from labels of bottles and comprised of chlorpyrifos (n=3), malathion (n=2) and azinphosmethyl (n=2). OP type was not determined in one patient. Patients were divided into being moderately or

Neurological diseases or co-existing diseases likely to confound testing were screened for via complete medical history. Patients were excluded if they had ingested alcohol or any other chemicals/ pharmaceuticals

Means and standard deviations are presented. Students t test and Pearson’s test were also applied; p < 0.05


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or peripheral neuropathy (such as paresthesia, numbness and tendon/plantar reflex dysfunctions) and comprised the test group

Electromyography (EMG) was performed via use of sterile concentric needle electrodes inserted (after the patient had relaxed) into the biceps, extensor digitorum communis of the hands, quadriceps, anterior tibialis and gastrocnemius of the legs. Patients were asked to maximally contract their muscles (once spontaneous contractions had passed) and the amplitude and duration of waves recorded. Both electrophysiological tests were performed 3 times per patient on the same day to generate a median value

severely poisoned depending on the extent of acute signs symptoms: (i) moderate - patients with cholinergic syndrome, CNS depression and no respiratory failure; (ii) severe - patients with cholinergic syndrome, CNS depression, and respiratory failure requiring mechanical ventilation

McCauley 2001 USA/Iraq 653 male and female US troops (mean age 39.0 years, SD 7.5) believed to be within a 50-km radius of the Khamisiyah Ammunition Storage Point and likely to have sustained low-level exposures to sarin/cyclosarin following ground-based detonation of chemical munitions at Bunker 73 between March 1 and March 15, 1991. NB. The authors refer to

The authors used two comparison groups: (i) 610 male and female military personnel (mean age 37.8 years, SD 7.3) deployed to the Southwest Asia theatre during the Gulf War (GW) combat period. The authors assume that these veterans would of had no known exposure to nerve agents but may have been exposed to the carbamate pyridostigmine bromide and/or

Telephone computer assisted interview survey was conducted from Oct 1998 to April 1999. The adapted GW telephone survey instrument queried subjects on their general health and symptoms during the time period in which the Khamisiyah detonations occurred (i.e. immediate or short term effects of sarin vapour exposure). This included a 24-item checklist of symptoms known to be associated with chemical warfare agents (CWA) exposures and symptoms not generally associated with exposure to anticholinesterase agents. The authors developed an

Self-reported neurological and neurophysiological signs and symptoms experienced during the first 2 weeks after the Ground War (which includes muscle twitching, muscle cramping/weakness, tingling of hands and feet) and current health symptoms (which includes tingling, burning, pins and needles, numbness or lack of feeling, loss of muscle strength in

The adapted GW telephone survey instrument was used to obtain more detailed information about troop movements in the Khamisiyah area including exposure to detonation of ammunition bunkers The US Department of Defence used dispersion plume modelling of the detonations to estimate the temporal-spatial movement of releases. The authors considered those

Age, gender, and region of residence. The authors also screened for exposures to biological and chemical factors including prescription and experimental medications taken in-theater. Contactable veterans were found to be significantly more likely to have college or advanced degrees, married, Caucasian and

Chi-squared tests were used to analyse categorical data. Logistic regression was used to estimate odds ratios and confidence intervals (which were adjusted for potential confounders); p-values were not reported. T-tests were used to analyse continuous variables (age)


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this group as the Khamisiyah (KHAM) population. The authors divide the 653 KHAM population into two subgroups: (i) 162 KHAM subjects who reported being involved or witnessing the Khamisiyah operations (KHAM witness); (ii) 405 KHAM subjects who reported not being involved or witnessing Khamisiyah operations (KHAM-non-witness); the remaining 86 subjects were unsure. NB. The source population comprised of 3219 veterans who were active or reserve status in the US Army between 01/08/90 – 30/06/91. However, only 2918 were contactable, to which 2388 (later corrected to 2834 due to 54 having given incorrect deployment information) were considered eligible. Of these

OP insecticides. The authors refer to this group as the Non-Khamisiyah (N-KHAM) population; (ii) 516 male and female military personnel (mean age 38.1 years, SD 8.3) who had not served in the GW theatre of operations but were either on active duty or were activated during the GW period. The authors refer to this group as the Non-deployed (ND) population (negative controls)

OP-affected case definition based on reporting ≥ 3 ocular and respiratory effects that commonly arise after aerosol or vapour exposure to low doses of OP CWA. Other items on the checklist included symptoms associated with low-level exposure to mustard agents. A second checklist sought information about veteran’s current health symptoms, and included more neurological specific items. NB. At the time of the telephone interview all veterans resided in one of five US states: Oregon, Washington, California, Georgia or North Carolina

arms or legs, loss of balance or co-ordination, and cramping, aches, pains or stiffness of muscles)

veterans within a 50km radius were likely to have sustained low-level exposures to sarin/cyclosarin (i.e. although the 50 km radius was broad, it probably includes personnel that were at greatest risk for airborne exposure to CWAs). The survey sought information about the nature of environmental exposures during the GW and poised questions about service duty, living conditions, combat exposures and heat and sand exposures. Exposures to biologic and chemical factors including prescription and experimental medications taken in-theatre were also assessed in detail

male. Participants compared to non-participants/ non-responders were significantly more likely to be Caucasian and on active rather than reserve duty.


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555 refused to participate resulting in 1779 interviews (77% participation rate)

McCauley (Cross-referenced from Spencer 2001)

2002 US/Iraq 653 male and female Desert Storm veterans present within a 50 km radius of Khamisiyah, Coalition-Occupied Iraq who may have been exposed to sarin /cyclosarin during first 2 weeks of March 1991. These were selected from a source population of 3219 deployed and non-deployed veterans. However, 301 were uncontactable. Of the 2388 deemed to be eligible, 555 refused to participate leaving a participant population of 1833. This represented a 76.7% participation rate.. 54 subjects who completed the interviews were subsequently excluded on the basis they provided incorrect deployment information. The

The authors adopted two comparison groups: (i) 610 Non-Khamisiyah male and female veterans deployed to SW Asia during the GW combat who did not serve in Khamisiyah; (ii) 516 Non-deployed male and female GW-era veterans (negative controls), who did not serve in SW Asia but were either currently active, reserved or retired

Subjects underwent a computer-assisted telephone interview conducted between October 1998 to April 1999 which comprised a symptom checklist that addressed health both in theatre and after return from S.W. Asia, including current health symptoms. The interview also contained questions on conditions that had been diagnosed by a physician since the Gulf War, employment status, hospitalisations, and disability). NB. A random sample of 150 veterans were recontacted six months later and asked identical questions

Various current health status outcomes (including peripheral neuropathy)

Exposure history was taken from a computer-assisted telephone interview questionnaire, which involved contacting veterans via a letter mailout and telephone. The adapted survey instrument involved gathering detailed information on the nature of GW-related environmental exposures, including information on personal movement in the Khamisyah area and exposure to detonation of ammunition bunkers). Telephone numbers were tracked as previously described in McCauley 2001

Region of residence, gender and age, race/ethnicity (based on group background characteristics). The authors compared veterans with contactable telephone numbers to those who could not be easily located and found the former veterans were more likely to have college or advanced degrees, be married, white and male (no more than 10% difference between the two groups) . The authors excluded subjects on the basis or not being enrolled in the Army or National Guard during the GW, being veterans of prior conflicts,

Factor analysis of self-reported health symptoms. ORs were reported for a study group to report being diagnosed with a particular health outcome


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authors noted there were significant differences in the background characteristics of the 1779 participants with respect to current region of residence, military status, gender, age, race, education

having language or hearing problems preventing interview for proceeding, being deceased. Comparison of eligible participants that responded to those that didn’t respond showed that they were more likely to be Veterans who agreed to participate were more likely to be older, female and Caucasian compared to non-participants

Miyaki 2005 Japan 23 male subway workers (mean age 47.4 years, SD9.8) exposed to sarin. Subjects were further subdivided into high and low exposure groups NB. A total of 36 subway workers (from a source population of 55 workers) participated in the study representing a 65% participation rate. Authors did not evaluate reasons for non-participation

13 referent subway workers (mean age 42.5 years, SD 11) not exposed to sarin. There were significant differences wrt age (compared to high exposure group) and scores of General Health Questionnaire (GHS) compared to exposed (p<0.05)

Computerised neurobehavioural tests which included assessment of stabilometry that used a strain-gauge type force platform first with the subjects eyes open and then closed for 60s. The following body-sway parameters were used: total length (i.e. the total sway length of the centre of foot pressure during the 60s measurement), X length (sway length in the medio-lateral direction), Y length (sway length in the anterior-posterior direction) and sway area (area enclosed within the envelope of the outer perimeter of the X-Y

Nervous system effects particularly in relation to neurobehaviour

[Dose response analysis] The authors used hospital status after poisoning based on replies to self-administered questionnaire to classify subjects into either high exposure group (hospitalised immediately after poisoning) and low exposure group (attended hospital as outpatients)

Exposure status, age, educational level, smoking, drinking, and GHQ score were among potential confounding variables evaluated. None of the workers had an illness or histories of disease known to affect the nervous system

Student’s t test to compare mean values of data between exposed and referent groups and one-way ANOVA to compare data among high vs low vs referent groups. Multiple logistic regression to control for possible confounding variables. The authors also combined the data with that of


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plot of the centre of foot pressure. NB. The authors noted that these tests were performed using same procedures as Nishiwaki 2001 i.e. by investigators who had no prior knowledge of subject’s exposure status

a previous study in rescue staff and police officers and reanalysed to increase the statistical power. This resulting in 80 exposed subjects (30-high, 60-low) and 65 referents

Nishiwaki 2001 Japan 54 (from an original 57) exposed subjects comprising of male rescue team staff members of the Tokyo Fire Department and police officers who arrived at the scene of the 1995 Tokyo subway sarin attack. Subjects were followed up between 34-45 months after the incident

52 age matched subjects without exposure taken from each office of each department (i.e. 29 male rescue team staff members and 23 police officers). Significant group differences were evident wrt height and body weight.

Vibration perception threshold (VPT) and stabilometry comprised two of four tests conducted by investigators who had no prior knowledge of subject’s exposure status; other tests conducted included neurobehavioural tests of psychomotor function, and psychometric testing. VPT tests were measured bilaterally at the index and middle finger using frequencies of 63, 125 and250 Hz via an AU-02B Rion Co. vibration sensation meter. Subjects placed a fingertip on the vibration meter and were subjected to a gradual increase of the vibration intensity at each frequency until sensation was felt. The threshold for each frequency at each finger was displayed in decibels. Stabilometry was performed to assess the chronic effects of sarin on equilibrium using the strain-gauge-type force platform

Chronic physical and psychiatric effects of sarin on the CNS, equilibrium, and on vibratory sensations in relation to traumatic stress symptoms

Self-administered questionnaire was used to collect information on the exposed subject’s hospitalisation after the incident. To assess the dose-effect relationship exposed subjects were divided into two groups according to their level of exposure. This was based on the severity of poisoning i.e. whether subjects were hospitalised immediately after poisoning (high-exposed group), or attended hospital as outpatients (low-exposed group). High-exposed subjects were further subdivided into a possible hypoxia-positive group (i.e. those who lost

Age, height, weight, smoking status, alcohol consumption, smoking status, and psychometric variables (IES score or GHQ score) for the stabilometry and vibration perception thresholds; exclusions for co-exposure to other chemicals such as solvents and reagents at work. The authors also screened for past histories of diseases that could affect the nervous system. NB. Three subjects were excluded during the selection process on the grounds of

Mean values underwent appropriate transformations to obtain a normal distribution. Students t-test was used to compare mean values of high exposed and referent groups. Welch’s method was used to compare mean values between low-exposed and referent groups. p< 0.05. Multiple regression analysis was applied to continuous variables and multiple logistic regression applied to discrete variables to control for possible


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at a sampling frequency of 20 Hz first with the subjects eyes open, followed by testing with their eyes closed for 60 secs. Subjects watched a small red circle positioned 3m away from where they were standing on a flat hard floor. The following body-sway parameters were assessed: total length, X length, Y length and sway area

consciousness or suffered from dyspnea immediately after poisoning) and a hypoxia-negative group. This was done to exclude the possibility that CNS effects were secondary outcome due to hypoxia resulting from coma and dyspnea

having amnesia not related to sarin exposure, or not providing information about their hospitalisation. For the stabilometry tests two subjects were excluded because they had low back pain or a past history of auditory ossicles fracture

confounding factors

Spencer 2001 US/Iraq 653 male and female Desert Storm veterans present within a 50 km radius of Khamisiyah, Coalition-Occupied Iraq who may have been exposed to sarin /cyclosarin during first 2 weeks of Mar 1991. These were selected from a source population of 3219 deployed and non-deployed veterans. However, 301 were uncontactable. Of the 2388 deemed to be eligible, 555 refused to participate leaving a participant

The authors adopted two comparison groups: (i) 610 Non-Khamisiyah male and female Desert storm veterans with no known potential exposure to nerve agents, 23.9% were activated reserve troops. A total of 203 veterans underwent Level I neurobehavioural assessments and 26 completed Level II neuro-physiological testing; (ii) 516 Non-deployed male and female GW-era veterans (negative controls),

A two-level clinical examination was performed. NB. The following information was abstracted from McCauley et al, under review – which was attached to the current study: (Subjects underwent a telephone interview which comprised a symptom checklist containing 24 items that addressed health both in theatre and after return from S.W. Asia, including current health symptoms. The interview also contained questions on conditions that had been diagnosed by a physician since the Gulf War, employment status, hospitalisations, and disability). Subjects who completed the telephone interview were recruited for

OP-associated persistent CNS damage (i.e. neurobehavioural deficits and EEG abnormalities) and neuromuscular and neurophysiological deficits

Exposure history was taken from a computer-assisted telephone interview questionnaire, which involved contacting veterans grouped into one of three US Department of Defence categories based on their proximity to the defined 50km radius of Khamisiyah detonations. NB. The following information in parentheses was abstracted from McCauley et al, under review – which was attached to the current study: (The adapted

Region of residence, gender and age, race/ethnicity. The authors compared veterans with contactable telephone numbers to those who could not be easily located and found the former veterans were more likely to have college or advanced degrees, be married, white and male (no more than 10% difference between the two groups) . The authors

Factor analysis of self-reported health symptoms. ORs were reported for a study group to report being diagnosed with a particular health outcome. Multivariate analysis of variance was used to assess differences among deployment groups with respect to. relative activity of EEG data; p-values are reported


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population of 1833. This represented a 76.7% participation rate. A large number of the Khamisiyah veterans’ region of residence clustered in the Southeastern US cf. West Coast. 94% were active, regular military vs. activated reserve troops. A total of 267 veterans underwent Level I neurobehavioural assessments and 42 completed Level II neuro-physiological testing. The authors noted that the deployed subjects (i.e. this Khamisiyah subgroup together with the non-Khamisiyah (comparison) subgroup) were slightly younger than those in the non-deployed subgroups (p=0.015). Of the deployed subjects, the Khamisiyah veterans were younger than those in the non-Khamisiyah group (p=0.010)

30.2% consisted of activated reserve troops. A total of 154 veterans underwent Level I neurobehavioural assessments and 28 completed Level II neuro-physiological testing

neurobehavioural testing (Level I), which employed a computerised neurobehavioural test battery. Level II testing comprised a focused clinical neuromuscular and neurophysiological examination i.e. electro-encephalography (EEG), upper and lower limb somatosensory evoked potential testing (recording relevant peak latency and amplitudes), and transcranial magnetic motor evoked potentials, with muscle recordings from abductor pollicis brevis, adductor digiti minimi, and tibialis anterior soleus (recording relevant onset latency and average peak amplitudes)

survey instrument involved gathering detailed information on the nature of GW-related environmental exposures, including exposures to biological and chemical factors, prescription and experimental medication taken in theatre and exposure to detonation of ammunition bunkers). Telephone numbers were tracked and located using Telematch, Equifax, IRS, Transunion and/or internet search engines

excluded subjects on the basis or not being enrolled in the Army or National Guard during the GW, being veterans of prior conflicts, having language or hearing problems preventing interview for proceeding, being deceased. Comparison of eligible participants that responded to those that didn’t respond showed that they were more likely to be white or on active vs reserve duty


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Stallones & Beseler

2002b US The study population comprised of 761 male/ female farm residents (i.e. operators and their spouses) mean age of 50.7 years (range 24 – 85, SD 13.6) selected from a multistage area sample of farms in an 8 county area of North-eastern Colorado (479 farms). This represented a 57% response rate from the 835 farms contacted. Terbufos was the most used crop insecticide (used by 20.8% of the residents) and applied at mean frequency of 1.2 times per year (SD, 1.3). The most used pesticides for livestock (by 37.1% residents) were defined as other OP/carbamates applied a mean frequency of 2.6 times per year (SD, 5.2). NB. The authors did not provide further

Respondents who answered “No” to whether they reported experiencing a pesticide related illness (692 respondents) were used as comparison groups

Personal interview/ questionnaire conducted during the slower agriculture seasons conducted separately for both spouse and farm operator. Data collected included general health questions e.g. medical history, pesticide-related illnesses (medically diagnosed) and more specific questions to assess neurological symptoms experienced in the past month. These 24 symptoms were ranked according to occurrence: 1= not at all, 2= a little, 3=moderately, 4= quite a bit, and 5= extremely

Neurological symptoms including muscle strength in limbs and hands, numbness and tingling in toes and fingers

Personal interview/ questionnaire performed during the slower agriculture seasons was conducted separately for both spouse and farm operator. Data collected included questions on farm characteristics (e.g. primary agricultural activities, types of pesticides used and their application i.e. aerial application, use of granules or sprayer, water-based centre pivot irrigation system), use of protective equipment, working hours and number of pesticide applications in the previous year. The authors noted that the OPs used on the farms (for crop and/or livestock) included terbufos, chlorpyrifos, phosmet, and dichlorvos. Other non-OP pesticides and herbicides were also used on the farm

Age, gender. The authors also screened for general health, race, SES, education and smoking habit. Reasons for non-participation were not followed-up but the authors compared farming operations in the eight counties with those who did participate and found that there were no differences

Odds ratios with 95% CIs were estimated from a univariate analysis for reporting a neurological symptom and experiencing pesticide-related illness. A Mantel-Haenszel χ2 test for trend was calculated for neurological symptoms that showed an elevated odds ratio. A univariate analysis was performed to obtain estimated odds ratios for the % of farm residents using the pesticide and reporting a pesticide related illness. Univariate analyses was also used to calculate ORs and 95% CIs for pesticide related illnesses and the method of application of pesticides. Any significant variables were included in (i) a


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information re: the breakdown of OPs for this application. A total of 69 respondents reported having ever experienced a physician diagnosed pesticide-related illness, which was used to generate groups for sub-analyses of possible associations with health outcome.

multivariate logistic regression analysis, modelling the probability of having reported a pesticide related illness (the best model verified by stepwise logistic regression; (ii) a multivariate conditional regression analysis; p-values are reported

6.A.4. Case series studies

Dahlgren 2004 US Family of seven (2 parents, aged 37 and 39 years, and 5 children, aged 1 to 11, with another in-utero at 5 months gestation) exposed to diazinon on 16 June 1999 following mistaken application to interior by pesticide company. Mother and youngest child were exposed 4h after application to a visibly wet floor with dermal and inhalation exposure. Other family members

N/A Medical history and examination (on first visit to clinic in Aug 1999). Examination was repeated on second visit to the clinic in Aug 2002 with a battery of tests that included neurophysiological testing of grip strength using the Jamar Dynamometer. A range of tests for neurological and neuropsychological assessment were also used

Neurological, neurophysiological and neuropsychological deficits

Exposure assessed via wipe, air and bulk sampling. Five wipe samples and one control sample were analysed in June 29 1999 by the California Pesticide Regulation. Healthscience Associates conducted wipe sampling between July 1999 to Feb 2000. Air sampling was performed on July 9 1999 and tested by DataChem (Utah) using NMAM 5600MOD. Bulk samples were

No information provided



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were exposed thereafter. The family left the home 2 days after exposure. The family returned home after 11 months since the initial exposure once the house was thoroughly cleaned

analysed from July 1999 to Feb 2000 using EPA methods. NB. Authors were unable to determine the quantity of diazinon (diluted) applied in the home. According to data sheets the concentrate comprised of 22.4% diazinon and 77.6% inert ingredients. A urine sample was tested for diazinon parent compound

6.A.5. Case reports Loh 2010 US/Iraq A 34 year old white

male senior Army explosive ordinance disposal (EOD) sergeant exposed to sarin in May 2004 while deployed in Iraq. The sergeant had been called to disarm an improvised explosive device (IED) after a blast was reported. The IED was placed into the enclosed vehicle that the sergeant was driving prior to the development of acute symptoms several minutes

n/a Non-diagnostic neurological, neurophysiological evaluations were performed. It is not clear when these tests were conducted but the authors noted that the subject returned to duty two weeks after the incident and these tests were done before the subject had a neuropsychological assessment which itself was performed eight months after he first started to experience chronic symptoms (which was 2 months following his return). The neurological assessment included a cranial nerve examination and tests of motor, reflex,

Neurological sequelae

No external/ environmental measures of exposure levels were performed. The IED was inspected and 3-5 cm3 of a colourless liquid was discovered in the vehicle and identified as sarin. Erythrocyte cholinesterase activity was measured to determine the level of acute sarin exposure

The authors reported that the subject had no significant past history except for sinus, haemorrhoid, and arthroscopic knee surgery. There was no significant family history and the subject denied tobacco, alcohol, or illicit drug use and was not taking any medications



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later sensation and gait examinations. No further details are provided. The neurophysiological examination included electromyography and nerve conduction tests. No further details are provided.

Soummer 2011 France A 24 year old North African man who attempted suicide by injecting chlorpyrifos in both forearms and ingested two psychoactive drugs (alprazolam (5mg) and olanzapine 100mg)). The patient was conscious on arrival and displayed several symptoms/signs consistent with acute OP poisoning (i.e. ACh receptor overstimulation/acute cholinergic crises) which lasted for 30 days. The patient received appropriate treatment to manage symptoms (e.g. atropine and pralidozime) but subsequently went into a coma and awoke six weeks after admission

n/a Manual and electrophysiological testing of proximal and distal muscles were conducted at various times: day 50, 120 and one-year. The authors did not elaborate on the tests or scoring system used in manual tests. For electrophysiological testing repetitive nerve stimulations over several frequencies were used (range not reported)

Neurological seqela including muscle weakness and denervation

Patient history and hospital admittance status (OP poisoning). Plasma butyrylcholinesterase and RBC AChE activities were assessed. Chlorpyrifos and its metabolites (3,5,6-trichloro-2-pyridinol (TCP), diethylphosphate (DEP), and diethylthiophosphate (DETP) levels were measured in plasma and urine via gas or liquid chromatography

Co-administration of psychoactive drugs alprazolam (5mg) and olanzapine 100mg



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6.B.1. Cohort studies Albers 2004b US 53 of 66 eligible

male and female chlorpyrifos-manufacturing workers (mean age 41.2 ± 7.5 years) employed on Sept 1, 1999, in buildings involved in chlorpyrifos production with known measureable exposure to chlorpyrifos (and no occupational exposure to other OP insecticides or other known/suspected neurotoxicants). Subjects were followed up 1-year after baseline examination (performed during a period of potential chlorpyrifos exposure)

60 of 74 eligible male and female chemical workers (mean age 41.3 ± 8.4 years) employed on Sept 1, 1999, in the Saran production building. These subjects were involved in manufacturing Saran (a clear plastic film wrapping material) and had no occupational exposure to chlorpyrifos or other known/suspected neurotoxicants. A total of 58 referents were followed up 1 year after baseline examinations

A questionnaire were used: the first conducted at baseline evaluation to elicit overt symptoms relevant to cognition and other potential neurological dysfunction (as well as to obtain demographic, medical history, family history of neurological diseases, etc); the second questionnaire was conducted one year later to update information. NB. A board-certified neurologist clarified neurological symptoms elicited in the questionnaire. Mental status was examined as part of the standard neurologic examination Mini-Mental State Examination (MMSE). The neurological examination included assessment of selected cranial nerve function, co-ordination, alternate motion rate, presence of abnormal movements, station, gait, tone, strength (proximal and distal), sensation (i.e. dual simultaneous stimulation, touch pressure, joint position, pin pain and vibration), muscle stretch reflexes, and primitive and pathologic reflexes. Results from the neurologic

The authors present health outcome data under titles that would suggest assessment of motor and sensory nerve effects. However, these outcomes are evaluated in the context of CNS abnormalities within the cortical, pyramidal and extrapyramidal tract as indicated by mental status symptoms/ motor/reflex abnormalities/ clinical encephalopathy (for cortical dysfunction), abnormal tone and gait and upper motor neuron distribution weakness (for pyramidal tract dysfunction), and 3 cardinal signs of Parkinson’s disease including abnormal gait and abnormal

Historic cumulative chlorpyrifos exposure estimates (from the time of initial employment to baseline examination) were established from industrial hygiene records and estimates of air levels based on historical personal air sampling data. Interim cumulative chlorpyrifos exposure (during the year between baseline and second examinations) was also determined. Biological indicators of exposure to chlorpyrifos was assessed via: analysis of the urinary excretion of 3,5,6 trichloro-2-pyridinol (TCP – a chlorpyrifos metabolite, reported as a weighted average of 4 overnight collections/creatinine concentration); plasma butyrylcholinesteras

Potential effects of employment in the chemical industry were controlled for via use of saran manufacturing referents; a general medical examination was performed to detect medical conditions that potentially produce nervous system abnormalities; acute OP poisoning

The following tests were used for comparing relevant clinical results for the chlorpyrifos group and the referent group at baseline and second examinations: t test for differences in means; Fischer exact test, or chi-squared test for association; a P value > 0.05 was considered significant. The authors did not adjust for multiple comparisons


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questionnaire and examination were used to develop specific ratings of the presence or absence of dysfunction within different areas of the CNS (cortical, pyramidal tract and extrapyramidal)

posture/postural instability (for extra-pyramidal tract dysfunction). NB. The cortical motor/reflex rating was not used to identify peripheral nervous system abnormalities but to identify possible cortical or upper motor neuron dysfunction

e activity (BuChE – monthly activities were assessed and averaged during same the intervals); and red blood cell cholinesterase activity (AChE – measured at baseline and at the second examination). Information on non-occupational exposures to insecticides and occupational and environmental histories (chemical exposures at home and work) were obtained via interview questionnaires. NB. Investigators were blinded to an individual subject’s group membership, and exposure histories

Albers 2004a US 53 of 66 eligible male and female chlorpyrifos manufacturing workers (mean age 41.2 years, SD 7.5), employed on 1 September 1999 in buildings involved in chlorpyrifos production). Subjects had

60 of 74 eligible male and female workers involved in manufacturing Saran (clear plastic film wrapping material) (mean age 41.3 years, SD 8.4). Referents were randomly sampled from those employed on 1

A standardised questionnaire was used to collect information on demographics, chemical exposures at home and work, family history of neurological diseases, personal medical history including medications, and social history including alcohol use and non-occupational exposure to insecticides. Neurological

Neuropathy. Subclinical or clinically evident neuropathy was defined using symptoms, signs and NCS results. ‘Probable’ neuropathy was defined as the presence of abnormalities consistent with a

Ambient chlorpyrifos exposure estimates were derived from industrial hygiene measurements. Interim cumulative chlorpyrifos exposure estimates (i.e. the period during baseline and follow-up) were derived from

Age, sex, height, weight, BMI (body mass index), skin temperature over nerve being tested; smoking in pack-years, socio-economic status, education level, anxiety were evaluated as

For NCS, data were inspected to determine whether transformation was necessary (to make them more normally distributed). Conduction velocity scores were transformed so


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known and measurable exposure to chlorpyrifos, but no occupational exposure to other OPs or other neurotoxicants. Baseline examination occurred during a period of potential chlorpyrifos exposure. 1-year evaluations were completed on all 53 subjects evaluated at baseline

September 1999 in the saran production building and had no occupational exposure to chlorpyrifos or to suspected neurotoxicants. 1-year evaluations were completed on 58 of the 60 referents evaluated at baseline

evaluations were performed by a neurologist to determine sensory (loss of feeling, paraesthesias, numbness or tingling, pins and needles sensation, imbalance) and motor symptoms (weakness), and identify neurological abnormalities (signs). Peripheral nervous system examination comprised of strength, station, gait, muscle stretch reflex and sensation evaluations. NB. Subjects were subjected to general medical examinations to detect medical conditions that potentially produce nervous system abnormalities. Nerve conduction studies (NCS) were performed on the dominant side of sensory and motor nerves. For the sensory nerves (median, ulnar and sural) sensory negative peak amplitude and onset latency were recorded, while for motor nerves (median and peroneal) the motor response amplitude, onset latency and F wave latency were recorded. Limb temperatures (palm and leg) were monitored and warmed when necessary to maintain temperatures above 32oC (palm) and 31oC (foot). Abnormalities were based on published values using comparable techniques.

sensory or sensorimotor neuropathy in at least two of the following three categories: (i) symptoms (includes reports of persistent or intermittent and symmetrical stocking or stocking-glove distribution numbness, tingling or sensory loss), (ii) peripheral sensation or (iii) decreased ankle reflexes (trace or absent). Subjects categorised with ‘possible’ neuropathy had a single appropriate abnormality among symptoms or reflexes. ‘Confirmed’ neuropathy required a diagnosis of possible or probable neuropathy and abnormal electrodiagnostic testing, which consisted of at least one abnormal NCS measure in two peripheral nerves.

industrial hygiene records. Historic cumulative chlorpyrifos exposure was estimated from the time of initial employment to the baseline examination (estimates of airborne levels based on historical personal air sampling data were also used). Biological chlorpyrifos exposure was assessed during the interim period and included analyses of: (i) the urinary excretion of the chlorpyrifos metabolite 3,5,6 trichloro-2-pyridinol (TCP) which was reported as an average of 4 overnight collections (and provided an estimate of daily chlorpyrifos exposure); (ii) monthly plasma butyrylcholinesterase (BuChE) activity; (iii) and red blood cell AChE activity both at baseline and the one year examination.

potential confounders; Medical conditions that could potentially produce nervous system abnormalities were also accounted for

that a higher value indicated faster conduction. Authors compared clinical, laboratory and nerve conduction study results (via t test for difference in means (p values given). Fisher’s exact and χ² tests were used to compare frequency count data and neurological symptoms, p values given. Logistic regression was used to calculate ORs for chlorpyrifos workers developing clinical signs or any diagnosable level of peripheral neuropathy at either examination compared to referent group at baseline. Mixed regression models were used to account for repeated measures on


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Summary amplitude and conduction velocity Z scores were calculated for motor and sensory nerves using NCS results obtained from Saran subjects

Subjects without symptoms or signs of neuropathy who had at least one abnormal NCS measure in two peripheral nerves were categorised as having subclinical neuropathy

Questionnaires were also used to determine chemical exposures at home and at work and possible non-occupational exposure to insecticides

individuals over time. Models (used to evaluate the interaction between group and time to determine whether chlorpyrifos exposure was associated with changes in NCS results over time) included the main effects of group and time and an interaction term

Albers 2007 US 53 male and female chlorpyrifos manufacturing workers (mean age 41.2 years, SD 7.5), employed on 1 September 1999 in buildings involved in chlorpyrifos production). Subjects had known and measurable exposure to chlorpyrifos, but no occupational exposure to other OPs or other neurotoxicants. Baseline examination occurred during a period of potential chlorpyrifos exposure.

60 male and female workers involved in manufacturing Saran (clear plastic film wrapping material) (mean age 41.3 years, SD 8.4) randomly sampled from those employed on 1 September 1999 in the saran production building). Referents had no occupational exposure to chlorpyrifos or to suspected neurotoxicants. 1-year evaluations were completed on 58 of the 60 referents

Nerve conduction studies (NCS) were performed on the dominant median, ulnar, and sural sensory nerves and the median and peritoneal motor nerves using conventional electrodiagnostic medicine techniques. For sensory nerves, antidromic sensory nerve action potential amplitude (baseline to negative peak), onset latency and negative peak latency were recorded. For motor nerves, compound muscle action potential amplitude (baseline to negative peak), onset latency and fastest F wave latency were recorded. Limb temperatures (palm and leg) were monitored and warmed when necessary to maintain temperatures above 32oC

Subclinical adverse effects indicative of peripheral neuropathy

Ambient chlorpyrifos exposure was estimated using air monitoring data and direct biological measurements. Industrial hygiene records were used to establish estimates of chlorpyrifos exposure during the year between the baseline and 1 year examinations (i.e. interim chlorpyrifos exposure) and chlorpyrifos exposure from the time of initial employment to the baseline examination (i.e. historic chlorpyrifos exposure). A

Age, sex, height, weight, BMI (body mass index), skin temperature over nerve being tested; smoking in pack-years, alcohol use (drinks/day), alcohol related behaviours, socio-economic status ( via estimated general ability), education level and anxiety were evaluated as potential confounders. The authors also screened for existing medical conditions that could account for

Initial analysis included comparison of results relevant to the evaluation of peripheral neuropathy at the baseline and the 1-year examinations. All analyses used linear mixed models with repeated measures to account for the longitudinal nature of the data. Each NCS variable was modelled as a function of interim and historic chlorpyrifos exposure, age


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1-year evaluations were completed on all 53 subjects evaluated at baseline. NB. The authors noted that the although subjects comprised a healthy group of workers an inclusive selection process was used to ensure that potentially vulnerable individuals were not excluded

evaluated at baseline

(palm) and 31oC (foot). Summary Z scores were calculated for motor and sensory nerves using referent subject baseline results. Amplitude Z scores used distal amplitude results. Conduction velocity Z scores used distal latency, terminal and extremity conduction velocity (which transformed latency measures so that a higher value reflected better performance). Mean baseline NCS results were within normal limits

mathematical formula was used to calculate historical and interim chlorpyrifos exposure estimates (based on historical personal air sampling data and geometric mean exposure levels calculations). Biological assessment of interim chlorpyrifos exposure was also performed using urine TCP metabolite of chlorpyrifos (reported as a weighted average of four overnight collections obtained during the year, and expressed as interim TCP/creatinine (Cr) (µg/g)). Plasma BuChE activity and red blood cell AChE activity were also measured, the results of which showed appreciable inhibition of B-esterases below the range where a physiologic effect on AChE exists

neuropathy and time, with additional covariates also considered. Regressions were performed using interim chlorpyrifos exposure and repeated using interim TCP/Cr. Akaike Information Criterion (AIC) was used to select the best model for each NCS variable (based on the lowest AIC). Models were subsequently created that allowed the effect of historic chlorpyrifos exposure on the NCS variable to be different among the following exposure groups: referents, chlorpyrifos subjects with historic chlorpyrifos exposure that was either ≤ 20 mg/m3 x days or > 20 mg/m3 days. Authors reported


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parameter estimates, which indicate the change in a nerve conduction measure per unit change (e.g. 1 mg/m3 x days) in chlorpyrifos exposure; 95% CI and p values reported

Starks 2012b US 678 male participants (mean age 61 years, SD 11.6) in the Agricultural Health Study (AHS), who were licensed pesticide applicators from Iowa and North Carolina. Subjects were sourced from private applicators enrolled between 1993-1997 who completed all AHS questionnaires (two self-administered that were completed as part of Phase 1; and a further two telephone based questionnaires completed in a 5 year follow-up as part of Phases 2 and 3). The sample was limited to applicators who had reported

No reference group was used because the authors considered that comparing pesticide applicators to non-applicators would preclude determination of whether specific OPs contributed to adverse PNS function

Testing was conducted in Iowa and NC between (Nov 2006- Mar 2007) and (Jan – Mar 2008) respectively. Neurological clinical examinations were performed by a blinded physician and followed standard protocols for vibration perception (128Hz) and proprioception of the great toes, Achilles deep tendon reflexes (bilaterally performed), Romberg test, tandem gait and postural tremor (unilaterally performed). Results were recorded as being normal, equivocal or abnormal. Abnormal results for tests performed bilaterally were defined on the basis of the absence of normality on one or both sides. For tests performed unilaterally an abnormal result was anything that was not normal (includes equivocal). Hand strength i.e. gross grip strength and key and

Neurological dysfunction

Data obtained from (i) Phase 1 self-administered questionnaires; (ii) Phase 2/3 telephone interviews performed in the 5 year follow-up; and (iii) Questionnaire administered on the day of neurological testing to ascertain pesticide use information on the past 12 months. These were used to create the following exposure variables for the 16 OP pesticides and four carbamate pesticides used by at least 50 neurological study participants: (a) ever use of any pesticide, and (b) cumulative lifetime days use (summary measure for lifetime

Potential confounders explored include age, height, state, smoking status, education, alcohol use, ear infection/ surgery, neurotoxic substance exposures and BMI. Subjects were excluded if they had medical conditions and treatments known to confound tests (e.g. diabetes, amyotrophic lateral sclerosis, multiple sclerosis, polio, cancer chemotherapy, dementia, brain tumour), drank ≥ 42 alcoholic beverages per

Logistic regression was used to estimate odd ratios of association between pesticide use and dichotomised test results. Exposure response was examined by creating a three-level variable for individual pesticides with the distribution of lifetime days of use split at the median among the pesticide users to create two exposure categories (< = median, and > median) with never-use as the referent category. Chi-squared tests for


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having higher lifetime use of 10 of 16 OP pesticides. NB. After exclusions, a total of 1807 AHS participants were initially eligible, and the final study participants thereby yields a 39% participation rate. NB. Among the 701 subjects who participated a further 23 were excluded

palmar pinch strength were assessed bilaterally using digital grip and pinch dynamometers. A total of six tests were performed and a mean z-score was calculated. Sway speed was measured using force plate stable platform with eyes open and closed. Average sway speed (nm/s) of four trials were reported separately for each condition. Vibrotactile threshold (log microns) of the great toe was measured bilaterally at 120 Hz under standard protocols using a vibration sensitivity device (Vibratron II) Electrophysiological testing of the dominant peroneal motor nerve were performed under standard procedures by one examiner. Parameters measured were distal motor amplitude (mV), distal and proximal motor latency (ms) and short F-wave latency (ms). Nerve conduction velocity was also calculated (m/s).

days use for (i) each pesticide or (ii) for any OP pesticides (iii) for any pesticide, (iv) having experienced an high pesticide exposure event. NB. Lifetime days use was created by multiplying no of days used per year by number of years. Information on whether subjects experienced a high pesticide exposure event (HPEE) was also collected

week, and had a physician-diagnosis of pesticide poisoning The authors also investigated potential confounding of the association between neurological outcomes and each pesticide by other pesticides

trend were used for exposure/dose response models with exposure levels assigned 0 (non-exposed), 1 (≤ median) and 2 (2 median). Analyses were restricted to pesticides with at least five exposed cases per category. Linear regression was used to examine associations between pesticide use and the continuous outcomes. Cumulative lifetime days of pesticide use variables were log10 transformed. For interpretation reasons, parameter estimates for distal motor/short F-wave latencies, sway speed and vibrotactile threshold were multiplied by -1 so that lower scores indicated poorer


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performance for all continuous outcomes. Confounding by multiple pesticides were assessed in both linear and logistic regression models for pesticide pairs with r ≥ 0.30

6.B.2. Case-control studies Crawford 2008 US

4926 white male applicators (median age 54 years) taken from 14229 eligible applicators enrolled in the Agricultural Health Study in 1993-1997 in Iowa and North Carolina. These were those who completed two questionnaires at enrolment and the 5 year follow-up interview and answered “YES” to questions about having hearing loss and also reported ever personally using pesticides. NB. From the 52393 applicators (82% of those eligible) who completed the first questionnaire

9303 eligible white male applicators (median age 45 taken from 14229 eligible applicators who completed two questionnaires at enrolment and the 5 year follow-up interviewandanswered “NO” to the question: “Do you have trouble hearing in one or both ears (without a hearing aid)?”

The authors conducted a 5 year follow-up telephone interview to collect information on self-reports of hearing loss, head injury requiring medical attention and hypertension. Subjects were asked: “Do you have trouble hearing in one or both ears (without a hearing aid)?”

Hearing loss The authors used questionnaires to collect information on frequency (days of use per year) and duration (years of use) of pesticide use. (enrolment and applicator questionnaire). Questionnaires were also used to obtain information on pesticide related poisoning events and subsequent medical attention and diagnosis of pesticide poisoning. Responses were dichotomised as ever/never. Pesticides were subdivided according to function (i.e. herbicides,

Subjects were excluded if they reported never using pesticides or whose hearing loss was attributable to a genital condition, or infection/injury, or if they were non-white or female (and due to few numbers). Following confounders were adjusted for: state, age, and exposure to noise (i.e. from grinding animal feed/metal, working in swine areas, driving gasoline tractors but no account of firearm use), solvents (i.e.

Logistic regression models were constructed using hearing loss as the dependent variable. Odd ratios and 95% CIs are reported. P values (two-sided) for trend for cumulative days of pesticide use were calculated, where applicable using a continuous variable defined by the midpoints of the levels of the categorical variable. Two-stage hierarchical logistic regression was used to increase


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at enrolment, 22915 (44%) completed the second applicator questionnaire, of which 16246 (73%) completed the 5 year telephone interview prior to further exclusions. The authors noted that non-participation in the telephone interview was largely due to subjects being non-contactable (12%) and refusals (10%) or death (4%). However, differences were observed between those who participated and those who did not and were adjusted for

insecticides, fungicides and fumigants), chemical type (i.e. insecticides were categorised as organophosphates, organochlorines or carbamates) and specific pesticides e.g. permethrin. The authors calculated cumulative days of use for each pesticide class by multiplying duration by frequency for each pesticide class, summing over all pesticides in the class and then categorising exposed subjects in tertiles in which those reporting zero exposure were used as the reference category. This was done for pesticide classes where at least 40% of the study population was exposed (which included organophosphates). Other approaches were used for classes were less than 40% of subjects were exposed (which did

gasoline, paint, solvent poisoning, other non-farm job solvents) and metals (i.e. non-farm job exposures to lead solder, lead mercury, cadmium, and other metals). The authors noted that education, personal protective equipment use, diagnosis of high blood pressure, smoking, head trauma and alcohol did not confound the relationship of hearing loss with cumulative pesticide exposure/ OP exposure. Due to there being limited information on lifetime use of noisy farm equipment the authors created a noise variable based on applicators current use of noise equipment

precision when evaluating multiple individual pesticides. Interactions between OP exposure or diagnosis of pesticide poisoning + (age, smoking, metal, noise or solvent exposures) were investigated by stratified analysis and further testing of models (p<0.15 for significance)


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not include organophosphates)

Jamal 2001 UK [Group 1]: 15 male and one female OP exposed farmers (mean age, 44.5 years, SD 11.4; range 23-67 years) regularly involved in sheep dipping (for at least 4 years using OP compounds i.e. diazinon, propetamphos, or chlorfenvinphos) were selected from the OPIN database of 200 sheep farmers. The authors reported that the farmers claim to have used these pesticides prior to the development of long term ill health. Subjects had ≥ 3 episodes of mild-moderate acute occupational OP poisoning, and other causes for their reported chronic illness had been excluded by doctors. Subjects were selected by random sampling from the Organophosphate Information Network (OPIN)

16 healthy age/sex-matched controls of mixed office-based occupations (mean age 44.9; SD 11.7; range 24-70). Controls were matched to farmer group 1 on an individual basis

[Symptoms]: Clinical symptoms (i.e. fatigability, weakness, paraesthesiae – sensation of tingling, prickling or numbness, spontaneous sensation of burning heat and cold, and pain) were scored by examiner as being either present (1) or absent (0). Scores were summated to give an overall range of 0-6. NB. The authors did not describe how symptom data were obtained. [Reflexes]: Scoring of five tendon reflexes (knee, ankle, supinator, biceps and triceps) on the right side, graded as being either normal (0), diminished (1) or absent (2) and summated to provide an overall score of 0-10 per subject. [Muscle power]: Assessment of power in the distal, intermediate and proximal groups of muscles in the right upper and lower limbs using an inverted MRC scale criteria, with normal score (0) and complete paralysis (5). All muscle scores were combined to an overall score ranging from 0 to 30. [Sensory examination]: Sensations of pin prick, vibration, touch and position on the right side were separately scored with

Peripheral and central nervous system abnormalities

Biochemical markers of recent OP exposure: (i) blood AChE and serum cholinesterase activity were determined by published methods. The authors noted that detailed exposure levels/history were not available

Age, excessive alcohol intake, occupation, taking medication with neurotoxic effects. The authors noted that other potential causes of PN were screened for and excluded (no further information was provided)

ANOVA was used to assess the presence of statistical differences between groups for parameters that were normally distributed (or Kruskal-Wallis ANOVA by rank otherwise). 95% CIs of mean difference between each farmer group and control was calculated for parameters showing a significant ANOVA test; pooled standard error from all groups was used. The mean for farmer group was considered different from the control mean wherever confidence interval did not include zero. p-value was calculated using a double-sided student t-test for parameters with


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database of 200 sheep farmers. [Group 2]: 16 asymptomatic male OP exposed farmers (mean age 42.3 years, SD 11.2; range 25-62 years) regularly involved in dipping sheep (for at least 4 years using OP compounds i.e. diazinon, propetamphos, or clorfenvinphos) with no history of symptoms of acute poisoning. Subjects were selected at random from an area covering the west of Scotland within 60 mile radius of Glasgow. 129 calls were made and 39 sheep farmers were contacted and 13 subjects did not participate (due to either refusal or the date being inconvenient). NB. In both groups the time since last exposure to sheep dip was in excess of four months; no subject had a previous history of exposure to other

normal (0), reduced below ankle (2) to reduced below elbow and knee (4). Combination of sensation scores into a single score ranged from 0 to 16 per subject. [Neurophysiological techniques]: included motor (and sensory) nerve conduction studies performed in the right median and common peroneal (or sural) nerves. The tests measured distal motor latencies, motor nerve conduction velocities, muscle action potential and sensory amplitudes and F wave and sensory latencies. Needle electromyography (EMG) was performed on the anterior and extensor digitorum the brevis muscles, and results were scored as from 0 (normal), up to 2 (> 50% polyphasia and/or frequent spontaneous activity). EMG scores for the two muscles were combined to a range from 0 to 4. [Quantitative sensory testing]: involved measuring thermal thresholds on the dorsum of the right foot and vibration perception threshold over the right first metatarsal bone. Multi-modality evoked potential (EP) were performed to examine EP latencies of pattern reversal

normal distribution (or Mann-Whitney for otherwise)


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toxic chemicals

(visual), brainstem auditory, and somatosensory nerves. Tests were performed by blinded investigators

Jamal 2002a UK 23 male and female sheep farmers/dippers (mean age 46.1 years, SD 11.0, range 22-62) exposed to OPs with definite or probable neuropathy. Neuropathy status was determined from the outcome of neuropathy scores derived from responses to the neuropathy symptoms questionnaire and sensory QST tests (first conducted during a previous second phase cross-sectional field study but repeated in this third phase hospital clinic study). An intermediate group comprising 34 male and female farmers (mean age 46.2 years, SD 10.6, range 30-64) were included who were categorised with possible neuropathy. NB.

15 male and female farmers (mean age 38.8 years, SD 13.3, range 20-66) exposed to OPs but almost certainly without neuropathy in third phase study

A neuropathy/ neurological questionnaire (based on the Mayo Clinic neurological symptom questionnaire and performed by a neurologist) focussed on symptoms occurring in the upper and lower limbs to detect possible chronic neurological effects, which may be associated with exposure to OPs. Questions on cranial nerve involvement were excluded. This was accompanied by a clinical assessment (based on Mayo Clinic recommendations, and performed by a neurologist), which assessed the presence of neurological signs (reflexes, sensation and muscle power) in both the upper and lower limbs. Muscle power was assessed by applying an inverse MRC scale, where a score of zero signifies normality and a higher score signifies increasing abnormality. Subjects were scored as having abnormal reflexes with the absence or reduction of certain reflexes, abnormal sensory signs if ≥ 2 modes of sensation were affected in the classic stocking-glove

Peripheral neuropathy (and neuropsychological deficits)

Questionnaire was used to collect data on recent exposure. Mean cumulative exposure was based on an exposure index (OPEXP) derived from the second phase study that utilised an exposure history assessment questionnaire and an occupational hygiene study. NB. OPEXP represents the sum of the concentration of urinary OP metabolites DEP (diethylphosphate) and DETP (diethylthiophosphate) (in units of nmol/mmol of creatinine) across dipping days, and is also the weighted sum of cumulative exposure to both concentrate (CONC) and dilute dip splash (SPLASH). Pilkington et al 2001. Occup Environ Med;58:702–710

Gender, age, alcohol consumption, inherent neuropsychological, characteristics, were considered. Data for QST scores were based on age dependent thresholds; the following used as exclusion criteria: taking medication e.g. for hypertension, diagnosed with rheumatoid arthritis, family history of high foot arches, carpal tunnel syndrome and radioculopathy

The authors calculated 95% confidence limits of normality for motor sensory latency, nerve conduction velocity, and potential amplitude to help set more severe criteria for neuropathy and increase the specificity. Decision criteria for overall abnormality were subsequently established. The number/proportion of subjects with neurological and/or neurophysiological deficits is reported. For neurological outcomes, subjects were considered to have significant signs if they had at least two of three abnormal signs. For nerve conduction studies, subjects


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685 subjects from the field study were invited to take part in this clinical study. However, of the 201 were deemed eligible only 95 responded to the questionnaire, 79 of which also attended the clinic (later amended to 72 due to exclusions)

pattern, or abnormal muscle power if there was any weakness in the distal part of the lower limb. Nerve conduction studies were carried out by a neurophysiologist using standard techniques on lower limbs of the right hand side (normally). Latency, peak-to-peak muscle action potential amplitude, nerve conduction velocity, F-wave latency and persistence were measured in motor conduction studies of median and common peroneal nerves. Peak latency, peak-to-peak amplitude, and nerve conduction velocity were measured in sensory studies of median and sural nerves. Subjects were scored as having sensory abnormalities if they scored both abnormal sural potential latency and amplitude. Motor function in the lower limb (peroneal nerve) was scored as abnormal if at least two out of three action potential variables (latency, amplitude, or conduction velocity) were abnormal. The authors considered subjects with either neurological signs or abnormal nerve conduction to have clinical neuropathy. Electromyography (EMG) and single fibre EMG

were considered to have significant nerve conduction deficits if either sensory function or motor function in the lower limb was abnormal


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(SFEMG) were conducted by a neurophysiologist. The EMG was recorded in the right extensor digitorum brevis, tibialis anterior, and extensor digiti communis. Each muscle was scored such that 0, indicated < 15% polyphasia and no spontaneous activity, up to a maximum score of 2, if the polyphasia > 35% with profuse spontaneous activity. SFEMG studies were performed on the right extensor digitorum communis muscle to investigate the integrity of the neuromuscular junction. The mean values from 10 measurements were recorded. Quantitative sensory tests (QSTs) were conducted by a neuroscientist and involved measuring hot and cold thresholds from the dorsum of the foot (to test for small peripheral nerve fibre function) and vibration threshold over the middle of the index metacarpal bone and first metatarsal bone (to test the large peripheral nerve fibre function). QST thresholds were assessed for abnormalities by comparing with age-dependent clinical reference values, which were calculated from 68 healthy volunteers (for thermal sensory tests) or from a relevant study of 100


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subjects (for vibration threshold tests). The authors define small fibre abnormality as abnormal hot or cold thresholds, and large fibre abnormality as abnormal vibration sensation threshold or abnormal sural nerve conduction. Neurophysiological sensory abnormalities were defined as abnormal sural conduction and one or more QST value(s). Tests were conducted on the right side of the body (unless otherwise contraindicated by right-side injury) and performed during standard pesticide use (i.e. between January 1998 and end May 1998). NB. The research team were blinded to subjects, OP exposure, neuropathy classification status and occupation; five different investigators performed different groups of tests; a nurse accompanied each subject to ensure no exposure information was passed to the clinical team

6.B.3. Cross-sectional studies Abdel-Rasoul 2008 Egypt

50 of 56 eligible male children aged between 9 and 18 years from five of 50 randomly chosen villages in the Shebin Elkom

50 children who never worked in the cotton fields selected from friends and relatives of the applicator children,

A detailed clinical medical examination and a complete neurological examination was administered by specialists. No further information was provided. Various tests

Neurobehavioural impairments and various neurological symptoms, which included numbness

A questionnaire was used (completed during the last week of the spraying season) to collect data on work history and

Age, education and BMI (wrt analyses of neurobehavioural outcomes)

The t-test was used to examine the differences between means. The χ2 test was used to examine the differences


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District hired by the local agricultural office to work as seasonal workers i.e. to spray pesticides on cotton crop in cotton fields via backpack applicators under the supervision of adult engineers and agricultural employees. Children were subdivided into two applicator groups: Younger (9-15 years of age, n=30); and Older (16-18 years of age, n=20). NB. Data relevant to the Older group is presented in this summary. The authors noted that pesticide mixtures were first prepared by the local agricultural office before being loaded onto the backpack sprayers. Reloading occurred multiple times/ day until the spraying is completed. Personal protective equipment were

living in the same community and attending the same schools were matched on age and education. Children were subdivided into two groups: Younger (9-15 years of age, n=30); and Older (16-18 years of age, n=20). NB. Data relevant to the Older group is presented in this summary

were also conducted to assess neurobehavioral function (which comprised the main health outcome of this study). A questionnaire was used (completed during the last week of the spraying season) to collect data on subjects medical history

exposure to pesticides. Serum or plasma AChE was determined to measure recent acute exposure to OP pesticides. The authors noted that pesticide application for the cotton crop are highly regulated in Egypt and specific guidelines are followed. Cotton fields are subject to 4 application cycles per season with each application lasting 5-11 days. OP pesticides (chlorpyrifos) are applied during the 2nd and 4th cycles from the end of June through to the 1st week of July and during the second week of August. NB. Non-OP pesticide treatments are used in the 1st and 2nd cycles. The amount of pesticide applied varies each day depending on the size of the field/ degree of pest infestation

between percentages. NB. Pearson’s correlation, Holm’s correction of p-values, and multiple linear regression were used in relation to neurobehavioural analyses


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not commonly used by the applicators

Albers 2004c US 53 of 66 eligible mostly male Dow Chemical Company employees (aged 18-65 years) involved in manufacturing chlorpyrifos (i.e. chlorpyrifos production and product formulation/ packaging)

60 of 74 eligible mostly male randomly chosen Dow Chemical Company employees (aged 18-65 years) involved in manufacturing saran (i.e. clear plastic-film wrapping material) with no current occupational exposure to chlorpyrifos or other neurotoxicants. NB. The authors noted that a few referents had occasionally worked in chlorpyrifos-exposed jobs in past years

Neurological evaluations were performed by a board-certified neurologist via a clinical interview to identify symptoms of neuropathy (e.g. loss of feeling, paraesthesias, numbness or tingling, imbalance or weakness). A neurological examination was also conducted to identify clinically evident signs of neuropathy, which included evaluation of: selected cranial nerve function, strength (including bilateral intrinsic hand and foot muscles), station, gait, co-ordination, alternate-motion rate, presence of abnormal movements (tremor, fasciculations), sensation (dual simultaneous stimulation, von Frey monofilament at index finger and great toe, joint position sensation at great toe, pin-pain at the index finger and great toes, vibration at index finger and great toe), muscle stretch reflexes (biceps brachii, brachioradialis, quadriceps, and gastrocnemius-soleus), and documentation of abnormal corticospinal tract signs. Nerve conduction studies were performed on the dominant side sensory

Subclinical or clinically evident peripheral neuropathy. This was defined using a combination of abnormalities from the categories of symptoms, signs and nerve conduction testing. The authors defined “Probable” clinical neuropathy as the presence of abnormalities consistent with a sensory or sensorimotor neuropathy in at least two of three categories: symptoms (i.e. reports of persistent/ intermittent and symmetrical stocking or stocking-glove distribution numbness, tingling, or sensory loss; peripheral sensation (i.e. evidence of symmetrical stocking or

Potential neurotoxic exposures were assessed by systematic review of the chemicals used, standard work procedures and historical industrial hygiene records, including personal air sampling data. Ambient chlorpyrifos exposure estimates were derived from industrial hygiene measurements for similarly exposed groups of workers. Historical cumulative chlorpyrifos exposure estimates (from the time of initial employment to the baseline examination) were derived from geometric mean exposure levels that were calculated for each of the above exposed groups. Biological exposure assessment included measurement of: urinary overnight excretion of the

Age, gender, anthropometric features, alcohol use, smoking history, medication use, and systemic illness such as diabetes mellitus were considered

Univariate descriptive statistics were examined for each data element to determine the mean, SD, range, and shape of the distribution. The authors compared clinical, laboratory, and electrophysiological results relevant to the evaluation of peripheral neuropathy for the chlorpyrifos group and the referent group (t-test for difference in means, Fisher exact test, or chi-square test for associations)


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(median, ulnar, and sural) and motor (median and peroneal) nerves. Antidromic sensory conduction studies recorded the following variables from digit II, V and the ankle: sensory response amplitude (baseline to negative potential peak), onset and negative peak latency. Motor conduction studies recorded the following variables from the abductor pollics brevis and extensor digitorum brevis muscles: motor response amplitude (baseline to negative peak), onset latency for the wrist, below-elbow and ankle stimulation sites, and F-wave latency for antidromic stimulation at the wrist and ankle. A median forearm conduction velocity was recorded. The authors noted that abnormalities were defined based on previously used values. Also, limb temperature (palm and leg) were recorded, monitored and maintained during the evaluation. Z-scores were calculated for motor and sensory nerves using nerve conduction results of referent subjects. Distal amplitude measures were used to calculate summary amplitude Z scores, while conduction velocity Z scores were calculated

stocking-glove sensory loss among tests of pin-pain, vibration, joint position, fine touch, or touch-pressure sensation); or decreased ankle reflexes (i.e. trace or absent). The authors defined “Possible” clinical neuropathy in subjects who had a single abnormality among symptoms, sensation, or reflexes. The authors defined “Confirmed” clinical neuropathy in subjects who had a diagnosis of possible or probable clinical neuropathy and abnormal electrodiagnostic testing (i.e. who had at least one abnormal nerve conduction measure in two peripheral nerves). The authors defined “subclinical” neuropathy in

chlorpyrifos metabolite 3,5,6-trichloro-2-pyridinol (TCP) (collected during the study period and normal operations, and standardised to urinary creatine (Cr) excretion i.e. TCP/Cr (µg TCP/g Cr)); monthly plasma butyrylcholinesterase (BuChE) activity (milliunits per ml) (averaged over the 4-month study period); and red blood cell cholinesterase (RBC AChE) (obtained on the day of the examination)


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using distal latency, terminal conduction velocity (for sensory nerves) and extremity conduction information after transforming latency measures (negative values), so that a higher value always indicated faster conduction. NB. Investigators examined workers on a given day without any indication to which group the subject belonged. Examinations took place during a 2-month period (during a period of potential chlorpyrifos exposure). Participants were also interviewed to collect relevant demographic, medical history and exposure data (including info on non-occupational exposure to insecticides)

subjects who had no symptoms or signs of neuropathy but did have at least one abnormal nerve conduction measure in two peripheral nerves. Isolated nerve conduction abnormalities were identified based on predefined definitions of abnormality

Bazylewicz Walczak

1999 Poland 26 female greenhouse workers (mean age 35.4 years, SD 7.9) performing standard gardening/ planting jobs employed at 3 large gardening enterprises. NB. The authors noted that exposure to OPs in the greenhouses was seasonal and cyclic, occurring in the first half of the

25 matched female canteen, kitchen and admin workers (mean age 36.0 years, SD 8.6) employed at 3 large gardening enterprises

Note: Peripheral neurological abnormalities compromised a minor part of a subjective symptom questionnaire, which itself was a component of a battery of tests used in the psychological assessment. The Finnish Subjective Symptoms Questionnaire – one of two questionnaires used in the Polish adaption of the Neurobehavioural Core Test Battery for psychological assessment performed between 9am and 2pm at the following

Neurobehavioural effects (primarily), which included assessment of peripheral neurological symptoms.

Exposure levels were assessed during March to May (the period of intensive spraying) via environmental tests i.e. measurements of ambient air exposures at the workplace and contamination of skin and clothes. Air sampling was carried out via the use of bacterial aspirators in an

Subjects were matched for age, education level (recognised confounders for neuropsychological testing) and place of habitation

Two-Way ANOVA was used to identify OP exposure effects dictated by (i) a single spraying season, or (ii) long term effects of exposure in the exposed vs. control group, or (iii) interaction between short and long term effects of exposure. Mean


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year and were dependent on the frequency of spraying in the individual cultures [sic]. Workers were restricted from entering the sprayed area for a period of 1-3 days

periods: Jan – Feb (the period preceding intensive application of pesticides in greenhouses); and June (the period following major spraying operations in greenhouses). NB. The authors noted that the period of intensive spraying was during March to May. The questionnaire collected information on the following peripheral neurological symptoms: paresthesia in limbs and hands (sensory) and limb weakness (motor) and possibly cranial nerve effects i.e. vertigo, sense of smell, taste, facial numbness, facial muscle changes/sensation

area of a greenhouse at 5 measuring sites during 4 random periods of the working day. Pesticide levels on clothing were determined on α-cellulose pads placed on garments, while levels on the skin were determined from skin washes. Samples were collected at the end of the working day and pesticide levels of all samples were determined via gas chromatography. Exposure assessment was conducted via Durhan & Welfe method (which takes into account both dermal and respiratory absorption routes). The most frequently used OPs were Dichlorvos, Methamidophos, Methidathion, Pirimiphos-Methyl), however additional exposure to carbamates, synthetic pyrethroids and dithiocarbamates also occurred

values and SDs for the NCTB tests were reported. Significance level α=10 used to determine neurobehavioural effects


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Farahat 2003 Egypt 52 of 64 eligible

male agriculture engineers (who worked in the fields and were responsible for the entire application procedure), and mixers and mechanics (who repaired spraying devices), with a mean age of 43.63 years (SD 5.51) working in the pesticide application departments at Berket El-Sabe district, Menoufiya Governate, Egypt. NB. Reasons for non-participation were due to exclusion or refusal. Subjects were required to remain in the field during the spraying season and worked six days per week during the period between June and September. Most participants reported never using protective clothing (88%) and none reported acute poisoning incidents requiring hospitalisation

50 of 63 eligible male clerks and administrators, mean age of 42.48 years (SD 5.54) never occupationally exposed to pesticides recruited from different departments of the Ministry of Agriculture

Note: Participants were examined during workdays during their break between 12-3 pm (a typical workday being from 8am -12pm and 3-7pm). Assessments included a medical history questionnaire and clinical examination, performed along with specific neurological tests for sensory and motor functions of: cranial nerves (for example, sense of smell, visual acuity, visual fields, pupillary reactions, extraocular movements, corneal reflexes, hearing and gag reflex - as categorised by authors); motor system (includes muscle status, muscle tone, muscle power, and coordination); reflexes (e.g. knee and ankle reflexes and plantar response); sensory system (i.e. superficial sensations, e.g. pain and touch), deep sensations (i.e. vibration), position and discriminate sensation (e.g. stereognosis, number identification, and two point discrimination). No further information was provided. A series of neurobehavioural tests were also administered

Neurobehavioural effects (that included sensory and motor neurological outcomes)

Occupational history questionnaire. Analysis of serum AChE and liver and kidney function (from blood samples taken from 45/52 exposed and 37/50 unexposed participants) was done after completion of questionnaire, clinical examination and neurobehavioural tests. The authors noted that the latter two tests were done to exclude participants with renal or kidney disease (higher risk of encephalopathy)

The authors screened for effects of age, education BMI, smoking, alcohol consumption and medical history. Subjects were excluded if they reported being seasonal workers; had less than 12 years education; had any of the following medical diagnosis: diabetes mellitus, liver or kidney disease, peripheral neuropathy, vitamin deficiency, anaemia and addiction; had long term treatment with psychotropic drugs; prior history of head injury resulting in loss of consciousness or recent exposure to other neurotoxic agents.

Prevalence (%) and odds ratios (95% CIs) of symptoms and signs. Odd ratios were computed from a logistic regression model. Confidence intervals for odds ratio were based on the likelihood ratio test (which the authors reported are known to be more accurate for small sample sizes). Holm’s modification of the Bonferroni correction equation was used to adjust for multiple comparisons; p-values are reported. Multiple regression analysis was conducted to adjust for confounders. Mean, SD and SEM also reported for comparisons of serum AChE levels in exposed and control groups


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either prior to or during the study. Pesticides were typically applied once every 15 days, with an initial treatment of insect repellent in the early cotton growth stages, followed by application of one or a combination of two to three OP pesticides (profenofos, chlorpyrifos, triaziphos and phorate), carbamate, or pyrethroid insecticides via knapsack sprayers (carrying 120 litres of pesticides) or motorised sprayers such as tractors (400-600 litres). Routes of exposure were either via inhalation, skin or ingestion (during machine repair)

Horowitz 1999 US 9 male fruit growers (aged 33 to 69) representing a small subgroup of a previous study population described in Stokes et al 1995; Occup Environ

The authors provide little information on the use and characteristics of the reference group except with regards to comparing sensory threshold and

Each subject underwent the following tests which were conducted off-season when subjects had been free of pesticide exposure for 3 - 7 months: a standard clinical neurological history (with emphasis on neuropathic symptoms); a physical examination;

Peripheral nerve abnormalities

The authors provide little information on exposure assessment aside from details of the study subjects OP exposure history.

Diabetes mellitus, neurological disorder, alcohol consumption

The authors noted that 95% confidence limits were established in matched controls. No further details of statistical tests are provided


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Med., 52:648-53. All subjects had the highest vibration indices in the original study. Subjects sprayed OPs (methyl parathion, tetraethyl pyrophosphate and azinphos-methyl) on fruit trees from mid-April to mid September. The author’s note that subjects were also exposed to non-OP chemicals e.g. fungicides albeit to a lesser extent. One subject reported an acute episode of OP poisoning 15 years before the study although subject completely recovered. Furthermore, exposure to chemicals varied with some using protective clothing and others not. Subjects underwent more intensive testing and examination cf. previous study and had been free of pesticide exposure for 3 - 7 months

electrophysiologic test values with 95% confidence limits established in age and sex matched controls

Computer-Aided Sensory Evaluation-Version 4 (Case IV) to assess sensory thresholds; the Tufts Quantified Neurological Evaluation (TQNE) to assess muscle strength, and electrophysiological studies i.e. nerve conduction velocity studies of upper and lower extremity nerves. Case IV is an automated microprocessor-controlled system that delivered quantitated and graded sensory stimuli over a range of intensities. Vibratory (VDT) and cooling (CDT) detection thresholds of the great toes and foot were determined via algorithms. The authors describe TQNE as a computer-directed quantitative assessment of muscle strength, in which the force (kg) of eight different muscle groups (shoulder, elbow and knee extensions and flexions, hip flexion, and ankle dorsiflexon) were measured bilaterally; grip strength, respiratory-forced vital capacity, expiratory volumes and manual dexterity (using a pegboard) were also evaluated. Electrophysiological studies measured the sensory and motor conduction velocities (CVs) and amplitudes of the


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evoked potentials (EPs) of the following nerves: median and ulnar nerves in both upper extremities; radial sensory CV upper extremity; motor CVs of the peroneal and posterior tibial nerves of the lower extremity; and the sural nerve CV of the opposite lower extremity. F-waves were measured for each motor nerve. The H-reflex was measured for one posterior tibial nerve

Hoshino 2008 Brazil

18 rural workers (5 male, 13 female) from the municipality of Teresopolis of Rio de Janeiro, aged between 16 to 59 years (mean age 39.6y) who had complaints of dizziness and occupational exposure to pesticides that included tamaron (aka methamidophos - used 27.1%) and folidol (aka parathion – used 33.3%). Other pesticides used included Manzate (54.2%), and Gramoxone (25%)

None. NB. Previously defined hearing normality criteria were used to characterise normal hearing thresholds in adults.

Three questionnaires: the first collected information on ear anatomic and physiological disorders; auditory and vestibular symptoms; and general health; the remaining two questionnaires sought information on dizziness symptoms in relation to work practices. Clinical examination included: otorhinolaryngological, audiological and vestibular examinations. Vestibular examination was done using the Contronics programme under previously defined principles.

Vestibular system alterations

Questionnaire was used to collect information on work practices. Exposure time was calculated based on the number of years the workers were active

The authors excluded subjects on the basis of the following risk factors: smoking, alcohol and drug abuse, metabolic, hormonal and neck disorders

Frequency of variables expressed as total number of subjects and percentages. The authors did not conduct any tests of significance

Kamel 2005

White male private pesticide

Controls were categorised as

The authors used two questionnaires at enrolment

Neurological symptoms, of

Questionnaire completed at

Age, state, education,

The authors calculated the


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applicators (mostly farmers) aged 18-75 years taken from the Agricultural Health Study (AHS) cohort in 1993-97 who at the time were applying for new or renewed licenses (due to the Iowa and North Carolina requirement for licenses to be renewed every 3 years). From the approximate 52,400 applicators (82% of those eligible) who completed the first questionnaire at enrolment, 44% completed the second applicator questionnaire, (which included questions on neurological symptoms). The authors noted those who completed the second questionnaire were similar to those who did not in most respects including prevalence of symptoms and pesticide exposure. Only

those applicators who experienced < 10 symptoms (low frequency). See study population column for further details

to collect information from applicators on demographic characteristics, lifestyle, medical history and pesticide use. The second questionnaire (completed a month after the first) included questions on neurological symptoms experienced during the year before enrolment. These questions were based on a previously established symptoms questionnaire (Q16), that was used to evaluate effects of occupational exposure to neurotoxicants. Twenty-three symptoms were reported. The authors created two measures reflecting the number of symptoms experienced at least once in the year before enrolment: (i) a continuous variable “the number of symptoms”, and (ii) a dichotomous variable “many symptoms” that compared the 20% of applicators who experienced ≥ 10 symptoms (cases) with the remaining 80% who experienced < 10 symptoms (controls)

which the following represented peripheral neurologic outcomes: numbness in hands/feet, twitches in arms/legs, weakness in arms/legs, poor balance, and tremor in hands, blurred/ double vision, changes in smell or taste and difficulty speaking

enrolment provided detailed information on lifetime pesticide use and exposure (i.e. frequency and duration of use of 50 specific pesticides) during the prior year. The authors considered several pesticide exposure measures: years of use of any pesticide; days per year; lifetime days of use (categorised into quartiles ranging from 0 to >500 days); and cumulative lifetime days for pesticide groups. NB. Pesticides were categorised according to function/ mode of use i.e. herbicides, insecticides (that were further categorised by chemical classes, which included OPs), fungicides, or fumigants

smoking and alcohol use. The authors also screened for medical history

percentage of study participants experiencing a particular frequency of neurologic symptoms in the year before enrolment to help categorise cases and controls (with symptom distributions dichotomised so that the positive category included between 5% to 15% of participants). For specific neurologic data, odd ratios were calculated by logistic regression for experiencing a specific neurological symptom with high frequency compared with low frequency. 95% CI were calculated (not shown)


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subjects who provided complete information on neurological symptoms (89% of those who returned the second questionnaire) were included (n=18,782). Applicators who provided incomplete information differed wrt, age, state and education. The symptoms listed in the questionnaire were based on a previously used and established questionnaire ‘Q16’. The responses were used to derive the applicators frequency of experiencing these symptoms at least once in the year before enrolment. “Cases” were categorised as those with a high frequency of symptoms i.e. ≥ 10 symptoms

Kamel 2007b US White male private pesticide applicators (mostly farmers) aged 18-

‘Controls’ were categorised as those applicators who experienced <

The authors used two questionnaires at enrolment to collect information from applicators on demographic

Various neurological symptoms that were categorised

Several measures of pesticide exposure were constructed from

Age, state, education, cigarette smoking, alcohol

Logistic regression was used to estimate associations of


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75 years taken from the Agricultural Health Study (AHS) cohort in 1993-97 who at the time were applying for new or renewed licenses (due to the Iowa and North Carolina requirement for licenses to be renewed every 3 years). From the approximate 52,400 applicators (82% of those eligible) who completed the first questionnaire at enrolment, 44% completed the second applicator questionnaire, (which included questions on neurological symptoms. The authors noted those who completed the second questionnaire were similar to those who did not in most respects including prevalence of symptoms and pesticide exposure. Only subjects who provided complete

10 symptoms (low frequency). The authors noted this comprised 80% of the applicators. (See study population column for further details)

characteristics, lifestyle, medical history and pesticide use. The second questionnaire (completed a month after the first) included questions on neurological symptoms experienced during the year before enrolment. These neurological symptoms were based on a previously established questionnaire (Q16) that was used to evaluate effects of occupational exposure to neurotoxicants). Twenty-three symptoms were reported. These symptoms were categorised a-priori in groups under several functional domains which included sensory (comprising of numbness or paresthesia, poor night vision, blurred or double vision, changes in smell or taste), and motor (comprising of twitches, weakness, poor balance, tremor and difficulty speaking). The authors used the motor group plus a group consisting of the two vision symptoms (i.e. poor night vision and blurred or double vision) in their evaluation of symptoms. Group symptom variables were created by summing, for each applicator, the number of times per year any symptom in the group was experienced, and then

a priori into groups representing several functional domains were evaluated. This included motor and vision domains (as well as affect, cognition and autonomic groups)

questionnaire data: reported duration (years) and frequency (days/year) of use for any pesticide in categories; cumulative days of use (created by multiplying duration times by frequency using midpoints of the reported categories and then categorising the product in quartiles, ranging from 0 to >500 days); cumulative lifetime days of use of pesticides in functional groups (insecticides, herbicides, fungicides and fumigants) or chemical subgroups of insecticides (OPs, organochlorines, carbamates and pyrethroids) were created by multiplying duration by frequency of use for each pesticide in the functional or chemical group, summing across pesticides in the group, and categorising the sum into never use and other

use. The authors also screened for medical history

symptom outcomes with pesticide exposure. Results were expressed as odd ratios with 95% CIs


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information on neurological symptoms (89% of those who returned the second questionnaire) were included (n=18,782). Applicators who provided incomplete information differed wrt, age, state and education. The symptoms listed in the questionnaire were based on a previously used and established questionnaire ‘Q16’. The responses were used to derive the applicators frequency of experiencing these symptoms at least once in the year before enrolment. “Cases” were categorised as those with a high frequency of symptoms i.e. ≥ 10 symptoms. The authors noted this comprised 20% of the applicators

dichotomising the distribution of sums so that the positive category included approximately 10% of the participants

categories of use. The authors also classified application methods likely to involve low or high personal exposure i.e. (aerial application, tractor boom, pre-applied to seed) and (airblast, mist blower, backpack sprayer, and hand spraygun) respectively to categorise applicators as using low or high exposure methods

Keifer

2000 US Male and female farm workers (mean age 28 years old)

Unexposed age, education and gender matched-controls comprised

Sensory and motor nerve conduction tests, vibration threshold tests of hand and foot, and repetitive

Neurophysiological (i.e. peripheral nervous system) and

Self-reported life-long work history, and reported home pesticide exposure

Demographics, alcohol intake, home pesticide exposure, health,

Multiple linear regression was used for continuous


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recruited from several orchards in the Wenatchee area. Subjects applied OPs (most prominent being azinphos-methyl) during the task of apple orchard thinning, and underwent two periods of evaluation: 137 workers took part in the first round of tests, which took place at end of the thinning season (April-August) in the Wenatchee area. 90 workers were tested a second time conducted 6-9 months later

of workers recruited from a non-agricultural workplace (i.e. garment manufacturing, hotels and restaurants). No further information was provided. NB. The authors did not specify the no. of controls used; it is presumed 137 controls were used to match the total no. of subjects

simulation electromyography were also performed. Other tests were conducted to assess possible neurobehavioural outcomes. Testing in round 1 was performed by trained interviewers and experienced testers who were blinded to the exposure status of the workers

neurobehavioural health outcomes

information was collected from a structured interview and work-calendar questionnaire. Worker’s recent thinning hours was used as the primary exposure variable. Biochemical measures of exposure were conducted twice (at the end of the exposure season when recent exposure would be at its cumulative peak, and 6-9 months later before beginning of the next season). The tests performed included measures of AChE levels in blood, paraoxonase, chlorpyrifos oxonase and diazinon oxonase activity (in most participants)

work and social history, Peabody Picture Vocabulary Score

outcome variables; logistic regression was used for some binomial variables and non-parametric chi square was used to evaluate ordinal variables. The Fischer exact test was applied to dichotomous variables; p<0.05 two-tailed testing was used for significance

Kilburn

1999 US 22 male and female patients (mean age 44.7 years, SD 13.0, range 34 to 77 years) representing 6% of 384 patients being evaluated in a neurotoxicology clinic for possible neurobehavioural

[Neurobehavioural function analysis]: 264 male and female referents (mean age 45 years, range 18 to 83 years) unexposed to neurotoxic chemicals were recruited at random from voter

Subjects completed a questionnaire which collected frequency data on 35 common health complaints scaled from rare (1) to frequent or daily (11). The assessment included a standard respiratory questionnaire. Complete physical and neurological examinations were also done.

CNS effects i.e. neurobehavioural impairments (that included assessment of neurophysiological function)

Three different measures were used: (i) detailed schedules of pesticide application for offices (exposed by spraying every 3 to 9 weeks); (ii) analysis of personal clothing or rugs; (iii) case histories of

Adjustments were made for age, sex and education, gender, height and other factors with significant coefficients. Authors collected information on possible confounding

Comparisons between exposed subjects and unexposed referents were made using the following equation: observed/predicted multiplied by 100 equals the


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effects of exposure to chemicals. Subjects were selected on the basis that they identified chlorpyrifos insecticides as the main chemical of exposure. (NB. The authors noted that other major shared chemicals included diazinon, carbamates, pyrethroids and a phenolic chemical)

registration polls in Arizona and Los Angeles and tested to generate predicted values

Neuropsychological and neurophysiological test batteries were conducted by staff at the clinic. Several neurophysiological tests were conducted (some of which were categorised under the neurobehavioural test battery) including: body balance (measured minimal sway speed with eyes open and closed); blink reflex (measured with surface electromyographic electrodes (EMG) on the eyelid sphincter muscle after stimulating the supraorbial nerve); and vibration sense threshold (disappearance) via the use of a 128Hz tuning fork (whereby the higher the threshold the greater the impairment); colour discrimination was measured with the desaturated Lanthony 15 hue test and scored by the method of Bowman (1982) Acta Ophthalmol, 60:907-16); visual field threshold was tested via a computerised recorder that mapped the central 30o of right and left eyes individually, expressed as sum of scores for 80 stimuli (db); hearing was measured in left and right ears with standard audiometers at stepped frequencies of 500-8000Hz. The sum of both deficits

exposure incidents. NB. The authors noted that no air sampling of chlorpyrifos or other OPs were conducted in the premises due to the time lapsed since exposure (9-108 months)

exposures i.e. other hazardous chemicals including pesticides and herbicides, alcohol, tobacco and drug use (illicit and prescription), unconsciousness, anaesthesia, head trauma and neurologic and medical histories

percentage of predicted value. This was done by calculating the predicted values for each test of each patient from stepwise linear regression equations, adjusted for factors with significant coefficients. Also analysis of variance was used to compare mean percentage of predicted neurobehavioural test scores (adjusted) and SD between exposed and unexposed, and to compare symptom frequencies in chlorpyrifos-exposed subjects. Statistical significance was defined as p<0.05


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was the hearing score. NB. Grip strength was also tested. Tested criteria were used to select values for blink reflex latency. Patients underwent multiple trials to select their best scores. Neuropsychological tests were also performed, as well as assessments of respiratory flows and vital capacities

Kimura 2005 Malaysia 76 of 80 male tobacco farmers (average age 45.1 years, SD 7.2) randomly selected from National Tobacco Board (NTB) register. Main pesticides used in the farms were either pyrethroids, dinitroaniline, organochlorines, carbamates, or the organophosphate methamidophos (trade name Tamaron). The authors noted that 26 farmers used Tamaron (although most farmers used two or more pesticides in combination)

38 of 40 male officers of the NTB (average age 45.1 years, SD 7.1) who did not handle pesticides or wet tobacco leaves

Tests were performed by an experienced clinical technician during work time. Nerve conduction studies were performed in the right forearm and lower limb. Maximal motor conduction velocity (MCV) of the median and tibial nerve, and sensory conduction velocity (SCV) of the median and sural nerves were measured according to standard techniques. Brain-evoked potentials and computerised static posturography were used to assess disorders within the CNS

Peripheral and central nervous system functions

Interviews were conducted prior to electrophysiological studies to obtain data on years of working with tobacco, and details on the type of pesticide and protective equipment used. This was followed by analysis of serum cholinesterase activity

Age, education, alcohol, smoking status were considered. The authors also screened for systemic conditions i.e. diabetes mellitus, and present illnesses

Students t-test was used to examine differences in various parameters including NCV, blood level of cholinesterase activities, and potential confounders

Konieczny 1999 Poland 35 male chemical plant workers of a plant producing

[Spirometry]: 22 healthy men, aged 20-62 years (mean

Spirometry was performed to estimate the following parameters (which aided

Impaired respiratory muscle function

Air chlorfenvinphos concentration in the production area

Age, smoking. The authors also screened for

Mean and SD of spirometry indices; absolute


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chlorfenvinphos aged 25-55 years (mean 42.1 years, SD 7.5). The authors noted that 22 subjects were smokers (17.3 pack-years, SD 11.6); and 13 (37.1%) were diagnosed with chronic bronchitis according to a BMRC questionnaire; none had atopic airways disease

39.6 years, SD 12.3), without occupational exposure to chemicals. The authors noted that 12 controls subjects were smokers (15.7 pack-years, SD 9.5); chronic bronchitis was diagnosed in 22.7% of subjects. [Repetitive nerve stimulation test]: 30 healthy males, aged 35-52 years (mean 47.5 years, SD 8.7) who were residents of the region provided normal amplitude differences for comparison with study subjects. [RBC AChE and plasma cholinesterase activity]: 33 healthy males, aged 20-55 years (mean 33.1 years, SD 12.9) who were residents of the region with no occupational exposure to chemicals

the assessment of PN-induced lung dysfunction): vital capacity (VC), forced expiratory volume in one second (FEV1), and FEV1/VC ratio (FEV1%), maximal expiratory flow at 25%VC (MEF25%), peak expiratory flow (PEF), gas transfer factor (TFLCO), specific airways conductance (sGaw), intrathroacic gas volume (ITGV), the ratio of residual volume to total lung capacity (RV/TLC) and maximal static inspiratory (MIP) and expiratory (MEP) mouth pressures at the functional reserve capacity and total lung capacity. The repetitive nerve stimulation test was performed using an electromyograph with surface electrodes, and involved stimulating the right ulnar nerve (forearm) at intervals with repeated stimuli at different frequencies. Compound action potentials of the hypothenar muscle (fingers) were recorded and contraction amplitude differences were estimated

was estimated via gas-liquid chromatography according to Polish standards. RBC AChE and plasma cholinesterase activity was determined spectrophotometically

conditions known to confound respiratory function i.e. chronic bronchitis and atopic airways disease

values of maximal inspiratory and expiratory pressure (MIP and MEP) in workers and controls. Unpaired Student’s t-test, with values of < 0.05 considered significant

Peiris-John 2002 Sri Lanka 30 male farmers (mean age 36.56 years, SD 11.4) engaged in regular spraying of OP

30 fishermen (mean age 37.2 years, SD 10.9) living close to subjects but not

Evaluations were conducted at two time points: during an intercultivation or “between” season (June/July 2000)

Neurophysiological abnormalities

OP exposure was estimated by an interviewer administered questionnaire and

Subjects were screened for confounding effects of age, BMI, extent of

Data was subjected to tests of significance via t-test: a paired t-


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pesticides, with a mean duration of exposure of 13.8 years. Farmers who resided in the Uda Walawe irrigation scheme of Southern Sri Lanka were on average directly exposed to pesticides for approximately 20 hours during a cultivation season. The authors noted that farmers had co-exposures to non-OP agents: all farmers had used AChE-inhibiting pesticides (OP and carbamate pesticides) within the preceding month and 80% had used anilides and 42% had used pyridine

involved in pesticide spray activities

and during a cultivation or ”during” season (April/May 2000). A detailed medical history was taken followed by a physical examination, which included a neurological evaluation that adhered to a fixed protocol (no further information was provided). The following neurophysiological tests were conducted by a tester blinded to subject’s exposure status: (i) nerve conduction studies using Neuropak EMG/evoked potential measuring systems via surface electrodes. Motor conduction velocity of the right median nerve (elbow to wrist) was measured by stimulating the nerve at the wrist and elbow, with active recording, reference and ground electrodes placed over appropriate positions to enable the distance between the two stimulation points (distance travelled) to be measured. The authors defined motor conduction velocity as the time taken for the impulse to travel the given distance. Sensory conduction velocity of the sural nerve was measured by antidromic stimulation of the calf muscle with the active recording and reference electrodes situated in appropriate positions. The

by measuring erythrocyte AChE activity. Hb-corrected levels were measured using approved tests. Additional information collected included details on farming activities and work history

farming activities and work, alcohol consumption and medical conditions e.g. diabetes mellitus. NB. Subjects were excluded if they had the following: vitamin deficiency, encephalopathy of known origin, long-term administration of psychotropic medication or previous injury resulting in deformity and/or other abnormality of the limbs interfering with peripheral sensations

test was used to compare pre-exposure and exposure data; an independent sample t test was used to compare data between farmers and controls. The difference between AChE levels during and between cultivation seasons was measured as the calculated difference


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authors defined sensory conduction velocity as the time taken for the impulse to travel the measured distance. Sensory and motor latency was defined as the time taken for the action potential to travel from the stimulating to the recording electrode; (ii) neuromuscular synapse testing was conducted via the use of a neuromuscular transmission monitor and involved physically immobilising the limb with a splint to avoid electrode movement during the test and stimulating the medial nerve in the same manner as previously described. The nerve was subjected to four stimulations at three time points with the action potential recorded using surface electrodes. The decrement in response was determined using the ratio of amplitudes of action potentials at different trains (rounds) of stimulation

Pilkington 2001 UK 612 male and female sheep farmers/farm workers (mean aged 45.1 years, SD 13.4) who had dipped sheep were recruited from databases maintained by the Ministry of Agriculture,

Low exposure groups for comparisons comprised of (i) 53 male and female farmers (mean age 39.2 years, SD 12.7) without sheep-dipping experience recruited from MAFF/ Scottish

Neurological assessments were conducted using a symptoms questionnaire in conjunction with a series of quantitative sensory tests. Symptoms were categorised into three groups relating to (i) muscle weakness (ii) sensory symptoms (iii) autonomic symptoms i.e. sweating, fainting and impotence.

Clinically detectable abnormalities of the peripheral and autonomic nervous system

Retrospective exposure information was obtained via use of an exposure history questionnaire administered by a trained interviewer and developed during the first phase of the study. The first study

Authors adjusted for the following potential confounders of neurological symptoms: age, sex, country and alcohol habit, exposure to vibration via occupation/recreationally, other

Linear logistic regression was used to calculate ORs for reporting symptoms and to test the significance of the differences between occupational groups and the effect of


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Fisheries and Food (MAFF) and the Scottish Office for farms in England, Wales and Scotland. This represented 293 of 335 eligible farms (prior to exclusions based on potential confounding conditions)

Office database; (ii) 107 ceramics factory workers (brick makers) recruited via the British Ceramics Confederation and selected from two companies based in Scotland and England

Sensory and motor symptoms of the upper and lower limbs were indicative of damage to the peripheral nervous system. Analysis was based on an overall dichotomous symptom indicator where a positive score was obtained for at least one symptom in sensory or motor symptom category or two symptoms in the autonomic group. Heat and cold threshold values were determined via the use of a stimulating probe (thermode) with attempts to maintain skin temperature at a constant 34ºC ± 0.2ºC when not being stimulated. Vibration threshold values were determined via the use of a vibrameter which stimulates receptors sensitive to vibration following standard methods. In all three quantitative threshold tests, the higher the threshold the less sensitive to stimuli and therefore evidence of nerve damage. NB. The neuropathy symptoms questionnaire also included questions about current exposure to vibration (i.e. from occupational and recreational use of tools/machinery, etc)

phase consisted of an occupational hygiene study of dipping practice at 20 farms. A simple dermal exposure model was derived for measured urinary OP metabolites (diethylphosphate DEP, and diethylthiophosphate DETP) and was compared with a hygienist’s observations of working practice. The model identified handling of the concentrate dip product as the principal source of exposure among dippers, alongside splashing with dilute dip in the bath. Data on the no. of days spent dipping and use of concentrate including protective measures were gathered, from which the cumulative exposure to OP dips (OPEXP – i.e. the sum of the urinary [DEP] and [DETP] across dipping days in nmol) was calculated as the weighted sum of

chemical exposures. Subjects were also screened for education, and having a disease or taking medication known to confound PN diagnosis. NB. Comparative group analyses showed differences in age, gender and alcohol consumption (the latter only between ceramic workers and farmer groups).

cumulative exposure. Scatter plots (that included a locally weighted scatter plot smoother (LOWESS)) were used to graphically compare the three sensory tests to aid comparison of trends. Multiple linear regression was used to investigate exposure–response relations among sensory test thresholds in the presence of potential confounding variables. Cumulative exposure variables were scaled in the regression analyses by their interquartile range across all subjects. A 5% level was used to determine significance


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cumulative exposure to both concentrate ((CONC) – an estimate of the total no. of concentrate handling events) and dilute dip splash (SPLASH) – cumulative time-weighted splash score based on hygienist’s observations of splashing during dipping). The authors also determined a simpler exposure variable (DAYS) defined as the total no. of dipping days. Estimates of the daily intensity of exposures to both concentrate and splash were derived by dividing OPEXP and its components CONC and SPLASH by DAYS

Srivastava 2000 India 59 male quinalphos (QP) manufacturing workers (mean age 30.0 years, SD 6.61). NB. The authors noted that these workers are also exposed to other chemicals e.g. raw materials and by-products of

17 eligible male control subjects (mean age 28.9 years, SD 4.5) not engaged in manufacture or handling of QP but employed as tea vendors, roadside hawkers, etc (sourced on the basis of being

Detailed clinical history of subjects were recorded via a structured interview. Each subject was clinically evaluated for general health status and possible abnormalities in several body systems including musculoskeletal and CNS by medically qualified doctors after 5-6 hrs of typical work exposure (in

Various CNS outcomes in particular reflex and neurobehavioural abnormalities

Detailed occupational history of subjects were recorded via a structured interview. AChE levels in blood were measured via a previously published method

Variables investigated included age, sex, height, weight, education, income, smoking and drinking habits, work history duration and previous neurological

Student’s t-test was used to test the significance of the difference in mean values between exposed and control subjects. Chi-square test was used to test the significance of prevalence of


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QP, e.g. organic solvents, o-phenylene diamine (OPD), Glyoxalic Methyl Ester Hemiacetal (GMHA), 2-hydroxyquinoxaline (2-HQ), diethyl thiaryl phosphorocyanide (DETCN), and xylene

situated near the manufacturing unit and having no direct or indirect exposure to OP pesticides)

QP manufacturing workers). Evaluation also included a haematological analysis. Clinical psychological tests were conducted via a battery of neuro-behavioural tests

conditions that could confound PN diagnosis. Both groups were similar wrt age, work history, physical activity at work, sex, education, and drinking and smoking habits (p>0.05), although exposed subjects tended to be more educated, smokers and alcohol drinkers

signs and symptoms in exposed and control subjects. Fisher’s exact test was used where the expected cell frequencies were > 5

Steenland 2000

US 193 current and former termiticide applicators (mean age 39.3 years, SD 9.4) who had reported using chlorpyrifos for a year or more in a 12 county area of North Carolina. NB. Some also used chlordane. The source population identified from pest control operators/ companies licensed between 1987-1997 comprised of 3605 pest control workers of which 2917 were screened and 688 were unscreened

Two non-exposed groups were used: (i) 106 age (within 5 years) and sex-matched controls (mean age 38.0 years, SD 9.7) who were friends of the exposed subjects. The authors did not clarify whether these friend controls were screened for their exposure to OPs; (ii) 83 from 856 age, sex and race-matched blue-collar North Carolina State employees (maintenance workers and correction officers) (mean age 42.6

A short 24-item self-administered questionnaire (modified version of the Hogstedt symptom questionnaire) was used to determine neurological symptoms experienced in the last month e.g. muscle weakness, numbness, tingling, imbalance, etc. The authors also conducted a series of neurological tests for both central and peripheral function. Tests for the latter included (i) a vibrotactile test conducted in the finger and non-dominant toe at two vibrational frequencies: 31.5 Hz and 125 Hz, over an intensity range of 90-160 dB; (ii) arm/hand tremor test,using a NIOSH-developed hand held device that measured

Central and peripheral nervous system abnormalities

Subjects were interviewed to obtain information on work history i.e. termiticide use and job history to determine whether subjects used other pesticides, including previous poisoning. Biological samples of urine were collected to measure levels of the metabolite TCP via gas chromatography in applicators who reported current chlorpyrifos exposure (n=105) and in non-exposed subjects (n=52). Buccal swabs were also collected for

The following demographic variables were checked for potential confounding effects and included in the final models: age, race, education, smoking, BMI, Subjects were interviewed to obtain information on other pesticide, solvent use. NB. Subjects were asked to refrain from alcohol consumption on the night before testing. Subjects were tested for

Symptom questionnaire responses were considered either continuous or dichotomised. Linear regression was used to compare neurological test data between subjects. Significance level was (α = 0.05)


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due to the companies either being out of business or refusing to provide information. 383 of the 2917 screened candidates were considered eligible i.e. worked as termiticide applicators for a minimum of one year. However, 239 (61%) of the 383 eligible participants were contactable and 153 of the contactable participants were tested (64%). Reasons for non-participation included refusals, ineligibility, and lack of availability. The authors sourced a further 206 applicators among the previously unscreened group. 43 subjects (21%) were subsequently scheduled for testing – reasons for non-participation included refusals, unavailability and inability to locate

years, SD 8.7) living in central North Carolina who had never worked (or been poisoned) with pesticides

visible tremor (i.e.1-6hertz) and non-visible tremor (e.g. 7-30 hertz) via accelerometers. Horizontal and vertical tremors were averaged; (iii) 30 sec postural sway test (analogous to the Romberg clinical test) using computer-controlled force platform and varied test conditions (i.e. eyes open/closed, hard/soft platform, one/two legs); (iv) vision tests of visual acuity and colour vision with the Farnsworth and Lanthony test used to discriminate congenital from acquired colour vision loss; (v) olfaction cross-cultural smell identification test where subjects were asked to correctly identify 12 microenscapsulated odours prepared by the Uni of Pensylvannia; (vi) nerve conduction velocity tests in the following three nerves of the dominant limbs (othrodromic motor responses in the peroneal nerve, and antidromic responses in the sural and ulnar sensory nerves); and (vii) clinical neurological examination performed on all subjects by two neurologists that evaluated pupils, eye movement, tremor, coordination, tone, strength, sensation, reflexes, station and gait

genotyping participants (all exposed and 55 non-exposed) with regards to paraoxonase polymorphisms at amino acid 54 of chromosome7 i.e. their ability to detoxify chlorpyrifos (as individuals homozygous for methionine exhibit lower serum concentrations of paraoxonase and subsequently at greater risk of chlorpyrifos oxon-induced neurotoxicity). This was done by first isolating the DNA, followed by PCR amplification of the enzyme gene sequences. Exposed subjects were subsequently classified as being either current applicators, former applicators, applicators with a susceptible genotype and previously poisoned applicators

this via completion of a saliva test performed on the morning of the test. Individuals who failed (i.e. had an alcohol saliva concentration of > 0.03% returned at a later time)


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6.B.4. Case series studies – none

6.B.5. Case reports – none Table 7. PERIPHERAL NEUROPATHY & NEUROMUSCULAR DYSFUNCTION RESULTS AND CONCLUSIONS

First Author




7.A.1. Cohort studies Kawana 2001 [VICTIMS OF THE 1995 TOKYO SARIN ATTACK FOLLOWED UP AFTER 2, 3 AND 5 YEARS]

Health Outcome Data: [Questionnaire-based] Frequently reported peripheral neuropathy-related symptoms (>10%) included muscle stiffness, dim vision and fatigue. NB. It is not clear whether reports of fatigue related to ‘muscle’ fatigue or ‘general tiredness’ (of which the latter would denote a non-neurological symptom). Reports of most physical symptoms did not change significantly from survey to survey (symptom frequency rate for muscle stiffness was 18.7 %, 14.1% and 14.7% at successive follow-ups, and 23.3, 25.7 and 25.1% for dim vision). The authors noted that most of the reported symptoms were medically unexplained (evidenced by the lack of medical treatment) according to medical diagnostic criteria. Results of comparing symptom incidence (%) of the current study with those reported by victims of two other groups exposed to sarin (i.e. Tokyo sarin attack studied by a non-governmental organisation (NGO) and the 1994 Matsumoto sarin attack), and a control group from Matsumoto city, showed that for muscle stiffness, the symptom incidence rate was highest in the NGO Tokyo victim group (31.8% of 655 victims) and lowest in the Matsumoto control group (8% of 87); the symptom incidence rate for the 5-year follow up of victims evaluated at St Luke’s Hospital (study subjects of the current analysis) was 14.7% of 191 victims; and 18.4 % of 88 victims for Matsumoto sarin attack. For dim vision, the symptom incidence rate was highest in the NGO Tokyo victim group (43.8% of 655 victims) and lowest in the Matsumoto control group (10.3% of 87); the symptom incidence rate for the victims evaluated at the St Luke’s Hospital was 25.1% of 191 victims; and 19.5% of 88 victims for Matsumoto sarin attack. The authors suggested that the reason for symptom differences between these groups may be due to the cohort receiving multiple interventions, including physical examination, counselling and laboratory work

Strengths Subjects known to have definite exposure to the OP sarin. Adequate sample size. Limitations Low and inconsistent response rate (declines with subsequent follow-ups). Exposure assessment was based solely on hospital admittance as a sarin victim. No objective health assessment used (based on subjective self-reports). Lack of quantitative

This study reported a higher incidence of ‘muscle stiffness’, and ‘dim vision’ in victims of the 1995 Tokyo sarin attack (up to 5 years) compared to referent Matsumoto control group. NB. Although dim vision could arise from dysfunction in the optic nerve it could also result from dysfunction in the eye or brain. Therefore, without further testing it would be imprudent to classify dim vision as a cranial nerve issue This study, does not provide


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measures of motor abnormalities. The follow-up period for the two other sarin victim groups is not specified in the symptom incidence rate comparisons No analysis of possible associations between exposure and risk of reported symptoms. Possible confounders not considered. No statistical tests of significance to rule out chance findings when comparing results between different study groups

any convincing evidence of peripheral neuropathy related symptoms due to various study limitations including the lack of an objective health assessment or analyses to determine possible associations

Miranda 2002a [INDIVIDUALS HOSPITALISED FOR ACUTE OP POISONING FOLLOWED UP AT: (I) HOSPITAL DISCHARGE i.e. 1-18 DAYS AFTER POISONING; AND (II) APPROXIMATELY 7 WEEKS AFTER POISONING] Exposure Data: Erythrocyte cholinesterase activity in one severely poisoned patient (with no altered consciousness) was 2.6IU/g Hb (85% below the lower limit of the normal level in the control group. Mean erythrocyte cholinesterase activity in control group > 28.0 IU/g Hb. Health Outcome Data: [Muscle strength tests] Compared to controls, grip and pinch strength were impaired among all OP-poisoned subjects at both examinations (particularly among those poisoned with neuropathic OPs which were the only group to produce results that reached statistical significance, (p<0.05))

Grip strength Pinch strength 1st

examination 2nd

examination 1st examination 2nd examination

Strengths Subjects received definite exposure to OPs. Uses a standardised and objective health assessment method. Adequate account of possible confounders. Reported a dose-dependent effect whereby impairment was more marked

This study reported motor neurological impairment (based on diminishing grip and pinch strength) in a cohort of subjects moderately and severely poisoned with known neuropathic and non-neuropathic OP pesticides. However, given that the maximum follow-up period was approximately


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Controls (n=39), mean (SD)

43.2 kg (5.9)

44.9 kg (6.5)

9.9 kg (1.9)

10.0 kg (1.6)

Poisoned with non-neurpathic OPs Moderate poisoning (n=12) Mean (SD) 41.5 kg (7.6) 42.8 kg (5.7) 9.1 kg (1.5) 9.7 kg (1.3) RC (CI)* -1.6 (-6.5, 3.3) -2.1 (-6.8, 2.6) -0.6 (-1.8, 0.7) -0.1 (-1.3, 1.1) Severe poisoning (n=6) Mean (SD) 37.8 kg (11.8) 39.3 kg (8.3) 9.3 kg (2.5) 9.3 kg (1.3) RC (CI)* -5.3 (-11.8, 1.1) -5.6 (-11.7, 0.6) -0.5 (-2.1, 1.1) -0.6(-2.2, 1.0) Poisoned with neuropathic OPs Moderate poisoning (n=25 for grip and 23 for pinch) Mean (SD) 39.2 kg (7.4) 41.0 kg (7.8) 9.0 kg (1.7) 9.2 kg (2.2) RC (CI)* -3.9 (-7.7, -0.1) -3.9 (-7.5, -0.3) -0.8 (-1.8, 0.1) -0.7 (-1.7, 0.2) Severe poisoning (n=16) Mean (SD) 34.4 kg (8.4) 33.7 kg (7.2) 8.3 kg (2.0) 7.7 kg (2.3) RC (CI) -8.8 (-13.1, -4.4) -11.3 (-15.4, -7.1) -1.6 (-2.7, -0.5) -2.2 (-3.2, -1.1)

*RC = age-adjusted regression coefficient or difference from controls with 95% CI Among subjects severely poisoned with neuropathic OPs, impairments were more marked for poisonings that were intentional rather than as a result of occupational accidents, which worsened at second examination (p<0.05)

Grip strength Pinch strength 1st examination 2nd examination 1st examination 2nd examination


43.2 kg (5.9)

44.9 kg (6.5)

9.9 kg (1.9)

10.0 kg (1.6)

Occupational poisoning (n=10) Mean (SD) 33.8 kg (9.4) 35.6 kg (6.6) 8.9 kg (2.0) 8.9 kg (1.0) RC (CI) -9.3 (14.1, -4.4) -9.3 (-14.1, -4.6) -1.0(-2.4, 0.3) -1.1 (-2.3, 0.0) Intentional poisoning (n=6) Mean (SD) 35.3 kg (7.0) 30.4 kg (7.5) 7.3 kg (1.6) 5.8 kg (2.7) RC (CI) -7.9 (-13.9, -1.9) -14.6 (-20.5, -8.7) -2.5 (-4.2, -0.8) -2.2 (-5.5, -2.6)

Analyses of mean changes of grip and pinch strengths (between first and second examinations) showed a significant decrease in performance for subjects intentionally poisoned with neuropathic OPs as compared with controls (p < 0.05):

Change in Grip Strength

Change in Pinch Strength


1.8 kg (3.6)

0.1 kg (1.2)

Occupational poisonings with non-neuropathic OPs (n=6) Mean (SD) 1.6 kg (9.5) 0.0 kg (1.8) RC (CI)* 0.2(-3.9, 4.4) 0.1 (-1.1, 1.2) Occupational poisonings with neuropathic OPs (n=10) Mean (SD) 1.8 (4.2) 0.0 (1.3) RC (CI) 0.0 (-3.0, 3.0) 0.1 (-1.0, 1.2) Intentional poisoning (n=6)

among those with severe poisonings due to neuropathic OPs. Limitations Small sample size. Loss of control subjects at second follow-up evaluation. Authors did not attempt to evaluate background differences between participants and non-participants. Exposure categories appear to be based on severity of poisoning i.e. the extent/severity of symptoms recorded and not measured levels of OPs (authors measure cholinesterase levels only in the control group and one severely poisoned patient). Potential confounders not addressed include: muscle training (since subject’s muscular load on hands/grip and pinch strength may be influenced by extent of muscular

7 weeks, the findings most likely represent symptoms of the intermediate syndrome or OPIDP, which is a well established sequale of acute OP intoxication (that arises 2-3 weeks after poisoning and often resolves within 6 to 12 months)


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Mean (SD) -4.9 kg (-7.3) -1.5 kg(3.2) RC (CI) -6.7 (2.9, 10.4) -1.6 (0.2, 3.0)

*Regression coefficient or difference from controls (RC) with 95% CI

training); depressive symptoms in suicidal subjects (may lower baseline strength). Suitability of control group given pesticide exposure in some control subjects

Miranda 2002b [PATIENTS HOSPITALISED FOR ACUTE OP POISONING; FOLLOWED UP AT: (I) HOSPITAL DISCHARGE I.E. 1-24 DAYS AFTER POISONING; AND (II) APPROXIMATELY 7 WEEKS AFTER POISONING] Exposure Data: Mean erythrocyte cholinesterase level in (a) severely poisoned men (n=9) was 8.0 IU/g Hb (range 2.6-17.1, SD 5.5) (b) moderately poisoned men (n=12) was 11.6 IU/g Hb (range 2.3-24.6, SD 5.9). Erythrocyte cholinesterase activity in one severely poisoned patient (with no altered consciousness) was 2.6IU/g Hb (85% below the lower limit of the normal level in the control group. Mean erythrocyte cholinesterase activity in control group > 28.0 IU/g Hb, SD 6.6 Health Outcome Data: [Quantitative sensory threshold (QST) tests]: {Non-neuropathic OP poisonings} (n=16): No differences in index or toe vibrotactile thresholds at either examination when compared to controls. Intra-individual thresholds mean changes between 1st and 2nd evaluations for index finger or big toe vibrometrey were also not different from controls. {Neuropathic OP poisonings} (n=40): No significant increases were detected for moderately or severely poisoned patients in vibrotactile threshold of index finger or big toe compared to controls. However, separate analysis of the group with severe poisonings revealed significantly increased toe vibrotactile thresholds of men with severe intentional poisonings in the second examination as compared with controls (p-values not reported):

Threshold of Dominant Big Toe

1st examination 2nd examination Controls (n=39), mean (SD) 0.58 log mic (0.36) 0.51 log mic kg (0.27)

Occupational (severe) poisoning (n=10) Mean (SD) 0.58 log mic 0.34) 0.43 log mic (0.28) RC (CI)* -0.01 (-0.25,0.22) -0.08 (-0.29, 0.13) Intentional (severe) poisonings (n=5) Mean (SD) 0.56 log mic (0.21) 1.1 log mic (0.47)

Strengths Subjects received definite exposure to OPs. Biological measures of OP exposure via cholinesterase activity (reported for 21 subjects). Adequate account of potential confounders including assessing co-exposures to other chemical agents, and occupational exposure to vibrational machinery. Uses a standardised and objective health assessment method. Limitations Small sample size. Loss of control subjects at second follow-up

This study reported significant sensory impairment of peripheral nerve (based on increased big-toe vibrotactile thresholds) in a cohort of subjects severely and intentionally poisoned with known neuropathic OP pesticides. No significant sensory impairment of vibrotactile thresholds were detected in association with occupational poisonings or less severe intentional poisonings with either type of OP. Given that the maximum follow-up period was approximately 7 weeks, the findings most likely represent symptoms of the


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RC (CI) 0.01 (-0.29, 0.29) 0.60 (0.33, 0.86) *RC = age-adjusted regression coefficient or difference from controls with 95% CI Significant threshold impairment was not detected in the index finger (regardless of poisoning agent or severity). Mean change difference in toe thresholds (i.e. difference in vibration thresholds between the two evaluations) was significantly higher in the intentional-poisonings subgroups compared to controls

Change in Threshold of Index Finger Big Toe

Controls (n=39), mean (SD) -0.06 log mic (0.23) -0.09 log mic (0.34) Occupational (severe) poisoning (n=10) Mean (SD) -0.08 log mic (0.29) -0.15 log mic (0.34) RC (CI) -0.02 (-0.19, 0.15) -0.05 (-0.33, 0.22) Intentional (severe) poisonings (n=5) Mean (SD) -0.06 log mic (0.14) 0.52 log mic (0.6) RC (CI) 0.04 (-0.19, 0.26) 0.60 (0.24, 0.97)

evaluation. Suitability of control group given pesticide exposure in some control subjects

intermediate syndrome or OPIDP, which is a well established sequale of acute OP intoxication (that arises 2-3 weeks after poisoning and often resolves within 6 to 12 months)

Miranda 2004 [PATIENTS HOSPITALISED FOR ACUTE OP POISONING, FOLLOWED UP AT: (I) HOSPITAL DISCHARGE; (II) APPROXIMATELY 7 WEEKS AFTER POISONING; (III) BETWEEN 24 AND 32 MONTHS (MEAN 28 MONTHS) AFTER POISONING]. Exposure Data: Erythrocyte cholinesterase activity in one severely poisoned patient (with no altered consciousness) was as previously described (no further information was provided). Health Outcome Data: [Muscle strength tests] {Non-neuropathic OPs}: Grip and pinch strength were slightly but not significantly lower than in the controls at examination I and II. At examination III grip strength had recovered and was not different from controls. However, pinch strength remained lower than controls for either category of poisoning (i.e. moderate/severe). {Neuropathic OPs}: Men with moderate poisonings appeared to follow a similar course as those noted for the non-neuropathic OP poisoning. Men with severe poisonings recovered part of their strength in examination III (especially grip strength), however, they remained significantly weaker than controls (p values not reported):

Grip strength (Kg) Pinch strength (Kg)

1st 2nd 3rd 1st 2nd 3rd Controls (n=28), mean (SD) 44.0 (6.2) 45.9 (6.6) 47.1 (6.3) 9.7 (2.1) 9.9 (1.9) 10.4 (1.8) Poisoned with neuropathic OPs Moderate poisonings (n=10) Mean (SD) 41.7 (8.3) 42.6 (6.3) 47.3 (7.1) 9.0 (1.5) 9.6 (1.4) 9.7 (1.9) Adjusted Difference (95% CI) -2.2

(-8.1 to 3.7) -3.1

(-8.2 to 2.0) -0.3

(-5.2 to 4.6) -0.6

(-2.0 to 0.9) -0.2

(-1.7 to 1.3) -0.8

(-2.0 to 0.4) Severe poisonings (n=5) Mean (SD) 39.3 (12.5) 40.3 (8.9) 47.6 (4.7) 9.4 (2.8) 9.2 (1.4) 9.4 (0.6)

Strengths Subjects received definite exposure to OPs. Uses a standardised and objective health. assessment method Adequate account of potential confounders. including assessment of co-exposures to other chemical agents. Accounted for non-participation in examinations II and III. Limitations Small sample size. Loss of control subjects at second and third follow-up evaluation. It is not clear

This study reported that sensory and motor impairments of the peripheral nerve (based on increased index finger vibrotactile thresholds and reduced hand strength) were apparent two years after subjects were severely poisoned with known neuropathic OP pesticides. The results show that some sensory and motor effects in men poisoned with OPs considered non-neuropathic, or moderate poisoning category of neuropathic OPs do resolve, which lends to symptoms consistent with the


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Adjusted Difference (95% CI) -4.7 (-12.3 to 3.0)

-5.6 (-12.3 to 1.0)

0.5 (-5.9 to 6.9)

-0.3 (-2.2 to 1.6)

-0.6 (-2.6 to 1.3)

-1.0 (-2.5 to 0.6)

Poisoned with neuropathic OPs Moderate poisonings (n=19) Mean (SD) 41.0 (7.8) 43.5 (6.4) 46.0 (8.1) 9.2 (1.7) 9.5 (2.1) 9.9 (1.8) Difference (95% CI) -3.0

(-7.8 to 1.7) -2.6

(-6.8 to 1.6) -0.8

(-4.8 to 3.1) -0.6

(-1.8 to 0.6) -0.4

(-1.6 to 0.8) -0.4

(-1.4 to 0.5) Severe poisonings (n=14) Mean (SD) 34.5 (8.6) 33.8 (7.4) 39.6 (5.5) 8.4 (2.0) 7.8 (2.4) 8.7 (0.8) Difference (95% CI) -9.6

(-14.7 to -4.5) -12.1

(-16.6 to -7.7) -7.1

(-11.4 to -2.9) -1.4

(-2.7 to -0.2) -2.0

(-3.3 to -0.8) -1.6

(-2.6 to -0.6) Results expressed as mean and SD, age adjusted regression coefficient (RC) with 95% CI Intra-individual mean changes in grip and pinch strength between examinations were not significantly different from controls. [Quantitative sensory threshold (QST) tests]: For vibrometry results, although thresholds for index finger and big toe were somewhat higher in study subjects than in controls for all categories of OP poisonings, these were significant for the index finger threshold of subjects with severe neuropathic poisoning at examination III (p values not reported).

Index finger (log microns) Big toe (log microns)

1st 2nd 3rd 1st 2nd 3rd Controls (n=28), mean (SD) 0.08 (0.27) 0.07 (0.25) 0.04 (0.21) 0.51 (0.33) 0.51 (0.22) 0.55 (0.26) Poisoned with neuropathic OPs Moderate poisonings (n=10) Mean (SD) 0.02 (0.36) 0.01(0.28) 0.08 (0.35) 0.47 (0.34) 0.49 (0.16) 0.56 (0.32) Difference (95% CI) -0.02

(-0.26 to 0.22) -0.04

(-0.21 to 0.14) 0.08

(-0.12 to 0.28) 0.00

(-0.23 to 0.24) 0.04

(-0.20 to 0.28) 0.06

(-0.16 to 0.27) Severe poisonings (n=5) Mean (SD) 0.11 (0.33) 0.02 (0.26) 0.12 (0.14) 0.60 (0.22) 0.67 (0.34) 0.70 (0.28) Difference (95% CI) 0.04

(-0.28 to 0.35) -0.05

(-0.27 to 0.18) 0.10

(-0.16 to 0.37) 0.10

(-0.20 to 0.41) 0.18

(-0.13 to 0.49) 0.16

(-0.12 to 0.43) Poisoned with neuropathic OPs Moderate poisonings (n=18) Mean (SD) 0.18 (0.43) 0.10 (0.24) 0.16 (0.25) 0.64 (0.35) 0.56 (0.41) 0.67 (0.21) RC (95% CI) 0.09

(-0.11 to 0.28) 0.02

(-0.12 to 0.17) 0.10

(-0.05 to 0.26) 0.12

(-0.07 to 0.31) 0.04

(-0.15 to 0.23) 0.11

(-0.06 to 0.28) Severe poisonings (n=14) RC (SD) 0.16 (0.24) 0.10 (0.14) 0.21 (0.28) 0.59 (0.29) 0.61 (0.48) 0.70 (0.43) Difference (95% CI) 0.07

(-0.14 to 0.17) 0.02

(-0.14 to 0.17) 0.19

(0.01 to 0.36) 0.07

(-0.14 to 0.27) 0.09

(-0.12 to 0.30) 0.13

(-0.06 to 0.32)

Intra-individual threshold mean changes in index finger and big toe vibrometry between examinations were not significantly different from controls

whether authors measure cholinesterase levels only in the control group and one severely poisoned patient or for all categories. Suitability of control group given pesticide exposure in some control subjects

pattern of OPIDP - a well established sequale of acute OP intoxication (that arises 2-3 weeks after poisoning and is known to resolve and/or persist for years). The authors noted that previous chronic OP exposures reported in some subjects did not appear to confound the results (as demonstrated by retrospective questionnaire evaluation)

Nakajima 1999 [VICTIMS OF THE MATSUMOTO SARIN ATTACK, FOLLOWED UP 3 WEEKS, 4 MONTHS, 1, 2 AND 3 YEARS AFTER THE SARIN INCIDENT]. NB. The authors evaluate symptoms reported after one and three years after the

Strengths Subjects received

This study reported an increased


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attack; the results of earlier/other surveys are reportedly published elsewhere: Nakajima et al, 1998. J Epidemol, 8:33-41. Health Outcome Data: [Questionnaire-based] {First year survey}: 58 sarin victims (28 men and 30 women) reported symptoms associated with sarin exposure, (mean age 45.9 years, SD ± 19.0); symptoms resolved in 260 sarin victims, mean age 48.5, SD ± 20.1). For 54 symptomatic subjects, 11 had been admitted, 26 were outpatients and 17 were non-patients. Significant differences in the percentage of symptoms were seen between admitted and outpatient groups .

Symptom Admitted patients (n=11)

Outpatients (n=26)

Non-patients (n=17)

Total (n=5

4) Asthenia (muscle weakness)

9(81.8)a** 5(19.2) 4(23.5) 18(33.3)b

* Shoulder stiffness 4(36.4) 9(34.6) 6(35.3) 19(3

5.2) Narrowing of visual field

1(9.1) 2(7.7) 0(0) 3(5.6)

Husky voice 0(0) 6(23.1) 2(1.8) 8(14.8)

aSignificant differences were noted between the group admitted and outpatients bSignificant differences were noted among the group admitted, non-, and outpatients *p<0.05; **p<0.01 Erythrocyte AChE activity (measured 4 weeks after the incident) was significantly lower in subjects reporting the following PN symptom one year after exposure (cf. subjects reporting no symptoms):

Symptom na E-AChE(IU/L)b Asthenia Yes = 9

No = 84 1.23±0.40 (p<0.01)

1.55±0.31 Shoulder stiffness Yes = 6

No = 87 1.32±0.36 1.54±0.32

Narrowing of visual field Yes = 1 No =92

1.4 1.52±0.33

Husky voice Yes = 3 No = 90

1.6, 1.5, 1.6 1.52±0.33

aNo. of subjects who had (yes) and did not have (no) symptoms bmean ±SD (or value when no’s were under 3)

definite exposure to an OP agent. Health examinations were performed (although it appears that this was not a consistently applied). Limitations Lack of unexposed controls. Background prevalence of sarin-related symptoms is unclear. Possible information bias of an overestimated risk of later sequelae in victim group due to over-recall of each symptom. Possible underestimation of risk due to lower questionnaire compliance in the victim group (36.1% vs. 54.5% in non-victim group). Clinical assessment of symptoms not fully described. No objective neurological tests used to validate

prevalence of chronic symptoms (asthenia, shoulder stiffness, and narrowing of visual fields) in victims one and three years after acute sarin poisoning.. In view of the various study limitations that includes the lack of objective data to verify the symptoms reported, this study provides very weak evidence of peripheral neurological symptoms in victims of Matsumoto sarin attack


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Postal code mapping of the location of subjects with symptoms showed that percentage of symptomatic subjects were greatest in the block where sarin was deliberately released (17.5%), followed by blocks north and north east; most victims were located in an elliptical area, with a 400m axis, north east of the site (data represented by illustration). {Three year survey}: The table below shows the number (and percentages) of all victims reporting peripheral neuropathy-related symptoms (incl. for ‘Victims A’ i.e. those with symptoms both one and three years after sarin incident) that were significantly greater than those reported in non-victims. The likelihood of these symptoms (i.e. asthenia, shoulder stiffness, and narrowing of visual fields) being reported (depending on whether subject was a victim or not) is significantly greater in victims than non-victims:

Symptom Non-victims 669 (%)

All victims 167 (%)

ORs (95% CI)

Victims A

ORs (95% CI)

Asthenia 11(1.6) 14(8.4) p<0.001 5.47 (2.44-12.29) 4 (2.4)

1.47 (0.47-4.64)

Shoulder stiffness* 25(3.7) 15(9.0) p<0.01 2.54 (1.31-4.94) 2 (1.2)

0.31 (0.08-1.23)

Narrowing of visual field

7(1.0) 6(3.6) p<0.05 3.52 (1.17-10.63) 1(0.6)

0.57(0.07-4.54)

Husky voice 7(1.0) 2(1.2) 1.15(0.24-5.57) 0 -

* Shoulder stiffness may be either a spinal or cranial nerve symptom as the inability to shrug shoulders can also be associated with a damaged accessory nerve. The authors also noted in their discussion the results of additional surveys plus clinical investigations, which found that three out of 31 people admitted were diagnosed to have sensory polyneuropathy with reducing nerve conduction velocity. The authors noted, however, that these victims were all included in the present survey.

health sequelae. Does not appear to account for possible exposure to other OP agents or medical conditions that could confound reported symptoms. Lack of further evaluation to confirm possible cranial nerve involvement in some non-specific symptoms i.e. “narrowing of visual field” and “husky voice”

7.A.3. Cross-sectional studies Jalali 2011

[PATIENTS WITH INTENTIONAL ACUTE ORGANOPHOPHOSPHATE POISONING] Exposure Data: Three patients were graded as having moderate poisoning and five with severe poisoning (three of which presented with intermediate syndrome). AChE activity was below normal level (4.2 U/ml) on admission and varied between 0.5 and 3.5 U/ml. The authors noted a highly significant negative correlation between AChE activity and severity of poisoning (p< 0.0001). Health Outcome Data: [Clinical evaluation?]: The authors summarised that all patients had sensory dysfunction, and motor dysfunction was present in 4 patients (one of which was exposed to chlorpyrifos). However, it is unclear whether this refers to electrophysiological tests only or is a general summary that includes earlier clinical outcomes. [EMG]: A sensory-motor peripheral polyneuropathy was identified that was predominantly a distal sensory deficit.

Strengths Subjects have known OP exposure Adequate control of potential confounders Electrophysiological testing of both nerves and muscle Limitations Small sample size

Patients poisoned with OPs displayed impaired peripheral nerve function particularly in the lower extremities. Electro-physiological tests showed greater sensory deficits. However, the lack of a dose response relationship together with the


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[NCV testing]: In the lower limbs, NCV (m/s) in the right and left tibial nerves were significantly lower than normal values (indicating an abnormality). NB. The authors did not discriminate motor from sensory NCV. The only other notable result occurred in the upper limb in which the NCV of the left ulnar nerve was significantly higher than the normal value (the authors are unable to explain this discrepancy):

Nerves Mean ± SD Normal value p value

R.Median 59 ± 6.07 56.7 ± 3.8 0.319 L.Median 59.4 ± 7.61 56.7 ± 3.8 0.375 R.Ulnar 64.4 ± 3.15 62.7 ± 5.5 0.128 L.Ulnar 65.6 ± 3.15 62.7 ± 5.5 0.034 R.Tibial 46.8 ± 9.16 54.9 ± 7.6 0.04 L.Tibial 44.6 ± 10.04 54.9 ± 7.6 0.023 R.Peroneal 47.4 ± 10.21 51.6 ± 4.1 0.294 L.Peroneal 48.7 ± 9.00 51.6 ± 4.1 0.137

Sensory latencies (ms) in each nerve were significantly higher in all eight patients compared to normal values (suggests a nerve abnormality):

Nerves Mean ± SD Normal value p value

R.Median 4.2 ± 0.71 2.4 ± 0.3 <0.0001 L.Median 3.77 ± 0.54 2.4 ± 0.3 <0.0001 R.Ulnar 3.23 ± 0.76 2.4 ± 0.3 =0.017 L.Ulnar 3.37 ± 0.63 2.4 ± 0.3 =0.003 R.Tibial 5.41 ± 0.83 3.5 ± 0.2 <0.0001 L.Tibial 5.22 ± 0.96 3.5 ± 0.2 =0.001 R.Peroneal 4.82 ± 0.75 2.9 ± 0.3 <0.0001 L.Peroneal 4.61 ± 0.64 2.9 ± 0.3 =0.004

The authors also report (graphically, were X = NCV(s), Y=sensory delay (ms)) that nerve dysfunction was significantly negatively correlated to sensory nerve latency (r=-0.558, r2=0.312 p< 0.0001) Wave amplitudes (mV) for all nerves were below normal values (which is suggestive of axonal loss):

Nerves Mean ± SD Normal value p value Motor Median 9.14 ± 1.86 13.2 ± 0.5 <0.0001 Ulnar 3.8 ± 1.4 5.8 ± 1.5 <0.0001 Tibial 2.3 ± 0.5 8.8 ± 3.4 <0.0001 Peroneal 2.1 ± 0.39 3.9 ± 1.2 <0.0001 Sensory Median 8.08 ± 3.2 15-50 *

Lack of controls (comparisons based on reference values) Subjects were tested after different time periods had lapsed since poisoning Expertise of the investigators performing tests is not reported

limitations of this study weaken the significance of these findings


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Ulnar 15.9 ± 3.56 15-50 * Tibial 6.02 ± 1.34 15-50 * Peroneal 7.3 ± 2.19 10.5 ± 6.1 <0.0001

*The authors reported that no p value could be calculated due to lack of a mean reference value Lower limbs were significantly more impaired than the upper limbs as indicated by the lower motor NCV and higher sensory nerve latencies (p<0.0001):

Limb Motor NCV mean (SD) m/s

Sensory nerve latency mean (SD) ms

Upper 62.1 (3.38) 3.35 (0.47) Lower 55.77 (5.86) 4.33 (0.64)

There was no statistically significant correlation between clinical severity or AChE activity and electrophysiological changes (no p-value reported)

McCauley 2001 [US TROOPS DEPLOYED IN KHAMISIYAH AND EXPOSED TO SARIN/CYCLOSARIN] Exposure Data: The authors noted that 50 of the 653 KHAM subjects reported that they had never been in Iraq (which the authors suggested questions the status provided by the US Department of Defence). Furthermore, the authors reported that at least 53 of the 610 N-KHAM subjects claim to have been within 50-km of Khamisiyah to which 15 reported being involved in or watching detonations Health Outcome Data: [Telephone interview-based survey]: {Symptoms experienced during the first 2 weeks after the Ground War}: No difference was found in reported symptoms between the Khamisiyah (KHAM) and non-Khamisiyah (N-KHAM) deployed groups when adjusted for confounders. Comparison between the two KHAM subgroups showed that KHAM witness subgroup were more likely to significantly report the following spinal and cranial nerve peripheral neuropathy related symptoms (among others) compared to KHAM non-witness subgroup (except for slurred speech which did not reach statistical significance):

Symptom Khamisiyah Witness (n=162)

Frequency (%)

Khamisiyah Non-witness (n=405)

Frequency (%)

OR (95% CI)**

Vision problems* 14.2 6.7 2.4(1.3 – 4.2) Muscle twitching 20.4 10.6 2.0(1.2 – 3.3) Muscle cramping/weakness 27.8 10.6 2.1(1.4 – 3.3) Tingling of hands/feet 23.4 11.1 2.5(1.5 – 4.0) Vertigo 9.3 3.6 2.71.3 – 5.8) Slurred speech 4.9 2.5 1.8(0.7 – 4.9) Hoarse voice 17.3 8.4 2.5(1.4 – 4.3)

Strengths Good sample size. Attempts to stratify exposure level and measure possible associations with reported health symptoms. Highlights US government’s possible misclassification of potential chemical warfare agents (CWA) exposure status in KHAM group subjects. Limitations Exposure assessment based on crude measures of exposure. Lacks objective health assessment. Possible information bias (since all KHAM

This study presents findings which suggest the development and persistence of peripheral neuropathy (PN) symptoms in individuals who witnessed/involved in the Khamisiyah detonations. This subgroup were almost twice as likely to report experiencing sensory symptoms both during the 2-week period in which the detonations occurred, and eight years after. However, in view of the possible sources of bias introduced into the study and the


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*This is too vague a symptom to be categorised within the cranial nerve category ** Adjusted for age and gender. {Current health symptoms}: Deployed veterans (KHAM and N-KHAM) reported significantly higher rates of nearly all health symptoms including those related to peripheral neuropathy (shown below) compared to non-deployed (ND) veterans. Furthermore, the KHAM witness subgroup showed significantly increased reports of ‘tingling and burning sensation of the skin’ (among other non PN-related symptoms) compared to the KHAM non-witness subgroup. NB. Previously reported symptoms suggestive of cranial nerve dysfunction were not among the current health symptoms reported.

Symptom Deployed (n=1263) vs

Non-deployed (n=516) OR (95% CI)*

Khamisiyah Witness (n=162) vs.

Khamisiyah Non-witness (n=405) OR (95% CI) †

Tingling, burning, sensation of pins and needles 2.2(1.6-3.2) 1.7(1.1-2.8) Numbness or lack of feeling 2.4(1.7-3.4) 1.4(0.9-2.2) Loss of muscle strength in arms and legs 3.4(2.4-4.9) 1.4(0.9-2.2) Loss of balance or coordination 2.8(1.8-4.3) 1.4(0.8-2.4) Cramping, aches, pains or stiffness of muscles 2.8(2.2-3.8) 1.4(1.0-2.1) - borderline

* Adjusted for age, gender and region of residence †Adjusted for age and gender.

subjects were informed by the Dept of Defence that they had potentially been exposed to low levels of chemical warfare agents (CWA), and US army training materials refer to symptoms associated with CWA exposure). Possible recall bias (since the Kham witness subgroup would be more interested in the study and differ in their ability to recall symptoms). Possible selection bias (i.e. sample may not be representative of the entire population of troops serving in the GW, since the sample was limited to individuals whose telephone numbers could be tracked using common search mechanisms; extensive tracking measures were not used). Potential for the differences in health symptoms reported during the

limitations associated with the exposure and health assessment, these findings should be interpreted with caution


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detonations and those currently experienced to be associated with differences in work exposures and not specifically the detonations themselves (due to differences in GW job codes). Non-specific exposures to chemical agents among deployed veterans


2002 [US DESERT STORM VETERANS DEPLOYED IN KHAMISIYAH AND EXPOSED TO SARIN/CYCLOSARIN] Health Outcome Data : [Telephone interview-based survey]: The authors calculated the likelihood (ORs) for reported medical conditions diagnosed by a physician since the Gulf War. Deployed troops [Khamisiyah subgroup and Non-Khamisiyah subgroup] were significantly more likely to report a diagnosis of several medical conditions including slipped disk or pinched nerve (OR=1.5, 95%CI (1.1-2.0) compared to non-deployed troops. NB. The authors considered that the reliability of this diagnosis was moderate (as indicated by the test-retest reliability coefficient (k=0.59)) – no explanation was provided re: how this coefficient was generated). However, the frequency of self-reported medically diagnosed peripheral neuropathy did not differ significantly between these groups OR=1.2, 95%CI(0.6–2.4). Hospitalisation rates among deployed and non-deployed troops did not differ.

Strengths 76.7%% participation rate. Addressed possible selection bias by comparing veterans with contactable telephone no’s with those who were unavailable or untraceable. Evaluated the reliability of self-reported medical diagnoses Limitations Sample not representative of the entire population of troops serving in the GW due to sample limited to individuals whose telephone numbers

This study does not provide any evidence of increased prevalence of peripheral-neuropathy related symptoms in Khamisiyah-based veterans exposed to sarin


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could be tracted using common search mechanisms. Exposure assessment based on subject’s deployment status, which was later shown to be subject to possible misclassification Lack of precise characterisation of exposure. Symptoms recalled eight years after the event No validation of self-reported medical diagnoses

Miyaki 2005

[SARIN-EXPOSED SUBWAY WORKERS OF THE 1995 TOKYO SARIN ATTACK FOLLOWED UP 7 YEARS AFTER THE INCIDENT] Exposure data: 5 exposed subjects were assigned to the high exposure group and 18 exposed subjects were assigned to the low exposed group. Health Outcome Data: [Stabilometry]: The authors noted that the stabilometry tests did not show any exposure related changes.

Subway workers Referents p for trend

Exposed High (n=5) Low (n=18) (n=52) Mean SD Mean SD Mean SD Mean SD Stabilometry (eyes open) Total length (cm)a 79.6 1.37 77.0 1.44 80.3 1.36 80.7 1.23 ns X length (cm)a 55.6 1.41 51.1 1.45 56.9 1.41 57.5 1.24 ns Y length (cm)a 44.5 1.38 46.3 1.48 44.0 1.36 43.9 1.30 ns Sway area (cm)a 2.93 1.50 2.90 1.58 2.94 1.50 2.78 1.48 ns Stabilometry (eyes closed)

Strengths Attempts to assess a dose-response relationship. Exposed groups received definite exposure to sarin (albeit to varying degrees) Adequate control of confounders. Used an objective health assessment . Health assessment was blinded to subjects exposure status Limitations Small sample size. Fails to evaluate

This study does not report any convincing data to suggest any link between sarin exposure and chronic peripheral neurological impairment in subway workers exposed at the time of the sarin attack


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Total length (cm)a 105.3 1.35 107.5 1.46 104.6 1.33 116.7 1.27 ns X length (cm)a 72.4 1.36 74.9 1.53 71.7 1.33 83.0 1.34 ns Y length (cm)a 60.8 1.35 60.9 1.34 60.8 1.37 63.6 1.27 ns Sway area (cm)a 3.84 1.49 3.02 1.40 4.13 1.48 4.46 1.57 0.057

ns = non-signficant This was also apparent when the data was combined with those of rescue staff and police officers of a previous study (Nishiwaki 2001).

possible selection bias from non-participating subjects Severity of exposure based on hospitalisation admission status obtained from questionnaire No objective exposure assessment conducted

Nishiwaki 2001 [SARIN-EXPOSED RESCUE TEAM STAFF MEMBERS AND POLICE OFFICERS OF THE 1995 TOKYO SARIN ATTACK FOLLOWED UP BETWEEN 34 – 45 MONTHS AFTER THE INCIDENT] Exposure Data: 25 exposed subjects were assigned to the high-exposed group and 29 exposed subjects assigned to the low-exposed group. Health Outcome Data: [Quantitative Sensory Threshold (QST) tests]: The authors noted that none of the parameters for the vibration perception thresholds had any relation to exposure

Exposed Referents High

(n=25) Low (n=29) (n=52)

Mean SD Mean SD Mean SD Vibration perception thresholds Right index finger (dB 63 Hz 3.8 7.1 4.1 4.7 3.0 6.0 125 Hz -1.5 7.4 -1.6 5.8 -2.1 5.9 250Hz 3.2 9.1 4.0 5.8 2.2 7.1 Right middle finger (dB) 63 Hz 4.4 7.4 3.4 7.2 1.4 7.4 125 Hz -1.3 5.7 -1.6 7.1 -2.7 6.9 250Hz 4.5 7.8 3.3 5.4 2.5 8.6 Left index finger (dB 63 Hz 4.9 5.5 2.6 6.4 2.0 7.5 125 Hz -0.9 6.2 -3.6 6.3 -3.5 7.0 250Hz 2.8 7.4 1.2 5.6 0.8 8.3

Strengths Attempts to assess a dose-response relationship. Exposed groups received definite exposure to sarin (albeit to varying degrees) Adequate control of confounders. Used an objective health assessment of sensory function Health assessment was blinded to subjects exposure status Limitations Small sample size. Severity of exposure based on hospitalisation admission status obtained from questionnaire (which does not account for

This study does not report any convincing data to suggest any link between sarin exposure and chronic peripheral neurological impairment in rescue workers and police who arrived at the scene of the sarin attack.


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Left middle finger (dB) 63 Hz 1.8 6.1 2.9 7.0 0.6 8.9 125 Hz -2.6 6.9 -2.4 6.2 -2.3 7.6 250Hz 2.0 7.4 0.9 6.3 1.3 9.5

[Stabilometry]: For the stabilometry the authors noted that total length, and X length with eyes open in the low-exposed group were significantly larger than those in the referent group even after adjustment. However, no dose-effect relationship was observed.

Exposed Referents High

(n=25) Low (n=29) (n=52)

Mean SD Mean SD Mean SD Stabilometry (eyes open) Total length (cm)a 84.3 1.4 96.7 1.2*,**,# 83.5 1.2 X length (cm)a 58.2 1.4 68.3 1.2*,**,# 57.3 1.2 Y length (cm)a 48.6 1.4 53.6 1.3 48.3 1.3 Sway area (cm)a 3.4 2.0 3.7 1.4 3.4 1.5 Stabilometry (eyes closed) Total length (cm)a 111.7 1.4 137.1 1.3* 114.5 1.3 X length (cm)a 75.2 1.4 91.6 1.4* 76.3 1.4 Y length (cm)a 66.3 1.5 81.4 1.4* 68.5 1.3 Sway area (cm)a 4.1 2.0 4.7 1.6 4.4 1.5

aGeometric mean and GSD (geometric standard deviation) *p<0.05 in Student’s t-test or Welch’s method compared to referents **p< 0.05 for variable of group (dummy) in multiple regression analysis; IES (Impact of Event Scale) score was included as psychometric variable #p<0.05 for variable of group (dummy) in multiple regression analysis; the GHQ score was included as psychometric variable instead of the IES score

possible differences in susceptibility to anticholinesterase effects due to PON-1 polymorphisms). No objective exposure assessment conducted. Does not provide complete quantitative sensory testing (e.g. no thermal threshold assessment). QST is not a completely objective sensory test

Spencer 2001 [US DESERT STORM VETERANS DEPLOYED IN KHAMISIYAH AND EXPOSED TO SARIN/CYCLOSARIN] Health Outcome Data : [Telephone-based interview survey]: {Acute symptoms} During the time period of the Khamisiyah detonations no symptom differences were evident between participants in the US Dept of Defence’s broadly defined Khamisiyah group and troops outside the defined area. However, differences were found between the small no. of individuals who witnessed detonations and others in the larger Khamisiyah group: 16 symptoms were reported in excess and were mostly mild immediate responses to low doses of OPs. The authors did not provide further information but refer readers to McCauley et al 2000. NB. No articles were published by McCauley in 2000 - it is likely that this was an editorial error and Spencer 2001 intended to cite an appended manuscript as explained

Strengths 76.7%% participation rate. Addressed possible selection bias by comparing veterans with contactable telephone no’s with those who were unavailable or untraceable. Used intensive

The positive peripheral neuropathy-related findings of this study (i.e. that deployed troops were more likely to report being medically diagnosed with a ‘slipped disk or pinched nerve’ cf. non-deployed


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below. {Current symptoms} The following information in parentheses was abstracted from ‘McCauley et al, under review’ – which was attached to the current study. A subsequent Pubmed search revealed that this manuscript has been published as ‘McCauley et al (2002). Illness Experience of Gulf War Veterans Possibly Exposed to Chemical Warfare Agents. Am J Prev Med; 23(3):200-6’: (Nine years following the Gulf War, deployed troops [Khamisiyah subgroup and Non-Khamisiyah subgroup] were significantly more likely to report a diagnosis of several medical conditions including slipped disk or pinched nerve (OR=1.5, 95%CI (1.1-2.0) compared to non-deployed troops. However, the frequency of self-reported medically diagnosed peripheral neuropathy did not differ significantly between these groups OR=1.2, 95%CI(0.6–2.4). Hospitalisation rates among deployed and non-deployed troops did not differ. [Clinical/Neurophysiological] Neurological and neuro-physiological examinations revealed no group differences among the three groups. The authors further report that within the group receiving examinations, the 7 veterans who witnessed Khamisiyah had similar neurological functioning to that of others who received clinical examinations. Lower and upper limb latencies for somatosensory evoked potentials were similar for veterans from each deployment group, although the authors noted that there was a definite age effect (p-value = 0.006 and 0.007 for lower and upper latencies)

telephone tracking systems to minimise possible selection bias. Adopted a two-tiered clinical assessment of reported symptoms (which included assessment of neurophysiological symptoms). Limitations Exposure assessment based on subject’s deployment status. Lack of precise characterisation of exposure. Small number of participants in the Level II neurophysiological tests. Level II testing was designed to test for signs of OP-associated persistent CNS damage. Symptoms recalled nine years after the event

troops) are limited by the fact that the frequency of self-reported medically diagnosed peripheral neuropathy was not significantly different between groups. More importantly, these findings were not specific to Khamisiyah-based troops and neurological and neurophysiological examinations did not reveal any group differences among the groups

Stallones & Beseler

2002b [FARM RESIDENTS EXPOSED TO OPS AND OTHER PESTICIDES] Health Outcome Data: [Questionnaire-based] Analyses of the odds for reporting re-occurring neurological symptoms in those who experienced a pesticide-related illness showed a significantly increased association between previous poisoning and current ‘difficulty moving fingers

Strengths Addressed possible selection bias with regards to type of farming operation used by those who refused and those who

The finding that farm operators who reported having experienced a pesticide-related illness had a 2-fold increased likelihood of


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and grasping things’ (OR=2.08[95% CI:1.06-4.09). Other peripheral neuropathy (PN)-related symptoms failed to reach statistical significance i.e. lack of coordination or loss of balance (OR=2.34, 95% CI(0.97-5.65); loss of muscle strength in legs and feet (OR=0.98, 95% CI(0.40-2.43) or arms and hands (OR=1.36, 95% CI(0.67-2.74), numbness or tingling in fingers lasting more than a day (OR=1.16, 95% CI(0.34-3.99) or toes (OR=2.61, 95% CI:1.00-6.78) although the authors imply that the latter was borderline (symptoms classified as being significantly different are reported as having CIs that do not include the value 1.0). The χ2 test for trend showed that ‘loss of muscle strength in legs or feet’ and ‘difficulty moving fingers or grasping things’ (among other non-PN related symptoms) were significantly associated with having a pesticide related illness (χ2 4.37, P = 0.0365 and χ2 11.40, P = 0.0007 respectively). In the remaining analyses, the authors did not provide data for associations with specific PN-related symptoms, but rather present data on the odds of reporting an acute pesticide-related illness with regards to the type of pesticide used and the method of pesticide application

took part in the study. Limitations 57% response rate. Exposure assessment based on self-reports. Exposure and health outcome analyses is not specific for OP exposure. Lacks objective measures of neurological symptoms (health outcome based on self reports). Lack of verification from physicians of pesticide poisonings. Reference population unclear. Authors noted that time sequence of exposure and outcome was flawed: pesticide poisoning reflected cumulative lifetime incidence while exposure information came from the previous year; the time frame for the neurological symptom assessment did not match that used to assess current

reporting ‘having difficulty moving fingers and grasping things’ should be considered with caution. This is because the above finding is not specific for OP exposure as subjects had co-exposure to other non-OP pesticides. Furthermore, the study failed to use objective measures of neurological assessment; lacks any physician-verified pesticide diagnosis; used crude/subjective measures of OP exposure, and had flawed time frames for both exposure and neurological symptom assessment. Therefore this study provides very limited evidence for the development of chronic peripheral neuropathy in acutely OP exposed individuals


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exposure. Co-exposures to other pesticides

7.A.4. Case series studies Dahlgren 2004 [FAMILY ACCIDENTALLY EXPOSED TO DIAZINON]

Exposure Data: A urine sample test was negative for diazinon parent compound. Wipe samples analysed ranged from 0.0048-0.9932 µg/cm2 in June 1999 and 0.2-1.1 µg/cm2 in Jul 1999 to Feb 2000. Air sampling data detected diazinon at concentrations of 1.9 µg/cm3 in the living room, 10 µg/cm3 in the playroom, and 2.7 µg/cm3 in the kitchen in Jul 1999 and 0.0037-0.0183 mg/m3 in Jul 1999 to Feb 2000. Bulk carpet samples ranged from 255 mg/kg (ppm) 24 days after exposure and 1.18-4.72 mg/kg (ppm) 6.5 months after exposure. Follow-up testing was positive for diazonin in two of 11 air, bulk and wipe samples (0.0004645 µg/cm2) for up to 8 months after exposure. Health Outcome Data: [Clinical examination] In addition to acute symptoms experienced soon after exposure, patients also experienced chronic symptoms evident after a 3-year follow-up, particularly in the children. Signs relevant to peripheral neuropathy are shown below, none of which arose in the adults. The authors did not report the results of the grip strength tests. The authors concluded that children are more susceptible to the effects of acute OP exposure. Chronic PN/NMD signs (clinical evaluation conducted 3 years later)

Patient 1 (Mother)

Patient 2 (Father)

Patient 3 (Daughter aged 11y at exposure)


Patient 5 (Son aged 6y at exposure)


Patient 7(Son in-u5m gestaexposure

CNS - - Episode of numbness on right hand and face, prolonged sway speed

Prolonged sway speed

Prolonged sway speed

- -

Muscular-skeletal

- - - - - - Weak tonoral musculat

Respiratory - - - - - - - GI - - - - - - - Eyes - - Abnormal colour

vision Abnormal colour discrimination

Abnormal colour discrimination

- -

Skin - - - - - - -

Strengths Relatively long follow-up period. Comprehensive assessment of external exposure levels. Limitations Biological measure of exposure based on testing diazinon parent compound in urine. No testing for diazinon metabolites or cholinesterase levels soon after exposure. Fails to report the results of grip strength tests

This study does not provide quantitative data in relation to any peripheral neuropathy symptoms. The authors do report a symptom of relevance although these were experienced by children. It is not possible to determine whether the adults experienced chronic peripheral neuropathy as a result of accidental acute exposure to diazinon based on the information provided


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No symptoms indicated by (-)

7.A.5. Case reports Loh 2010 [ARMY SERGEANT EXPOSED TO SARIN FROM AN IMPROVISED EXPLOSIVE DEVICE]

Exposure Data: The authors report that the subject had an erythrocyte cholinesterase activity of 2.88 U/ml (0.46 δ pH unit/h), which represented a 39% depression from a baseline of 0.75 δ pH unit/h drawn prior to deployment to Iraq. This returned to normal one month after exposure. Health Outcome Data: [Clinical neurological examination] The subject’s acute symptoms that occurred several minutes after exposure to sarin included confusion, dyspnea (shortness of breath), blurred vision, and a severe headache. The subject complained of episodic dyscoordination and imbalance when being tested several months after the incident. The authors reported that the subject’s cranial nerve examination was unremarkable, as was the motor, reflex sensation, and gait examinations. [Electrophysiological examinations] The subject also had normal electromyography and nerve conduction study results. No further information was provided

Strengths Conducted objective clinical examinations Partial attempt to consider potential confounders Limitations Lack of detail on the neurological tests conducted and the level of clinical expertise of the examiner No indication of the sarin external dose to which the sergeant was exposed

No evidence of chronic peripheral neuropathy-related outcomes

Soummer 2011 [SUICIDE ATTEMPT IN A 24 YEAR-OLD MALE VIA SUBCUTANEOUS INJECTION OF CHLORPYRIFOS] Exposure Data: The authors noted that there were persistent reports of a pesticide-like odour coming from the patient. Plasma butyrylcholinesterase activity remained low (<500 IU/L, N:8000-18,000) until day 60 and increased slightly on day 100 (2134 IU/L) suggesting prolonged poisoning. Low RBC AChE activity (1138 IU/L, N:2800-5200) on admission normalised by day 60. Chlorpyrifos was still detectable in the serum on day 20. Urine metabolite concentrations of TCP, DEP and DETP were elevated on day 30 and reached values of occupational exposure on day 50 and normalised by day 1000. The concentration of chlorpyrifos in the forearm muscle was measured at 44 ug/kg on day 50 Health Outcome Data: {Day 50} - electrophysiological testing of proximal muscles yielded results that were suggestive of an Intermediate Syndrome-like pattern; denervation of distal muscles was suggestive of OPIDN. {Day 120 (hospital discharge) }– the subject remained tetraparetic (weakness in all limbs), although proximal muscle testing exhibited some improvement (scoring: 4/5) and distal muscle testing showed persistent severe deficits (scoring: 0/5). Electrophysiological testing revealed increased amplitudes of proximal muscle contractions with disappearance of 20 Hz-decremental responses. Distal upper limb muscles improved with signs of reinnervation, while distal lower limb

Strengths Comprehensive assessment of internal dose of OP exposure Limitations Provided minimal information on electrophysiological tests It is unclear the extent to which coadministration of psychoactive drugs may have affected the clinical

This study provides evidence of chronic peripheral neuropathy in a patient acutely poisoned with chlorpyrifos. However, the strength of this finding is limited by the subjects co-adminstration of two psychoactive drugs which may have impacted on the manual strength tests (which are


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muscles remained in a fibrillatory status with typical denervation. {One year follow-up} – persistent severe muscle weakness was observed which the authors reported as being suggestive of irreversible OP-related peripheral neuropathy. The authors did not elaborate further on whether this was assessed manually or electrophysiologically. NB. The authors considered that the course of the patients symptoms/signs (i.e. delayed cholinergic phase, prolonged coma and prolonged neurological toxicity with overlapping electrophysiological phases suggestive of intermediate syndrome and distal neuropathy) were extreme and severe. They suggested this may be due to there being a prolonged and continuous release of chlorpyrifos from the body fat and forearm muscles which acted as reservoirs

outcomes subjective)


7>B.1. Cohort studies Albers 2004b [CHLORPYRIFOS MANUFACTURING WORKERS, FOLLOWED-UP ONE YEAR AFTER BASELINE EXAMINATIONS]

Exposure Data: Study subjects had a mean duration of work in chlorpyrifos exposed areas of 9.72 years, a historic cumulative chlorpyrifos exposure of 64.16 mg/m3*days, and an interim cumulative chlorpyrifos exposure of 6.13 mg/m3*days. Urine TCP/Cr was much higher than controls 192.2 µg/g-Cr, and the average BuChE activity was 7155 mu/mL. All these measures were significantly different to controls (P<0.0001). AChE levels at baseline and second examination were 6923 and 7149 mu/mL respectively. This was not significantly different to controls. For control subjects the mean duration of work in chlorpyrifos exposed areas was 0.10 years, and referent subjects had a historic cumulative chlorpyrifos exposure of 0.69 mg/m3*days, an interim cumulative chlorpyrifos exposure of 0.00 mg/m3*days, and urine TCP/Cr of 6.2 µg/g-Cr. The average BuChE activity was 8183 mu/mL, and AChE levels at baseline and second examination were 6966 and 7253 mu/mL respectively. Health Outcome Data: NB. Although sensory and motor tests were conducted as part of the neurological assessment (questionnaire and clinical evaluation), these results were used to develop specific ratings for the subsequent analyses of CNS dysfunction. Applicable results (which incidentally did not reach statistical significance) were as follows: [Questionnaire] At baseline, two subjects (1 chlorpyrifos, 1 referent; P > 0.99) reported intermittent symmetric sensory symptoms, which disappeared at the second evaluation. At baseline, two subjects (1 chlorpyrifos, 1 referent; P > 0.99) reported a problem with balance (1 as a child and the other as a transient difficulty lasting 1 day). No subject reported a change in balance or gait since baseline. No subject reported persistent, bilateral extremity weakness at either evaluation. [Clinical examination] The neurological results at baseline and at the second examination were generally comparable with the exception of the frequency at which palmomental reflexes were identified. However, this was not significant (see below): Comparison of clinical examination results relevant to the CNS evaluation for chlorpyrifos (CPF) and referent subjects at

Strengths Uses investigators/ personnel blinded to individual subject’s group status and exposure history Good follow-up response rate (80% and 98%). Demonstrated the lack of acute OP poisoning in chlorpyrifos-workers (due to similar AChE levels evident in both groups). Uses historical exposure data in a population of workers that enable reliable exposure measurements. Uses validated diagnostic tests to confirm reported symptoms.

This study evaluates disorders derived from CNS neuropathologies i.e. neuropsychological abnormalities and parkinsonism due to dysfunction within different areas of the CNS (cortical, pyramidal tract and extrapyramidal). It does not report on peripheral neuropathy (PN) or neuromuscular dysfunction (NMD) except in relation to findings used to develop specific ratings for subsequent CNS analysis, which themselves were either unremarkable or non-significant.


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baseline (CPF n= 53; referent n= 60) and second evaluations (CPF n= 53; referent n= 58) Measure Baseline No. P-value* Second No. P-vaCranial nerves Impaired eye movements CPF

Referent 0 0

CPF Referent

0 0

Abnormal nystagmus CPF Referent

0 0

CPF Referent

0 0

Motor system** Proximal weakness CPF

Referent 0 0

CPF Referent

0 0

Increased tone CPF Referent

0 0

CPF Referent

0 0

Abnormal station or gait CPF Referent

0 0

CPF Referent

0 0

Muscle stretch reflexes** Pathologically increased (4+ at knees or ankles)

CPF Referent

0 0

CPF Referent

0 0

Primitive and atavistic reflexes** Palmomental CPF

Referent 7 5

0.54 CPF Referent

12 13

>0.9

Chaddock CPF Referent

0 0

CPF Referent

0 0

Babinski CPF Referent

0 0

CPF Referent

0 0

*Fisher’s exact two-tailed test **Motor or reflex signs in italics were used in the determination of clinical encephalopathy

Limitations Small sample size. Does not account for healthy worker effect

Therefore, there is no evidence for PN/NMD as the evaluation is too limited to draw conclusions

Albers 2004a [CHLORPYRIFOS MANUFACTURING WORKERS, FOLLOWED UP ONE YEAR AFTER BASELINE EXAMINATIONS] Exposure Data: Chlorpyrifos workers had a historic cumulative chlorpyrifos exposure of 64.16 mg/m3*days, and worked in exposed areas for a duration of 9.72 years, with an interim chlorpyrifos exposure of 6.13 mg/m3*days. Measures of urine TCP/Cr were 192.2 µg/g-Cr with an average BuChE of 7155 mu/ml over the study year. Subjects had an estimated chlorpyrifos exposure of approx. 576-627 µg/day. AChE activities were similar to referents at both examinations. Referents had a historic cumulative chlorpyrifos exposure of 0.69 mg/m3*days, and worked in exposed areas for a duration of 0.01 years, with an interim chlorpyrifos exposure of 0.00 mg/m3*days. None of the referents had any identifiable exposure to chlorpyrifos in their jobs during the period of observation. Measures of urine TCP/Cr were 6.2 µg/g-Cr with an average BuChE of 8183 mu/ml over the study year. AChE activities were similar to chlorpyrifos workers at both examinations Health Outcome Data: [Questionnaire]

Strengths Subjects had measurable external and biological exposure to an OP (chlorpyrifos) with no occupational exposure to other OP insecticides or other potential neurotoxicants. TCP daily excretion measures and AChE activities

This study of peripheral neuropathy in chemical workers exposed to chronic low doses of chlorpyrifos does not appear to support an association. However, the one-year follow-up period may have compounded detection of clinical (and possibly


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No subject reported persistent sensory or motor symptoms suggestive of neuropathy at baseline, although intermittent symptoms of stocking or stocking-glove distribution were reported in one subject from each group, which subsided at the one year evaluation. No subject reported persistent extremity weakness at either evaluation. [Clinical neurological examination] A few neurological signs of peripheral neuropathy were identified (i.e. symmetrical sensory signs of numbness or tingling, vibration, touch pressures and decreased or absent ankle reflexes), which were not significantly different between each group (although at the one year examination, 5 referents had symmetrical sensory signs and diminished vibration sensation cf. none in the chlorpyrifos group (p=0.06)). [Nerve conduction studies] No significant group differences for any measure at baseline were apparent. At the one-year evaluation, there was a borderline significant group difference for peroneal motor distal latency, which was slightly prolonged among chlorpyrifos subjects cf. referents (4.9 v 4.6 ms, p=0.07). For the change over one-year, there were borderline significant group differences in the median motor and sural sensory amplitudes (i.e. 1.5 v 0.6 mv, p=0.08 and 2.1 v 0.1µv p=0.09 respectively) with a larger increase (indicating better performance) among chlorpyrifos subjects cf. to referents.

Group n One year Change Mean (SD) p value* Mean (SD) p value*

Median sensory Amplitude (μv) CPF 53 32 (12.8) 0.52 2.1 (3.8) 0.91 Referent 58 33 (13.6) 2.0 (5.2) Terminal CV (m/s) CPF 53 52.9 (7.0) 0.74 −1.9 (4.2) 0.93 Referent 58 53.3 (6.7) −1.� (4.5) Distal latency (ms) CPF 53 3.5 (0.5) 0.69 0.2 (0.22) 0.84 Referent 58 3.5 (0.5) 0.2 (0.21) Ulnar sensory Amplitude (μv) CPF 53 30 (12.3) 0.68 3.0 (6.0) 0.65 Referent 58 31 (14.5) 3.6 (7.1) Terminal CV (m/s) CPF 53 55.3 (5�5) 0.75 −2.2 (4.4) 0.71 Referent 58 54.9 (5.2) −2.5 (4.2) Distal�latency (ms) CPF 53 3.3 (0.3) 0.97 0.1 (0.2) 0.80 Referent 58 3.3 (0.3) 0.1 (0.2) Sural sensory Amplitude (μv) CPF 53 20 (�.2) 0.20 2.1 (6.2) 0.09 Referent 58 18 (6.8) �.1 (6.4) Terminal CV (m/s) CPF 53 48.6 (4.7) 0.34 −1.8 (5.1) 0.70 Referent 58 47.7 (4.7) −2.2 (4.1) Distal latency (ms) CPF 53 3.7 (0.3) 0.50 0.2 (0.3) 0.82 Referent 58 3.7 (0.3) 0.2 (0.3) Median motor Amplitude (mv) CPF 53 10.9 (�.2) 0.81 1.5 (2.7) 0.08

indicated that chlorpyrifos subjects were exposed to low doses (daily chlorpyrifos exposure estimate was approximately three orders of magnitude higher than estimates in household exposures derived from a similar study, and 30% (range 0-250%) of that received by a typical subject exposed during a working day at the permissible exposure level of 200 µg/m3; also, AChE activities did not differ significantly between groups or at the one year evaluations). Adequate account of confounders. Authors controlled for potential factors associated with employment in chemical industry via use of referents working in saran manufacturing. Investigators were blinded as to the individual subject’s group status and their exposure

subclinical) peripheral nerve health effects


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Referent 58 10.7 (3.4) 0.6 (2.5) Forearm�CV (m/s) CPF 53 55.5 (3.9) 0.63 −1.2 (3.4) 0.18 Referent 58 55.1 (3.6) −2.0 (3.5) Distal latency (ms) CPF 53 3.8 (0.6) 0.57 0.3 (0.3) >0.99 Referent 58 3.7 (0.6) 0.3 (0.�) F wave latency (ms) CPF 53 28.3 (2.1) 0.92 0.5 (0.9) 0.69 Refer�nt 58 28.3 (2.5) 0.6 (1.4) Peroneal motor Amplitude (mv) CPF 53 6.3 (2.7) 0.83 −1.0 (2.1) 0.81 Referent 58 6.4 (2.4) −1.1 (1.7) Distal latency (ms) CPF 52 4.9 (0.8) 0.07 0.2 (0.6) 0.18 Referent 58 4.6 (0.6) 0.1 (0.3) F wave laten�y (ms) CPF 50 49.2 (3.8) 0.22 0.7 (1.9) 0.18 Referent 58 50.3 (5.2) 1.4 (2.9)

*t test for difference in means (adjusted for unequal variance between groups). CV, conduction v�locity.

The number of subjects fulfilling any of the 4 different diagnostic categories of neuropathy were not significantly different between groups at either evaluation. The odd ratios for developing symptoms (or conventional clinical signs) of neuropathy did not differ significantly among chlorpyrifos subjects at either evaluation cf. referent group at baseline. Similarly, the odd ratios for developing any diagnosable level of peripheral neuropathy were not significantly increased among chlorpyrifos subjects at either examination cf. referent group at baseline. Outcome No.* OR† OR (group) (CPF at baseline) OR (time) (referent at one year) OR (group*time) (CPF at one year)Neurological evaluation Symptoms of neuropathy‡ 2 1.00 1.13 (0.01, 90.55) 1.03 (0, 40.34) § Symmetrical sensory signs‡ 12 1.00 0.84 (0.12, 5.24) 1.32 (0.27, 7.01) 0.29 (0, 113.48) Trace or absent ankle reflexes 13 1.00 0.55 (0.10, 3.13) 1.32 (0.82, 2.12) 0.55 (0.10, 3.��) Diagnostic categories¶ Subclinical neuro�athy 4 1.00 1.13 (0.07, 18.60) 1.04 (0.06, 17.37) 1.13 (0.70, 18.60) Possible neuropathy 19 1.00 0.62 (0.17, 2.24) 0.87 (0.41, 1.87) 0.30 (0.06, 1.50) Probable neuropathy‡ 4 1.00 1.13 (0.01, 90.5 2.09 (0.11, 126.39) § Confirmed neuropathy 4 1.00 0.46 (0, 6.02) 1.04 (0.07, 14.74) § *Total number of positive responses at baseline and at one year. †Referent group at baseline. ‡Approximate odds ratios using repeated measures logistic regres�ion with the normal distribution on sparse data. §Not estimable due to small number of abnormalities (complete or partial separation). ¶ See text for definitions and diagnostic criteria.

Longitudinal analysis of the effect of chlorpyrifos exposure on NCS results over time were not significantly different between groups pooled over the two examinations, although numerous significant differences existed among all subjects combined between the baseline and one year examination.

history. There was a high participation rate in chlorpyrifos subjects and referents (i.e. 80% and 81 % respectively). Authors used standard clinical outcome variables with known sensitivity and specificity and outcome measures with established reliability and precision. Limitations Small sample size. Follow-up period was only one year which may preclude detection of neuropathic changes. Does not account for healthy worker effects. May have been worthwhile categorising the daily chlorpyrifos internal exposure estimates given the wide range of exposure levels (0-250%) Authors did not perform QST


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First Author



Group (p value) Time (p value) Group*time (p value)

Median sensory Amplitude (μv) −2.41 (0.13) 2.26 (<0.01) −0.09 (0.91) Terminal CV (m/s) −0.64 (0.51) −2.34 (<0.01) −0.09 (0.91) Distal latency (ms) 0.03 (0.65) 0.19 (<0.01) 0.01 (0.88) Ulnar sensory Amplitude (μv) −1.20 (0.30) 3.72 (<0.01) −0.68 (0.59) Terminal CV (m/s) −0.05 (0.91) −2.98 (<0.01) 0.28 (0.73) Distal latency (ms) 0.01 (0.78) 0.15 (<0.01) 0.01 (0.88) Sural sensory Amplitude (μv) −0.65 (0.75) 0.24 (0.04) 1.97 (0.10) T�rminal CV (m/s) 0.41 (0.34) −2.55 (<0.01) 0.39 (0.66 Distal latency (ms) −0.05 (0.26) 0.21 (<0.01) 0.01 (0.84) Median motor Amplitude (mv) −0.71 (0.63) 0.61 (<0.01) 0.87 (0.08) Forearm CV (m/s) −0.61 (0.65) −1.88 (<0.01) 0.72 (0.28) �istal latency (ms) 0.09 (0.31) 0.39 (<0.01) −0.01 (0�88) F wave latency (ms) 0.34 (0.26) 0.6 (<0.01) −0.09 (0.68) Peroneal motor Amplitude (mv) −0.16 (0.82) −1.15 (<0.01) 0.08 (0.82) Distal latency (ms) 0.1 (0.11) 0.09 (<0.01) 0.16 (0.08) F wave latency (ms) 0.31 (0.99) 1.39 (<0.01) −0.�2 (0.19) Summary Z scores Motor amplitude −0.27 (0.64) −0.14 (0.98) 0.28 (0.18) Motor conduction −0.57 (0.37) −0.08 (0.85) 0.21 (0.52) Sensory amplitude −0.35 (0.41) −0.01 (0.44) 0.2 (0.�7) Sensory conduction −0.02 (0.72) 0.15 (0.93) −�.24 (0.68) *Potential covariates included age, height, weight, sex, body mass index, temperature over nerve being tested, education level, anxiety, smoking pack-years, and estimated general ability

Albers 2007 [CHLORPYRIFOS MANUFACTURING WORKERS, FOLLOWED UP ONE YEAR AFTER BASELINE EXAMINATIONS] Exposure Data: Chlorpyrifos subjects had a historic cumulative chlorpyrifos exposure of 64.16 mg/m3 x days, and worked in exposed areas for a duration of 9.72 years, with an interim chlorpyrifos exposure of 6.13 mg/m3 x days. Measures of urine TCP/Cr were 192.2 µg/g-Cr. Subjects had an estimated chlorpyrifos exposure of approx. 576-627 µg/day. AChE activities were similar to referents at both examinations. Referents had a historic cumulative chlorpyrifos exposure of 0.69 mg/m3 x days, and worked in exposed areas for a duration of 0.01 years, with an interim chlorpyrifos

Strengths Subjects had measurable external and biological exposure to an OP (chlorpyrifos) with

In view of the fact that the analyses gave conflicting significant results (one suggesting deteriorating electrophysiologica


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First Author



exposure of 0.00 mg/m3 x days. None of the referents had any identifiable exposure to chlorpyrifos in their jobs during the period of observation, although, six had past chlorpyrifos exposure ranging from 3.1 to 15.98 mg/m3. Measures of urine TCP/Cr were 6.2 µg/g-Cr. AChE activities were similar to study subjects at both examinations. Health Outcome Data: [Electrophysiological testing] {Interim chlorpyrifos exposure}: Median sensory terminal conduction velocity was significantly associated with the interim TCP/Cr level (parameter estimate = -0.0032 (95% CI, -0.0064 to -0.00004; (p=0.05), suggesting deteriorating performance (decreasing velocity) with increasing interim TCP/Cr level i.e. median sensory terminal conduction velocity decreases by 0.0032 m/s for each increase of 1 µg/g Cr. NB. This was also apparent for workers with exposures exceeding 20 mg/m3 x days. However, a single borderline significant association suggesting improved performance (increasing amplitude) with increasing interim exposure was observed between median motor amplitude and interim chlorpyrifos exposure (parameter estimate = 0.11 (95% CI, -0.014 to -0.23; p=0.08). {Historic chlorpyrifos exposure} Three out of 20 NCS outcomes showed a significant association with historic chlorpyrifos exposure. Median motor forearm conduction velocity, median motor F wave latency, and summary Z score for sensory conduction showed significant associations with historic exposure. All three suggested an adverse effect (decreasing velocity, increasing F wave latency, and decreasing summary Z score with increasing historic chlorpyrifos exposure)

Parameter estimate

95% CI p value

Median motor Forearm CV (m/s) -0.013 -0.024 to -0.0010 0.03 F wave latency (ms) 0.0068 0.00044 to 0.013 0.04 Summary Z scores Sensory conduction -0.010 -0.020 to -0.00044 0.04

Workers with the highest historic chlorpyrifos exposures (exceeding 20 mg/m3 x day) showed only minor, statistically non-significant relationships between historic cumulative exposure and NCS results. A significant interaction between education and historic chlorpyrifos exposure was observed in which highly educated subjects showed increased median F wave latency and decreasing summary Z score for sensory conduction (both indicating deteriorating electrophysiological performance) with increasing historic exposure cf. subjects with less education who showed no effect (data not shown)

no occupational exposure to other OP insecticides or other potential neurotoxicants. TCP daily excretion measures and AChE activities indicated that chlorpyrifos subjects were exposed to low doses. Conducted a dose-response analysis. Used sensitive quantitative indicators of neuropathy (although none of the statistically significant associations involved the sural NCS measures, which are considered among the most sensitive indicators of sensory neuropathy). Authors noted the adoption of an inclusive selection process to ensure that potentially vulnerable individuals were not excluded. Adequate control of confounders for PN Authors controlled

l function and the other suggesting enhanced electrophysiological function with increasing interim chlorpyrifos exposure) and the absence of significant dose-effect relationships among subjects with historic chlorpyrifos exposure exceeding 20 mg/m3 x day, it is suggested that no causal association exists between exposure to chlorpyrifos over many years during the manufacturing process and subclinical neuropathy


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for potential factors associated with employment in chemical industry via use of referents working in saran manufacturing. Limitations Small sample size. Follow-up period was only one year which may preclude detection of neuropathic changes

Starks 2012b

[PRIVATE PESTICIDE APPLICATORS WITH HIGH LIFETIME USE OF OP PESTICIDES] Exposure Data:

OP pesticide % of participants reporting ever use of pesticides Any 98 Malathion 77 Dimethoate < 10 Tebupirimfos < 10 Tetrachlorvinphos < 10

The authors did not provide data on the duration or level of OP pesticide exposure, or proportions of participants reporting use of carbamates. Assigned exposure levels (based on the median value) used in the exposure response modelling is not presented. This may have been included in ‘Supplementary Material’ which for some reason was not part of the original article. Health Outcome Data: NB. These following effects were not specific to OPs, as some associations were also observed with carbamate use. [Neurophysiological clinical examination] Ever use of 13 of the 16 OP pesticides (excluding malathion, phorate and terbufos) was significantly associated with one or more of the six neurophysiological clinical outcomes (except Romberg test) as shown below. Applicators had an approximate two to three fold increased odds of having an abnormal result. However, ever use of acephate, diazinon, and (ethoprop or parathion) were inversely associated with having an abnormal result for tandem gait, postural tremor

Strengths Large sample size. Authors evaluated background characteristics of eligible non-participants. Ensured study participants had no previous history of OP poisoning. Comprehensive assessment of peripheral nerve function. Health assessment performed by blinded experts. Adequate control of potential confounders and assessed confounding effects of multiple exposures.

This study reported findings that suggest pesticide applicators with long term exposure to OPs may have increased odds of experiencing neurophysiological clinical abnormalities. However, the evidence provided from more quantitative measures do not appear to support these findings. The study limitations weaken the reliability of the data presented


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and toe proprioception respectively (data italicised):

Exposure Ankle reflex (abnormal =109) normal = 554)

Postural tremor (abnormal =117) normal = 547)

Tandem gait (abnormal =180) normal = 586)

Toe proprioception (abnormal =62) normal = 603)

Toe vibration (abnormal =120)normal = 554)

OR 95% CI OR 95% CI OR 95% CI OR 95% CI OR 95% CI Acephate 0.67 ns 1.39 ns 0.60 (0.37,0.97) -- ns 0.54 ns Chlorpyrifos 0.81 ns 1.03 ns 1.21 ns 2.35 (1.28, 4.31) 1.45 ns Coumaphos 1.40 ns 0.93 ns 1.05 ns 2.03 (1.06, 3.90) 1.27 ns Diazinon 1.24 ns 0.63 (0.41, 0.96) 1.07 ns 0.67 ns 1.02 ns Dichlorvos 1.35 ns 0.81 ns 2.29 (1.41,3.71) 2.73 (1.53, 4.86) 1.94 (1.32, 3.31Dimethoate 1.20 ns 1.90 (1.01, 3.54) 1.26 ns 1.86 ns 1.66 ns Disulfoton 0.95 ns 1.95 (1.19, 3.18) 0.86 ns 0.39 ns 0.67 ns Ethoprop 1.03 ns 2.16 (1.35, 3.47) 0.98 ns 0.36 (0.14, 0.93) 1.08 ns Fonofos 1.47 ns 1.07 ns 1.18 ns 3.06 (1.79, 5.25) 1.62 ns Malathion 1.37 ns 0.88 ns 0.82 ns 1.05 ns 1.20 ns Parathion 0.95 ns 0.99 ns 0.73 ns 0.34 (0.14, 0.81) 1.30 ns Phorate 0.68 ns 0.81 ns 1.05 ns 1.67 ns 0.99 ns Phosmet 2.87 (1.52,5.44) 0.64 ns 1.71 ns 2.82 (1.47, 5.42) 0.73 ns Tebupirimfos 2.01 (1.00, 4.05) 2.17 (1.18, 4.00) 1.00 ns 1.84 ns 0.86 ns Terbufos 0.95 ns 0.89 ns 1.08 ns 1.25 ns 1.02 ns Tetrachlorvinphos 1.13 ns 1.36 ns 1.01 ns 2.35 (1.11, 4.98) 2.15 (1.11, 4.17

-- = Results from models wih < 5 exposed cases ns = lower bounds were under 1.00 NB. The above test models were adjusted for either age (years, BMI (kg/m2), height (cm) or US state The authors did not present any numerical data for dose-response modelling but summarised that the three-level exposure models used to test for trends, were generally consistent with the ever-use models. Dose-response models for toe proprioception had significant tests for trend for chlorpyrifos, dichlorvos, fonofos, phorate, and phosmet, which all (except for dichlorvos) showed a monotonic increase in ORs. For ankle reflex, a significant monotonic increase in ORs was observed for phosmet. For postural tremor, test for trend were significant and ORs increased monotonically for dimethoate, ethoprop, and tebupirimfos. For toe vibration, dichlorvos had a significant dose-response for trend. The authors did not observe any significant increases in risk between quantitative function PNS tests (i.e. hand strength, sway speed and vibrotactile threshold) and pesticide use (and do not provide any numerical data). However, reduced risks were observed for the following (although the authors did not specify whether these were statistically significant) [Hand strength] Ever-use and lifetime days of phosmet use were associated with greater hand strength

Limitations Lack of a control group. Exposure assessment was based on estimated cumulative lifetime exposure that was based on self-reported data. Possible healthy worker bias due to requirement of all AHS questionnaire completion. Low participation rate Electrophysiological test of motor nerve only Authors fail to provide all the necessary numerical data. Multiple comparisons may led to some chance findings


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[Sway speed] Both measures of parathion, tebupirimfos were associated with lower sway speed (i.e. better sway function) with eyes open [Electrophysiological testing] The authors observed more associations consistent with reduced risk of electrophysiological nerve abnormality from pesticide use although very few were statistically significant. Reduced risk estimates were observed between distal motor amplitude and ever use/log10 lifetime use of phorate and tebupirimfos. Dose response models for phorate and and tebupirimfos were consistent with a monotonic risk reduction for distal motor amplitude. Acephate, phorate and lifetime days of all OPs showed shorter F-wave latency (i.e. lower risk). Increased risk of nerve abnormalities were observed with diazinon that showed greater distal motor latency. The authors noted that these risks increased with increasing use. None of the nerve conduction velocity data were significant (and is therefore omitted from the table below, which shows adjusted regression coefficients for significant data only):

Exposure Distal motor amplitude (mV) (n=664)

Distal motor latency (ms)a (n==655)

Short F-wave latency (ms)a (n=544)

β 95% CI β 95% CI β 95% CI Acephate Ever-use Lifetime days 0.73 (0.21, 1.25) Diazinon Ever-use -0.12 (-0.24, 0.00) Lifetime days -0.07 (-0.15, 0.00) Phorate Ever-use 0.47 (0.06, 0.88) 0.91 (0.16, 1.66) Lifetime days 0.28 (0.04, 0.52) 0.73 (0.29, 1.17) Tebupirimfos Ever-use 0.71 (0.05, 1.37) Lifetime days 0.43 (0.02, 0.84) Summary variables Lifetime days to ALL OPs 0.53 (0.02, 1.02)

aThe authors multiplied scores by -1 so that lower scores indicated poorer test results Tests were adjusted for age, height, foot temperature and state of residence Dichlorvos and coumaphos use were found to be correlated but had minimal significant effects on ORs (> 10% reduction) for tests that included both pesticides (e.g. toe proprioception).

7.B.2. Case-control studies Crawford 2008 [PESTICIDE APPLICATORS REPORTING HEARING LOSS]

Strengths Large sample size

This study observed


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Exposure Data: Subjects were exposed to several pesticides including the following organophosphates: chlorpyrifos, coumaphos, diazinon, DDVP, fonofos, malathion, parathion, phorate, terbufos, and trichlorfon (among other classes of pesticides) Health Outcome Data: [Telephone interview questionnaire] The authors reported that lifetime days use of any pesticide were modestly associated with hearing loss but there was no evidence of a trend with cumulative exposure. Analyses of exposure to specific pesticide classes showed that OPs had the strongest associations with hearing loss of any insecticide type with a 17% significant increase in odds in the highest quartile of exposure (which contrasts with non-significant increased ORs for carbamates, organochlorines and pyrethroids). However the p-value for trend with cumulative OP exposure was not significant p=0.08.

Exposure to Organophosphates*

Case (n)

% Control (n)

% OR** 95% CIs

None 734 15 1522 16 1.0 referent 2.5-38.75 1392 29 2575 28 1.12 1.00-1.26 39-129.5 1371 28 2602 28 1.09 0.97-1.23 >129.5 1393 28 2568 28 1.17 1.03-1.31 Trend p=0.08

*Variable based on reported non-intensity-adjusted cumulative lifetime days of use **ORs adjusted for state, age, solvent, noise and metal exposures The authors conducted an analysis of ever use of individual OPs (based on intensity weighted cumulative days of use) using traditional logistic regression and observed the following associations/trends with hearing loss:

Exposure Case (n)

% Control (n)

% OR* 95% CIs

Cumulative lifetime days of chlorpyrifos use None 2533 55 4927 57 1.0 referent 1-63 677 15 1270 15 1.08 0.96-1.21 64-235 676 15 1230 14 1.13 1.01-1.27 >235 672 15 1243 14 1.15 1.02-1.29 Trend p=0.07 Missing 365 633 Cumulative lifetime days of coumaphos use None 4.35 90 7743 91 1.0 referent Any 466 10 805 9 1.07 0.94-1.22 Missing 425 755 Cumulative lifetime days of DDVP use None 3933 86 7646 88 1.0 referent

Accounted for a few possible confounders of hearing loss Evaluated potential selection bias between applicators who completed the questionnaires/ 5 year follow-up and those who did not and adjusted for potential confounders. Used an internal control group (although controls were also exposed to pesticides) Limitations Subjects received co-exposure to other classes of pesticide Exposure and health assessments were based on self-reports. (The authors noted that it is likely that there was underreporting of hearing deficits due to possible stigma associated with hearing loss). Used an indirect measure of noise exposure which could result in misclassification

increased odds of self-reported hearing loss among licensed pesticide applicators in Iowa and North Carolina. The findings suggest a weak association with insecticide use, particularly OPs which demonstrated a 17% increase in odds in the highest category of OP exposure. However, there was no strong evidence of a positive trend with cumulative lifetime OP exposure and the limitations of this study weaken the strength of the conclusions made


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1-202 319 7 507 6 1.08 0.91-1.25 >202 313 7 493 6 1.16 1.00-1.36 Trend p=0.06 Missing 341 657 Cumulative lifetime days of diazinon use None 3646 77 7186 80 1.0 referent 0.5-54.6 372 8 619 7 1.23 1.06-1.42 55-161.8 344 7 574 6 1.13 0.97-1.32 >161.8 364 8 590 7 1.25 1.07-1.46 Trend p=0.01 Missing 200 334 Cumulative lifetime days of fonofos use None 3370 73 6831 78 1.0 referent 1-68.25 375 8 658 8 1.02 0.88-1.18 68.26-242.83 431 9 301 7 1.30 1.13-1.50 >242.83 423 9 612 7 1.19 1.03-1.37 Trend p=0.04 Missing 327 601 Cumulative lifetime days of malathion use None 1423 30 3258 37 1.0 referent 0.88-57.75 1027 22 1992 22 1.09 0.98-1.21 58-212 1116 24 1825 20 1.32 1.18-1.46 >212 1128 24 1851 21 1.20 1.08-1.34 Trend p=0.09 Missing 232 377 Cumulative lifetime days of parathion use None 4306 91 8328 93 1.0 referent Any 417 9 636 7 1.21 1.01-1.40 Missing 203 339 Cumulative lifetime days of phorate use None 2998 63 644 72 1.0 referent 1-54.6 417609 13 847 9 1.21 1.06-1.37 54.83-176.4 539 11 874 10 1.10 0.96-1.24 >176.4 593 13 834 9 1.25 1.10-1.41 Trend p=0.004 Missing 187 304 Cumulative lifetime days of terbufos use None 2647 58 5255 61 1.0 referent 1.25-91 626 14 1168 1168 0.96 0.86-1.09 91.2-348.1 630 14 1155 1155 1.00 0.89-1.13 >348.1 674 14 1115 1115 1.17 1.04-1.31 Trend p=0.01


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Missing 349 610 *ORs adjusted for state, age, solvent, noise and metal exposures NB. Trichlorfon was not included due to low number of exposed subjects (59) For each OP, the associations with hearing loss were significantly elevated for at least one category of use (except for coumaphos). More than 20% increase in odds was observed for malathion, fonfos, diazinon, phorate and parathion. Significant trends (p<0.05) were noted for diazinon, fonofos, phorate and terbufos.

Jamal 2001 [TWO GROUPS OF OP-EXPOSED FARMERS INVOLVED IN SHEEP DIPPING WITH OR WITHOUT PREVIOUS ACUTE OP POISONING] Exposure Data: All subjects in both farmer groups had ≥ 4 years exposure to OPs. The time since last exposure to sheep dip was in excess of 4 months. There were no differences in enzyme activities (i.e. RBC AChE and serum cholinesterase) between farmer groups and control. Health Outcome Data: [Clinical evaluation] Median clinical scores for symptoms, reflexes and sensation were significantly abnormal in Group 1, and to lesser extent Group 2 cf. controls (i.e. increased symptom scores). Clinical motor symptoms did not reach statistical significance. [Quantitative sensory threshold (QST) testing] Significant increases were observed in mean vibration and cold perception threshold in both groups; mean heat perception threshold significantly increased in Group 1 only. [Electromyography (EMG)] Most farmers in both groups (i.e. group 1, n=14; group 2, n =11) showed motor conduction abnormalities on the EMG (i.e. presence of polyphasic units without spontaneous activity), which were more notable in the distal muscles. These chronic neurogenic changes were more pronounced in farmer group 1.

Control (n=16) Farmers, group 2 (n=16) Farmers, group 1 (n=16)

CI (95%)

CI (95%) Kruskal-Wallis test

Unit Median Median Farmers Controls p Median Farmers Controls p p Clinical scores Symptoms 0.0 1.0 0.0 2.0 0.023 3.5 3.0 4.0 0.0 0.000 Reflexes 0.0 1.0 0.0 1.0 0.015 1.5 0.9 3.0 0.0 0.001 Muscle power 0.0 0.0 - - - 0.0 - - - NS Sensory examination

0.0 1.0 0.9 3.0 0.001 6.0 3.0 7.0 0.0 0.000

EMG 0.0 1.0 1.0 1.0 0.000 1.0 1.0 2.0 0.0 0.000

Strengths Investigators were blind to the outcomes obtained from the clinical examinations/ investigations conducted by other teams. Used 0.975 as the confidence level for each comparison of means/median value to keep the probability of Type I error at or below 0.05. Used an objective and comprehensive health outcome assessment method (i.e. nerve conduction studies, quantitative sensory testing and EMG). Accounted for possible confounding due to occupation by using test sites that did not have calluses (which would impact on

This study provides evidence of peripheral nerve damage in the form of distal axonopathy involving motor and sensory nerves in farmers with or without clinical symptoms (it being more pronounced in the former group). The authors used both subjective and objective measures of assessing peripheral nerve abnormalities.


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Quantitative sensory testing Vibration µm 0.38 2.28 0.26 3.07 0.004 2.10 0.25 5.18 0.0 0.003 Hot threshold oC 1.65 1.70 -1.0 2.0 NS 5.90 1.0 6.35 0.0 0.003 Cold threshold oC 0.25 1.55 0.15 1.85 0.001 0.70 0.25 3.15 0.0 0.000

CI= confidence interval of the mean difference in medians between each farmer group and the control group NS= Not significant Statistical differences between the two groups were assessed by Kruskal-Wallis ANOVA by rank p values for differences between medians calculated using the Mann-Whitney test (non-parametric) [Nerve conduction studies] The distal motor, F-wave and sensory latencies were significantly increased in both upper and lower limbs in the two farmer groups. Mean compound action potential amplitudes and nerve conduction velocities were all significantly reduced in both farmer groups compared to controls. Mean latencies of the evoked potentials (EP) were not significantly increased at the 1% level with the exception of the P100 latency in farmer group 2. NB. EP refers to responses involving recording or stimulation of the CNS and are therefore not discussed further.

Controls (n=16) Farmers, group 2 (n=16) Farmers, group 1 (n=16) CI (95%) t-test CI (95%) t-test ANOVA Unit Mean SE Mean SE Farmers Controls p Mean SE Farmers Controls p p Lower limb (motor) SDML ms 3.97 0.15 4.87 0.22 0.25 1.54 0.003 4.84 0.25 0.22 1.51 0.007 0.006 FMNCV m.s-1 51.6 1.54 45.2 1.30 -10.0 -2.7 0.004 45.5 1.32 -9.8 -2.4 0.006 0.005 Amplitude mV 6.09 0.58 2.75 0.31 -5.5 -1.2 0.000 2.91 0.55 -5.4 -1.0 0.001 0.000 F-wave latency

ms 48.0 1.20 56.5 1.23 4.9 12.1 0.000 59.0 1.14 7.4 14.6 0.000 0.000

Lower limb (sensory) Sural nerve latency

ms 3.05 0.07 3.96 0.08 0.37 1.44 0.000 4.09 0.20 0.5 1.6 0.000 0.000

Sural nerve amplitude

µV 12.0 1.32 6.0 0.51 -7.5 -4.4 0.001 3.3 0.56 -10.2 -7.1 0.000 0.000

Upper limb (motor) SDML ms 3.43 0.12 3.78 0.10 -0.41 1.12 NS 3.92 0.08 -0.27 1.25 NS 0.003 FMNCV m.s-1 59.2 1.40 56.5 1.23 -5.8 0.3 NS 54.1 0.88 -8.2 -2.1 0.005 0.014 Amplitude mV 10.9 1.23 5.23 0.42 -8.6 -2.6 0.001 4.47 0.75 -9.4 -3.4 0.001 0.000 F-wave latency

ms 27.6 0.44 29.1 0.43 -0.3 3.0 NS 30.1 0.37 0.70 4.25 0.001 0.000

Upper limb (sensory) Median nerve latency

ms 2.71 0.05 3.04 0.10 -0.22 0.88 NS 3.07 0.06 -0.2 0.9 NS 0.001

Median nerve amplitude

µV 22.5 2.09 9.8 0.68 -15.8 -9.7 0.000 8.3 1.40 -17.3 -11.1 0.000 0.000

Evoked potential latencies Visual – P100

ms 97.4 1.31 103.1 1.23 2.48 9.09 0.004 97.7 1.26 -3.6 3.0 NS 0.002

Brainstem Auditory I

ms 1.65 0.04 1.70 0.02 - - - 1.64 0.04 - - - NS

I-III ms 2.24 0.04 2.20 0.05 - - - 2.08 0.07 - - - NS III-V ms 1.86 0.09 1.65 0.07 -0.44 0.03 NS 1.96 0.08 -0.13 0.34 NS 0.028

sensory tests). Also for vibration threshold measurements used a method that is not influenced by thickness of skin Limitations Small no. of subjects in each group. Possible selection bias due to the characteristics of farmer group 1 (i.e. individuals who decided to join the OPIN and were therefore likely to be more severely affected, although it is noted that the authors were specifically looking for farmers with symptoms). Unavailability of detailed exposure levels/history. It is not clear how clinical symptom data were obtained. The authors do not appear to have accounted for smoking which is considered to exacerbate PN symptoms, and exposure to vibrational machinery.


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Somatosensory Erb

ms 10.2 0.15 10.8 0.25 - - - 10.7 0.24 - - - NS

Spine ms 13.9 0.25 14.6 0.25 - - - 14.5 0.26 - - - NS Cortex ms 19.6 0.23 20.2 0.33 - - - 20.2 0.30 - - - NS

SDML = Shortest distal motor latency; FMNCV = Fastest motor nerve conduction velocity CI= confidence interval of the difference in medians between each farmer group and the control group NS= Not significant Statistical differences between groups were assessed by ANOVA P values for differences between means were calculated using the Student’s t-test

Multiple comparisons increasing the risk that some would be statistically significant by chance

Jamal 2002a [OP-EXPOSED SHEEP FARMERS/DIPPERS WITH DEFINITE, PROBABLE OR POSSIBLE NEUROPATHY] Exposure Data: Sheep farmers categorised with definite or probable neuropathy had a mean cumulative exposure of 1758 OPEXP, SD 1486, range 12-6088; Farmers categorised as having possible neuropathy had the highest mean exposure based on the exposure index (OPEXP) derived in the second study (i.e. 4364, SD 8011, range 68-44010). Controls had a mean cumulative OP exposure of 1349 OPEXP, SD 1638, range 10-6534. Health Outcome Data: [Questionnaire] NB. Results of the neuropathy questionnaire and the QST thresholds from the hospital based investigation were used to classify the subjects into three different groups for subsequent neurological and neurophysiological data analysis. [Clinical examination] The authors noted in their methods that cranial nerve examinations were not performed. However, in their results the authors reported that no subject in any group had abnormal cranial signs. [Nerve conduction studies] Sensory abnormalities defined as abnormal sural conduction and one or more abnormal QST values were found more often than motor deficits (both symptomatically and neurophysiologically). For example, 18% of 72 with sensory abnormalities compared to 3% of 72 with abnormal motor nerve conduction. [Neuromuscular junction testing] The low numbers for SFEMG results indicate that there was little abnormality in the neuromuscular transmission. Small fibre populations appeared to be more affected than large fibre populations. [Quantitative sensory threshold (QST) testing] Abnormality of small nerve fibre function (assessed by hot or cold sensation threshold), was three times more common

Strengths Authors adopted comprehensive measures to minimise observer/ investigator bias. Questionnaires were screened to ensure consistency of recording. Employed a comprehensive battery of investigations to provide indices of the whole peripheral nerve fibre population (i.e. sensory and motor function, large and small nerve fibre function and neuromuscular transmission). Appropriate experts were used to perform specific tests. Limitations Health outcomes not analysed in relation to

This study was not designed to investigate associations between OP exposure and peripheral nerve abnormalities but rather was designed to aid the clinical interpretation of neuropathic outcomes in farmers exposed to OPs. The use of a crude measure of cumulative exposure (based on recall and empirical estimates derived from an exposure model) together with the lack of correlation between neuropathic symptoms and increasing OP exposure, suggest caution should be used to interpret the conclusion that


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(65% of 72) than abnormal large fibre function (assessed by vibration threshold or sural nerve function (21% of 72) Summary of neurological and neurophysiological findings

Subject group n Neurological signs

(n (%))

Nerve conduction

(n (%))

Neurological signs or nerve

conduction (n (%))

EMG (n (%))

SFEMG (n (%))

Sensory abnormality

(n (%))

Motor abnormality

(n (%))

Small fibre abnormality

(n (%))

Large fibre abnormality

(n (%))

No neuropathy

15 0(0) 1(7) 1(7) 3(21) 0(0) 0(0) 0(0) 0(0) 1(7)

Possible neuropathy

34 3(9) 7(21) 10(29) 10 (30) 5(15) 6(18) 0(0) 26(76) 7(21)

Probable or definite neuropathy

23 4(17) 8 (35) 12(52) 11(52) 1(4) 7(30) 2(9) 21(91) 7(30)

Total 72 7(10) 16 (22) 23(32) 24 (35) 6(8) 13(18) 2(3) 47(65) 15(21) The results did not show that cumulative exposure to OPs correlated with the presence of neuropathy (as there was very little difference in the exposures experienced in the no and probable/definite groups), although the authors noted that the study was not designed to examine this. Incidence of clinical neuropathy increased from the no neuropathy to probable/definite neuropathy group (table below shows the number of subjects in each group with abnormal findings). Symptoms (n(%)): hospital based clinical study

Subject group n Sensory symptoms Motor symptoms No neuropathy 15 0 (0) 0 (0) Possible neuropathy 34 4 (12) 4 (12) Probable or definite neuropathy 23 18 (78) 11 (48) Total 72 22 (31) 15 (21)

Subjects with abnormal QST (n(%)): hospital based clinical study

Subject group n Hot threshold Cold threshold

Vibration threshold

No neuropathy 15 0 (0) 0 0 (0) Possible neuropathy 34 12 (35) 22 (65) 8 (24) Probable or definite neuropathy 23 14 (61) 17 (74) 5 (22) Total 72 26 (36) 37 (62) 13 (18)

NB. QST score was dependent on comparison with an age dependent threshold NB. The authors also reported on neuropsychological and autonomic nervous system effects.

increasing OP exposure Reported incidence of abnormalities and not measures of association. Small number of subjects in each group. Low response rate in subjects from probable or definite neuropathy group and the possible neuropathy group

farmers chronically exposed to OPs developed neuropathy predominantly among the small fibre sensory peripheral nerve population

7.B.3 Cross-sectional studies Abdel-Rasoul

2008 [CHILDREN AGED 16-18 YEARS WORKING AS PESTICIDE APPLICATORS SPRAYING PESTICIDES ON COTTON CROP] Exposure Data: Children reported working for a mean of 7.2 years (SD 1.5 years), working for a mean of 21.4 days (SD 6.7) during the current application season. Mean AChE activity levels were significantly lower in applicators compared

Strengths Performed biological assessment of OP exposure.

The primary aim of this study was to determine whether increasing exposure to OP


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to control group (239.8, SD 60.0 IU/L and 283.1, SD 61.6 IU/L respectively; t =3.6; p< 0.05). Health Outcome Data: [Clinical neurological examination] An increase in neurological symptoms was observed in the applicator group compared to the control group. Significantly more participants in the applicator group had numbness (18%) (among other non-PN related symptoms) compared to control participants (2%) (p < 0.05). No further analyses specific to peripheral neuropathy-related symptoms were conducted (numbness was grouped along with other symptoms as neurological).

Reported neurological symptoms were verified by specialist. Limitations Small sample. Exposure assessment based on self-reported data. Exposure was not OP specific (other pesticides and growth enhancers were applied during the application period). AChE measurement was performed at one time point after the spraying season (this fails to provide a measure of the AChE variability of individuals). Inadequate account of confounders for PN Does not fully describe neurological examinations performed (only the results)

pesticides was associated with larger neurobehavioural deficits in children seasonally exposed to OP pesticides. This study was not designed to evaluate the development of peripheral neuropathy related symptoms in exposed subjects. Despite this, data from a sub-analysis of neurological symptoms revealed a significantly higher number of exposed subjects with ‘numbness’ compared to controls. However, given that these children were exposed to other non-OP pesticides during the application season limits the strength of this finding

Albers 2004c [CHLORPYRIFOS MANUFACTURERS] Exposure Data: Chlorpyrifos subjects had no significant exposures to other chemicals with neurotoxic properties, but had significantly longer duration of work in chlorpyrifos-exposed areas compared to referents (9.72 vs. 0.01 years; P < 0.0001) and higher historical cumulative chlorpyrifos exposure (64.16 mg/m3 vs. 0.69 mg/m3 per day; P < 0.0001). Chlorpyrifos subjects also had significantly higher measures biological exposure cf. referent subjects i.e. urine TCP/Cr

Strengths 80% and 81% participation rate for study subjects and referents respectively.

This well-designed study found no significant differences in the frequency of either clinical or


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(108.6 vs. 4.3 µg TCP/g Cr, P <0.0001) – the authors noted that the urine TCP/Cr levels in saran subjects calculates to a daily chlorpyrifos dose of 0.22 µg/kg per day that is similar to the amounts observed in the US adult general population. Chlorpyrifos subjects had lower average plasma BuChE activity cf. referents (7281 vs. 8176 mU/ml, P <0.003). RBC AChE activities were similar to referent group (6923.19 vs. 6966.77 mU/ml). NB. The authors noted that the estimated daily urine excretion of TCP suggested an average daily chlorpyrifos exposure of approx 17% (range 0-115%) of that received by a typical subject exposed during a working day at the permissible chlorpyrifos exposure level of 200 µg/m3. Health Outcome Data: [Questionnaire interview] There were no significant (P < 0.05) group differences for the number of subjects reporting symptoms [Clinical examination ] There were no significant (P < 0.05) group differences for the number of subjects reporting symptoms or for the presence of neurological signs i.e. station, gait, co-ordination, alternate-motion rate, presence of abnormal movements (tremor, fasciculations), numbness, tingling or sensory loss, decreased ankle reflexes. [Nerve conduction studies] No significant group differences were found for any of the nerve conduction measures. All mean values were comparable to published laboratory normal control values (based on subjects known to be healthy without underlying disorders associated with neuropathy). The number of subjects fulfilling the outcome criteria for neuropathy (i.e. probable, possible or confirmed clinical neuropathy) did not differ significantly between groups

Definition of abnormality Group N Mean (SD) P-value*

Sensory Median Amplitude (μV) <10.0 Chlorpyrifos 53 29 (12.8) 0.42

Referent �0 31 (12.3) Terminal conduction velocity (CV) (m/s) <50.0† Chlorpyrifos 53 54.8 (6.7) 0.70

Referent 60 55.3 (7.0) Distal latency (ms) >3.7 Chlorpyrifos 53 3.3 (0.4) 0.66

Referent 60 3.3 (0.5) Ulnar Amplitude (μV) <10.0 Chlorpyrifos 53 27 (11.0) 0.68

Referent 60 28 (11.1) Terminal CV�(m/s) <50.0† Chlorpyrifos 53 57.5 (5.5) 0.88


Referent 60 3.2 (0.3) Sural Amplitude (μV) <6.0 Chlorpyrifos 53 18 (5.9) 0.77

Referent 60 18 (7.2)

Employed a healthy population of workers (i.e. with little evidence of systemic disease). Selected an appropriate reference population that allowed effects of employment in the chemical industry to be controlled. Deployed a comprehensive exposure assessment. Workers had measureable occupational exposure to chlorpyrifos. Investigators were blinded to a subject’s exposure status during health and exposure assessment. Used standard clinical criteria to define neuropathy. Adequate account of main confounders for PN Limitations Small sample size

subclinical signs of peripheral neuropathy in subjects chronically exposed to occupational levels of chlorpyrifos and unexposed referents. The small sample size does question the significance of these findings, however the authors noted that quantitative measures of sensory and motor nerve function did have sufficient power to detect small group differences suggestive of subclinical neuropathy


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Terminal CV (m/s) <40�0 Chlorpyrifos 53 50.4 (5.0) 0.64 Referent 60 50.0 (5.1)

Distal latency (ms) >4.2 Chlorpyrifos 53 3.5 (0.3) 0.38 Referent 60 3.5 (0.3)

Motor Median Amplitude (mV) <4.2 Chlorpyrifos 53 9.4 (3.1) 0.27

Referent 60 10.1 (3.5) Forearm CV (m/s) <49.0 Chlorpyrifos 53 56.6 (4.1) 0.50


Referent 60 3.4 (0.6) F-wave latency (ms) >31.8 Chlorpyrifos 53 27.9 (2.3) 0.70

Referent 60 27.7 (2.7) Peroneal Amplitude (mV) <2.5 Chlorpyrifos 53 7.2 (3.6) 0.79


Referent 60 4.5 (0.6) F-wave latency (ms) >56.0 Chlorpyrifos 51 48.7 (4.2) 0.97

Referent 60 48.7 (4.9) * t-test for difference in means (adjusted for unequal variance between groups). † Terminal sensory conduction velocity based on 95th percentile onset latency value of 3.2 ms and 2.8 ms for median and ulnar nerves, respectively

Bazylewicz Walczak

1999 [GREENHOUSE WORKERS ASSESSED BEFORE AND AFTER OP/PESTICIDE APPLICATION] Exposure Data: Plant workers had a mean duration of exposure to OPs of 11.9 years (SD 6.8, range 1-24) and the daily cumulative exposure for combined dermal and inhalatory routes ranged from 0.006-1.386 mg (mean 0.177) equivalent to 0.001-0.009% of toxic dose (mean 0.001), which the authors noted is classified as a low a exposure level based on WHO recommendations. NB. The authors only report data on exposure assessment for the greenhouse workers and not for control subjects. Health Outcome Data: [Questionnaire] The authors did not include/report on peripheral neurological symptoms in their analysis of the effects of single spraying seasons or long-term exposure (or interactions between the two factors). The only findings of relevance that are reported are from the subjective symptom questionnaire results comparing the first and second examinations in exposed and control subjects, it is assumed that the mean differences were not significant as PN effects were not included in further analyses (which was conducted for significant findings only). NB. The mean number of peripheral neurological symptoms were higher in greenhouse workers compared to controls. Mean values and SD for NCTB-tests from the 1st and 2nd examinations of exposed and control groups

Name of test variable Exposed group N=26 Control group N=25 Examination 1 Examination II Examination 1 Examination II Mean SD Mean SD Mean SD Mean SD

Subjective Symptoms Questionnaire Neurological symptoms – periph* 1.96 1.68 1.81 1.67 1.40 1.35 1.36 1.58

Strengths Deployed a comprehensive assessment of external exposure levels. Limitations Small study group. Exposure assessment appears to be conducted on plant workers only and not in controls. Provides no information on the subject selection procedure and response rates. Report of peripheral neurological effects are subjective and has not been further evaluated to

The aim of this study was to assess the behavioural effects of chronic exposure to OP pesticides. The evaluation of peripheral neurological abnormalities comprised a minor part of a subjective symptom questionnaire, which itself was a component of a battery of tests used in the psychological assessment. There was a non-significant reduction in the number of peripheral


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* it is assumed that these peripheral symptoms refer to questions relating to paresthesia in limbs and hands (sensory) and limb weakness (motor) and possibly cranial nerve effects i.e. vertigo, sense of smell, taste, facial numbness, facial muscle changes/sensation, as the authors did not specify what constitutes peripheral neurological symptoms

control for recall bias. Inadequate control of confounders for PN-related symptoms Failed to account for plant workers co-exposures to other pesticide compounds

neurological symptoms after comparing the first and second examinations suggests the season had no effect

Farahat 2003 [AGRICULTURAL ENGINEERS, MIXERS AND MECHANICS WORKING IN PESTICIDE APPLICATION DEPARTMENTS] Exposure Data: Exposed subjects had a mean work duration of 18.04 years (SD 8.29) compared to controls who had a mean work duration of 17.08 years (SD 4.37). Mean level of serum AChE in exposed subjects was 87.34, U/ml which was significantly lower than controls 108.25 U/ml; p=0.0001. Health Outcome Data: [Questionnaire/clinical exam] Of the peripheral neurological symptoms evaluated, ‘numbness’ was the only symptom that was significantly higher in the exposed than the control group:

Neurological symptoms† and signs†† Exposed (n=52) n(%)

Control (n=50) n(%)

OR 95% CI Holm adjusted pvalue*

Numbness 13 (25) 1(2) 16.33 3.95 – 303 0.003 Tremors 8(15) 1(2) 8.91 1.55 – 169 0.101 Muscle power 5(10) 1(2) 5.21 0.80 – 102 0.281 Ankle reflex 6(12) 1(2) 6.39 1.04 – 123 0.225 Knee reflex 9(17) 3(6) 3.28 0.91 – 15.50 0.281 Superficial sensation (pain, touch) 12(23) 3(6) 4.70 1.38 – 21.69 0.101 Deep sensation (vibration) 1(2) 0(0) - - 0.489

* Significant after using Holm’s modification of Bonferroni correction (6 symptoms and 5 signs) †Have you ever had a history of any of the following symptoms? ††Detected by clinical examination The authors noted that most of the reported neurological symptoms and signs were significantly higher in older workers, which they suggest is due to their longer working duration in comparison to younger workers (data not shown). The authors note that cranial nerve examinations were conducted, however no further details on cranial nerve signs or symptoms were reported. Serum AChE showed no significant differences between those with positive and negative symptoms or signs, however

Strengths 79% and 81% response rates among controls and study subjects respectively. Attempts to evaluate dose related effects. Adequate control of confounders for PN Limitations Small sample size. Subjects exposed to other pesticide agents including carbamates and pyrethroids. Inadequate description of clinical neurological tests performed. Use of AChE limited by the fact that carbamates also inhibit AChE.

The limitations of this study weaken the validity of thesignificantly increased incidence of ‘numbness’ in Egyptian pesticide applicators cf. control subjects. This is because the pesticide treatment protocol included the use of other non-OP pesticides and the study fails to account for the effect of these multiple co-exposures. Furthermore, the authors considered the use of AChE as a measure of OP exposure but given that the applicators also used carbamate, which is known to inhibit AChE, makes this assessment non-specific for OP


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the authors reported a significant trend of lower AChE levels with increasing exposure duration (β= -1.18, SE 0.19, 95% CI -1.55 to -0.81)

exposure

Horowitz 1999 [SUB-SAMPLE OF FRUIT GROWERS WITH > 20 YEARS OP EXPOSURE AND HIGH VIBRATION THRESHOLD INDICES] Exposure Data: 7 subjects had >20 years of OP exposure (i.e. range from 36 to 55+ years in apple, pear and peach groves in Ulster County, New York). The remaining two subjects had less exposure i.e. 16 years in one farmer, and incidental childhood exposure followed by 5-years direct exposure in the same farmer’s son. Health Outcome Data: The authors present only descriptive data. [Clinical examination]: 5 subjects were neurologically normal. The remaining 4 subjects had reduced sensation in the lower extremities in a stocking distribution (i.e. sensations of touch, pain, temperature, vibration, position and ankle jerk). [Sensory thresholds]: Abnormal vibratory detecting thresholds (VDT) were apparent in one subject with reduced vibration and position sensations; and abnormal cooling detecting thresholds (CDT) apparent in two subjects with reduced touch, pain and temperature sensations. NB. Abnormal VDTs were not restricted to those with reduced vibration sensations. [Quantitative muscle strength]: Results for Tufts Quantified Neurological Evaluation (TQNE) were normal in all subjects for all modalities. [Electrophysiologic studies]: Motor-nerve CVs and amplitudes of the EPs were normal in all subjects, except for slowing of the carpal tunnel nerve in three subjects. Other reported findings include slowed F-wave in three subjects; one with a normal examination and two with reduced sensations to touch, pain and temperature; a slowed H-reflex was apparent in the latter two subjects with reduced sensations. Slowed upper extremity sensory CVs in one subject with reduced sensations, and reduced amplitude of sensory EPs in one subject with normal clinical examination and in three of four subjects with clinical sensory deficits. A single subject with reduced sensation to all modalities (lower extremities) had normal sensory EP amplitudes in the upper extremities. Four subjects with normal clinical examinations had normal sural MAXCV, MINCV, and normal amplitude of the sensory EPs. Three subjects with clinical sensory deficits had slowed MAXCV, slowed or absent MINCV, and two had reduced amplitudes of the sensory EPs The authors concluded that four of the nine fruit growers with long-term exposure to OP pesticides had clinical evidence of mild peripheral neuropathic dysfunction which they consider is not attributable to any other cause. In each of these four subjects the clinical dysfunction consisted of reduced sensation in the lower extremities in a “stocking” distribution.

Strengths Performed extensive clinical and subclinical testing of peripheral neurological health outcomes Limitations Analysis conducted in only nine subjects. Lack of referent subject details. Limited/crude exposure assessment that appears to be based on self reports. Failed to account for co-exposures to other non-OP chemical compounds. Reported on the number of subjects with/without peripheral neurological signs but fails to present the actual values measured. Lack of tests of significance reported for parameters measured.

There is evidence for peripheral neuropathy in some fruit growers with more than 20 years exposure to OPs. However, they were a selected group from a previous study of very few individuals with abnormal vibration thresholds, and there being several study drawbacks


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Results from electrophysiological tests in all four subjects with clinical abnormalities and computerised sensory testing in three of the four subjects corroborated the presence of peripheral neuropathic dysfunction. These tests did not reveal significant subclinical involvement in those subjects whose clinical neurological examinations were normal

Hoshino 2008 [RURAL WORKERS EXPOSED TO ORGANOPHOSPHORUS COMPOUNDS AND COMPLAINING OF DIZZINESS] Exposure Data:

Variables n % Years on the job (years) 0-10 4 22.2 11-20 4 22.2 21-45 10 55.6 Daily work hours <10 3 16.7 >10 15 83.3

Health Outcome Data: [Audiometric assessment]: The authors reported that 11 subjects (61.1%) had normal hearing (of which 7 subjects were aged between 16-35 years). The remaining 7 subjects (38.8%) had altered tone test results (of which 4 subjects had decreased performance at 6kHz and 8 kHz, and 3 subjects had sensorineural hearing loss). Five of these subjects were aged between 46-59 years. Only two subjects had bilateral hearing loss. All subjects had speech audiometry within normal ranges. [Vector electronystagmography (VENG)]: 16 subjects were found to have peripheral irritative vestibular syndrome (PIVS) (as indicated by altered nystagmus test results/ positional vertigo and hyperreflexia (overactive reflex) The authors further evaluate the relationship between VENG results and working practice ascertained from questionnaire. 10 subjects (8 with altered test results and 2 with normal test results) claimed that their dizziness worsened while working, and thirteen workers (12 subjects with altered and one subject with normal test results) reported that the chemical products used affected their balance. The authors reported that age did not influence the VENG results, in contrast to the number of years on the job, daily number of hours of exposure and failure to use personal protection equipment (PPE) which were associated with a larger number of subjects with altered vestibular examinations:

No of subjects with NVE*

No of subjects with PIVS

Total

Age range (y) 16-35 2 6 8

Strengths Objective health assessments were conducted that appear to have been validated via previous use Adequate control of potential confounders Limitations Lack of controls (aside from using the number of subjects with normal vestibular results) Exposure assessment based on self-reports of work duration/times; there being no verification of levels subjects were exposed to Subjects used/ were exposed to other non-OP pesticides of which manzate was the most common Inadequate description of vestibular tests performed. Data analysis was very basic (based on comparison of

The authors concluded that their study provides evidence that OP pesticides promote vestibular and hearing disorders. However, given the limitations associated with this study including co-exposures to other non-OP pesticides and its reliance on self-reports of work history duration, and the lack of a referent population these findings would need to be verified by a more robust evaluation


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36-45 0 3 3 46-59 0 7 7 Years on the job 0-10 0 4 4 11-20 0 4 4 21-45 2 8 10 Working hours <10 0 3 3 >10 2 13 15 PPE Yes 0 2 2 No 2 14 16

*NVE = Normal vestibular examination NB. % are not included in this table

percentage frequencies between subjects with normal and altered vestibular test results; there being no test of significance)

Kamel 2005 [PRIVATE PESTICIDE APPLICATORS/FARMERS CATEGORISED WITH A HIGH FREQUENCY OF SYMPTOMS (CASES)] Exposure Data: Both cases and controls were exposed to OPs. The authors used frequency of symptoms (rather than duration of exposure) to classify subjects as either cases or controls. The following describes the % of subjects in each group that fall into median, lowest and highest exposure quartiles respectively: 29% of cases (highest number of subjects) had between 51-500 cumulative lifetime days of OP exposure, 9 % with 0 days, and 6% with > 500 days. This compares with 27% of controls with 51-500 cumulative lifetime days of OP exposure, 14 % with 0 days, and 4% with > 500 days. Health Outcome Data: [Questionnaire] Table below shows the odd ratio of experiencing the following peripheral neurological symptoms (includes both spinal and cranial nerve symptoms) with high frequency compared with low frequency. These estimates were for the highest category of lifetime days of use of organophosphates. However, it should be noted that these symptoms were also found to be associated with other pesticide groups.

Symptom OR* Numbness in hands or feet 2.67 Twitches in arms or legs 2.34 Weakness in arms or legs 1.82 Poor balance 1.77 Tremor in hands 2.00 Blurred/double vision 1.87 Changes in smell or taste 1.83

Strengths Very large sample size. Evaluated the effect of other non-OP pesticide groups on health outcome. Addressed the potential for selection bias. Adequate control of confounders for PN Limitations All information on exposure and disease states was taken from self reports. No p-values reported. Used an internal control population for comparison (who had both symptoms and

This study made internal comparisons of exposed individuals from the same population and observed positive associations in applicators with a high frequency of reporting several peripheral neurological symptoms (both spinal and cranial nerve effects) compared to those with a low frequency of symptoms. However, the fact that both the exposure assessment and health outcome were based on


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Difficulty speaking 1.94 * Estimates for which the 95% CI excluded 1.00 In the remaining analyses, symptoms are dichotomised into either “many” or “no” symptoms, which thereby precludes any further evaluation of specific peripheral neurologic symptoms

exposure to OPs)

subjective measures (i.e. self reports) suggests these findings should be interpreted with caution

Kamel 2007b [PRIVATE PESTICIDE APPLICATORS/FARMERS CATEGORISED WITH A HIGH FREQUENCY OF SYMPTOMS] Exposure Data: 3306 cases (91%) had ever used OPs compared to 13184 (87%) controls. Health Outcome Data: [Questionnaire] Data specific to OP exposure and peripheral neurologic symptoms were apparent only for the analysis of possible associations between symptoms groups (defined a priori into several neurologic functional domains) and cumulative use of pesticides among applicators. NB. The data was presented in a graph. Use of OPs was associated with approximate 2-fold increased risk of experiencing symptoms related to motor function i.e. twitches, weakness, poor balance, tremor and difficulty speaking and vision i.e. blurred or double vision and poor night vision (although this was slightly lower than for the motor group) . These effects were not specific for OPs i.e. was apparent for other types of insecticide e.g. organochlorines.

Strengths Very large sample size. Evaluated the effect of other non-OP pesticide groups on health outcome. Adequate control of confounders for PN Limitations All information on exposure and disease states was taken from self-reports. Spinal and cranial nerve effects are grouped within the same category Used an internal control population for comparison (who had both symptoms and exposure to OPs). OR for relevant analyses presented graphically. No p-values reported. Discordant definition of sensory and motor

The finding of an association between motor system and vision outcomes and use of OPs is limited by the lack of specificity of effect given that other pesticides generated similar findings.The subjective nature of the exposure and health assessment is offset by the very large sample size of the study.


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symptom categories

Keifer 2000 [APPLE ORCHARD FARMERS SUBCHRONICALLY EXPOSED TO OPS] Exposure Data: Mean no. of years of farm work in farm workers was 2.26, SD 5.40 and 2.73, SD 6.7 in Round 1 and 2 respectively. The authors noted that the exposure estimate of reported hours of recent thinning was validated by a significant and strong inverse correlation with AChE activity. Health Outcome Data: The findings on the effects of exposure on nerve conduction and repetitive stimulation electromyography are not reported in this paper. The authors refer readers to previously published work (Engel et al 1997 – a poster presentation at the American Public Health Association Meeting in Nov 1997, New York City) and note that ongoing related work is planned for publication by the same author. Other findings that were planned for publication included the relationship between exposure and R-R interval testing (measures autonomic responses). The authors present the coefficients for effect i.e. exposure and confounders in multilinear regression for the variables of interest. The effect of the previous season of thinning did not significantly predict the performance of vibration threshold tests of the hand and foot. The authors refer readers to another publication for their findings on the analysis of the peripheral nervous system data from the exposure round of testing (Engel et al 1998, Arch Environ Health 53(1):7-13 – this was covered in the previous COT 1999 report), and briefly summarise that no significant differences were observed between exposed workers and controls

Strengths Testers were blinded to subject’s exposure status. Authors incorporated biochemical measures of exposure. Limitations Poorly designed study. Participation rate decreased significantly in second evaluation. Authors fail to present the data on biochemical levels of exposure. Study fails to present any quantitative data on peripheral nerve outcomes and do not describe the neurophysiological tests conducted. Cumulative exposure assessment based on crude measures (self reported work history). Workers previous work experience not verified. Inconsistency in tests conducted between two evaluations.

The limitations evident in the design, analysis and presentation of this study, question the reliability of the study’s findings, which prevent further comment on the significance of any peripheral neuropathy outcomes reported in this study


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Lack of appropriate control for farmers’ fatigue during tests (as some tests were conducted after work). Control subjects reported exposure to some farm work

Kilburn 1999 [PATIENTS EVALAUTED AT A NEUROTOXICOLOGY CLINIC FOLLOWING EXPOSURE TO CHLORPYRIFOS] Exposure Data: Patients were exposed either: outdoors via jobs as a agricultural worker or pesticide applicator (for a duration ranging from 1 day to between 5 to 6 years); indoors (in offices or homes where chlorpyrifos containing pesticides had been applied either repeatedly over days, months and up to 2 years, or were applied only once. Concentrations of pesticides applied in offices ranged from 0.25 to 24.1% (50% concentrate was used to make dilutions). Chlorpyrifos concentrations in clothing ranged from 0.21 to 0.40 µg/ft2 and in rugs ranged from 23.7 to 125 µg/ft2. Health Outcome Data: [Questionnaire] Comparison of symptom frequencies (on a ten-point scale) showed significant elevations for 33 of 35 symptoms in exposed subjects versus unexposed referents. The most elevated symptoms (peripheral neurological – spinal and cranial nerve-related) included itching, numb white fingers/Raynaud’s phenomena (data not shown), loss of balance and decreased smell.

Symptom Exposed (22) [mean ±SD]

Unexposed (264) [mean ±SD]

p value

Skin itching 6.5 ± 3.5 3.1 ± 2.7 0.0001 Loss of balance 6.4 ± 2.5 2.3 ± 1.9 0.0001 Decreased smell 4.5 ± 3.1 2.1 ± 1.9 0.0001

[Clinical examination] The authors noted that 11 patients had peripheral neuropathy manifested by weakness and sensory changes often with a history of painful extremities (data represented in qualitative terms). NB. No information was provided regarding the prevalence of PN in controls. Three of these patients had the onset of simple partial motor or temporal lobe seizures after exposure, and also had abnormalities of peripheral neuropathy. Physical and neurological examinations showed asymmetrical motor weakness and abnormal hyperactive, deep tendon reflexes in 11 subjects, and all had elevated thresholds for detecting 128 Hz vibration (no further information provided).

Strengths Performed both subjective and objective assessment of reported neurological symptoms Adequate control of confounders for PN Limitations Small number of study subjects. Acute and chronic-exposed subjects grouped into one category (i.e. range of exposure duration). Exposure assessment based on residues on clothes, rugs, estimates and personal history with no account of possible degradative effects. Definitions of what constitutes

This study observed impairments of body balance, visual fields, colour discrimination, hearing and grip strength and the following possible sensory effects (itching and decreased smell) in individuals exposed acutely and chronically to chlorpyrifos sprayed inside homes and offices. However, limitations of the study suggest these findings are interpreted with caution


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17 of 20 patients (two were not tested) had abnormalities in three or more quadrants of their visual fields; and in six patients the fields were constricted. Hearing was diminished in 4 patients. The authors categorise the following data under the physiological component of neurobehavioural tests:

Physiological test domain Exposed (22) [mean ±SD]


p value

Sway-balance (eyes open) (cm/s) 155.1 ± 87.7 100.2 ± 20.0 0.0001 Sway-balance (eyes closed) (cm/s) 237.9 ± 48.8 103.1 ± 26.8 0.0001 Blink reflex R-1 ms right 115.4 ± 18.2 108.8 ± 15.2 0.060 Blink reflex R-1 ms right 110.6 ± 18.6 105.2 ± 13.5 0.127 Grip strength (right) (kg) 76.3 ± 19.2 99.3 ± 17.5 0.0001 Grip strength (left) (kg) 77.3 ± 21.8 99.1 ± 17.5 0.0001

Visual fields right 83.9 ± 15.3 100.0 ± 22.9 0.005

Visual fields left 81.8 ± 16.6 101.1 ± 21.7 0.0005

Colour score right 156.1 ± 39.3 102.6 ± 51.1 0.0001

Colour score left 141.9 ± 26.1 102.6 ± 51.3 0.0001

Age, education and neurobehavioural tests of exposed and unexposed as mean percentage of predicted, (adjusted) and SD compared by ANOVA NB. Patients also demonstrated psychological and respiratory impairments Comparison of the mean percentage of predicted neurobehavioural test scores (adjusted), showed significantly impaired balance with eyes open and closed, and significantly impaired grip strengths in exposed subjects compared to unexposed referents (NB. Grip strength is first introduced as a health outcome variable in the results section, and the authors did not indicate the units; it is assumed to be in kg). Other significant differences indicating impaired function included colour discrimination and visual field performance in the right and left eyes – see Table below. Blink reflexes did not differ significantly between groups.

neurological vs. neurobehavioural examination is blurred e.g. grip strength included in neurobehavioural assessments while EMG is included in neurological examinations

Kimura 2005 [TOBACCO FARMERS] Exposure Data: Years working with tobacco farms in farmers ranged from 1 - >20 years. Pesticides were applied up to 5 times per week and more than 8 times per month. Serum cholinesterase activity for the tobacco farmers did not differ significantly from controls (data not shown). Health Outcome Data:

Strengths Conducted an objective health assessment. Adequate control of confounders for PN

This study did not observe any significant peripheral nerve system effects in tobacco leaf farmers exposed to the OP


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[Nerve conduction study] MCV and SCV data for Tamaron (OP) users did not differ significantly from controls. [Posturography] The authors did observe significant CNS effects (postural sway increases) in these users: 0-1 Hz of postural sway in the anterior-posterior direction with eyes open was significantly increased (p<0.05) indicating effects on vestibulo-cerebellar system

Limitations Relatively small sample size. Control group considerably smaller. No assessment of external levels of OP exposure. Farmers had co-exposure to other non-OP pesticides that had inhibitory cholinesterase activity. Failed to account for possible effects of nicotine poisoning from tobacco leaves

methamidophos (Tamaron). However, the study provides evidence for disequilbrium, although this was evaluated in the context of CNS impairment

Konieczny 1999 [CHEMICAL PLANT WORKERS PRODUCING CHLORFENVINPHOS] Exposure Data: The duration of employment in chlorenvinphos plant workers ranged from 1-15 years (mean 9.0, SD 4.3). Mean air chlorfenvinphos concentration estimates ranged from 0.0008-0.0018 mg/m3 (which did not exceed the maximum allowable concentration of 0.01 mg/m3. The authors noted (in the papers abstract only) that the erythrocyte AChE activity was similar to that observed in people unexposed to chemicals. No further information on was provided. Health Outcome Data: NB. Some of the following data was reported in the abstract only: [Spirometry] Spirometric investigations showed an elevated intrathoracic gas volume (ITGV), which was 136% of the mean value of 24 workers with an abnormally high index. There was also a decreased specific airway conductance (sGAW), which was 58.5% of the mean standard of 11 workers with an abnormal index. NB. The authors noted that an increased intrathoracic gas volume results in flattening of the diaphragm and shortening of muscle fibres that weakens the muscle force. Significant functional changes were found in the respiratory muscles in which both the maximal inspiratory pressures (MIP) and maximal expiratory pressures (MEP) were significantly lower in the chlorfenvinphos workers compared to controls i.e. MIP = 97.2 ± 28.3 cm H2O vs. 120.7 ± 31.7 cm H2O; and MEP = 113.9 ± 44.2 cm H2O vs. 154.4 ± 40.2 cm H2O respectively (p< 0.01). A possible dose-related effect was also observed in a subanalysis of workers years of exposure: workers with > 10 years of chlorfenvinphos exposure had a significantly lower MIP compared to workers with

Strengths Conducted objective health assessment and internal and external exposure assessment Limitations Small sample size. No explanation for the use of three different referent groups for comparisons. Fails to provide quantitative data on the results of AChE activity and electrophysiological tests, which were performed in hand

This study reported impaired respiratory muscle function in men occupationally exposed to the OP chlorfenvinphos. The authors noted that weakness of respiratory muscles could be due to pathologic muscle changes or fatigue as well as impairments of the nervous system. The electro-neurophysiological evaluations of the respiratory muscles were inferred from tests


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< 10 years work exposure i.e. 87.2 ± 28.06 cm H2O, (n = 17) vs. 106.6 ± 26.8 cm H2O, (n=18) respectively (p < 0.05). [Neuromuscular transmission testing] No disturbances in neuro-muscular transmission were observed although some disturbances were found in 3 workers via electrostimulating myasthenic testing (no further information was provided). No quantitative neurophysiological data was provided.

Also the study sought to evaluate respiratory muscle function) Inadequate control of confounders for PN

conducted on the hypothenar muscle of the finger and ulnar nerve. Therefore, this suggests that the reported lack of disturbance in neuro-muscular transmission requires more conclusive evidence in the actual muscles before dismissing any neurologic aetiological component

Peiris-John

2002 [FARMERS REGULARLY EXPOSED TO OP PESTICIDES] Exposure Data: Mean AChE levels in farmers did not differ from controls between or during the cultivation season (i.e. mean levels for both groups between the cultivation season (i.e. measured at June/July) were 27.92 U/g, SD 3.21 vs. 26.52U/g, SD 2.64 respectively), although levels in both groups were significantly reduced at the second evaluation (during season) compared to the first (i.e. mean levels in farmers and controls measured in April/May were 26.6 U/g, SD 3.88 and 24.9 U/g, SD 2.57 respectively; p< 0.01). Health Outcome Data: [Clinical examinations] There were no abnormalities in subjects, who were considered to be healthy (no further information was provided). [Nerve conduction studies] Between cultivation seasons, sensory conduction velocity in farmers was significantly higher than controls, whereas the motor conduction velocity was significantly lower between cultivation seasons compared with controls. The authors suggested that the unexpectedly higher sensory conduction velocity in farmers during the intercultivation season (i.e. June/July) could be indicative of damaged peripheral nerves becoming highly sensitive although such an assertion would require further verification.

Cultivation Season Between During

Measurement n Mean SD Mean SD t Value† P Value‡

Strengths Neurophysiological testing included assessment of neuromuscular transmission. Investigators were blinded to subjects exposure status Adequate control for confounders of PN Limitations Small sample size. Selection process not described. Failed to account for farmers co-exposure to other pesticides. Use of AChE activity as biological measure of OP exposure is

The finding that farmers chronically exposed to low-level OP pesticides (among others) had significantly altered sensory and motor effects should be considered in context with the limitations of this study


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Sensory conduction velocity (m/s) Farmers 28 44.99 7.47 35.5 9.28 4.56 < 0.01 Controls 25 40.65 8.11 35.7 7.88 2.17 0.04 t value§ 2.02 -0.07 P value0.93 0.04 װ Motor conduction velocity (m/s) � Farmers 26 51.08 3.83 51.00 5.65 -0.06 0.94 Controls 26 53.36 4.07 51.90 4.38 1.54 0.13 t value§ -2.03 -0.63 P value0.52 0.04 װ

†t value comparing between and during cultivation seasons ‡P value comparing between and during cultivation seasons § t value comparing farmers and controls P value comparing farmers and controls װ The authors also observed a significant reduction in sensory conduction velocities in both farmers and controls during the cultivation season compared with the intercultivation seasons (April/May). However, no difference in sensory and motor conduction velocities in both groups were seen during the cultivation seasons (June/July). Motor conduction velocities were similar in the farmers during and between cultivations seasons. [Neuromuscular transmission testing] No significant differences were observed between farmers and controls for evaluation of sensory/motor latencies or neuromuscular transmission during and between cultivation seasons

compromised by the fact that farmers were also exposed to carbamates (which inhibit AChE). Confusing description of cultivation seasons No description of clinical neurological examinations performed

Pilkington 2001 [SHEEP FARMERS/WORKERS WHO HAD DIPPED SHEEP] Health Outcome Data: NB. Only data relating to peripheral neurological symptoms are presented below. [Questionnaire] With regards to prevalence of reported symptoms, autonomic symptoms were most often reported within all groups followed by sensory symptoms, then muscle weakness symptoms of the upper and lower limbs.

Symptom group %

Muscle weakness Ceramics 0.0 Farmers not sheep dippers 1.9 Sheep dippers 7.8 Sensory Ceramics 1.9 Farmers not sheep dippers 5.7 Sheep dippers 11.6

Strengths Large study population. Several exposure measures used i.e. cumulative exposure and exposure intensity. Employed an objective assessment of sensory neurological symptoms i.e. validated QST tests. Adequate participation rates 88% and 62% among sheep and pig farmers

Subsequent analyses of symptom prevalence were based on an overall symptom indicator that combined peripheral and autonomic symptoms together thereby precluding further evaluation of possible associations between OP exposure and PN symptom prevalence. Sensory threshold results showed


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[Quantitative sensory threshold testing] For comparison of mean thresholds between groups, after correcting for age there was no evidence of differences in mean hot and vibration thresholds among groups. However, there was evidence that cold thresholds among sheep dippers were consistently higher across ages than among farmers who were not sheep dippers (data represented graphically). For the assessment of the relationship between cumulative exposure and sensory thresholds, without adjusting for covariates the authors noted relatively weak positive linear effects of the cumulative exposure indices (OPEXP and DAYS) and both cold and vibration thresholds. However, after adjusting for confounders (age, sex, and country effects) there was no evidence of a significant positive association between either of the two cumulative exposure indices and the three sensory thresholds. However, there was a small but significant negative association between cumulative exposure and hot thresholds, which was equivalent to a 6% decrease in hot sensory threshold per 74 days dipped.

Exposure Hot QST‡ (x effect) ORs (95% CI)

Cold QST‡ (x effect) ORs (95% CI)

Vibration QST‡ (x efORs (95% CI)

Unadjusted for covariates DAYS 0.99(0.93 to 1.05) 1.04(1.00 to 1.09) 1.06(1.01 to 1.12) OPEXP 1.01 (0.95 to 1.07) 1.04(1.01 to 1.09) 1.06(1.01 to 1.12) Adjusted for covariates DAYS 0.94(0.89 to 1.00) 0.99 (0.95 to 1.03) 1.01 (0.96 to 1.06) OPEXP 0.96(0.91 to 1.01) 0.99 (0.96 to 1.03) 1.01 (0.96 to 1.05)

p<0.05 ‡Adjusted for age, sex, country and occupational group Variables DAYS and OPEXP represent cumulative exposure indices for exposure to OPs and days dipped, scaled by the interquartile range of the values across all subjects: 74 days for DAYS; and 2350 nmol.mmol-1.days for OPEXP Analyses of the effect of average intensity of exposure and duration of exposure showed that concentrate handling intensity was significantly associated with vibration threshold. Intensity of splashing and duration of exposure were not significantly associated with other response variables.

Average exposure intensity Hot QST† p values

Cold QST† p values

Vibration QST† p values

AVCONC 0.819 0.594 0.031 AVSPLASH 0.710 0.130 0.276 AVOPEXP 0.714 0.166 0.067

† Adjusted for age, sex, country, occupational group and total days dipped Variables AVCONC, AVSPLASH, ad AVOPEXP correspond with average exposure intensity to concentrate, splash, and both respectively Estimated effects from a linear regression model that includes the effect of concentrate handling intensity (ever versus never), adjusted for total days dipped (and other covariates i.e. age, sex, country, occupational group) showed those who acted as concentrate handlers had 20% higher cold threshold (p=0.05). Adjusted for covariates, cold thresholds among sheep dippers who had not handled concentrate remained 41% higher than among farmers who were not sheep dippers

respectively; 60% and 80% among ceramic workers. Adequate control of confounders for PN Limitations Cumulative exposure to OP dips based on self-reports of exposure and exposure modelling. QST was the only neurophysiological test performed. Multiple comparisons

evidence of higher cold sensation thresholds among sheep dippers exposed to OPs compared with non-exposed farmers. There was no evidence of an effect of low-level cumulative exposure on sensory thresholds. However, further statistical analyses identified exposure to dip concentrate as an important exposure factor in relation to having higher sensory thresholds


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Multiplicative effects for Hot QST

OR (95% CI) Cold QST

OR (95% CI) Vibration QST OR (95% CI)

Sheep dippers v: Farmers not sheep dippers 1.08 (0.73 to 1.64) 1.41 (1.07 to 1.86) 1.06 (0.75 to 1.49) Ceramic workers 1.47 (1.05 to 2.04) 1.22 (0.97 to 1.54) 1.19 (0.89 to 1.57) DAYS (total days dipped) x 74-1 days 0.93 (0.88 to 0.99) 0.98 (0.95 to 1.02) 0.99 (0.95 to 1.04) Handling concentrate Ever v never 0.92 (0.71 to 1.21) 1.20 (1.00 to 1.45) 1.09 (0.87 to 1.37)

Srivastava 2000 [QUINALPHOS MANUFACTURING WORKERS] Exposure Data: Mean whole blood AChE levels in QP workers were not statistically different from controls i.e. 24.27 ± SD 11.21 µmol/min/100 ml (range 1.8 to 45.2 µmol/min/100 ml) vs. 24.41 ± SD 12.60 µmol/min/ 100 ml (range 6.6 to 40.6 µmol/min/ 100 ml). Study subject’s work history spanned 5.7 years (SD 4.4) compared to 3.35 years (SD 3.9) in control subjects. Health Outcome Data: NOTE. The authors classify the following outcomes under CNS effects: [Clinical neurological exam] Subjects exposed to QP showed significantly higher prevalence of abnormal plantar and ankle reflex. Knee reflexes showed non-significant effects

Variables Exposed (n=59)

Control (n=17)

P value

Subjects with abnormal reflexes Plantar 26 (49.1%) 0 <0.05 Ankle 20 (37.7%) 1 (5.8%) <0.05 Knee 8 (15.1%) 0 >0.05

The authors noted that 10 of the 26 subjects with abnormal plantar reflexes had diminished responses, and 8 subjects showed an equivocal response, with no response elicited in the remaining 8 subjects. For 20 subjects with abnormal ankle jerk reflexes, 8 showed a diminished response , 8 showed an equivocal response and no response was elicited in the remaining 4 subjects

Strengths Biological measure of exposure to OP. Employed objective clinical health assessment (for PN symptoms) that was conducted by trained individual. Adequate control of confounders for PN Limitations Small sample size. Questionable referent population/selection process. Exposure assessment based solely on AChE levels in blood with no evaluation of external/ambient levels of OP exposure. Lack of analysis of the effect of co-exposures to other

The finding that quinalphos manufacturing workers chronically exposed to the OP quinalphos showed a higher prevalence of abnormal reflexes of the foot and ankle should be interpreted with caution given the inadequate exposure assessment and lack of account of subjects co-exposures to other potentially neurotoxic chemicals during the manufacturing process


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chemicals in the manufacturing process. No neurophysiological assessment of peripheral neuropathy symptoms

Steenland 2000

[TERMITICIDE APPLICATORS EXPOSED TO CHLORPYRIFOS AND CHLORDANE] Exposure Outcome Data: Applicators (n=191) applied termiticides for a mean of 2.4 years (SD,2.2), however 34 % had also applied chlordane for a mean of 7.0 years, SD (6.3). 90% of applicators (173) applied other pesticides for a mean of 2.5 years (SD 2.5). 76 and 126 applicators reported having used termiticides within the last week and current year respectively. Mean urinary TCP levels in 65 applicators who applied termiticide in the last week was higher than in 40 applicators who did not (629 ug/L, SD1536.6; and 119 ug/L, SD 196 respectively). This contrasts with the low-level detected in 52 nonexposed subjects): urinary mean TCP level = 6.2 ug/L, SD 6.1) which the authors noted is within the normal range of US adult population (two orders of magnitude lower than that of the termiticide workers). Eight applicators reported having experienced previous poisoning episodes. Health Outcome Data: The paroxonase gene was genotyped in 184 applicators, 10% (18) of whom were homozygous for the gene responsible for potentially producing low levels of paraoxonase and thus had an increased susceptibility to the effects of chlorpyrifos. A similar proportion of non-exposed subjects also had this genotype. [Symptom questionnaire] Exposed subjects reported significantly more symptoms experienced in the last month than nonexposed subjects. These include: dizziness and loss of strength in limbs (p <0.05). The authors noted that four questions relating to loss of strength in arms, fingers, hands and legs were highly correlated with each other (pairwise Spearman correlation coefficients 0.40-0.60). The poisoned subgroup had significant associations for dizziness and arm/hand/leg weakness (among others) compared to nonexposed group (p<0.05). No significant trends were associated with TCP. NB. For the objective assessments below, the authors reported only p values and do not present any quantitative data. [Clinical examination] There was no significant differences between exposed and no-exposed subjects for abnormal upper extremity tremor, vibration sensitivity, pin prick tests, gastroc soleous reflex or Romberg test. TCP level was not a significant predictor of any clinical outcome. Poisoned subgroup had significantly decreased wrist vibration and pinprick sensitivity (p = 0.003 and 0.002 respectively) compared to non-exposed group. The susceptible genotype subgroup (n=18) had an increased odds of an abnormal gastroc soleus reflex (p=0.01) and Romberg test (p=0.04).

Strengths Adequate sample size Used two non-exposed groups that included friend controls. Use of two neurologists trained to conduct comparable methods. Conducted a range of tests to evaluate neurological function. Used extensive methods to locate uncontactable candidates. Adequate account of potential confounders. Exposure assessment included internal evaluation Limitations Possible selection bias due to low participation rate among contactable

This study provides very limited evidence of neurological effects in termiticide applicators exposed to chlorpyrifos. This is largely due to the subjects having substantial co-exposure to chlordane and other pesticides, and subjects not being representative of all the exposed workers.


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First Author



Tremor – No significant variables were significantly related to tremor. Vision tests – the authors eliminated 24 subjects with congenital loss of colour vision. No significant difference was observed between exposed and nonexposed subjects, although trend of poor colour vision with increased levels of TCP for one eye was observed (p=0.0006). Visual acuity did not differ between exposed or non-exposed groups. Smell tests – there were no differences in the ability to correctly identify odours between exposed and non-exposed groups, which was not correlated with the TCP level. [Posturography] Sway – Length and area of sway were correlated with a Spearman correlation coefficient of 0.60-0.70 for 12 sway tests. Exposed subjects did not perform as well as the non-exposed groups which was significantly different for length of sway on a hard surface with eyes open (p=0.04). This was more pronounced when compared to non-exposed friends (p=0.05). Separate comparisons of the exposed group to each nonexposed groups revealed more sway deficits (although these were not significant). TCP level significantly predicted some sway variables but this was no-longer significant after removal of a highly exposed applicator. No significant differences were observed for poisoned subgroup or those with susceptible genotypes. Subjects exposed prior to 1998 had significant associations for more area of sway with eyes open regardless of platform type compared to non-exposed subjects (soft/hard platform p=0.03, 0.05). The duration of chlorpyrifos exposure was also significantly correlated with length of sway for the right leg (p=0.04). [Sensory threshold] Vibrotactile sensitivity – Exposure did not decrease vibrotactile sensitivity for either finger or toe (tested at 31.5 and 125 Hz). However, exposure was associated with a significantly increased sensitivity for toe tested at 125 Hz. This was also apparent for those formely exposed (p=0.04); and for the finger in those susceptible to the effects of chlorpyrifos (p=0.05). TCP was not a predictor of vibrotactile sensitivity. [Nerve conduction studies] No significance differences were observed between applicators and the combined non-exposed group for peroneal, ulnar or sural nerve conduction velocity or amplitude. However, few significant differences were found when subgroups of the exposed subjects and non-exposed groups were compared separately: applicators had significantly less ulnar amplitude than the NC state employees (p=0.03); analysis of exposed applicator subgroups revealed no significant differences from the non-exposed group combined (except for the eight poisoned men who had significantly higher ulnar amplitude compared to non-exposed); significantly lower ulnar amplitude (p=0.03) but faster sural nerve conduction (p=0.05) were observed in the currently exposed applicators compared to the NC state employees. Tests for trends among applicators by duration of exposure (to either chlorpyrifos, chlordane or other pesticide) or urinary TCP levels were not significant

eligible participants; there being no evaluation of whether non-participants differed from subjects who participated. Friend controls may represent a select group of more sociable subjects. Age of 90% applicators was young (> 50) which may preclude detection of delayed neurotoxic effects. Applicators co-exposure to other pesticides including chlordane which preceded chlorpyrifos as the main pesticide used against termites. Ulnar nerve served as a substitute in some subjects where it was difficult to measure nerve conduction in sural sensory.

Table 8. EXPOSURE SCENARIO AND PERIPHERAL NEUROPATHY AND NEUROMUSCULAR DYSFUNCTION


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Section 8.A. ACUTE EXPOSURE WITH CHRONIC EFFECTS Table 8.A.1. Cohort studies

Test Name First Author and Date

Kawana 2001

Miranda, 2002a

Miranda, 2002b

Miranda, 2004

Nakajima, 1999

Questionnaires + St Luke's Symptom Questionnaire

Nakajima symptom questionnaire survey +

Muscle strength Grip strength + + Pinch strength + + Quantitative sensory tests Cutaneous tactile quantitative vibration thresholds - index finger

- +

Cutaneous tactile quantitative vibration thresholds - toe


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Table 8.A.3. Cross-sectional studies

Test Name

First Author and Date

Jalali 2011

McCaculey, 2001

Miyaki 2005

Nishiwaki 2001

Spencer, 2001

Stallones & Beseler, 2002b

Questionnaires McCauley Telephone Interview Symptom Survey + -

Stallones & Beseler Neurological Symptoms Questionnaire

+

Clinical evaluation signs/symptoms +

Sensory Nerve Conduction Studies (NCS) +

Motor NCS +

Quantitative Sensory Tests

Vibration Threshold - Hot Thermal Threshold Cold Thermal Threshold EMG + Evoked Potential Somatosensory - Muscle (hands, leg and foot) -


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Test Name

First Author and Date

Jalali 2011

McCaculey, 2001

Miyaki 2005

Nishiwaki 2001

Spencer, 2001

Stallones & Beseler, 2002b

Stabilometry - -

Table 8.A.4. Case series studies

Test Name First Author

and Date Dahlgren 2004

Clinical examination - Muscle strength

Grip strength not reported

Table 8.A.5 Case Reports

Test Name First Author and

Date Loh

2010 Soummer

2011 Clinical neurological examination -

Muscle strength +


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Test Name First Author and

Date Loh

2010 Soummer

2011 Electrophysiological NCS - EMG - Repititive nerve stimulation +

Section 8.B. CHRONIC LOW-LEVEL EXPOSURE Table 8.B.1. Cohort studies


Albers 2004b

Albers 2004a

Albers 2007

Starks 2012b

Questionnaires

Albers neurological questionnaire - -

Clinical examinations

Albers clinical neurological evaluation - -

Starks neurological clinical exam +

Muscle strength Hand strength -


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Albers 2004b

Albers 2004a

Albers 2007

Starks 2012b

Stabilometry - Sensory Nerve Conduction Studies (NCS)

Negative peak amplitude - - Onset latency - - Conduction velocity Z scores - +

Summary Z scores (sensory conduction) - +

Motor NCS Motor response amplitude - - - latency - - + F-wave latency - + - Conduction velocity + - Summary Z scores (motor conduction) -

QST/Vibrotactile threshold testing -

Table 8.B.2. Case control studies



Crawford 2008

Questionnaires


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Crawford 2008

Jamal Mayo Clinic based neurological symptom questionnaire

+

Telephone interview +

Jamal Clinical Evaluation Symptoms (PN) + Reflexes + + Sensation + + Muscle power (via inverse MRC scale) - +

Motor Nerve Conduction Studies (NCS)

Distal motor latencies + + Conduction velocities + + Muscle action potential amplitude + +

F-wave latency + + Sensory NCS Sensory amplitude + + Sensory latency + + Quantitative Sensory Tests (QST)

Vibration threshold + + Hot thermal threshold + + Cold thermal threshold + +


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Crawford 2008

EMG + + Neuromuscular junction test Single Fibre EMG (SFEMG) +


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Table 8.B.3(i). Cross-sectional studies


Abdel-Rasoul 2008

Albers 2004c

Bazylewicz Walczak

1999

Farahat 2003

Horowitz 1999

Hoshino 2008

Kamel 2005

Kamel 2007b

Questionnaires Albers Clinical Symptoms Interview

-

Finnish Symptoms -

Farahat Symptoms +

Kamel Symptoms + +

Pilkington Symptoms

Kilburn Symptoms

Hoshino +

Steeland

Clinical neurological examination

+ - + +

Vision/Smell tests

Muscle Strength

Grip strength Tufts Quantified Neurological Evaluation

-

Spirometric investigations

Sensory Nerve Conduction Studies

Conduction velocity - +

Latency - Amplitude - + Z-scores - Motor NCS Conduction velocity - +


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Abdel-Rasoul 2008

Albers 2004c

Bazylewicz Walczak

1999

Farahat 2003

Horowitz 1999

Hoshino 2008

Kamel 2005

Kamel 2007b

Latency - Amplitude - - F-wave latency - + H-reflex + Z-scores - Quantitative Sensory Tests (QSTs)

Hot Cold + Vibration + Neuromuscular transmission tests

Repetitive Nerve Stimulation

Audiometric examination +

Vestibular examination (electronystagmography)

+

Posturography Tremor tests

Table 8.B.3(ii). Cross-sectional studies continued


Kiefer 2000

Kilburn 1999

Kimura 2005

Konieczny 1999

Peris-John 2002

Pilking-ton

2001

Srivas-tava 2000

Steen-land 2000

Questionnaires Albers Clinical Symptoms Interview

Finnish Symptoms

Farahat Symptoms

Kamel Symptoms


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Kiefer 2000

Kilburn 1999

Kimura 2005

Konieczny 1999

Peris-John 2002

Pilking-ton

2001

Srivas-tava 2000

Steen-land 2000

Pilkington Symptoms +

Kilburn Symptoms +

Hoshino

Steeland +

Clinical neurological examination

+ - + +

Vision/Smell tests -

Muscle Strength

Grip strength + Tufts Quantified Neurological Evaluation

Spirometric investigations +

Sensory Nerve Conduction Studies

-

Conduction velocity - + -

Latency - Amplitude + Z-scores Motor NCS - Conduction velocity - + -

Latency - Amplitude - F-wave latency H-reflex Z-scores Quantitative Sensory Tests (QSTs)

Hot - Cold + Vibration - + + -


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Kiefer 2000

Kilburn 1999

Kimura 2005

Konieczny 1999

Peris-John 2002

Pilking-ton

2001

Srivas-tava 2000

Steen-land 2000

Neuromuscular transmission tests

Repetitive Nerve Stimulation

- - -

Audiometric examination +

Vestibular examination (electronystagmography)

Posturography + + + Tremor tests -


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Table 9. OVERVIEW OF PERIPHERAL NEUROPATHY AND NEUROMUSCULAR DYSFUNCTION TESTS 1. The subdivision of tests into different domains is based predominantly on the information given in papers that have been summarised, reference books and grey literature (See bibliography). The information is summarised here to aid members in interpreting the studies examining peripheral neuropathy and neuromuscular dysfunction. Clinical Neurological Evaluation 2. A neurological evaluation is a complete clinical assessment of the nervous system, which seeks to detect possible abnormalities by determining the function of relevant components of the nervous system. Peripheral neuropathy (PN) and neuromuscular dysfunction (NMD) are detected via the use of several standard and well validated tests as illustrated in Figure 1. Subject’s history 3. Prior to any tests being conducted, a subjective assessment of possible health effects is conducted by taking the subject‘s history. This is necessary to help determine the type of neuropathy and possible causes. Subject history involves collecting data on symptoms (to identify patterns and its development and progression over time), family history of PN/NMD, and any existing medical conditions, viral diseases or medications being taken. 4. The next stage of the evaluation is an objective assessment of possible PN/NMD health effects, which involves conducting a clinical (physical) neurological examination and is followed by more specific diagnostic tests. NB. Blood tests and urine screening can also be conducted to help rule out/identify potential causes and measure levels of toxic substances respectively. Neurological examination


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5. The clinical neurological exam is a physical examination of the nervous system. It can be conducted by any medical health care provider without specialist knowledge in neurology. It seeks to detect any signs or symptoms related to neurological abnormalities (in this case PN and NMD) and follow a standard protocol. Subsequent neurophysiological tests are rendered worthless in the absence of the clinical information provided from these examinations. The table below details components of the neurological exam that are used to detect signs of PN/NMD. Test Description

Motor function exam Tests major muscle groups (i.e. shoulder, arms, hips and legs) with regard to their motor function,

movement (e.g. tremor, fasciculations), muscle tone and strength. A muscle wasting inspection helps reveal lower motor neuron signs of dysfunction i.e. as a result of lesions in the muscle, nerve, plexus, root or anterior horn cells. Unless severe, reduced muscle tone is hard to detect. Increased muscle tone is easier to detect and more significant and is indicated by spasticity or rigidity. Manual hand muscle strength is often evaluated using the Medical Research Council (MRC) Scale, and graded on a scale from 0 to 5 (whereby 0 = no active range of motion and no palpable muscle contraction; and 5 = full active range of motion and normal muscle resistance. However, manual muscle testing is considered to be limited due to the scale being ordinal and the subjective nature of the examiner’s scoring. To create more quantitative assessments of hand muscle strength, examiners use dynamometers, which are more sensitive and provide a continuous scale. Dynamometer measurements are usually based on grip and pinch strength, and the most commonly used (due to its reliability and validity) is the Jamar Dynamometers. The amount of muscle strength loss is determined by comparing the results with normative data. However, these tests are still considered to be psychophysical as they rely on the subject’s participation i.e. there is no way to differentiate the results of subjects who are biased (whether consciously or subconsciously) toward an abnormal study from those with organic disorders. Coordination, gait, and station are also assessed in a motor examination, which provides information on motor function and the more subtle aspects of brain function. Damage to any part of the nervous system can be reflected in gait e.g. upper motor neurone damage, cerebellar dysfunction, true vertigo, etc. To test gait abnormalities, the physician observes the subject walking on toes, heels, and in tandem, hopping on either foot, getting up from a chair and squatting. Coordination tests are mainly directed to assessing cerebellar function and involve assessing the


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Test Description strength needed to maintain certain positions, as well as the smoothness, accuracy, rhythmicity and speed of movement. Station refers to the ability to maintain an erect posture and relates to balance and imbalance (disequilibrium). To test for imbalance the subject is asked to stand both with eyes open and closed with a narrow base of support (feet close together). The physician records any excessive sway (falling to one side), or marked worsening in the ability to stand when the eyes are closed. Excessive sway with the eyes open is common with cerebellar (bidirectional) or vestibular (unidirectional) problems. However, inability to maintain an upright posture may also be associated with reduced muscular strength thereby highlighting a potential peripheral nerve or muscular issue. Excessive sway with eyes closed may be indicative of a disorder of conscious proprioception (i.e. joint position sense as occurs with peripheral neuropathy or certain central conditions) and is known as a Romberg sign

Sensory function exam This is performed to evaluate sensation i.e. sensory responses to the following types of stimulation: light touch, pain (via pin-prick test), vibration, and temperature. A proprioception assessment is often conducted (with eyes open or closed) to determine a subject’s sensation of limb position and movement i.e. their sense of the relative position of neighbouring parts of the body and the strength of effort being employed in movement.

Reflexes Various deep tendon reflexes are tested, which provide useful information on potential problems with sensation and/or motor function (plus other neurological dysfunctions). A reflex hammer is often used to determine the extent of damage to the following reflexes: masseter, biceps and triceps tendon, knee tendon, ankle jerk and plantar.

6. Testing of the 12 pairs of cranial nerve that emerge directly from the brain are summarised in the table below. NB. Other exams (not considered here) include behavioural, cognitive and mental status examinations. No Nerve Sensory or

motor Function Dysfunction (signs/ symptoms)† Tests††/ notes

I* Olfactory S Smell Altered smell sensations Smell tests where a subject blocks one nostril and is asked to identify a known odour placed under the open nostril (and vice versa)


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No Nerve Sensory or motor

Function Dysfunction (signs/ symptoms)† Tests††/ notes

II* Optic S Vision Visual field deficits, blurring, dim vision, blindness (incl. colour), blindspots

Visual acuity/field tests. Visual impairment can be quantified in terms of: (i) acuity – the amount of detail a person sees compared to normal (20/20) vision. Common in people with optical vision loss (i.e. caused by irregularities in the eyes refractive surfaces or ocular media). Visual acuity can be normal in people with optical vision loss but may be accompanied with reduced contrast sensitivity (i.e. an inability to discern objects and fine detail under reduced contrast or low contrast conditions); (ii) visual field – the area that a person can see (measured as an angle in degrees). A 180 degree range is considered normal. Less is abnormal. Common in people with neural vision loss and can be accompanied with reduced acuity. More specific tests can be performed to discriminate neural from optical losses of vision. Pupillary light reflex tests: The optic nerve serves as the afferent limb of this reflex. The photosensitive ganglion cells sense incoming light and convey information to the optic nerve. The swinging flashlight test can discriminate an optic nerve pathology. Colour vision involves photoreceptors (eye); retinal ganglion cells/optic nerve (cranial nerve) and visual cortex (brain). NB. Vision is also important for balance and thus intertwines with effects of the vestibular system/vestibulocochlear nerve

III Oculomotor M Eye movements. **Pupil dilation

Droopy eyelid, abnormal eye movements leading to double vision **Pupil dilated (non-reactive to light), blurred vision

Eye muscle tests (e.g. ocular movement and optokinetic nystagmus tests). NB. The oculomotor nerve has autonomic fibres that innervate the ciliary muscles and the


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sphincter muscles of the iris. Pupillary reflex tests (e.g. pupillary light reflex test and accommodation reflex test). For the pupillary light reflex test the oculomotor nerve represents the efferent limb responsible for constricting the pupil (via constriction of the sphincter muscle). It does this by synapsing with parasympathetic ganglion. For the accommodation reflex test the oculomotor nerve is responsible for changing the shape of the lens (via constriction of the ciliary muscle). NB. Because the somatic and autonomic components of oculomotor nerve intertwine with each other there are no specific tests to discriminate autonomic from somatic nerve eye effects.

IV Trochlear M Eye movements Double vision, impaired downward gaze

See tests used for Oculomotor nerve (III)

V Trigeminal S/M Facial sensations/ mastication. Blink reflex (sensory).

Pain in scalp, forehead, eyes, nose, lips and jaw Impaired facial sensation Reduced corneal (blink) reflex Chewing problems

Light touch, pain and temperature tests conducted on forehead, cheeks or chin. Corneal reflex test (aka blink reflex but involves physical stimulation of the cornea)

VI Abducens M Eye movements Abnormal/loss of eye movements leading to double vision

See tests used for Oculomotor nerve (III)

VII Facial S/M Taste/ facial expression/ mouth opening. Blink reflex (motor). **Lacrimation/salivation

Facial weakness Reduced taste Loss of corneal reflex **Altered lacrimation/ salivation

Facial asymmetry and involuntary movement checks Taste tests

VIII Vestibulococlear (auditory/acoustic)

S Comprises of cochlear nerve (which carries information about hearing), and the

Vertigo (one of three main types of dizziness)

Vertigo (illusion of movement) is the chief symptom of damage to the vestibular system, which serves to stabilise the eyes (among other functions). There are two


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vestibular nerve (carries information about rotation, gravity and balance)

main causes of vertigo: (i) peripheral vertigo that arises from damage to either the inner ear receptor, or vestibulocochlear nerve; and (ii) central vertigo due to damage to vestibular signalling processing centres of the brain. Vestibular function tests are conducted to differentiate between peripheral and central vestibular forms of dysfunction and include eye position and nystagmus tests (which in peripheral vertigo is unidirectional, horizontal/rotary) and other clinical neuro-opthalmological, orthoptic and otological tests

Hearing loss/ tinnitus (often accompanies peripheral vertigo). NB.Hearing loss has multiple causes: age, gender, acoustic trauma (i.e. very loud noise, e.g. farmers exposed to noisy farm equipment), inner ear disease, head injury, inherited conditions, ruptured ear drum, and otoxicity (i.e. exposure to a chemical or physical agent such as a microbe that produces a toxic response in the auditory or vestibular systems). More than 200 chemical substances are known to cause hearing loss. These include exposure to solvents and heavy metals, side effects of medications such as antibiotics and antineoplastics, and various pesticides1. Hearing loss can also be accompanied with vertigo, nausea or gait instability

Hearing loss/deafness can be categorised in two ways: (i) Conductive deafness is hearing loss caused by defects/obstruction in the conduction system (i.e external auditory canal, tympanic membrane, middle ear or the ossicles); (ii) Sensorineural deafness is hearing loss caused by damage to the inner ear/cochlea (sensory hearing loss), the auditory/vestibulocochlear nerve (neural hearing loss) or the brain. Assessment of hearing loss includes a physical examination via an otoscope to evaluate the ear canal and tympanic membrane. Air conduction tests are also performed and involves stimulating the ear with air to test hearing quality and the integrity of various auditory components. Bone conduction tests such as the Rinne/Weber tests are simple hearing tests

1 Literature (animal or human) on pesticide exposure and hearing loss is sparse. Most evidence come from case reports that reported permanent nerve damage to the vestibular (movement/balance) and cochlear (auditory) components of the eighth cranial nerve


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. that use recognisable sounds or tuning fork. These bone conduction tests can be used to distinguish between sensorineural and conductive deafness. To differentiate cochlear (inner ear) disease from direct VIII-nerve involvement in sensorineural hearing loss, further laboratory tests can be performed such as brain stem audiometry)

Imbalance (another type of dizziness). There are many causes of imbalance which include, otoxicity, ear, head and neck injuries, migraines, infections, hearing loss, depression, alcohol abuse, seizure disorders, motion sickness or sensitivity. These disturb the control systems that maintain the upright posture.

Normal balance requires the integration of proprioceptive2 inputs (e.g. sensation from the feet), and visual, and vestibular (inner ear) inputs. The visual and skeletal system (wrt the muscles, joints and their sensors) work together with the vestibular system to maintain orientation and balance. Imbalance is often referred to as unsteadiness or disequilibrium. NB. It can also be consequent to a peripheral nerve defect (e.g. motor weakness) and numbness in feet (sensory nerve issue), vision (potential cranial nerve issue). Individuals with peripheral neuropathy have imbalance because they cannot feel the floor beneath their feet. This is because the small nerves in the feet and toes are often the first to be affected resulting in numbness in the feet. This imbalance can be especially severe on unstable surfaces and when the subject attempts to walk in darkness, as visual cues become important.

2 Proprioception is the sense of the relative position of neighbouring parts of the body and the strength of effort employed in movement; the awareness of movement is derived from muscular, tendon and articular sources. Proprioception is either felt consciously or unconsciously and involves activation of a “proprioreceptor” i.e. a specific nerve (stimuli) receptor that is located in the sensory neurons of the inner ear and stretch receptors in muscles and joint supporting ligaments. Once activated information is sent to the brain via sensory receptors.


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Various tests of sensation, motor function tests (including gait, station and co-ordination that assess fine and gross motor skills) are conducted (See above). More specific tests for imbalance include computerised dynamic posturography, voltage-acutated nerve conduction thresholds and oculomotor testing.

IX Glossopharyngeal S/M Taste, swallowing Difficulty speaking, swallowing. **Impaired blood pressure regulation

Gag response Palatal, guttural articulation

X Vagus S/M Taste, swallowing and voice control. **Respiratory, cardiac, digestive and circulatory reflexes

Hoarseness, vocal cord paralysis. **Cardiac and respiratory instability **Stomach atonia

See tests used for Glossopharyngeal nerve

XI Accessory M Shrugging, head movement

Inability to shrug shoulders or turn head sideways

Test for impairments

XII Hypoglosal M Tongue movement, swallowing, speech

Difficulty speaking and swallowing Tongue inspections

* Nerves I and II emerge from the cerebrum (i.e. the upper part of the brain with its two hemispheres). The remaining ten nerves (12 in total) emerge from the brainstem (i.e. the lower part of the brain that joins with the spinal cord) **These nerves also carry parasympathetic/sympathetic nerves for autonomic stimulation of various glands and smooth muscles in structures of the head and face † Most of these symptoms/signs can have other causes i.e. that are not due to impaired cranial nerve function. “Dizziness”, which is sometimes referred to as imbalance or instability, is a subjective and non-specific symptom that is difficult to diagnose due to it being a common complaint in different diseases. ††These cranial nerve examinations/tests are part of the neurological examination (i.e. physical tests used to identify possible cranial nerve dysfunction that are not conducted in a specialised laboratory). Diagnostic tests 7. Two types of diagnostic tests are typically used to evaluate PN and NMD: Electrodiagnostic tests and Quantitative Sensory Tests (QSTs). These tests are performed by physicians specialising in neurology are sometimes referred to as neurophysiological


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tests. These tests can distinguish axonal from dyemyelinating neuropathies and rely on relevant history and neurological examination information being obtained to avoid misdiagnoses. Furthermore, due to the invasive nature of some of these tests clinical history and examination enables these tests to be tailored to an individual’s/ or investigator’s needs. Other tests reported in the review that were used to assess/diagnose conditions that may have a peripheral nerve component include posturography, Tufts quantitative neuromuscular exam (TQNE) and spirometry (although the latter is not generally considered to be a reliable measure of PNS function). Electrodiagnostic Tests 8. Electrodiagnostic tests represent truly objective extensions of the physical evaluations of neurological function. These tests are sometimes referred to as electrophysiological tests and have been technically categorised as those that either record biological response to stimulation (e.g. nerve conduction studies (NCS)) or record bioelectrical activity (e.g. electromyography (EMG), electronystagmography (ENG) and audiometry). These electrodiagnostic tests assess the function of nerves, muscles or neuromuscular junction by measuring their electrical activity. They are often performed in conjunction with each other. Nerve conduction studies (NCS) 9. These tests are sometimes referred to as nerve conduction velocity (NCV) test – are typically performed before an EMG because they are less invasive and less uncomfortable. NCS test the function of large fibre motor and sensory nerve populations, by recording the speed at which signals (electrical impulses) travel along these nerves. This speed is known as the nerve conduction velocity (or waveform conduction speed). It is suggested that both sensory and motor nerves are evaluated in at least one arm and one leg, and preferably in at least two segments of the nerve i.e. proximal and distal. Factors known to affect NCV results (non-pathological) include: age, which in adults is associated with a slowing down of NCV after 60 years; and body temperature, whereby temperatures less than 34oC are associated with a slowing down of NCV and an increased amplitude of the waveform action potential (see below). Keeping limbs warm to a surface temperature of at least 32 oC or adding 5% to conduction velocity per oC below 34 oC are approaches to circumventing the confounding effect of body temperature.


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10. Approaches to assessing nerve conduction in portions of nerves that are inaccessible to direct assessment using conventional techniques (i.e. nerves that are more proximal – near the spine) are referred to as late response (F-wave and H-reflex testing). These two additional studies are complementary to NCS and are performed during the same subject evaluation Test Description

Motor nerve conduction study A stimulating electrode is placed over a large myelinated motor nerve, while an active

electrode is placed over the muscle of interest and a reference electrode is placed distal to the muscle. A low intensity electrical impulse is introduced to stimulate the nerve, which then fires and produces electrical signals (i.e. a depolarisation wave) that travels along axons and eventually depolarises nearby muscle fibres resulting in an electrical response in the muscle, which is detected and recorded by a computer. This waveform or action potential is amplified to generate the compound motor action potential (CMAP) which represents the sum of individual muscle fibre action potentials. From the CMAP several response parameters can be determined: (i) waveform amplitude or size of the response (mV), is a function of the total number of fibres stimulated and the synchronicity of the impulse. Normative values for various motor nerves are shown below. A decreased amplitude is indicative of axonal loss; (ii) latency (ms) is defined as the time interval between stimulation and depolarisation onset i.e. the time taken for the electrical impulse to travel from the stimulation site to the recording site. Latency is often recorded at two separate sites in relation to the portion of the nerve: proximal (closer to the spinal cord) and distal (further away from the spine). This is done to enable calculation of conduction velocity (and as such requires that the electrical stimulation is applied on the skin at various points along the course of a motor nerve being tested). Two types of latency are determined: peak latency is the time interval from stimulation to the peak of the CMAP; and onset latency is the time interval from stimulation to onset of the CMAP. Normative values for various motor nerves are shown below. Increased distal latency is indicative of a motor nerve abnormality. It indicates that nerve conduction is slowed, which is associated with demyelination of the nerve. (iii) motor conduction velocity (m/s) is calculated by measuring the distance between the stimulating and recording electrodes and dividing by the latency i.e. the time taken for the electrical impulses to travel between electrodes. The calculation is also described as


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Test Description Distance (A-B)/ Time (A-B) – where A = Proximal; B= Distal. Normative values for various motor nerves are shown below. Slow motor nerve conduction velocity is considered to be suggestive of an abnormality e.g. demyelination.

Sensory nerve conduction study For the sensory NCS, the electrical stimulation is applied near a sensory nerve of interest and the electrical response is recorded from a distant site along the nerve (e.g. active and reference electrodes are in the form of a ring placed around the finger). Unlike the motor NCS, the stimulation may be antidromic i.e. running against the normal (orthodromic direction of impulse conduction). Similar to motor NCS, the response is detected and recorded by a computer and the resulting compound sensory action potential (CSAP) enables the following response parameters to be determined: (i) amplitude (µV). Negative peak amplitude is sometimes determined and represents the peak amplitude within the portion of the waveform below zero. Normative values for various sensory nerves are shown below. A low/reduced amplitude is indicative of axonal loss; (ii) latency (ms). Similar to motor NCS, peak and onset latency are determined. Normative values for various sensory nerves are shown below. Increased latency is indicative of a sensory nerve abnormality. (iii) sensory conduction velocity (m/s) is calculated is calculated in a similar manner as for motor NCS. Normative values for various sensory nerves are shown below. Slow sensory nerve conduction velocity is considered to be suggestive of an abnormality e.g. demyelination. Sural nerve testing is considered particularly important as an abnormal sural response is generally accepted as one of the most sensitive indicators of sensory neuropathy. NB. Sensory NCS can only evaluate large myelinated nerve fibres. Therefore, QST can be used to evaluate small and unmyelinated nerve fibres.

Normal data for motor and sensory nerve conduction studies (Misulis & Head, 2003)

Nerve Distal* latency Nerve conduction velocity Amplitude Motor NCS


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Test Description

Median ≤ 3.8 ms @ 7 cm ≥ 50 m/s ≥ 5 mV Ulnar (below elbow) ≤ 3.1 ms @ 7 cm ≥ 50 m/s ≥ 5 mV Ulnar (across elbow) ≥ 50 m/s ≥ 5 mV Radial ≤ 3.4 ms @ 6 cm ≥ 50 m/s ≥ 5 mV Peroneal ≤ 6.0 ms @ 8 cm ≥ 40 m/s ≥ 2.5 mV Tibial ≤ 5.0 ms @ 10 cm ≥ 40 m/s ≥ 2.5 mV Sensory NCS Median ≤ 3.5 ms @ 13 cm ≥ 55 m/s ≥ 10 µV Ulnar ≤ 3.2 ms @ 11 cm ≥ 54 m/s ≥ 1 µV Radial ≤ 2.8 ms @ 10 cm ≥ 18 µV Sural ≤ 4.2 ms @ 14 cm ≥ 42 m/s ≥ 4 µV

NB. Healthy nerves conduct signals with greater speed and strength compared to damaged ones * Latency of the distal portion of a sensory/motor nerve

F-wave study This is a late response (i.e. appears after the principal action potential (AP)) and tests the conduction of the portion of a motor axon that is proximal to the stimulation site. A similar procedure to the motor NCS is used with the exception of the electrical stimulation, which is applied on the surface of the skin near a nerve in a manner that sends impulses both distally (orthodromically – so that APs travel toward the muscle) and proximally (antidromically – so that APs travel towards the soma of the motor neuron). This is achieved by turning the stimulating electrodes (anode/cathode) in opposite directions. F-wave study also has different machine settings and a separate stimulation (to obtain a larger number of responses). The reason why the F-wave is considered a late response is because the antidromic movement of APs results in the eventual depolarisation of the dendrites of the soma, which itself leads to the passive spread of electric potentials (i.e. electrotonic conduction of depolarisation) back to the axon hillock (specialised part of the soma that connects to the axon). A new AP is then transmitted back to the muscle causing activation of the motor end-plate (highly excitable region of the muscle fibre plasma membrane) and later APs in


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Test Description muscle fibres. The response parameter measured is the latency, which is defined as the time interval from stimulus to the onset of the F-wave. NB. The shortest of 10 successive F-waves is taken as the point to be measured.

H-reflex This is another late response, and has been described as the electrophysiological counterpart of the Achilles tendon reflex. The H-reflex measures the conduction through afferent and efferent fibres in the monosynaptic reflex arc and involves stimulating the tibial nerve and is most easily recorded from the calf muscle i.e. assessing the gastrocnemius /soleus muscle complex in the calf, although other muscles are also tested in rare instances. The H-reflex appears at a lower intensity of stimulation compared to the CMAP because the associated muscle afferents are larger and have a lower threshold for electrical activation compared to the alpha motorneurons involved in the CMAP. The H-reflex is usually performed bilaterally due to symmetry of responses being an important criterion for abnormality. It is usually absent when the F-wave and other nerve conduction studies are abnormal. Normal latency values ≤ 35 ms and the amplitude differs widely between individuals.

Repetitive nerve stimulation (RNS) This is a special type of NCS that tests the function/integrity of the neuromuscular junctional synapse. RNS is considered a sensitive method for detecting neuromuscular transmission abnormalities. After choosing the nerve to be stimulated, responses (CMAP amplitudes) are recorded from a muscle supplied by the nerve. CMAP amplitudes are recorded at baseline and during a train of 5-6 stimuli delivered at 2-5 Hz (or higher). Decrements in amplitude between the first and last stimulation of more than 10% are considered indicative of abnormal NMJ function.

11. Nerve conduction studies can also be used to diagnose cranial nerve dysfunctions. Only three cranial nerves are readily tested in this way: the trigeminal (V), facial (VII), and spinal accessory (XI) nerves. The latter two nerves (facial and spinal accessory nerves) are commonly tested because they travel superficially, which allows easy access to electrical stimulation from the surface. However, the trigeminal nerve is evaluated using blink reflex activity and information extrapolated from this (NB. The facial nerve can also be evaluated using the blink reflex).


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Blink reflex test 12. The blink reflex test (which is called the corneal reflex when performed in a cranial nerve exam) is an electrically evoked analogue of the corneal reflex. It is initiated by stimulating the supraorbital branch of the trigeminal nerve. Surface electrodes are used to stimulate the nerve and to record the evoked muscle action potentials. An active electrode (G1) is placed on the lower medial aspect of the orbicularis oculi (muscle responsible for closing the eyelid), a reference electrode (G2) 2 cm lateral to (G1), and a ground electrode under the chin. For the assessment of facial synkinesis (i.e. involuntary muscular movements) two pairs of recording electrodes are placed on the same side of the face, one pair over the orbicularis oculi and the other over the orbicularis oculi or platysma (neck muscle). The response propagates into the pons and branches to the lateral medulla of the brain. It then branches to innervate the ipsilateral (same side) and contralateral (opposite side) orbicularis oculi via the facial nerve. Two responses are evaluated, (i) an ipsilateral R1 latency (which represents the conduction time along the trigeminal (or facial) nerves and pontine relay and (ii) bilateral R2 latency, that reflects the excitability of interneurons and synaptic delay in addition to the axonal conduction time (which is longer and inherently variable from one trial to the next). The eyelid closure of the blink is associated with the R2 response. Prolonged latencies of R1, ipsilateral R2 and contralateral R2 are suggestive of afferent defects. In facial nerve lesions there is a delay in the reflex latency only on the affected side, regardless of the side of stimulation. Electromyography (EMG) 13. This is considered an essential part of a complete electrophysiological assessment. EMG provides information about the integrity of the muscles and its innervations by assessing the muscles response to motor nerve signals. It compares the muscle’s electrical activity at rest and when contracted. EMG can help toward differentiating between muscle and nerve disorders and help define the aetiology of muscle weakness i.e. differentiate between true muscle weakness and reduced use due to pain or lack of motivation. Due to its invasive nature it is usually performed subsequent to less invasive tests, or after an equivocal PN diagnosis. Experts suggest that EMG should be done in at least one proximal and one distal muscle in at least one arm and one leg to enable the extent of axonal involvement to be determined and to allow detection of conduction block and demyelination. Test Description

Needle electromyography (NEE) This measures the electrical activity of muscles via the insertion of a needle electrode into

the muscle of interest. The needle comprises of inner reference and recording electrodes


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Test Description which detect electrical activity that is displayed either on an oscilloscope or screen, or is recorded through a microphone speaker (enabling muscle/nerve function to be heard). The EMG record is based on the potential difference between the recording and reference electrodes. After taking recordings at rest, the subject is asked to contract the muscle and the needle records waveforms generated by selected muscles during voluntary contraction (aka motor unit action potentials). Each individual waveform represents activation of the muscle fibres that belong to an individual motor unit (i.e. one motor neuron and all the muscle fibres it innervates). The recorded waveform informs on form/function of motor units, which changes with various pathologies. Normal motor units should generate bi- or triphasic responses with a duration of 10 ms or less. Abnormal EMG results are indicated by longer duration units (i.e. signs of blocking or slowing down of responses to nerve stimulation) and polyphasic potentials.

Single fibre EMG (SFEMG) SFEMG is similar to NEE except that it uses special equipment and recording needles with a slightly different filter setting. SFEMG specifically tests the function of the neuromuscular junction by recording and identifying APs from individual nerve muscle fibres. Needles are inserted into the muscle of interest and the subject supplies low-level constant effort sufficient to active one motor unit near the recording electrode. Two response parameters are measured: (i) Neuromuscular jitter – defined as the variability in time between activation of the motor nerve and generation of the muscle fibre AP. Jitter reflects the normality of the nerve-muscle transmission and involves measuring time variability between APs from two muscle fibres in the same voluntarily activated motor unit, followed by stimulation of the motor axon and measuring the variability between stimulus and APs in the responding muscle fibres. Although normal jitter values vary between muscles and among muscle fibres within individual muscles, they generally range from 10 to 50 µs. They are reported as either the mean jitter among all pairs of APs recorded during voluntary activation or axonal stimulation, or reported as the % of pairs (or APs) in which either blocking was seen or jitter was normal. Abnormal jitter values are indicated by abnormally increased jitter (which is determined via statistical analysis) or by blocking whereby nerve activation fails to elicit a muscle AP.


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Test Description (ii) Muscle fibre density – defined as the measurement of the mean number of muscle fibres belonging to the same motor unit. Fibre density is determined by inserting the SFEMG electrode needle at a number of different sites during voluntary activation of a motor unit. Fibre density is subsequently reported as the mean number of fibres/motor units at 20 recording sites for each muscle tested. Increased fibre density can be indicative of myopathies, axonal neuropathies/radiculopathies (due to reinnervation) and age-related fibre diameter decrease which results in more fibres being packed into a smaller space.

Electronystagmography (ENG) 14. This test is used to assess balance or the vestibular-ocular reflex (i.e. the relationship between eye movement and the inner ear). It tests the function of the vestibular system by examining nystagmus (involuntary eye movement). Electrodes are placed around the eyes and a computer monitors nystagmus that is either spontaneous or induced under the following conditions i.e. while: the subject focusses on a visual target; hot or cold water is placed in the ear canal; or the head is manipulated into different positions. ENG is useful in differentiating peripheral from central causes of dizziness. In Video or Vector Electronystagmography (VENG), the subject wears video recording goggles and is asked to follow moving or stationary objects with their eyes only (or cool/warm air is gently blown into their ear canals). Eye movements recorded by an infrared camera are analysed using VENG software. Audiometry 15. This tests the vestibulococlear nerve (VIII) and provides a more precise measurement of hearing. The test involves the subject wearing headphones attached to an audiometer and pure tones (sound wave vibrations/Hz) of controlled intensity (loudness/dB) delivered to one ear at a time. The subject then indicates when a sound is heard and minimum intensity required to hear each tone is recorded. 16. Auditory brainstem response (ABR) audiometry is a neurological test of auditory brainstem function in response to auditory (click) stimuli. It is the most common application of auditory evoked responses. Tiny earphones are placed in the ear canal and


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sound is amplified through the earphones. The elicited auditory nerve’s response to these sounds (waveform response) is measured by surface electrodes placed on the vertex of the scalp and ear lobes. The signal travels along the auditory pathway from the cochlea of the inner ear to the brainstem regions (i.e. cochlear nuclear complex).The amplitude of the signal is averaged and charted against the time. The waveform peaks are labelled I-VII and normally occur within a 10ms time period after a click stimulus presented at high intensities (70-90 db normal hearing level). ABR waves I and II correspond to true action potentials and are generated by the peripheral and central portion of cranial nerve VIII respectively. Later waves may reflect postsynaptic activity in major brainstem auditory centres. 17. NB. Vestibular evoked myogenic potential (VEMP) testing is used to evaluate whether the saccule (i.e. sensory cells in the inner ear that translate head movements into neural impulses) and the inferior vestibular nerve are intact and functioning normally. During VEMP testing, headphones are placed over the ears and small electrodes are attached with an adhesive to the skin over the neck muscles. Sound stimulates the saccule, traverses the vestibular nerve and ganglion to reach the vestibular nucleus in the brainstem. From there, impulses are sent to the neck muscles via the medial vestibulospinal tract (MVST). When sound is transmitted through the headphones, the electrodes record the response of the muscle to the vestibular stimuli. Quantitative Sensory Tests 18. Quantitative sensory threshold (QST) tests are also known as cutaneous sensory perception thresholds and represent another type of diagnostic neurophysiological test. They are specifically used to assess damage to large and small sensory fibre nerve endings which detect changes in vibration and temperature respectively. The procedure is non-invasive and involves the application of either a hot, cold or vibrational stimulus and use of a computer testing system to measures how the nerves react to vibration and changes in temperature e.g. Computer-Aided Sensory Evaluation-Version 4 (Case IV). A subject’s sensory threshold is classically defined as the level of stimulus intensity necessary for a sensation to be just detectable. To determine the sensory threshold, the subject may be presented with a series of stimuli of different magnitudes and asked whenever a stimulus is detected. A stimulus is detected when the subject perceives an alteration of the baseline “internal noise”. The test results are then compared with normative values. NB. When establishing normal values, researchers have found that age, sex, and site of the stimulation can affect sensory thresholds. 19. Sensory threshold testing is a psychophysical test of the neurophysiological function of large and small nerves and, therefore, lacks the objectivity of NCS. There is currently no way to differentiate the results of subjects who are biased (whether consciously or subconsciously) toward an abnormal study from those with organic disorders. Results are subject to changes due to


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distraction, boredom, mental fatigue, drowsiness, or confusion, which should be monitored given that a routine QST usually takes about 1-2 hours to complete. Although QST are generally used to assess sensory nerve impairment they actually test the integrity of the entire sensory neuraxis from receptors to brain and are therefore of no localising value. These tests are used on the basis that dysfunction of the peripheral nerves or CNS may give rise to abnormal QST. Therefore, it is generally accepted that the results of QSTs should not be used as the sole criterion to diagnose a peripheral neuropathy but rather used in conjunction with either clinically evident signs obtained via neurological examination or electrodiagnostic results. Test Description

Thermal Threshold These test the function of small (afferent) peripheral nerve fibre populations (which can be either

myelinated or unmyelinated) including specialised and sensitive receptors. Hot temperatures test the function of unmyelinated C fibres, while cold temperatures test the function of the thinly myelinated Aδ fibres. Increased thresholds are indicative of abnormalities.

Vibration Threshold These test large fibre afferent peripheral nerve fibres i.e. the thickly myelinated Aβ fibres, including specialised sensitive mechanoreceptors. Increased thresholds are indicative of abnormalities.

Other Tests Test Description

Posturography Is a set of techniques used to quantify postural control in an upright stance during static (still) or

dynamic (moving) conditions. It measures the ability to balance (i.e. quantification of the vestibular-spinal component of body balance) while standing on a stable or unstable platform (aka force plate) that record vertical forces exerted on its surface over time. Static posturography (aka stabilometry) uses static force platforms to analyse the ability of subjects to maintain balance during unperturbed sway. Subjects stand on a fixed instrumented platform that is connected to sensitive detectors (force and movement transducers) capable of detecting tiny oscillations of the body. The following components of balance are measured: centre


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Test Description of pressure (COP); centre of gravity (COG) and sway. COP refers to the integrated pressure field that is exerted on a supporting surface (e.g. foot). It changes through movement as different parts of the foot are loaded i.e. when a person walks COP moves from the heel to the toes. The COG is located at the midpoint of the base of support (i.e. when standing is found approximately in the middle of the sacrum. It is difficult to quantify as it is subject to change in posture and therefore is often measured with COP. Types of body-sway parameters assessed include: (i) anterior-posterior direction (x-axis – forward and backward); (ii) medial-lateral direction (y-axis – side to side); and (iii) vertical direction (z-axis). Stabilometry is performed under different conditions i.e. with feet together, separated or on one foot, eyes open or closed, or using a hard or foam force plate surface. Stabilometry is limited by its inability to convey information about the motor and sensory mechanisms that may be involved in postural control. Dynamic (computerised) posturography (aka test of balance) enables quantification of the ability to maintain balance in non-static conditions. It also enables quantification of a subject’s vestibular system by way of visual references (either eyes open or closed) in a moving environment. The subject’s movement is perturbed via use of a movable platform with movements transmitted to the computer in real time. Various types of dynamic posturographic techniques exist which include the sensory organisation test (SOT) – that analyses the balance performances of a subject during six conditions in which somatosensory and visual inputs can be selectively altered; limits of stability test (LOS); motor control test (MCT); and the adaptation test (ADT).

Tufts Quantitative Neuromuscular Exam (TQNE)

A standardised tool for measuring muscle strength (and pulmonary function). TQNE is a computer-directed quantitative assessment of muscle strength, in which the force (kg) of eight different muscle groups (shoulder, elbow and knee extensions and flexions, hip flexion, and ankle dorsiflexon) are measured bilaterally, including grip strength. NB. Respiratory-forced vital capacity, expiratory volumes and manual dexterity (using a pegboard) are also evaluated.

Spirometry Standard pulmonary function tests (PFTs), measuring lung function, specifically the measurement of the amount (volume) and/or speed (flow) of air that can be inhaled and exhaled. The following parameters are measured: vital capacity (VC), forced expiratory volume in one second (FEV1), and FEV1/VC ratio (FEV1%), maximal expiratory flow at 25%VC (MEF25%), peak expiratory flow


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Test Description (PEF), gas transfer factor (TFLCO), specific airways conductance (sGaw), intrathroacic gas volume (ITGV), the ratio of residual volume to total lung capacity (RV/TLC) and maximal static inspiratory (MIP) and expiratory (MEP) mouth pressures at the functional reserve capacity and total lung capacity. The ITGV is the total amount of air in the thorax post expiration. Increases are associated with a weakening of muscle force. Maximal static inspiratory/expiriatory mouth pressure (i.e. MIP and MEP respectively (cm H2O)) provide a measure of respiratory muscle function.


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Figure 1. Schematic illustration of the basic approaches and tests used to evaluate peripheral neurological disorders. These neurophysiological tests assess the function of peripheral nerves, muscle or the neuromuscular junction.

Cranial nerve function

Motor function

Sensory function

Reflex function

NMJ function

Key

Other nerve function tests


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Bibliography URLs 1. Hearing Loss. Columbia University. Department of Otolaryngology: www.entcolumbia.org/hearloss.html 2. Electrodiagnostic testing. North American Spine Society. Public Education Series: www.spine.org 3. Foundation For Peripheral Neuropathy: www.foundationforpn.org 4. MedlinePlus – Audiometry: www.nlm.nih.gov/medlineplus 5. Nerve Conduction Studies – Cranial nerves: www.teleemg.com 6. Peripheral Neuropathy: www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001619/ 7. The Merck Manual. Neurologic disorders: www.merckmanuals.com/professional/neurologic_disorders.html


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TOX/2012/26 Annex 8 COMMITTEE ON TOXICITY OF CHEMICALS IN FOOD, CONSUMER PRODUCTS AND THE ENVIRONMENT EVALUATION OF AUTONOMIC NERVOUS SYSTEM EFFECTS OVERVIEW OF COT 1999 REPORT 1. The COT evaluation published in 1999 can be found in Chapter 7 of the report paragraphs 7.71 to 7.73 (COT 1999). 2. The COT noted in the executive summary of the report that the data on the effects on the autonomic nervous system (ANS) were insufficient to allow any firm conclusions to be drawn. The Working Group considered that the observations suggesting autonomic dysfunction needed further investigation by more rigorously designed studies in larger samples of subjects. OVERVIEW OF LITERATURE PUBLISHED AFTER COT 1999 REPORT Definition of Autonomic Nervous System Dysfunction 3. The COT 1999 report does not provide a definition of autonomic nervous system (ANS) dysfunction. However, information from the published literature suggests that autonomic lesions resulting in ANS dysfunction can arise either in the central nervous system (CNS) or peripheral nervous system (PNS). Centrally-mediated autonomic dysfunction occurs when parts of the CNS that process and integrate stimuli from the body and external environment are damaged i.e. the hypothalamus, nucleus of the solitary tract, reticular formation, amygdala, hippocampus and olfactory cortex, which are subsequently unable to initiate appropriate responses in preganglionic nerves. Peripherally-mediated autonomic dysfunction arises when the sympathetic and parasympathetic neurones that emanate from the brain and spinal cord are damaged and unable to send messages to the viscera, vascular smooth muscle, endocrine and exocrine glands, the immune system and soft tissues (Freeman, 2007). NB. ANS abnormalities can also be caused by damage to the receptors on the effector cells. 4. Members are reminded that because the ANS innervates virtually all of the organs of the body, disorders arising in the ANS can cause autonomic insufficiency or failure in any system of the body. Therefore, the symptoms produced are multiple and varied, often affecting functions not considered by neurologists. For this reason, and because of the non-specific nature of many autonomic symptoms (i.e. they can have a non-neurological cause), very few symptoms can be considered to be suggestive of autonomic dysfunction in the absence of objective confirmation by autonomic testing. For example, although ‘dizziness’ can have an autonomic nerve aetiology (e.g. presyncope due to orthostatic hypotension (OH)), it can also be


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caused by cranial nerve abnormalities (i.e. due to vertigo or disequilibrium/ imbalance associated with cranial nerve VII dysfunction). 5. Detailed information of the studies summarised in this section can be found in Annex 8, which present information on the study design used (Table 6), and the results, strengths/weaknesses and conclusions (Table 7). Acute Exposure with Chronic Peripheral Neuropathy and Neuromuscular Dysfunction 6. For the purpose of this review, acute exposure is defined as a single incident where the exposure is continuous and may last up to a number of days, e.g. four days. 7. Seven epidemiological studies provide data on the chronic effects of acute exposure to OPs in relation to the potential development of autonomic nervous system (ANS) dysfunction. These investigations comprised of two cohort studies (Kawana et al 2001, Nakajima et al 1999), four cross-sectional studies (McCauley et al 2001, Spencer, 2001, Stallones and Beseler, 2002b, Ohtani et al 2004) and one case series (Dahlgren et al 2004). There were no case-control studies or case reports evaluating persistent ANS dysfunction following acute OP exposure. 8. Acute exposure to OPs arose largely from poisoning episodes. Two studies examined the effects of acute exposure to OPs insufficient to cause acute toxicity (McCauley et al 2001, Spencer, 2001). 9. Poisoning (or attempted poisoning) incidents were either:

a. accidental i.e. following occupational exposure (Stallones and Beseler, 2002b) or residential exposure (Dahlgren et al 2004); or

b. intentional i.e. the result of deliberate chemical attacks during warfare (McCauley et al 2001; Spencer, 2001) or terrorism (Nakajima et al 1999, Kawana et al 2001, Ohtani et al 2004).

10. Members should note that the 1994/1995 sarin terrorist attacks in Japan and the 1991 Gulf War Khamisiyah incident are included in the current evaluation (despite not being evaluated in the COT 1999 report) because they represent defined exposure incidents where a large number of individuals received definite acute exposure to specific OP chemical agents. 11. Various medical conditions are known to be associated with ANS dysfunction/failure [Annex 8, Table 9]. These include autonomic neuropathies (e.g. diabetes, systemic amyloidosis), immune-mediated neuropathies (e.g. caused by Guillain Barre syndrome, HIV/AIDS), inherited nerve disorders, degenerative conditions (e.g. multiple system atrophy, Parkinson disease with autonomic failure, and diffuse Lewy body dementia), cancer (e.g. multiple myeloma), disorders involving scarring and hardening (sclerosis) tissues, pulmonary hypertension, pure autonomic failure and surgery or injury involving nerves. Medications can also cause autonomic failure e.g. chemotherapeutic agents and anticholinergic medications.


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Most of the studies evaluated in this report did not specifically set out to assess disorders of the ANS and, therefore, did not necessarily attempt to control for exposures or conditions known to confound development of ANS dysfunction i.e. taking account of a subject’s exposure to autonomically-active agents such as caffeine. Unless otherwise stated, studies tended to account for the possible confounding effects of age, gender, education, occupation, socioeconomic status, lifestyle, existing medical conditions, and exposure to other neurotoxic agents often in relation to peripheral sensory and motor nerve function. 12. Paragraphs 13 to 36 provide key detailed summaries of these studies. NB. For each summary, the first paragraph briefly describes the study design, with the main findings and conclusions outlined in subsequent paragraphs. Cohort studies Victims of the Sarin Terrorist Attacks in Japan 13. Nakajima et al used an author-generated questionnaire survey to collect symptom data on 2052 residents of the 1994 Matsumoto sarin attack (Nakajima et al 1999) (Table 6.A.1, Annex 8). All victims were Matsumoto inhabitants living or staying in the area at the time of the incident (i.e. within 1050 metres north to south and 850 metres east to west of the spot where about 12 litres of sarin was released on the night of June 27). The authors used an internal comparison group i.e. non-victims defined as residents who were not diagnosed with definite muscarinic and/or nicotinic symptoms immediately after the incident. Subjects were followed up at various times after the attack, which ranged from three weeks up to three years (although the current study evaluated symptoms reported one and three years after the attack - the results of the earlier surveys were reported in an earlier paper (Nakajima et al 1998)). Erythrocyte and serum AChE activity levels were measured only in victims admitted to hospital four weeks after sarin exposure. Health examinations were also performed (albeit inconsistently i.e. for those who requested an examination). No objective neurological tests were used to validate symptoms or collect data on the background prevalence of the sarin-related symptoms, and the authors accounted for the confounding effects of age and gender only. 14. Postcode mapping revealed that the percentage of symptomatic subjects were greatest in the block where sarin was released (Nakajima 1999, Table 7.A.1, Annex 8). Of the 318 victims and 919 non-victims who responded in the first year survey (60.3% participation rate), nine victims reported having ‘asthenopia’ (eye strain), which was associated with a significantly lower erythrocyte AChE activity compared to victims who did not report having this symptom (p<0.05). A total of 167 victims and 669 non-victims responded to the three-year survey, there being a lower compliance in the victim group (36.1% of the victims immediately after the incident) compared to the non-victim group (54.5%). Victims (n=40) were 9.7 times more likely to report symptoms of ‘asthenopia’ compared to non-victims (n=21) (p<0.01), and 6.1 times more likely to report ‘blurred vision’ (p<0.001). The authors confusingly note there were no differences in ‘palpitation’ symptoms between victims and non-victims but then report that victims were more likely to report having experienced ‘palpitations’ compared to non-victims (OR=4.10[1.17-14.33]; p<0.05)


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n=5,5 respectively). Victims who experienced symptoms both one and three years after the incident were more likely to report ‘blurred vision’ and ‘asthenopia’ (OR=3.21[1.44-7.16]n=10, p<0.01; 2.39[1.17-4.86]n=12, p<0.05 respectively) compared to controls (n= 13 and 21 respectively). 15. The authors concluded that there was a possible association between sarin exposure and chronic symptoms reported in victims. However, the authors did not systematically conduct objective health assessments (or present the results for subjects who requested them). Consequently, in the absence of objective verification of these symptoms it would be imprudent to assume that they represented a definite disorder of the ANS as they are quite non-specific (i.e. they could also have a non-autonomic nerve aetiology). With regards to study design, there was no unexposed comparison group and the study did not attempt to control for potential confounding exposures. Furthermore, the background prevalence of these symptoms was unclear and there was no attempt to control for possible selection bias from the low questionnaire compliance (especially in victims) or account for possible information/ measurement bias caused by over-recall of symptoms in the victim group. 16. Kawana et al, also used an author-generated questionnaire to investigate whether 582 victims of the 1995 Tokyo sarin subway attack treated locally in St Luke’s International Hospital suffered chronic symptoms after two, three and five years (Kawana et al 2001) (Table 6.A.1, Annex 8). Three referent groups were used in a comparative analysis of symptom incidence: these comprised of two sarin-exposed referents (i.e. 88 Matsumoto sarin victims, and 655 sarin victims of the Tokyo incident analysed by a non-governmental organisation (NGO)), and an unexposed group of 87 subjects taken from controls used in a study of the Matsumoto 1994 attack (Nakajima et al 1998). The questionnaire response rate was low and inconsistent, which declined from 48.6% to 32.8% in subsequent follow-ups. This was not analysed further to rule out selection bias. Exposure assessment was based on subject’s admittance to the hospital as a victim. The authors did not attempt to stratify the level of exposure or conduct any form of biological exposure assessment. No objective health assessments were performed and the authors did not account for any potential confounders. 17. ‘Tiredness of eyes’ and ‘difficulty focussing’ were among the more frequently reported symptoms (for e.g. in 1997, the percentage of St Luke subjects reporting these symptoms was 37.5% and 21.2% respectively) (Kawana 2001, Table 7.A.1, Annex 8). The symptom frequency rates did not change significantly from survey to survey, except for ‘abdominal pain’ which decreased from 7.4% in 1997 to 2.4% in 1998 (p<0.05). Other less frequently reported symptoms included diarrhoea, dizziness, nausea, difficulty breathing, appetite loss, sudden palpitations, and chest tightness. The frequency rate for ‘tiredness of the eyes’ was highest for the NGO Tokyo victim group (60.9%), followed by victims of the current study of St Luke’s Hospital subjects at (39.3%), and was lowest in the Matsumoto control group (14.9%). The authors did not report whether these differences were significant. This trend was also apparent for most of the other reported symptoms. The authors suggested that the symptom differences between these groups may be due to the St Luke’s cohort receiving multiple interventions i.e. physical examinations, counselling and laboratory work.


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18. There were several limitations associated with this study. These included the lack of account of any potential confounders, questions over the appropriateness of the external comparison groups (as the authors did not elaborate on whether subjects were matched), there was no attempt to assess possible recall bias, and no use of measures of association i.e. odd ratios or relative risks. Additionally, in the absence of further testing it would be imprudent to classify most of these symptoms (except for palpitations) as definite autonomic nerve disorders as they could also result from dysfunction in other systems unrelated to the ANS. This study, therefore, provides a more descriptive account of self-reported chronic symptoms in sarin victims, which prevents any meaningful inferences from being drawn. Cohort studies summary 19. Two cohort studies investigated the long-term effects of acute OP exposure i.e. sarin poisoning in victims of the two terrorist attacks in Japan (Nakajima et al 1999, Kawana et al 2001). Both studies reported a significantly increased prevalence of symptoms suggestive of ANS dysfunction compared to controls (e.g. palpitations) although most were non-specific visual, respiratory and gastrointestinal symptoms. These findings relate to symptoms obtained using subjective health assessments alone i.e. questionnaire surveys. No data from objective and routinely applied neurological examinations were reported in either study to verify these symptoms and corroborate the existence of autonomic abnormalities. Cross-sectional studies Victims of Sarin Terrorist Attacks in Japan 20. Ohtani et al. used a self-rated questionnaire survey in their five-year follow-up assessment of somatic and mental symptoms in victims of the 1995 Tokyo sarin subway attack (Ohtani et al 2004) (Table 6.A.3, Annex 8). The eligible population comprised of 565 victims who were treated at St Luke’s International Hospital emergency wards for acute sarin intoxication and were sent letters asking them to participate, of which 170 responded (30% response rate). Sixty-four victims agreed to participate (11% participation rate) however, data was available for only 34 victims. The authors did not further evaluate reasons for the low response and participation rates, but it appears that the limited number of available data for victims who agreed to participate was related to having to do face-to-face interviews (this is not clearly explained). No referent group was used for comparative purposes. Somatic symptoms evaluated included the following potentially autonomic-related effects: tightness in the chest, palpitations, nausea, diarrhoea, abdominal pain, loss of appetite, dizziness, easily tired eyes, blurred vision, and difficulty focussing. The severity of these symptoms were scored using a three-point scale (2=severe; 1=mild; 0=none). The authors do not appear to have accounted for any potential confounding exposures or conditions. 21. Victims tended to report experiencing a range of symptoms that included ‘palpitations’ (n=14), and ‘blurred vision’ (n=20) (Ohtani 2004, Table 7.A.3, Annex 8).


6

Highly rated symptoms included ‘blurred vision’ (found in 63% of subjects); ‘difficulty focussing’ (60%); and ‘eyes tend to become easily tired’ (56.7%). No further information regarding symptoms suggestive of ANS dysfunction was provided. 22. There are several drawbacks of this study that weaken the strength of these findings. Firstly, the assessment was self-administered and no objective tests were conducted to verify the reported symptoms (most of which are quite non-specific) or measures undertaken to address potential recall bias. Secondly, the lack of attempt to control for the low response and participation rates suggests possible selection bias cannot be ruled out. Other limitations relating to the absence of a control group, lack of accounting of confounding factors or use of statistical tests to rule out chance findings prevent any meaningful conclusions from being drawn. US Farm Residents 23. Stallones & Beseler based their evaluation of neurological symptoms in a study population of 761 US farm residents (operators and their spouses) on data collected through a non-standardised questionnaire interview (Stallones and Beseler, 2002b) (Table 6.A.3, Annex 8). Subjects were selected from a sample of farms (479) in an eight county area of North Eastern Colorado. This represented a 57% response rate (835 eligible farms were initially contacted). Subsequent comparisons of farming practices between farms that refused and those that participated were found to be similar. Exposure assessment was based on data collected via a questionnaire in relation to farm characteristics (provided from operators only), use of pesticide equipment, working hours and number of pesticide applications conducted in the previous year. Subjects reported using mainly terbufos (used by 20.8% of subjects) and other OPs (e.g. chlorpyrifos, phosmet, and dichlorvos)/carbamates (used by 37.1% of subjects) to treat crops and livestock respectively. NB. Other non-OP pesticides/herbicides were also used albeit to a lesser extent. Neurological assessment comprised of questions relating to symptoms experienced in the past month (ranked according to occurrence), and having any physician-diagnosed pesticide related illnesses. Sixty-nine subjects reported having ever experienced a physician-diagnosed pesticide illness and were defined as ‘cases’. The remaining 692 respondents (who answered ‘No’) were used as internal controls. Age and gender were found to be significant variables and were adjusted for in subsequent analyses. 24. Subjects with previous pesticide poisoning had a 2.8-fold increased likelihood of reporting ‘heart palpitations without exertion’ compared to controls (95% CI[1.22-6.54]) (Stallones & Beseler 2002b, Table 7.A.3, Annex 8). Chi-squared test for trend revealed that having a pesticide-related illness was significantly associated with ‘difficulty driving due to feeling dizzy or tired’ (P=0.0368). However, it is not possible to say the latter symptom is associated with ANS dysfunction (unlike dizziness associated with pre-syncope/orthostatic hypotension, which typically arises when an individual changes position from supine to upright). 25. The finding of ‘increased heart palpitations’ in exposed subjects is limited by the fact that subject’s had co-exposure to other non-OP pesticides, and the physician-verified pesticide poisoning was based solely on self-reports; there being


7

no objective tests to verify the symptoms or exposures reported. Furthermore, the authors noted that the time-sequence of exposure and outcome was inconsistent, since pesticide poisoning reflected cumulative lifetime incidence while information on pesticides used on the farm came from the previous year. US Desert Storm Veterans Deployed in Khamisiyah, Iraq 26. McCauley et al, and Spencer report different findings of the same study of 653 US troops believed to have been within a 50 km radius of the Khamisiyah Ammunition Storage Point. These troops are believed to have sustained low-level exposure to sarin/cyclosarin following ground detonation of chemical munitions during the first two weeks of March 1991 (McCauley et al 2001, Spencer, 2001). 27. McCauley et al used a telephone computer-assisted survey to ascertain neurological and neurophysiological symptoms experienced during the first two weeks of the Ground War in Khamisiyah, Iraq (March 1991), and at the time the study was conducted (approximately eight years later) (McCauley 2001 Table 6.A.3, Annex 8). The authors were able to contact 2918 (90.6%) of 3219 veterans in their sampling pool. Contactable veterans were found to be significantly more likely to have college or advanced degrees, be married, Caucasian and male. Only 2388 (81.8%) of the subjects reached by phone were subsequently eligible and of these, 555 (19.0%) refused to participate resulting in 1833 completed telephone interviews (i.e. a 77% participation rate). Participants compared to non-participants/ non-responders were significantly more likely to be Caucasian and on active rather than reserve duty. The final study participant population comprised of 1779 interviews (due to 54 being excluded for providing incorrect deployment information). The 653 Khamisiyah-deployed subjects were subdivided according to whether they witnessed the explosions/operations or not (i.e. 162 KHAM-witness and 405 KHAM non-witness respectively) – 86 subjects were unsure. Two groups were used for comparisons: 610 military personnel who were deployed elsewhere and assumed to have no known exposure to nerve agents (i.e. non-Khamisiyah aka. N-KHAM); and 516 non-deployed military personnel (ND). Comparative analysis revealed significant differences between groups in relation to current region of residence, gender, age, race, and military status (i.e. whether regularly active or activated reserve). Data on potential confounding exposures during the duty were also collected, although these did not relate to possible effects on ANS function. No objective assessments of exposure and health outcomes were conducted. The authors categorised the level of sarin exposure within the 50km radius as ‘low’. However, this does not account for the fact that the troops closest to the chemical munitions would have received greater exposures than those further away. The authors used trained interviewers and a standardised interview technique. 28. After adjusting for confounding effects of age, gender and region of residence, the authors found that during the first two weeks after the Ground War the KHAM-witness subgroup were significantly twice as likely to report having experienced acute symptoms of ‘nausea, abdominal cramping, runny nose, increased urination, and sweating of hands of feet’ than the KHAM non-witness group (95% CIs were greater than the value one; p-values not reported) (McCauley 2001, Table 7.A.3, Annex 8). NB. These symptoms were considered to be mild acute health effects of


8

acute exposure to low-levels of OPs and not demonstrative of episodes of acute illness consistent with high exposure to AChE inhibiting agents. However, eight years later, the reported symptoms in the KHAM group as a whole were not significantly different from the N-KHAM. Similarly, KHAM group differences were not apparent, although the KHAM-witness subgroup were more likely to significantly report non-specific symptoms of ‘bloody diarrhoea’ compared to KHAM-non witness subgroup (OR=3.1[1.6-6.0]). All deployed veterans (KHAM and N-KHAM) were significantly more likely to report experiencing several non-specific symptoms compared to non-deployed veterans (ND) i.e. dizzy spells, choking sensations, shortness of breath/problems breathing, bloody diarrhoea, eye irritation/sensitivity, (ORs ranged from 2.2 – 4.3). The only reported symptom suggestive of ANS dysfunction i.e. ‘fainting’ did not reach statistical significance (OR=2.5[0.9-8.4]). 29. This study does not present any convincing evidence to suggest the development and persistence of symptoms suggestive of abnormalities in the autonomic nervous system (ANS) in individuals who witnessed/involved in the Khamisiyah detonations. The study is limited by potential exposure misclassification for some of the KHAM and N-KHAM subjects (who are reported to have been categorised by the US Department of Health): about 8% of those classified as KHAM subjects reported never being in Iraq, and about 9% of the N-KHAM subjects claimed to have been within the 50km radius of Khamisiyah, with a third of these being involved or watching the detonations. Furthermore, the KHAM subjects had been notified of their potential exposure to chemical warfare agents and were also informed on what the likely symptoms were (which thereby potentially introduced information and recall bias). Other limitations include possible selection bias arising from the sampling process, as it was limited to individuals with easily accessible telephone numbers, and the absence of objective autonomic tests to substantiate reported symptoms. However, this study is noted for its assessment of the long term effects of an acute low-level exposure to an OP. 30. In a related study, Spencer 2001 extends the above findings of McCauley et al (2001) to report on a two-tiered clinical assessment of subjects who completed the telephone interview, and who were subsequently recruited for neurobehavioural testing (Level I) and a focused clinical neuromuscular and neurophysiological examination (Level II) (Spencer, 2001) (Table 6.A.3, Annex 8). In this study, subjects are classified as either Khamisiyah (KHAM), Non-Khamisiyah (N-KHAM) or Non-deployed (ND). The authors noted that the telephone interview sought information on hospitalisations and conditions that had been diagnosed by a physician. Furthermore, an adapted survey instrument was used to gather detailed information on the nature of confounding Gulf War-related exposures, and telephone numbers were tracked using more extensive tracking measures to reduce possible selection bias. No objective tests for autonomic dysfunction were conducted. 31. Interview survey results were reported in an attached manuscript entitled “McCauley et al under review”, which was subsequently published in McCauley et al (2002). The findings revealed no differences between subjects in the KHAM group and N-KHAM group during the time period of the Khamisiyah detonation (although an unreported sub-analysis revealed that mild immediate responses to OPs were evident in a small number of KHAM subjects who witnessed the detonations compared to those who did not). Nine years after the incident, KHAM subjects did


9

not differ from other deployed troops on reports of any medical conditions or hospitalisations (Spencer 2001 and McCauley 2002, Table 7.A.3, Annex 8). However, deployed troops were significantly 1.7 times more likely to report a diagnosis of a several medical conditions that included ‘high blood pressure’ compared to non-deployed troops (95%CI[1.3-2.4]), although this is not specific to sarin-exposed Khamisiyah based troops. Cross-sectional studies summary 32. The cross-sectional studies provided very limited evidence for the development of persistent ANS abnormalities. This is because the positive findings of ‘palpitations’ as observed in two studies (Stallones & Beseler 2002b; Ohtani et al 2004) were based solely on self-reported data – no objective tests were conducted to verify the reported symptoms, or confirm that autonomic abnormalities indeed existed. Furthermore, the other reported symptoms were largely non-specific i.e. they could also have a non-ANS cause and one study did not use a comparison group (Ohtani et al 2004). Case series US Family Acutely Poisoned From Residential Diazinon Exposure 33. One case series reported possible chronic autonomic effects in a US family accidentally exposed to diazinon following a pesticide company’s mistaken application to the family house interior (Dahlgren et al 2004) (Table 6.A.4, Annex 8). A comprehensive assessment of external exposure levels were performed via wipe, air and bulk sampling. The authors also tested urine samples for the diazinon parent compound, which was found to be negative. The authors did not test for diazinon metabolites or cholinesterase levels. A battery of health tests were conducted three years after the initial exposure, which included a neurological clinical examination (no further information was provided) and neurophysiological and neurobehavioural tests. 34. In addition to acute effects experienced soon after exposure, the chronic effects observed three years later included ‘blurred vision’ in both the mother and father (Dahlgren 2004, Table 7.A.4, Annex 8). The authors categorised this as a “CNS outcome”. Other effects suggestive of ANS dysfunction also arose in two children. 35. This study observed a chronic symptom suggestive of an ANS abnormality in adults accidentally exposed to diazinon in the home. However, it should be noted that although ‘blurred vision’ can be a sign of ANS dysfunction (if associated with ciliary muscle defects) it can also be associated with dysfunction/abnormality in the eye (e.g. cornea), optic nerve or brain. Consequently, the absence of further autonomic testing does limit the strength of this finding. Chronic Effects Following Acute Exposure Discussion Summary


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36. Most of these studies were limited in their attempts to reduce bias and account for potential confounders of ANS dysfunction. Evidence for the development of possible chronic ANS abnormalities in acutely exposed individuals came from one case series and to lesser extent from two cohort and two cross-sectional studies. No case-control study or case report examined the long-term effects on the ANS of acute exposure to OPs. The evidence from the single case-series was based on a clinical examination that reported ‘blurred vision’ in exposed adults three years after the diazinon poisoning incident (Dahlgren et al 2004). However, no further specific autonomic tests were conducted, which would have corroborated the existence of an autonomic abnormality (since blurred vision can also have non-neurological causes). Study design limitations aside, the two cohort studies (Nakajima et al 1999; Kawana et al 2001) and the two cross-sectional studies (Stallones & Beseler 2002b; Ohtani et al 2004) that observed self-reported symptoms of ‘palpitations’ and other non-specific visual, respiratory and GIT symptoms in OP-exposed individuals did not conduct any objective tests to substantiate these effects. The two cross-sectional studies of army officials acutely exposed sarin at levels that were considered insufficient to cause acute toxicity did not observe an increased prevalence of ANS dysfunction in exposed subjects (McCauley et al 2001; Spencer, 2001). Chronic Exposure with Chronic Effects 37. For the purpose of this review, chronic exposure is defined as a multiple repeat incident with a variable length of exposure (i.e. lasting for one season up to a lifetime). 38. Eleven epidemiological studies evaluated the chronic effects of prolonged low-level exposure to OPs in relation to the development of possible ANS abnormalities. These comprised of one case-control study (Jamal et al 2002a) and 10 cross-sectional studies (Bazylewicz-Walczak et al 1999, Kilburn, 1999, Srivastava et al 2000, Steenland et al 2000, Pilkington et al 2001, Farahat et al 2003, Cox et al 2005, Kamel et al 2005, Kamel et al 2007b, Abdel Rasoul et al 2008). No cohort study, case series or case reports evaluated the chronic effects of low-level long-term exposure to OPs. 39. The exposure to OPs was largely occupational, in which subjects either worked as:

a. chemical plant workers manufacturing quinalphos (Srivastava et al 2000);

b. farmers/farm workers involved in either sheep dipping (Pilkington et al 2001, Jamal et al 2002a), or working as pesticide applicators in farms/orchards (Kamel et al 2005, Kamel et al 2007b), cotton-fields (Farahat et al 2003, Abdel Rasoul et al 2008), or lawns and houses (Steenland et al 2000);

c. greenhouse workers (Bazylewicz-Walczak et al 1999). 40. Residential exposure to OPs was also examined (Cox et al 2005), although one study used a mixed cohort of individuals exposed either occupationally (i.e. via


11

job as agricultural worker or pesticide applicator), or residentially (i.e. at offices or at home) (Kilburn, 1999). 41. Most of the studies examining chronic low-level OP exposure did not specifically set out to assess disorders of the ANS and, therefore, did not necessarily attempt to control for exposures or conditions known to confound development of ANS dysfunction e.g. taking account of a subject’s consumption or exposure to autonomically-active agents such as caffeine. Unless otherwise stated, studies tended to account for some or all of the possible confounding effects of age, gender, education, occupation, socioeconomic status (SES), lifestyle, existing medical conditions, and exposure to other neurotoxic agents. 42. Paragraphs 43 to 77 provide key detailed summaries of these studies. NB. For each summary, the first paragraph briefly describes the study design, with the main findings and conclusions outlined in subsequent paragraphs. Case-control studies Sheep Farmers 43. Jamal et al sought to clinically interpret the neuropathic outcomes of 72 sheep farmers/dippers nested within a previous cross-sectional field study (that had invited 685 subjects to participate) (Jamal et al 2002a) (Table 6.B.2, Annex 8). The farmer’s previous neuropathy status was determined from the outcome of neuropathy scores derived from responses to questionnaire and sensory tests conducted in the field study. The current study repeated the neurological symptom assessment and quantitative sensory test (QST) measurements in the clinic to firstly re-categorise subjects into neuropathy groups and subsequently verify these assignments with more specific neurophysiological tests. Ninety-five of the 201 eligible subjects responded to the standardised questionnaire (47% participation rate), but only 79 also attended the clinic (39% attendance rate). Reasons for non-participation included travelling distance, and for non-attendance included lack of work cover, and delayed clinical study time. Despite these low rates, the authors reported that that there was no evidence to suggest that the non-attendees differed in any significant way than those attending. Information on chronic neurological symptoms occurring in the upper and lower limbs were collected via a neurologist-administered and previously established (and modified) Mayo Clinic neuropathy/neurological symptom questionnaire. NB. It is apparent from the results that this included questions on autonomic symptoms, however, the authors did not further elaborate on this. Symptom scores were derived according to a previously used method. This was followed with a hospital-based clinical assessment of neurological signs (none of which related to autonomic dysfunction) that was scored according to severity of symptoms, in which a higher score signified increasing abnormality. The final study participant group comprised of 72 subjects (due to exclusions). Twenty-three subjects were subsequently categorised as having “definite or probable” neuropathy, with 34 subjects as “possible” and 15 as “no” neuropathy (the latter used as the control referent group). Group comparative analysis revealed that age was lower in the “no” group. Exposure assessment involved calculating a mean cumulative exposure metric based on a crude exposure index “OPEXP” that used exposure


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history and occupational hygiene data, as well as urinary OP metabolite concentration data. OPEXP also represented the weighted sum of cumulative exposure to concentrate and dilute dip splash. The “possible” group had the highest mean cumulative exposure followed by the “definite or probable” group, and then the controls. NB. The authors did not report the level of significance and attributed the high value in the “possible” group to the presence of a few very highly exposed people. Finally, it appears that the neurophysiological tests were conducted to detect sensory and motor abnormalities (not ANS abnormalities); a diagnosis of ‘clinical neuropathy’ was defined as a subject having both neurological signs and nerve conduction abnormalities. Comprehensive measures were used to minimise investigator/observer bias. Subjects were excluded to control for confounding effects of various medical conditions/neurophysiological abnormalities. 44. The authors reported (in their discussion) that autonomic nervous system symptoms were commonly reported by the no-neuropathy and possible neuropathy group, but less commonly by the probable neuropathy group. There is no further description of what these symptoms were. However, the authors present a table that contains data suggesting the reverse i.e. for autonomic symptom scores of 0.5 or above 20%, 26% and 61% of subjects in the no, possible and probable/definite neuropathy reported autonomic symptoms respectively (Jamal 2002a, Table 7.B.2, Annex 8). These symptoms were reported more commonly than sensory or motor symptoms and it is suggested that the ANS may be potentially more vulnerable than motor and sensory nerves. 45. The findings of this study are limited by the fact that the authors did not detail the type of autonomic symptoms being evaluated, and report conflicting information regarding the prevalence of autonomic symptoms within the different neuropathy groups. The suggestion that the ANS may be more vulnerable than motor and sensory nerves is based solely on subjective data. However, it should be noted that this study was designed to validate previous peripheral neuropathic outcomes in farmers exposed to OPs, which therefore may have precluded its ability to demonstrate an association between OP exposure and chronic ANS dysfunction. Cross-sectional studies 46. This comprised the largest group of studies to investigate the development of possible ANS dysfunction in individuals chronically exposed to low-levels of OPs. Three of the 10 cross-sectional studies incorporated objective health assessments into their design (mainly as part of a clinical examination). Quinalphos Manufacturers 47. Srivastava et al examined various health risks in 59 Indian quinalphos (QP) manufacturing workers who had a self-reported mean work exposure history of approximately six years (Srivastava et al 2000) (Table 6.B.3, Annex 8). Seventeen controls were selected who were not engaged in QP manufacturing but worked for three years outside the manufacturing unit as tea vendors and roadside hawkers. The authors noted this lower number was due to only a few control subjects being eligible. However, it is reported that both groups were similar with respect to age,


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work history, physical activity at work, sex, education, and drinking and smoking habits (p>0.05), although exposed subjects tended to be more educated, smokers and alcohol drinkers. The authors did not evaluate external/ambient levels of OP exposure but used a pretested structured interview to collect information on subjects occupational history, and also measured AChE activity. Mean blood AChE levels in QP workers were not statistically different from controls. A medical doctor conducted a general clinical evaluation on subjects after a typical days work to determine the presence of any abnormalities, which included assessment of their cardiovascular, respiratory, gastrointestinal, genitounrinary, ophthalmic and dermal systems. No further details are provided. Neurophysiological and neuropyschological tests were also conducted. The authors accounted for most general confounders, including previous neurological conditions. 48. The authors present the clinical findings on a system-wide basis i.e. for skin, respiratory and gastrointestinal tract which yielded non-significant results compared to unexposed controls (Srivastava et al 2000), Table 7.B.3, Annex 8). 49. The lack of data on ANS dysfunction in the QP workers was partly due to the study’s system-wide assessment of health outcomes, which is compounded by the fact that the methods were not described in detail. The study was further limited by lack of account of subject’s co-exposure to other chemicals e.g. potentially neurotoxic raw materials and by-products of QP manufacture, and its use of an inappropriate and small control group. Sheep Farmers 50. Pilkington et al examined clinically detectable abnormalities of the nervous system in 612 UK sheep farmers/workers who had dipped sheep from 1970 (Pilkington et al 2001). Two low OP exposure groups were used for comparisons: 53 farmers without sheep-dipping experience and 107 ceramic factory workers recruited from two companies based in either England or Scotland. The response rate based on the number of eligible sheep and pig farms contacted was 88% and 62% respectively. Reasons for non-participation included being disinterested or too busy, or being excluded due to ineligibility. Participations rates for the ceramic factory workers recruited from England and Scotland were 60% and 80% respectively. Comparative group analyses showed differences in age, gender and alcohol consumption (the latter being higher in ceramic workers). The current study was an exposure-response design that utilised retrospective exposure data collected from a previous ‘first phase’ study. This earlier study used an exposure history questionnaire and an occupational hygiene study to obtain data on dipping practice in 20 farms. A dermal exposure model based on the two urinary OP metabolites (diethylphosphate (DEP), and diethylthiophosphate (DETP)) was derived that identified ‘handling of dip concentrate’ and ‘splashing of dilute dip’ as principal sources of exposure among farmers. The authors calculated the following exposure variables: (i) cumulative exposure to OP dips (OPEXP) defined as the weighted sum of cumulative exposure to both concentrate and dilute dip splash; (ii) DAYS defined as the total number of dipping days; and (iii) daily intensity of exposure to both concentrate and splash (Table 6.B.3, Annex 8). NB. No biological assessment of exposure was conducted. A technician-administered questionnaire (based on the


14

Mayo Clinic questionnaire) was used to obtain data on neurological symptoms that included those of the ANS i.e. sweating, fainting and impotence, which were subsequently scored via an overall dichotomous symptom indicator; a positive score in the autonomic group was obtained when there were at least two symptoms. NB. Other symptom groups included sensory and muscle weakness. This questionnaire was performed in conjunction with a series of quantitive sensory threshold (QST) tests. No clinical assessment of autonomic function were conducted. The authors did not account for factors likely to confound assessment of ANS dysfunction. 51. Autonomic symptoms comprised the most prevelant of reported symptoms within all groups (followed by sensory and muscle weakness). Symptom prevalence was highest among sheep dippers (28.4%) and lowest in ceramic workers (10.3%) (Pilkington 2001, Table 7.B.3, Annex 8). The statistical significance of these findings is not reported. NB. Subsequent analyses of symptom prevalence was based on an overall symptom indicator that was not specific for autonomic nerve symptoms, therefore it was not possible to further evaluate the effect of low-level cumulative exposure on the odds of reporting abnormal autonomic symptoms. 52. These findings show that sheep farmers had higher rates of self-reported autonomic symptoms compared to other workers. However, use of an overall symptom indicator (that included sensory and motor symptoms) precluded analysis of odds of reporting autonomic nerve-related effects due to cumulative OP exposure. The lack of objective verification of the reported symptoms and the exposure assessment based on self-reported data and exposure modelling suggests these findings should be considered with caution. Pesticide Applicators (Farms/Orchards) 53. Kamel et al used a symptoms questionnaire to evaluate neurological outcomes in US private pesticide applicators (Kamel et al 2005). Subjects (n=18, 782) were mostly farmers applying for new or renewed licences and were taken from the Agricultural Health Study (AHS) cohort of 52,400 applicators (Table 6.B.3, Annex 8). The questionnaire was based on a previously established neurological questionnaire (Q16). Subjects provided complete information on neurological symptoms experienced during the year before enrolment. Applicators who provided incomplete information (n=2,603) differed in terms of age, education and state. The authors used the applicator’s questionnaire responses to classify subjects into two groups based on their frequency of symptoms rather than their level of exposure. Twenty-three symptoms were reported. Cases comprised of subjects with a high frequency of symptoms i.e. 10 or more symptoms (20% of applicators), while subjects who experienced less than 10 symptoms (low frequency) were used as internal controls (80% of applicators). The authors constructed several measures of pesticide exposure from the questionnaire data, which included cumulative lifetime days of pesticides, in which OPs comprised one of four subdivisions of chemical class of insecticides. The authors did not identify the specific OPs to which subjects were exposed. No biological measures of exposure were conducted. A greater proportion of control subjects had no cumulative lifetime days of OP exposure compared to cases (14% vs. 9% respectively), while the number of subjects experiencing higher levels of exposure (i.e. from 1 – 500 cumulative lifetime days) was comparable in both groups (ranged from 11% – 29%). However, slightly more


15

cases had more than 500 cumulative lifetime days of OP use compared to controls (i.e. 6% vs 4% respectively). The authors used logistic regression to calculate the odds of experiencing a particular neurological symptom with high frequency compared with low frequency. Data was adjusted for age, state, education and smoking and alcohol consumption. 54. Several neurological symptoms were found to be associated with the highest category of lifetime days of use of OPs (Kamel 2005, Table 7.B.3, Annex 8). These included symptoms suggestive of autonomic nerve dysfunction (i.e. excessive sweating, fast heart rate, loss of consciousness) and other more non-specific symptoms (e.g. dizziness, nausea, loss of appetite, and poor night vision). These all had significant odd ratios between 1.77 and 1.95 (no 95% CIs or p-values were provided except from noting that the CIs excluded 1.0). However, these findings were not specific to OP exposure as they were also associated with other pesticide groups. 55. Despite the use of internal comparisons of exposed individuals from the same population, the authors observed positive associations in applicators with a high frequency of reporting several autonomic nerve symptoms compared to those with a low frequency. The significance of this finding is limited by the subjective nature of both the exposure and health outcome assessment and the potential for recall bias. 56. Kamel et al re-analysed the above data in a separate publication by categorising the neurological symptoms a priori into groups representing several functional areas that included an autonomic domain and also an affect, cognition, motor and vision domain (Kamel et al 2007b) (Table 6.B.3, Annex 8). Each domain was based on the previously established Q16 questionnaire. The autonomic domain included the following symptoms: nausea, loss of appetite, excessive sweating and fast heart rate; Symptoms of ‘dizziness’ and ‘loss of conciousness’ were included the ‘other’ domain that also comprised of symptoms of headache, fatigue and insomnia. NB.The ‘other’ group were not further analysed as it was considered too heterogeneous to provide interpretable results. 57. The authors observed an approximate 1.75-fold increased risk of experiencing symptoms relating to autonomic dysfunction among cases from the use of OPs compared to controls (Kamel 2007b, Table 7.B.3, Annex 8). However, this result was presented graphically and was not specific for OPs (as the authors also evaluated the effect of other non-OP pesticide groups on the development of neurological symptoms). Furthermore, in the absence of more objective tests it would be imprudent to categorise some of these symptoms as being autonomic symptoms (e.g. nausea and loss of appetite which can have non-peripheral nerve cause). Therefore, this reanalysis does not provide further conclusive evidence. Pesticide Applicators (Cotton Fields) 58. Two studies examined ANS-related effects of long term low-level exposure to OPs in subjects who routinely applied pesticides to cotton fields. 59. Farahat et al assessed neurological function in 52 of 64 eligible Eygptian agricultural engineers, mixers and mechanics who worked for approximately 18 years in pesticide application departments at Berket El-Sabe district of Menoufiya


16

Governate, Egypt (Farahat et al 2003) (Table 6.B.3, Annex 8). This represented an 81% participation rate among eligible subjects. Reasons for non-participation were due to exclusions or refusal. An external comparison group of fifty male clerks and administrators from different departments of the Ministry of Agriculture who had no occupational exposure to pesticides were used as controls. The response rate among controls invited to participate was 79%. Both groups were comparable in terms of socioeconomic class, area of residence and work. Other demographic characteristics were not significantly different between either group. A questionnaire was used to ascertain occupational history information. The engineers applied the pesticide to the cotton crops while mechanics repaired spray devices. The pesticide application was done once every 15 days with an initial treatment of insect repellent followed by application of one (or a combination of two to three) OP pesticide(s), and other non-OP insecticides, which included carbamates and pyrethroids. NB. Seasonal applicators were not included in the study. All cotton field workers reported having to remain in the field during the spraying seasons (June to September) and working six days per week. Mean serum AChE levels in exposed subjects was significantly lower than controls (p=0.0001), although all were within normal limits. The authors reported that serum AChE was found to be significantly associated with duration of exposure, and suggested that ageing of AChE due to long term inhibition may account for the lower levels in exposed participants. Both a questionnaire and clinical examination were used to collect neurological symptom data and signs respectively. The latter assessment included specific tests of the sensory and motor system, and reflexes. The authors did not specify whether this included tests of autonomic function. No further details regarding the adminstration of these tests were provided. The authors screened for many potential confounding conditions or exposures including tea and coffee consumption. 60. The authors group both reported symptoms and signs detected together making it difficult to distinguish between the two types of health assessment. Exposed subjects were approximately nine times more likely to significantly report symptoms/signs of ‘dizziness’ than controls (p=0.008) (Farahat 2003, Table 7.B.3, Annex 8). However, it would be imprudent to classify ‘dizziness’ as an ANS outcome in the absence of more specific tests (as it can also arise from cranial nerve dysfunction and inner ear abnormalities). Other symptoms and signs (e.g. blurred vision) although higher in exposed participants were not significant. 61. These findings should be interpreted with caution in view of the study’s crude measure of external exposure, and its failure to account for the effect of multiple exposures to non-OP pesticides (which includes carbamates that also inhibits AChE; which thereby questions the specificity of this outcome for OP exposure). The authors did, however, allude to the possibility that the observed associations were age-related due to a significant higher number of older workers reporting symptoms and signs compared to younger workers. They suggested this could be due to older workers having a longer duration of work (and thus exposure to pesticides) compared to younger workers (data not shown). 62. Consequently, the same research group investigated the prevalence of various neurological symptoms in 50 of 56 eligible Eygptian children aged between nine and 18 years who worked for approximately seven years as seasonal workers helping the agricultural engineers spray pesticides onto cotton crops (Abdel Rasoul


17

et al 2008) (Table 6.B.3, Annex 8). The authors subdivided the children (randomly chosen from five of 50 villages within the Shebin Elkom District) into two applicator groups: a younger group comprising of 30 children aged nine to 15 years, and an older group of 20 children aged 16-18 years. Six children declined to participate yielding a response rate of 89.3%. A control group of 50 children who were friends and relatives of the applicator children and had never worked in the cotton fields were used to match on age, education and location. Group comparisons showed that both lived in the same community and attended the same schools. Both had similar demographics except for the BMI of control children being significantly higher than the applicator children (p<0.05). A questionnaire was used to ascertain their work history and pesticide exposure. Exposed children in the older group reported working for an average of approximately 21 days during the current season application. OP pesticides (i.e. chlorpyrifos) was applied in tandem with other non-OP pesticides. As in the previous study, the mean AChE activity levels were significantly lower in applicators compared to controls (p<0.05), although these were within the normal range. Members should note that the assessment of neurological symptoms (via a neurological examination) comprised an adjunctive component of a much larger examination of neurobehavioural deficits in these children. The authors did not elaborate on the clinical neurological examinations performed by specialists, and account for age, education and BMI with regards to being confounders for the neurobehavioural outcomes. 63. The authors observed a significantly higher number of exposed subjects with ‘blurred vision’ and ‘dizziness’ (i.e. 28% vs. 24% respectively compared to controls (8%) (p<0.05)) (Abdel Rasoul 2008, Table 7.B.3, Annex 8). No further analyses specific to autonomic nerve symptoms were conducted. Therefore, this study provides very limited data, and is further compounded by the self reports of external exposure, lack of account of the multiple co-exposures to other non-OP pesticides and the fact evaluation of ANS dysfunction was not a primary study aim. Pesticide Applicators (Lawns/Houses) 64. Steenland et al evaluated neurological function in current and former termiticide applicators who reported using chlorpyrifos (or chlordane, which was banned in 1998) for a minimum of one year in a 12-county area of North Carolina, US (Steenland et al 2000) (Table 6.B.3, Annex 8). The source population comprised of 3605 pest-control workers, of which 3123 were screened. A total of 520 workers were found to be eligible, however only 193 (representing a 37% participation rate) were tested. Reasons for non-participation included subjects being uncontactable, unavailable, non-compliant, or not willing to participate. The authors did not evaluate potential differences between participants and non-participants. Two non-exposed groups were used for comparisons: (i) 106 age- and sex- matched friend controls and, (ii) 83 age-, sex- and race-matched blue collar workers living in the area. The authors deployed both subjective and objective methods to assess exposure. This comprised of an interview (to ascertain whether subjects were current or former applicators, with or without a history of pesticide poisoning) and biological monitoring of the chlorpyrifos metabolite, TCP, via gas chromatography to discriminate current (n=128) from former (n= 63) chlorpyrifos applicators and the non-exposed group. Subjects were also genotyped for paraoxonase polymorphisms via PCR


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amplification of DNA obtained from buccal swabs. A total of 18 applicants (10% of those tested) were found to have the polymorphism for increased susceptibility to chlorpyrifos effects. A similar proportion of non-exposed subjects also had this genotype. The mean duration of chlorpyrifos exposure among applicators was 2.4 years, although subjects also had years of exposure to chlordane and other pesticides. Eight subjects reported having previously been poisoned. A short standardised symptom questionnaire was used to determine neurological symptoms experienced in the last month which included questions on ‘dizziness’ and ‘heart palpitations’. A series of neurological tests for both central and peripheral nerve function were also conducted, however, none appeared to assess autonomic nerve function. The authors accounted for general confounding effects of age, race, education, smoking BMI and exposure to other chemicals but this was in relation to central, motor (somatic) and sensory nerve function. 65. The authors did not present any quantitative data and reported only on the p-values. For the symptom questionnaire, significant differences were observed between exposed (n=191) and non-exposed groups (n=189): all exposed subjects (and previously posioned subgroup(n=8)) reported more symptoms of ‘dizziness’ than the non-exposed group (p<0.05) (Steenland 2010, Table 7.B.3, Annex 8). The association with ‘heart palpitations’ was considered to be borderline (0.05 < p < 0.10), however, it was the only symptom that showed a significantly increased trend with duration of chlorpyrifos exposure (p=0.01). 66. This study provides very weak evidence of ANS dysfunction in termiticide applicators chronically exposed to chlorpyrifos. This is because the positive association found for reports of ‘heart palpitations’ and increasing duration of chlorpyrifos exposure was based solely on subjective health assessment. Also, it would be imprudent to classify ‘dizziness’ as a ANS symptom in the absence of additional information/testing. Furthermore, subjects had substantial co-exposure to other pesticide agents, and the low participation-rate suggests possible selection bias cannot be ruled out. Greenhouse Workers 67. Bazylewicz-Walczak et al assessed neurological symptoms as part of a much larger examination of neurobehavioural effects in 26 Polish greenhouse workers who performed standard gardening/planting jobs for approximately 12 years (range was from one to 24 years) (Bazylewicz-Walczak et al 1999) (Table 6.B.3, Annex 8). No further details of the selection process was provided. Workers were exposed to OPs on a seasonal and cyclic basis that occured in the first half of the year. Dichlorvos, methamidophos, methidathion and primiphos-methyl were the most frequently used OPs, although additional exposure to carbamates, synthetic pyrethroids and dithiocarbamates also occured. The authors conducted a comprehensive assessment of ambient levels of exposure during March to May (the most intensive spraying period). This involved measuring levels in air, and contamination of skin and clothes, followed by estimation of daily cumulative exposure to OPs via the Durhan & Welfe method, which takes into account both dermal and respiratory absorption routes. No biological measures of exposure were performed. The daily cumulative exposure in plant workers was between 0.001-0.009% of the acutely


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toxic dose, which the authors noted is classified as a low exposure level based on WHO recommendations. The authors do not appear to have performed an assessment of OP exposure in the 25 controls (matched for age, sex, education and habitation) as no exposure data are reported. Controls worked as canteen, kitchen and administrative workers at the same gardening enterprises as the greenhouse workers. An adapted subjective symptoms questionnaire was used as part of the neurobehavioural core test battery (NCTB) for psychological assessment, which collected information on several symptoms including ‘fast heart beats’, ‘sweating’, ‘poor appetite’, ‘diarrhoea’, ‘constipation’ and ‘stomach ache’. This was performed twice: before and after the intensive spraying season. The authors did not elaborate on whether the questionnaire was blindly conducted. The authors used two-way (factor) analysis of variance to identify OP exposure effects. 68. Comparison of the first and second examinations in exposed subjects revealed a significant increase in the number of ‘gastrointestinal symptoms’ compared to controls (as indicated by the higher mean values of the NCTB tests i.e. 0.65 and 1.08 in exposed vs 1.04 and 0.88 in controls) (Bazylewicz-Walczak 1999, Table 7.B.3, Annex 8). However, the authors did not specify the symptoms included under the gastrointestinal (GIT) category. A further analyses of the effects of a single spraying season or long-term exposure (or both) on ‘neurobehavioural’ effects was conducted, and the authors observed possible interactive effects of both short and long term exposure on GIT symptoms (F-value of 5.63; p=0.02). 69. In conclusion, the increased GIT symptoms reported in greenhouse workers after the spraying season are general/non-specific. In the absence of more objective tests, it is not possible to conclude that these were definite symptoms of ANS dysfunction. It is noted that the assessment of neurological symptoms comprised an auxiliary study objective, as neurobehavioural effects was the overall health outcome evaluated. The study is limited by its small sample size and the lack of account of co-exposure to other pesticides. Indoor Exposure (Office or Residential) 70. Cox et al examined the development of various adverse health effects in US residents whose homes were illegally sprayed with methyl parathion (MP) over an eight-year period (Cox et al 2005) (Table 6.B.3, Annex 8). The US EPA commissioned sampling of homes or businesses where illegal spraying took place (conducted over a two year period). This involved obtaining wipe samples of flat surfaces in different rooms and measuring MP levels via gas chromatography, and calculating an average for all rooms combined. MP levels developed by US Environmental Protection Agency and The Agency for Toxic Substances and Disease Registry were used to risk-stratify the homes. All residents were informed by the local health department of their results prior to enrolment. The eligible study population comprised of 140 residents whose homes had levels of MP greater than 150 µg/100 cm2. The mean age of residents was 28 years (the analysis included children aged under 18 years that comprised 39.2% of the exposed group). Nineteen residents did not participate resulting in a population of 121 study participants. Reasons for non-participation were due to residents having work/school commitments, being away, or children living with other parents. The


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control group comprised of 170 residents (from 197 eligible) whose homes had either undetectable levels of MP or levels <15 µg/100 cm2 (children comprised 43.4% of the group). Twenty-seven controls did not participate for the same reasons as for the exposed study group but also because they knew that their homes had low levels of MP. Both groups were similar in terms of gender, age and average number of hours spent indoors (approximately 15.5 hours per day). However, differences were apparent in relation to tobacco exposure, frequency and duration of homes being sprayed. Plasma-, RBC- and whole-blood cholinesterase activity were measured and found to be similar between the two groups. Health screening included a questionnaire that detailed various symptoms within the respiratory, cardiovascular, neuropsychiatric, genitourinary, and GIT systems. NB. The authors assigned ‘dizziness’ and ‘blurred vision’ to the neuropsychiatric category and included ‘heat and cold intolerance’ and ‘excessive sweating’ in a miscellaneous category. A clinical examination performed over a six month period was conducted for all participants in the exposed group, and was offered to those in the control group. Positive symptoms were defined as those that either occurred after the initial spraying, and lasted for a minimum of one month or were recurrent. NB. Symptoms that occurred prior to spraying but worsened afterwards were also considered positive. The chronic symptoms evaluated and used to assign subjects as having ‘chronic toxicity’ included ‘blurred vision’ and ‘abdominal pain’ (the authors did not elaborate any further except to note that expert physicians used predetermined protocols and were blinded to the subject’s exposure status). Chronic OP toxicity was classified as being either ‘unlikely’, ‘possible’, ‘probable’, or ‘high’, based on a specified number of symptoms reported and supportive clinical signs. For toxicity ranking purposes, signs and symptoms were excluded if they were easily explained by other disease states, tobacco use/exposure or other confounders. 71. There were no significant differences in symptom reporting between groups regardless of whether the outcome was examined on a systemwise basis or as specific symptoms within each category (p>0.05) (Cox 2008, Table 7.B.3, Annex 8). The lack of difference was also apparent in the findings from the physical assessment of chronic toxicity. 72. Several factors may have contributed to the null finding observed. The study did not stratify the data according to age, which would have otherwise helped address any possible developmental factors. Possible exposure misclassification bias cannot be ruled out as the authors did not account for subject’s variable cleaning habits. Furthermore, by averaging out the room MP levels, the study did not account for the variable amount of time individuals spent in each room. Also excluding analysis of the carpet and upholstry may have reduced the accuracy of exposure assessment. The study was subject to possible selection bias as the participants were aware of their exposure status, which influenced control subject participation. 73. Kilburn et al assessed neurological function as a component of a much wider investigation of neurobehavioural impairments (Kilburn, 1999) (Table 6.B.3, Annex 8). Twenty-two of 384 US patients who were being evaluated in a neurotoxicology clinic for exposure to various neurotoxic chemicals were selected on the basis that they identified chlorpyrifos as the main chemical of exposure. Other major chemicals subjects were exposed to included diazinon, carbamates, pyrethroids and phenolic


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chemicals. Patients had different exposure scenarios i.e. either occupational or residential, although 86% of patients were exposed via the office or home. The duration of indoor exposures ranged from a single one day incident to chronic repeat events spanning two years. The authors estimated the concentration of pesticides applied in offices based on spraying schedules, and measured the residues in clothing and rugs. However, no air sampling of chlorpyrifos or other OPs were conducted in premises due to the time elapsed since exposure (which ranged from nine to 108 months). Concentrations of chlorpyrifos were assumed to be low as demonstrated by levels used in offices to make dilutions (ranged from 0.5% to 24% of concentrate) and the levels found in clothing and rugs (ranged from 0.21 to 0.40 µg/ft2 and 23.7 to 125 µg/ft2 respectively). Two hundred and sixty-four referents with no exposure to neurotoxic chemicals were randomly recruited from voter registration polls and used to generate predicted values for subsequent health assessment comparisons. The authors did not compare exposure levels between these two groups. All subjects were similar in most respects except for education. Other potential confounders explored included age, sex, height and other test determinants. A previously established questionnaire was used to collect frequency data on 35 common health complaints that were scaled from one to 11 depending on the extent of occurrence. A standard respiratory questionnaire was also included and subjects were screened for several potential confounding exposures and conditions. The authors conducted complete physical and neurological examinations which included assessment of respiratory flows and vital capacities. 74. Comparison of symptom frequencies between exposed patients and unexposed controls showed significant elevations for thirteen potential ANS-related symptoms (p values were generally = 0.0001) (Kilburn 1999, Table 7.B.3, Annex 8). ‘Loss of consciousness’ yielded a mean score value of 2.2 and 1.3 for exposed and unexposed participants respectively (p=0.02). Other increases were observed for ‘palpitations’ (5.6 vs. 2.1), and ‘dry mouth’ (6.0 vs. 3.2). The remaining ten symptoms were somewhat non-specific for the ANS e.g. dizziness, loss of libido, chest symptoms (e.g. shortness of breath, chest tightness, burning in the chest) or GIT symptoms (e.g. nausea, swollen stomach, indigestion, loss of appetite). The authors did, however, further evaluate ‘shortness of breath’ and noted that a higher percentage of exposed subjects experienced this upon exercise; the prevalence increased with increasing intensity of exercise (i.e. at rest (26%); while walking (47%); and climbing stairs (68%) vs. 4%, 8% and 37% in unexposed subjects p =0.0001, 0.0001, and 0.008 respectively). The authors did not report any ANS-related outcomes of the physical and neurological examinations. 75. This study observed increased frequencies of symptoms suggestive of ANS dysfunction in individuals exposed to chlorpyrifos largely in the home and office. However, the significance of this finding is limited partly because this is based solely on subjective data with many of the symptoms being non-specific (the exceptions being ‘shortness of breath upon exercise’, ‘loss of consciousness’, ‘palpitations’ and ‘dry mouth’). Other drawbacks of the study relate to the assessment of both acute and chronic exposed subjects, the small sample size, and the fact chlorpyrifos levels were measured in clothing and rugs without accounting for possible degradation. Furthermore, the subject’s co-exposure to other non-OP pesticides that may add or synergise with the effects of chlorpyrifos were also not accounted for.


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Cross-sectional studies summary 76. Overall, the findings from the cross-sectional studies provide very weak evidence of an increased prevalence of ANS abnormalities in subjects chronically exposed to low-levels of OP pesticides and chemicals. No studies performed tests specifically designed to assess autonomic nerve function. Most of the evidence came from six studies that used symptom questionnaires as their only method of health assessment, and they generally observed an increased prevalence of ANS-related symptoms in exposed subjects (Bazylewicz-Walczak et al 1999; Kilburn et al 1999; Steenland et al 2000; Pilkington et al 2001; Kamel et al 2005; 2007b) However, most of these symptoms were non-specific, with five studies reporting symptoms considered to be more suggestive of ANS dysfunction i.e. heat intolerance, excessive sweating, fast heart rate/ palpitations, shortness of breath upon exercise, dry mouth, fainting/loss of consciousness, and impotence). Two studies did not use a questionnaire but deployed a clinical examination to assess neurological dysfunction in exposed subjects (Srivastava et al 2000; Abdel Rasoul et al 2008). Neither however provided an adequate description of the clinical tests conducted. Srivastava et al did not observe any evidence of respiratory, GIT or skin abnormalities in subjects exposed to quinalphos. Abdel Rasoul et al reported an increased prevalence of blurred vision (and dizziness, although in the absence of autonomic testing it would be imprudent to consider this as an ANS-related symptom). Two studies provided both self-reported symptom and clinical data and both found no evidence of ANS dysfunction (Farahat et al 2003; Cox 2005). Chronic Effects Following Chronic Low-Level OP Exposure Discussion Summary 77. Most of the studies evaluating the development of persistent effects on the ANS in individuals exposed to chronic low-levels of OPs had issues associated with controlling for potential confounders and bias. The majority of evidence is suggestive as it is based on data collected via self-reported questionnaires used in six cross-sectional studies (Bazylewicz-Walczak et al 1999; Kilburn et al 1999; Steenland et al 2000; Pilkington et al 2001; Kamel et al 2005; 2007b) and one case-control study (Jamal et al 2002a). The symptoms evaluated in these questionnaires were largely non-specific for the ANS; a few were more suggestive of ANS dysfunction (i.e. fast heart rate, palpitations, fainting/loss of consciousness/lightheadedness, heat intolerance, excessive sweating, and shortness of breath (upon exercise), impotence and a dry mouth). Four studies performed objective health assessments (Srivastava et al 2000; Farahat et al 2003; Cox et al 2005; Abdel Rasoul et al 2008), with a positive association being reported in one of them i.e. an increased frequency of ‘blurred vision’ (and dizziness) in OP exposed 16-18 year old pesticide applicators compared to controls (p<0.05) (Abdel Rasoul et al 2008). However, no study used tests that were specifically designed to assess autonomic function.


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Overall Summary 78. Tables (i) and (ii) below summarise the main outcomes, and documents whether the study reports evidence of ANS-related symptoms (i.e. significant positive findings) or observed no evidence/ no significant evidence of ANS-related symptoms (negative findings) as described in this section. NB. The findings are categorised according to whether they were obtained from objective or subjective health assessments and are irrespective of the quality of the study. 79. It is not possible to make any firm conclusions on the evidence reported in these studies. This is largely due to the lack of rigorous testing of the ANS via methods specifically designed to assess autonomic function. The evidence is universally subjective relying on individual symptoms (most of which were not entirely specific symptoms of the ANS) or non-validated symptom questionnaires. No studies provide objective assessment of direct or indirect autonomic function. These studies would have been better regarded had they: (i) conducted a multiple/composite assessment of autonomic symptoms (rather than individual symptoms) to enhance sensitivity and specificity; (ii) used a subjective tool that had been validated against objective measures of autonomic function (e.g. the orthostatic grading scale or the composite autonomic symptom scale). None of the studies were particularly noteworthy in terms of quality as most had limitations associated with their study design in relation to the measures used to reduce bias and confounding. As such, the positive results which suggest that individuals exposed to OPs experience a wide range of symptoms that could be related to autonomic dysfunction, should be interpreted with caution. These findings, accord with the COT 1999 conclusions with regard to the lack of comprehensive tests evaluating the long-term autonomic health effects of acute and chronic OP exposure. Acute OP exposure and autonomic abnormalities 80. Positive evidence for palpitation symptoms in OP exposed subjects was consistently observed in four studies i.e. two cohort (Nakajima et al 1999; Kawana et al 2001) and two cross-sectional studies (Stallones & Beseler 2002b; Ohtani et al 2004). However, none of these studies performed tests to objectively confirm or refute these reports of abnormal heart beats in OP exposed groups. Furthermore, the weaknesses apparent in these studies would suggest these findings should be interpreted with caution. Consistent positive evidence for other possible ANS-related symptoms were apparent for symptoms associated with the visual and gastrointestinal systems. However, many of these symptoms are non-specific (e.g. blurred vision, nausea, and diarrhoea); therefore, in the absence of more specific probe questions/tests to confirm that they are indeed ANS-related, it would be imprudent to categorise them as ANS symptoms. Objective data was reported in one case-series (Dahlgren et al 2004) but the study did not document the clinical tests used, and the associated outcome ‘blurred vision’ is known to also have non-autonomic nerve causes. Chronic OP exposure and autonomic abnormalities


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81. On balance, the available studies tended to consistently report positive findings for ANS symptoms related to the cardiovascular system (i.e. increased heart rates, palpitations, pre-syncope/lightheadedness and fainting), abnormal sweating, and male sexual dysfunction in subjects chronically exposed to OPs compared to controls. However, these were based a small number of studies, none of which performed objective tests to confirm or refute the above symptoms. Other non-specific symptoms that could potentially be related to ANS dysfunction (e.g. dizziness, and various gastrointestinal effects) were also commonly reported in studies. However, objective evidence for these non-specific symptoms (obtained from the few clinical examinations conducted) were generally unsupportive/inconclusive. The lack of corroborative evidence from specific autonomic tests suggests other non-neurological causes cannot be ruled out.


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Table (i). Crude summary of the study findings for chronic effects of acute exposure. ANS OUTCOME OP EXPOSURE Positive


Negative findings reported by authors

CONCLUSIONS

VISUAL Limited suggestive evidence for visual symptoms

Subjective assessment

Tiredness of eyes Sarin poisoning Kawana 2001 Ohtani 2004

Difficulty focussing Sarin poisoning Kawana 2001 Ohtani 2004

Asthenopia (eye strain)

Sarin poisoning Nakajima 1999

Blurred vision Sarin poisoning Nakajima 1999 Ohtani 2004

Continuous eye irritation/sensitivity

Sarin exposure McCauley 2001


Blurred vision Diazinon poisoning Dahlgren 2004

Limited number of studies. Evidence to suggest signs of blurred vision in exposed subjects

GIT Subjective assessment

Diarrhoea Sarin poisoning Kawana 2001 Ohtani 2004

Limited suggestive evidence for GIT symptoms Abdominal pain Sarin poisoning Kawana 2001

Ohtani 2004

Bloody diarrhoea Sarin exposure McCauley 2001

Nausea Sarin poisoning Kawana 2001 Ohtani 2004

Appetite loss Sarin poisoning Kawana 2001 Ohtani 2004

CARDIOVASCULAR Subjective assessment

Palpitations Sarin poisoning Kawana 2001 Nakajima 1999 Ohtani 2004

Limited suggestive evidence for palpitations

OP exposure Stallones & Beseler 2002b

Fainting McCauley 2001

Insufficient evidence

High blood pressure

Spencer 2001 (McCauley 2002)


RESPIRATORY Subjective assessment

Difficulty breathing/ Shortness of breath

Sarin poisoning Kawana 2001 McCauley 2001

Inconclusive evidence for respiratory symptoms

Choking sensation Sarin exposure McCauley 2001

OTHER Subjective assessment

Dizziness Sarin poisoning Kawana 2001 Ohtani 2004

Inconclusive evidence for symptoms of dizziness

Sarin exposure McCauley 2001

OP exposure Stallones & Beseler 2002b

Chest tightness Sarin poisoning Kawana 2001 Ohtani 2004

Limited suggestive evidence for symptoms of chest tightness


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Table (ii). Crude summary of the study findings for chronic effects of chronic low-level exposure ANS OUTCOME OP EXPOSURE Positive


Negative findings reported by authors

CONCLUSIONS

VISUAL Subjective assessment

Blurred vision Methyl parathion exposure

Cox 2005 Inconclusive evidence for visual symptoms OP exposure Farahat 2003

Poor night vision OP exposure Kamel 2005


Blurred vision OP exposure Abdel Rasoul 2008

Farahat 2003 Inconclusive evidence for signs of blurred vision Methyl parathion

exposure Cox 2005

GIT Subjective assessment

General GIT symptoms

OP exposure Bazylewicz-Walczak 1999

Limited suggestive evidence for GIT symptoms Diarrhoea Methyl parathion

exposure Cox 2005

Vomiting Methyl parathion exposure

Cox 2005

Nausea Methyl parathion exposure

Cox 2005

OP exposure Kamel 2005 Kamel 2007b

Chlorpyrifos exposure

Kilburn 1999

Stomach cramps Methyl parathion exposure

Cox 2005

Swollen stomach Chlorpyrifos exposure

Kilburn 1999

Loss of appetite OP exposure Kamel 2005 Kamel 2007b

Chlorpyrifos Kilburn 1999 Indigestion Chlorpyrifos Kilburn 1999


General GIT symptoms

Quinalphos exposure

Srivastava 2000

No evidence for GIT symptoms

Abdominal pain Methyl parathion exposure

Cox 2005

SEXUAL Subjective assessment

Loss of libido Chlorpyrifos exposure

Kilburn 1999 Limited suggestive evidence for sexual dysfunction Impotence OP exposure Pilkington

2001

SECRETOMOTOR Subjective assessment

Dry mouth Chlorpyrifos exposure

Kilburn 1999 Insufficient evidence for secretomotor effects

CARDIOVASCULAR Subjective assessment

Fast heart rate/ palpitations

OP exposure Kamel 2005 Kamel 2007b

Limited suggestive evidence for cardiovascular symptoms


Kilburn 1999 Steenland 2000

Steenland 2000

Fainting/loss of consciousness

OP exposure Kamel 2005 Pilkington 2001


Kilburn 1999

Lightheadedness Chlorpyrifos exposure

Kilburn 1999

SUDOMOTOR Subjective assessment

Heat intolerance Methyl parathion exposure

Cox 2005 Limited suggestive evidence for


27

Excessive sweating

OP exposure Kamel 2005 Kamel 2007b Pilkington 2001

sudomotor effects

RESPIRATORY Subjective assessment

Runny nose Methyl parathion exposure

Cox 2005 Inconclusive evidence for respiratory symptoms

Cough Methyl parathion exposure

Cox 2005

Shortness of breath (or upon exercise)


Kilburn 1999


General respiratory symptoms

Quinalphos exposure

Srivastava 2000


OTHER Subjective assessment

Autonomic symptoms

OP exposure Jamal 2002a

Dizziness Methyl parathion exposure

Cox 2005 Limited suggestive evidence for symptoms of dizziness

OP exposure Farahat 2003 OP exposure Kamel 2005 Chlorpyrifos exposure

Kilburn 1999


Steenland 2000

Chest tightness Chlorpyrifos exposure

Kilburn 1999 Insufficient evidence for chest symptoms

Chest burning


Dizziness OP exposure Abdel Rasoul 2008

Limited suggestive evidence for dizziness OP exposure Farahat 2003

Skin (general) Quinalphos exposure

Srivastava 2000



28

Table 6: AUTONOMIC NERVOUS SYSTEM STUDY DESIGN DETAILS First Author





Measure of Exposure

Risk factors/ bias / confounders



6.A.1. Cohort studies Kawana 2001 Japan 582 male and

female sarin patients (mean age ranged from 36.6 ± SD 13.6 years to 39.7 ± 13.4 years old) treated at St Luke’s International Hospital, Tokyo, on the day of the attack (i.e. 20 March, 1995). Subjects were followed up 2, 3 and 5 years following the sarin attack (i.e. 1997, 1998 and 2000). Response rates were 48.6%, 35.3% and 32.8% (corresponding to 283 victims in 1997, 206 in 1998 and 191 in 2000 i.e. the current analysis) respectively

Three groups were used for a comparative sub-analysis of symptom incidence: two groups comprised of subjects exposed to sarin i.e. (i) 655 victims of Tokyo sarin attack studied by a non-governmental organisation (NGO); and (ii) 88 victims of the 1994 Matsumoto sarin attack); and one group of unexposed subjects (iii) 87 Matsumoto controls of the 1994 Matsumoto sarin attack

St Luke’s symptom questionnaire, containing 33 five-choice Likert scale items i.e. 14 physical symptoms (that included diarrhoea, dizziness, abdominal pain, nausea, difficulty breathing, appetite loss, sudden palpitation, feeling of squeezing one’s chest), 8 eye-related symptoms (that included tiredness of the eyes and difficulty focussing) and 11 psychological symptoms

Psychological, physical and eye related symptoms reported at follow-up. These were used in part as the diagnostic criteria for post-traumatic stress disorder

Subject’s status as a victim of exposure to the 1995 sarin attack in Tokyo’s subway system (following admittance to St Luke’s International Hospital for treatment)

None considered Symptom frequencies/ percentages were reported for St Luke’s Hospital sarin patients at each follow-up period and compared using Fisher’s exact test (i.e. 1997 vs. 1998, and 1998 vs. 2000); p values reported *p < 0.05; **p < 0.1; symptom incidence (%) compared in 3 groups exposed to sarin, and one control group

Nakajima 1999 Japan

[1 year survey]: 318 male and female victims of the Matsumoto sarin attack (taken from 1237

[1 year survey]: 919 male and female respondents identified as non-victims i.e. were

Questionnaire-related surveys were conducted 3 weeks, 4 months, 1, 2 and 3 years after the sarin incident to clarify symptoms and complaints. The

Various symptoms associated with sarin exposure were investigated. Symptoms

Residence in the Matsumoto area, 1050 metres north to south and 850 metres east to west where about 12

Age and gender Evaluation focussed on symptom prevalence/likelihood of reporting symptoms one


29

First Author





Measure of Exposure



respondents of an original cohort of 2052 people). Sarin victims were identified as those diagnosed with definite muscarinic and/or nicotinic symptoms immediately after the sarin incident (starting from evening of 27th to 28th June 1994). NB. The authors did not specify the mean age of all victims one year after exposure but only the mean age of the victims who were symptomatic (i.e. victims who reported symptoms associated with sarin one year after exposure) and for those for which symptoms resolved (Table 10). Subjects were all inhabitants living or staying in the area of the sarin attack in Matsumoto City. [3 year survey]: 167 sarin victim respondents (taken from 836 respondents); mean age 44.4 years, SD ±19.8).

not diagnosed with definite muscarinic and/or nicotinic symptoms immediately after the sarin incident. [3 year survey]: 669 respondents identified as non-victims (mean age 54.2 years, SD ±21.5)

questionnaire collected data on health status including: having a sarin toxicity diagnosis or experiencing muscarinic/nicotinic symptoms immediately after the sarin exposure; experiencing delayed symptoms; hospitalisations and consultations with doctors; and current symptoms. Health examinations were conducted after each survey for those requesting consultations (no further information was provided)

relating to autonomic nervous system abnormalities included blurred vision, asthenopia (eye strain), and palpitations

litres of sarin were released on the night of June 27, 1994, with the sarin release site in the centre. Erythrocyte and serum AChE activity levels were measured in victims admitted to hospital 4 weeks after sarin exposure

and three years after the attack. Chi-square test was used in the 1-year survey to test the significance of the prevalence of symptoms reported one year after sarin exposure during medical consultations, (p values were reported). In the 3-year survey chi-squared test was used to evaluate differences in prevalence of symptoms between two groups (victim and non-victim) and that of people with or without each symptom during the surveys (p values were reported). NB. Fisher’s exact test was used for small group numbers. One-way ANOVA was used to test the difference in erythrocyte and serum AChE activity at an early stage of intoxication


30

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Measure of Exposure



27 of these victims had been hospitalised immediately after the incident, 109 had consulted doctors and 31 victims did not consult any doctor

between people with or without symptoms (p values were reported). ORs and 95% CIs were calculated for symptoms in victim group

6.A.2. Case-control studies – none

6.A.3. Cross-sectional studies McCauley 2001 USA/Iraq 653 male and

female US troops (mean age 39.0 years, SD 7.5) believed to be within a 50-km radius of the Khamisiyah Ammunition Storage Point and likely to have sustained low-level exposures to sarin/cyclosarin following ground-based detonation of chemical munitions at Bunker 73 between March 1 and March 15, 1991. NB. The authors refer to this group as the Khamisiyah (KHAM) population. The authors divide the 653 KHAM

The authors used two comparison groups: (i) 610 male and female military personnel (mean age 37.8 years, SD 7.3) deployed to the Southwest Asia theatre during the Gulf War (GW) combat period. The authors assume that these veterans would of had no known exposure to nerve agents but may have been exposed to the carbamate pyridostigmine bromide and/or OP insecticides. The authors refer to this group as the Non-Khamisiyah (N-KHAM) population;

Telephone computer assisted interview survey was conducted from Oct 1998 to April 1999. The adapted GW telephone survey instrument queried subjects on their general health and symptoms during the time period in which the Khamisiyah detonations occurred (i.e. immediate or short term effects of sarin vapour exposure). This included a 24-item checklist of symptoms known to be associated with chemical warfare agents (CWA) exposures and symptoms not generally associated with exposure to anticholinesterase agents. The authors developed an OP-affected case definition based on reporting ≥ 3 ocular and respiratory effects that commonly arise after aerosol or vapour exposure to low doses of

Self-reported neurological and neurophysiological signs and symptoms experienced during the first 2 weeks after the Ground War (which included vision problems, nausea, abdominal cramping, increased hunger, increased salivation, runny nose, heart palpitations, increased urination, sweating of hands or feet, coughing, tearing of the eyes) and current health symptoms that included dizzy spells, fainting, choking

The adapted GW telephone survey instrument was used to obtain more detailed information about troop movements in the Khamisiyah area including exposure to detonation of ammunition bunkers The US Department of Defence used dispersion plume modelling of the detonations to estimate the temporal-spatial movement of releases. The authors considered those veterans within a 50km radius were likely to have sustained low-level exposures to sarin/cyclosarin (i.e.

Age, gender, and region of residence. The authors also screened for exposures to biological and chemical factors including prescription and experimental medications taken in-theater. Contactable veterans were found to be significantly more likely to have college or advanced degrees, married, Caucasian and male. Participants compared to non-participants/ non-responders were significantly

Chi-squared tests were used to analyse categorical data. Logistic regression was used to estimate odds ratios and confidence intervals (which were adjusted for potential confounders); p-values were not reported. T-tests were used to analyse continuous variables (age)


31

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Measure of Exposure



population into two subgroups: (i) 162 KHAM subjects who reported being involved or witnessing the Khamisiyah operations (KHAM witness); (ii) 405 KHAM subjects who reported not being involved or witnessing Khamisiyah operations (KHAM-non-witness); the remaining 86 subjects were unsure. NB. The source population comprised of 3219 veterans who were active or reserve status in the US Army between 01/08/90 – 30/06/91. However, only 2918 were contactable, to which 2388 (later corrected to 2834 due to 54 having given incorrect deployment information) were considered eligible. Of these 555 refused to participate resulting in 1779 interviews (77% participation rate)

(ii) 516 male and female military personnel (mean age 38.1 years, SD 8.3) who had not served in the GW theatre of operations but were either on active duty or were activated during the GW period. The authors refer to this group as the Non-deployed (ND) population (negative controls)

OP CWA. Other items on the checklist included symptoms associated with low-level exposure to mustard agents. A second checklist sought information about veteran’s current health symptoms, and included more neurological specific items. NB. At the time of the telephone interview all veterans resided in one of five US states: Oregon, Washington, California, Georgia or North Carolina

sensation, problems breathing, wheezing, coughing, shortness of breath, bloody diarrhoea, continuous eye irritation or sensitivity

although the 50 km radius was broad, it probably includes personnel that were at greatest risk for airborne exposure to CWAs). The survey sought information about the nature of environmental exposures during the GW and poised questions about service duty, living conditions, combat exposures and heat and sand exposures. Exposures to biologic and chemical factors including prescription and experimental medications taken in-theatre were also assessed in detail

more likely to be Caucasian and on active rather than reserve duty.

McCauley (Cross-

2002 US/Iraq 653 male and female Desert

The authors adopted two

Subjects underwent a computer-assisted

Various current health status

Exposure history was taken from a

Region of residence,

Factor analysis of self-reported


32

First Author





Measure of Exposure



referenced from Spencer 2001)

Storm veterans present within a 50 km radius of Khamisiyah, Coalition-Occupied Iraq who may have been exposed to sarin /cyclosarin during first 2 weeks of March 1991. These were selected from a source population of 3219 deployed and non-deployed veterans. However, 301 were uncontactable. Of the 2388 deemed to be eligible, 555 refused to participate leaving a participant population of 1833. This represented a 76.7% participation rate. 54 subjects who completed the interviews were subsequently excluded on the basis they provided incorrect deployment information. The authors noted there were significant differences in the background characteristics of the 1779 participants with respect to current

comparison groups: (i) 610 Non-Khamisiyah male and female veterans deployed to SW Asia during the GW combat who did not serve in Khamisiyah; (ii) 516 Non-deployed male and female GW-era veterans (negative controls), who did not serve in SW Asia but were either currently active, reserved or retired

telephone interview conducted between October 1998 to April 1999 which comprised a symptom checklist that addressed health both in theatre and after return from S.W. Asia, including current health symptoms. The interview also contained questions on conditions that had been diagnosed by a physician since the Gulf War, employment status, hospitalisations, and disability). NB. A random sample of 150 veterans were recontacted six months later and asked identical questions

outcomes (including high blood pressure)

computer-assisted telephone interview questionnaire, which involved contacting veterans via a letter mailout and telephone. The adapted survey instrument involved gathering detailed information on the nature of GW-related environmental exposures, including information on personal movement in the Khamisyah area and exposure to detonation of ammunition bunkers). Telephone numbers were tracked as previously described in McCauley 2001

gender and age, race/ethnicity (based on group background characteristics). The authors compared veterans with contactable telephone numbers to those who could not be easily located and found the former veterans were more likely to have college or advanced degrees, be married, white and male (no more than 10% difference between the two groups) . The authors excluded subjects on the basis or not being enrolled in the Army or National Guard during the GW, being veterans of prior conflicts, having language or hearing problems preventing interview for proceeding, being deceased. Comparison of eligible

health symptoms. ORs were reported for a study group to report being diagnosed with a particular health outcome


33

First Author





Measure of Exposure



region of residence, military status, gender, age, race, education

participants that responded to those that didn’t respond showed that they were more likely to be Veterans who agreed to participate were more likely to be older, female and Caucasian compared to non-participants

Ohtani 2004 Japan 34 male and female victims of the 1995 Tokyo subway sarin attack who responded to a letter sent out to 565 victims (i.e. those treated for acute sarin intoxication) and consented to participate in the investigation. NB. 170 victims responded and 64 agreed to participate. Mean age of subjects was 43.9 (SD 13.3) years (male, 50.2 (SD 11.9) years; female, 39.6 (SD 10.4) years). Victims were followed up at the St Luke’s International Hospital in Tokyo 5 years after the incident.

None specified Self-rating questionnaire survey evaluating 34 subjective somatic and mental symptoms developed by the staff of St Luke’s International Hospital. Mental symptoms were divided into PTSD symptoms and non-specific (post-sarin poisoning) mental symptoms. NB. The authors also compared the scores of a self-reported symptom checklist (i.e. the Impact of Event Scale-Revised) with the results of Clinician-Administered PTSD Scale.

Somatic and mental symptoms, and post-traumatic stress disorder. Somatic symptoms suggestive of abnormalities in the ANS included: tightness in the chest, palpitation, nausea, diarrhoea, abdominal pain, loss of appetite, dizziness, easily tired eyes, blurred vision, and difficulty focussing

Exposure was implicated by subjects sarin victim status and treatment at St Luke’s International Hospital emergency wards for acute sarin intoxication. No further information was provided

None specified. The authors did not further evaluate the reasons for the low response and participation rates. It is not clear why data was available for only 34 of the 64 victims who agreed to participate. However, the authors noted in the discussion that the limited number of participants was due to face-to-face interviews

A three-point scale was used to assess the severity of symptoms: severe was rated 2 points, mild 1, and none 0. No further statistical analysis was performed on physical symptoms


34

First Author





Measure of Exposure



Spencer 2001 US/Iraq 653 male and female Desert Storm veterans present within a 50 km radius of Khamisiyah, Coalition-Occupied Iraq who may have been exposed to sarin /cyclosarin during first 2 weeks of Mar 1991. These were selected from a source population of 3219 deployed and non-deployed veterans. However, 301 were uncontactable. Of the 2388 deemed to be eligible, 555 refused to participate leaving a participant population of 1833. This represented a 76.7% participation rate. A large number of the Khamisiyah veterans’ region of residence clustered in the Southeastern US cf. West Coast. 94% were active, regular military vs. activated reserve troops. A total of 267 veterans underwent Level I neurobehavioural assessments and

The authors adopted two comparison groups: (i) 610 Non-Khamisiyah male and female Desert storm veterans with no known potential exposure to nerve agents, 23.9% were activated reserve troops. A total of 203 veterans underwent Level I neurobehavioural assessments and 26 completed Level II neuro-physiological testing; (ii) 516 non-deployed male and female GW-era veterans (negative controls), 30.2% consisted of activated reserve troops. A total of 154 veterans underwent Level I neurobehavioural assessments and 28 completed Level II neuro-physiological testing

A two-level clinical examination was performed. NB. The following information was abstracted from McCauley et al, under review – which was attached to the current study (and republished as McCauley et al 2002): Subjects underwent a telephone interview which comprised a symptom checklist containing 24 items that addressed health both in theatre and after return from S.W. Asia, including current health symptoms. The interview also contained questions on conditions that had been diagnosed by a physician since the Gulf War, employment status, hospitalisations, and disability. Subjects who completed the telephone interview were recruited for neurobehavioural testing (Level I), which employed a computerised neurobehavioural test battery. Level II testing comprised a focused clinical neuromuscular and neurophysiological examination

OP-associated persistent CNS damage (i.e. neurobehavioural deficits and EEG abnormalities) and neuromuscular and neurophysiological deficits.

Exposure history was taken from a computer-assisted telephone interview questionnaire, which involved contacting veterans grouped into one of three US Department of Defence categories based on their proximity to the defined 50km radius of Khamisiyah detonations. NB. The following information in parentheses was abstracted from McCauley et al, under review – which was attached to the current study: (The adapted survey instrument involved gathering detailed information on the nature of GW-related environmental exposures, including exposures to biological and chemical factors, prescription and experimental medication taken in theatre and exposure to detonation of ammunition bunkers). Telephone numbers

Region of residence, gender and age, race/ethnicity. The authors compared veterans with contactable telephone numbers to those who could not be easily located and found the former veterans were more likely to have college or advanced degrees, be married, white and male (no more than 10% difference between the two groups) . The authors excluded subjects on the basis or not being enrolled in the Army or National Guard during the GW, being veterans of prior conflicts, having language or hearing problems preventing interview for proceeding, being deceased. Comparison of eligible participants that responded to

Factor analysis of self-reported health symptoms. ORs were reported for a study group to report being diagnosed with a particular health outcome


35

First Author





Measure of Exposure



42 completed Level II neuro-physiological testing. The authors noted that the deployed subjects (i.e. this Khamisiyah subgroup together with the non-Khamisiyah (comparison) subgroup) were slightly younger than those in the non-deployed subgroups (p=0.015). Of the deployed subjects, the Khamisiyah veterans were younger than those in the non-Khamisiyah group (p=0.010)

were tracked and located using Telematch, Equifax, IRS, Transunion and/or internet search engines

those that didn’t respond showed that they were more likely to be white or on active vs reserve duty

Stallones & Beseler

2002b US The study population comprised of 761 male/ female farm residents (i.e. operators and their spouses) mean age of 50.7 years (range 24 – 85, SD 13.6) selected from a multistage area sample of farms in an 8 county area of North-eastern Colorado (479 farms). This represented a 57% response rate from

Respondents who answered “No” to whether they reported experiencing a pesticide related illness (692 respondents) were used as comparison groups

Personal interview/ questionnaire conducted during the slower agriculture seasons conducted separately for both spouse and farm operator. Data collected included general health questions e.g. medical history, pesticide-related illnesses (medically diagnosed) and more specific questions to assess neurological symptoms experienced in the past month. These 24 symptoms were ranked according to occurrence: 1= not at all, 2= a little, 3=moderately, 4=

Neurological symptoms including heart palpitations without exertion, and difficulty driving due to dizziness/tiredness

Personal interview/ questionnaire performed during the slower agriculture seasons was conducted separately for both spouse and farm operator. Data collected included questions on farm characteristics (e.g. primary agricultural activities, types of pesticides used and their application i.e. aerial application, use of granules or sprayer, water-

Age, gender. The authors also screened for general health, race, SES, education and smoking habit. Reasons for non-participation were not followed-up but the authors compared farming operations in the eight counties with those who did participate and found that

Odds ratios with 95% CIs were estimated from a univariate analysis for reporting a neurological symptom and experiencing pesticide-related illness. A Mantel-Haenszel χ2 test for trend was calculated for neurological symptoms that showed an elevated odds ratio. A


36

First Author





Measure of Exposure



the 835 farms contacted. Terbufos was the most used crop insecticide (used by 20.8% of the residents) and applied at mean frequency of 1.2 times per year (SD, 1.3). The most used pesticides for livestock (by 37.1% residents) were defined as other OP/carbamates applied a mean frequency of 2.6 times per year (SD, 5.2). NB. The authors did not provide further information re: the breakdown of OPs for this application. A total of 69 respondents reported having ever experienced a physician diagnosed pesticide-related illness, which was used to generate groups for sub-analyses of possible associations with health outcome

quite a bit, and 5= extremely

based centre pivot irrigation system), use of protective equipment, working hours and number of pesticide applications in the previous year. The authors noted that the OPs used on the farms (for crop and/or livestock) included terbufos, chlorpyrifos, phosmet, and dichlorvos. Other non-OP pesticides and herbicides were also used on the farm

there were no differences

univariate analysis was performed to obtain estimated odds ratios for the % of farm residents using the pesticide and reporting a pesticide related illness. Univariate analyses was also used to calculate ORs and 95% CIs for pesticide related illnesses and the method of application of pesticides. Any significant variables were included in (i) a multivariate logistic regression analysis, modelling the probability of having reported a pesticide related illness (the best model verified by stepwise logistic regression; (ii) a multivariate conditional regression analysis; p-values are reported


37

First Author





Measure of Exposure



6.A.4. Case series studies

Dahlgren 2004 US Family of seven (2 parents, aged 37 and 39 years, and 5 children, aged 1 to 11, with another in-utero at 5 months gestation) exposed to diazinon on 16 June 1999 following mistaken application to interior by pesticide company. Mother and youngest child were exposed 4h after application to a visibly wet floor with dermal and inhalation exposure. Other family members were exposed thereafter. The family left the home 2 days after exposure. The family returned home after 11 months since the initial exposure once the house was thoroughly cleaned

N/A Medical history and examination (on first visit to clinic in Aug 1999). Examination was repeated on second visit to the clinic in Aug 2002 with a battery of tests that included neurophysiological testing, neurological and neuropsychological assessment

Neurological, neurophysiological and neuropsychological deficits

Exposure assessed via wipe, air and bulk sampling. Five wipe samples and one control sample were analysed in June 29 1999 by the California Pesticide Regulation. Healthscience Associates conducted wipe sampling between July 1999 to Feb 2000. Air sampling was performed on July 9 1999 and tested by DataChem (Utah) using NMAM 5600MOD. Bulk samples were analysed from July 1999 to Feb 2000 using EPA methods. NB. Authors were unable to determine the quantity of diazinon (diluted) applied in the home. According to data sheets the concentrate comprised of 22.4% diazinon and 77.6% inert ingredients. A urine sample was tested for diazinon parent compound




38

First Author





Measure of Exposure



6.A.5. Case reports – none


6.B.1. Cohort studies – none

6.B.2. Case-control studies Jamal 2002a UK 23 male and

female sheep farmers/dippers (mean age 46.1 years, SD 11.0, range 22-62) exposed to OPs with definite or probable neuropathy. Neuropathy status was determined from the outcome of neuropathy scores derived from responses to the neuropathy symptoms questionnaire and sensory QST tests (first conducted during a previous second phase cross-sectional field study but repeated in this third phase hospital clinic study). An intermediate group comprising 34 male and female farmers (mean age 46.2 years, SD 10.6, range 30-64)

15 male and female farmers (mean age 38.8 years, SD 13.3, range 20-66) exposed to OPs but almost certainly without neuropathy in third phase study

A neuropathy/ neurological questionnaire (based on the Mayo Clinic neurological symptom questionnaire and performed by a neurologist) focussed on symptoms occurring in the upper and lower limbs to detect possible chronic neurological effects, which may be associated with exposure to OPs. It is apparent (from the results) that this included questions on autonomic symptoms, however the authors did not further elaborate on this. Questions on cranial nerve involvement were excluded. The questionnaire results were subsequently scored. This was accompanied by a clinical assessment, which assessed the presence of neurological signs (reflexes, sensation and muscle power) in both the upper and lower limbs. NB. The research team were blinded to subjects, OP exposure, neuropathy classification status and occupation; five different investigators performed different groups of tests; a

Peripheral neuropathy (and neuropsychological deficits)

Questionnaire was used to collect data on recent exposure. Mean cumulative exposure was based on an exposure index (OPEXP) derived from the second phase study that utilised an exposure history assessment questionnaire and an occupational hygiene study. NB. OPEXP represents the sum of the concentration of urinary OP metabolites DEP (diethylphosphate) and DETP (diethylthiophosphate) (in units of nmol/mmol of creatinine) across dipping days, and is also the weighted sum of cumulative exposure to both concentrate (CONC) and dilute dip splash (SPLASH).

Gender, age, alcohol consumption, inherent neuropsychological characteristics were considered. Data for QST scores were based on age dependent thresholds; the following used as exclusion criteria: taking medication e.g. for hypertension, diagnosed with rheumatoid arthritis, family history of high foot arches, carpal tunnel syndrome and radioculopathy

Symptom scores were derived at the Institute of Occupational Medicine in an identical manner to the scoring scheme defined for the second phase study, and was not age dependent. The authors were careful to avoid generating false positive scores for missing data.


39

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Measure of Exposure



were included who were categorised with possible neuropathy. NB. 685 subjects from the field study were invited to take part in this clinical study. However, of the 201 were deemed eligible only 95 responded to the questionnaire, 79 of which also attended the clinic (later amended to 72 due to exclusions)

nurse accompanied each subject to ensure no exposure information was passed to the clinical team

Pilkington et al 2001. Occup Environ Med;58:702–710

6.B.3. Cross-sectional studies Abdel-Rasoul 2008 Egypt

50 of 56 eligible male children aged between 9 and 18 years from five of 50 randomly chosen villages in the Shebin Elkom District hired by the local agricultural office to work as seasonal workers i.e. to spray pesticides on cotton crop in cotton fields via backpack applicators under

50 children who never worked in the cotton fields selected from friends and relatives of the applicator children, living in the same community and attending the same schools were matched on age and education. Children were subdivided into two groups: Younger (9-15 years of age, n=30); and Older

A detailed clinical medical examination and a complete neurological examination was administered by specialists. No further information was provided. Various tests were also conducted to assess neurobehavioral function (which comprised the main health outcome of this study). A questionnaire was used (completed during the last week of the spraying season) to collect data on subjects medical history

Neurobehavioural impairments and various neurological symptoms, which included blurred vision and dizziness

A questionnaire was used (completed during the last week of the spraying season) to collect data on work history and exposure to pesticides. Serum or plasma AChE was determined to measure recent acute exposure to OP pesticides. The authors noted that pesticide application for the cotton crop are

Age, education and BMI (wrt analyses of neurobehavioural outcomes)

The t-test was used to examine the differences between means. The χ2 test was used to examine the differences between percentages. NB. Pearson’s correlation, Holm’s correction of p-values, and multiple linear regression were used in relation to


40

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Measure of Exposure



the supervision of adult engineers and agricultural employees. Children were subdivided into two applicator groups: Younger (9-15 years of age, n=30); and Older (16-18 years of age, n=20). NB. Data relevant to the Older group is presented in this summary. The authors noted that pesticide mixtures were first prepared by the local agricultural office before being loaded onto the backpack sprayers. Reloading occurred multiple times/ day until the spraying is completed. Personal protective equipment were not commonly used by the applicators

(16-18 years of age, n=20). NB. Data relevant to the Older group is presented in this summary

highly regulated in Egypt and specific guidelines are followed. Cotton fields are subject to 4 application cycles per season with each application lasting 5-11 days. OP pesticides (chlorpyrifos) are applied during the 2nd and 4th cycles from the end of June through to the 1st week of July and during the second week of August. NB. Non-OP pesticide treatments are used in the 1st and 2nd cycles. The amount of pesticide applied varies each day depending on the size of the field/ degree of pest infestation

neurobehavioural analyses

Bazylewicz Walczak

1999 Poland 26 female greenhouse workers (mean age 35.4 years, SD 7.9) performing standard gardening/ planting jobs employed at 3

25 matched female canteen, kitchen and admin workers (mean age 36.0 years, SD 8.6) employed at 3 large gardening enterprises

Note: Symptoms suggestive of abnormalities in the ANS compromised a minor part of a subjective symptom questionnaire, which itself was a component of a battery of tests used in the

Neurobehavioural effects (primarily), which included assessment of possible autonomic nerve symptoms.

Exposure levels were assessed during March to May (the period of intensive spraying) via environmental tests i.e. measurements of

Subjects were matched for age, education level (recognised confounders for neuropsychological testing) and place of

Two-Way ANOVA was used to identify OP exposure effects dictated by (i) a single spraying season (season), or (ii)


41

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Measure of Exposure



large gardening enterprises. NB. The authors noted that exposure to OPs in the greenhouses was seasonal and cyclic, occurring in the first half of the year and were dependent on the frequency of spraying in the individual cultures [sic]. Workers were restricted from entering the sprayed area for a period of 1-3 days

psychological assessment. The Finnish Subjective Symptoms Questionnaire – one of two questionnaires used in the Polish adaption of the Neurobehavioural Core Test Battery for psychological assessment performed between 9am and 2pm at the following periods: Jan – Feb (the period preceding intensive application of pesticides in greenhouses); and June (the period following major spraying operations in greenhouses). NB. The authors noted that the period of intensive spraying was during March to May. The questionnaire collected information on the following potential autonomic nervous system symptoms: sweating, poor appetite, diarrhoea, constipation, stomach ache

ambient air exposures at the workplace and contamination of skin and clothes. Air sampling was carried out via the use of bacterial aspirators in an area of a greenhouse at 5 measuring sites during 4 random periods of the working day. Pesticide levels on clothing were determined on α-cellulose pads placed on garments, while levels on the skin were determined from skin washes. Samples were collected at the end of the working day and pesticide levels of all samples were determined via gas chromatography. Exposure assessment was conducted via Durhan & Welfe method (which takes into account both dermal and respiratory absorption routes). The most frequently used OPs were Dichlorvos, Methamidophos, Methidathion,

habitation long term effects of exposure in the exposed vs. control group (group), or (iii) interaction between short and long term effects of exposure. Mean values and SDs for the NCTB tests were reported. Significance level α=10 was used to determine neurobehavioural effects


42

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Measure of Exposure



Pirimiphos-Methyl), however additional exposure to carbamates, synthetic pyrethroids and dithiocarbamates also occurred

Cox 2005 US

121 residents (from 140 who were eligible) whose homes were illegally sprayed with methyl parathion over an 8 year period and had levels of MP > 150 µg/100 cm2.. Mean age 28 (range from ≤ 5 to ≥ 60 years). Children < 18 years comprised 39.2%; < 18yrs (61%). Subjects voluntarily participated and 19 (13.6%) of those eligible for the exposure group chose not to participate. Reasons for non-participation were largely as follows: inability to leave work/school; being away from the home; children now living with other parents. NB. All subjects knew the results of their home MP

170 residents (from 197 who were eligible) whose homes were illegally sprayed with MP over an 8-year period and had non-detectable or minute levels of MP (<15 µg/100 cm2). Mean age 28 (range from ≤ 5 to ≥ 60 years). Subjects voluntarily participated and 27 (13.7%) of those eligible for the control group chose not to participate. Reasons for non-participation included prior knowledge of low MP levels

Prior to screening residents were informed by the local health department of the results for their home sampling of MP. Health screening was performed for individuals from homes with MP levels > 150 µg/100 cm2, and <15 µg/100 cm2 (offered). Residents from homes with intermediate levels of MP who expressed health concerns in relation to MP exposure were also evaluated but their data were not included in the primary data analysis. All medical evaluations were performed by one of four expert physicians using predetermined protocols. Physicians were blinded to the level of MP in the home. All subjects received a detailed review of systems (an inventory of 53 different symptoms), medical history and physical examination. This was conducted over a 6 month period. Symptoms were included as positive if they reported to occur after the initial spraying and lasted at least 1 month or were described as

Various adverse health effects/symptoms grouped into the following categories: respiratory (which included runny nose, cough, wheezing, shortness of breath); cardiovascular (which included high blood pressure); neuropsychiatric (which included dizziness and blurred vision); genitourinary (which included involuntary urination and impotence)l; gastrointestinal (which included nausea, vomiting, heartburn, stomach pain/cramps, constipation, diarrhoea, and excess salivation); and a miscellaneous

Homes or businesses where illegal spraying occurred were sampled for MP contamination by the US EPA. 10 cm2 wipe samples of flat surfaces in different rooms were obtained and analysed using gas chromatography/ mass spectrometry. Mean levels of MP were calculated for each home by averaging results for all rooms in the home. NB. Carpets and sofas were not sampled. Blood was drawn from subjects to determine cholinesterase activity. Levels of MP used to risk stratify the homes was developed by the US EPA and ATSDR. The author’s note that levels were derived using an animal LOAEL of 1mg/kg/day and a

For toxicity ranking purposes signs or symptoms were excluded if they were easily explained by other disease states, tobacco use/exposure or other confounders (no further info provided). The authors compared demographic factors and exposure histories and noted no significant differences in sex distribution, mean age or age distribution, average number of hours spent in homes. However, a higher percentage of exposed group subjects smoked and were exposed to

Comparison of exposed and control group data. Continuous data were compared using ANOVA for multiple groups and t-test when only two data sets were involved. Bonferonni-Dunn test was used to determine significance within multiple groups. Exposed subjects were divided into quartiles which were evaluated by logistic regression for the presence of toxicity and the number of subjective complaints. Significance was defined at the 95% level


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sampling prior to enrolment to study

recurrent. Symptoms that were present prior to spraying but worsened afterwards were also included. Subjects were subsequently ranked as to whether they had chronic OP toxicity based on the following specific criteria: 1 or two symptoms (unlikely); 3 or more symptoms (possible); 3 or more symptoms with supportive physical findings or 4 or more symptoms with no supportive physical findings (probable); and 4 or more symptoms with supportive physical findings (high)

category (which included heat or cold intolerance, excessive sweating, and watery eyes)

safety factor of 10 for humans. Max [air] and [surfaces] calculated. Sampling was conducted over a 2-year period. Subjects spent an average of 15.5 hours a day in the home.

second-hand tobacco smoke. Control group subjects reported their homes being sprayed more times and over a longer period than those from the exposure group. Furthermore, homes from the exposure group were reported as being sprayed more recently than those from the control group.

Farahat 2003 Egypt 52 of 64 eligible male agriculture engineers (who worked in the fields and were responsible for the entire application procedure), and mixers and mechanics (who repaired spraying devices), with a mean age of 43.63 years (SD 5.51) working in the pesticide application departments at Berket El-Sabe district, Menoufiya Governate, Egypt. NB. Reasons for non-participation were due to exclusion or

50 of 63 eligible male clerks and administrators, mean age of 42.48 years (SD 5.54) never occupationally exposed to pesticides recruited from different departments of the Ministry of Agriculture

Note: Participants were examined during workdays during their break between 12-3 pm (a typical workday being from 8am -12pm and 3-7pm). Assessments included a medical history questionnaire and clinical examination, performed along with specific neurological tests for sensory and motor functions of: cranial nerves; motor system; reflexes; sensory system. The authors did not specify whether this included tests for autonomic function. No further information was provided. A series of neurobehavioural tests were also administered

Neurobehavioual effects (that included sensory and motor neurological outcomes)

Occupational history questionnaire. Analysis of serum AChE and liver and kidney function (from blood samples taken from 45/52 exposed and 37/50 unexposed participants) was done after completion of questionnaire, clinical examination and neurobehavioural tests. The authors noted that the latter two tests were done to exclude participants with renal or kidney disease (higher risk of encephalopathy)

The authors screened for effects of age, education, BMI, smoking, alcohol and tea/coffee consumption and medical history. Subjects were excluded if they reported being seasonal workers; had less than 12 years education; had any of the following medical diagnosis: diabetes mellitus, liver or kidney disease, peripheral neuropathy, vitamin deficiency,

Prevalence (%) and odds ratios (95% CIs) of symptoms and signs. Odd ratios were computed from a logistic regression model. Confidence intervals for odds ratio were based on the likelihood ratio test (which the authors reported are known to be more accurate for small sample sizes). Holm’s modification of the Bonferroni correction equation was used to adjust for


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refusal. Subjects were required to remain in the field during the spraying season and worked six days per week during the period between June and September. Most participants reported never using protective clothing (88%) and none reported acute poisoning incidents requiring hospitalisation either prior to or during the study. Pesticides were typically applied once every 15 days, with an initial treatment of insect repellent in the early cotton growth stages, followed by application of one or a combination of two to three OP pesticides (profenofos, chlorpyrifos, triaziphos and phorate), carbamate, or pyrethroid insecticides via knapsack sprayers (carrying 120 litres of pesticides) or motorised sprayers such as tractors (400-600 litres).

anaemia and addiction; had long term treatment with psychotropic drugs; prior history of head injury resulting in loss of consciousness or recent exposure to other neurotoxic agents

multiple comparisons; p-values are reported. Multiple regression analysis was conducted to adjust for confounders. Mean, SD and SEM also reported for comparisons of serum AChE levels in exposed and control groups


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Routes of exposure were either via inhalation, skin or ingestion (during machine repair)

Kamel 2005

White male private pesticide applicators (mostly farmers) aged 18-75 years taken from the Agricultural Health Study (AHS) cohort in 1993-97 who at the time were applying for new or renewed licenses (due to the Iowa and North Carolina requirement for licenses to be renewed every 3 years). From the approximate 52,400 applicators (82% of those eligible) who completed the first questionnaire at enrolment, 44% completed the second applicator questionnaire, (which included questions on neurological symptoms). The authors noted those who completed the second questionnaire were

Controls were categorised as those applicators who experienced < 10 symptoms (low frequency). See study population column for further details

The authors used two questionnaires at enrolment to collect information from applicators on demographic characteristics, lifestyle, medical history and pesticide use. The second questionnaire (completed a month after the first) included questions on neurological symptoms experienced during the year before enrolment. These questions were based on a previously established symptoms questionnaire (Q16), that was used to evaluate effects of occupational exposure to neurotoxicants. Twenty-three symptoms were reported. The authors created two measures reflecting the number of symptoms experienced at least once in the year before enrolment: (i) a continuous variable “the number of symptoms”, and (ii) a dichotomous variable “many symptoms” that compared the 20% of applicators who experienced ≥ 10 symptoms (cases) with the remaining 80% who experienced < 10 symptoms (controls)

Neurological symptoms, of which the following represented outcomes suggestive of abnormalities in the ANS: dizziness, nausea, loss of appetite, excessive sweating, fast heart rate, poor night vision, loss of consciousness

Questionnaire completed at enrolment provided detailed information on lifetime pesticide use and exposure (i.e. frequency and duration of use of 50 specific pesticides) during the prior year. The authors considered several pesticide exposure measures: years of use of any pesticide; days per year; lifetime days of use (categorised into quartiles ranging from 0 to >500 days); and cumulative lifetime days for pesticide groups. NB. Pesticides were categorised according to function/ mode of use i.e. herbicides, insecticides (that were further categorised by chemical classes, which included OPs), fungicides, or fumigants

Age, state, education, smoking and alcohol use. The authors also screened for medical history

The authors calculated the percentage of study participants experiencing a particular frequency of neurologic symptoms in the year before enrolment to help categorise cases and controls (with symptom distributions dichotomised so that the positive category included between 5% to 15% of participants). For specific neurologic data, odd ratios were calculated by logistic regression for experiencing a specific neurological symptom with high frequency compared with low frequency. 95% CI were


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similar to those who did not in most respects including prevalence of symptoms and pesticide exposure. Only subjects who provided complete information on neurological symptoms (89% of those who returned the second questionnaire) were included (n=18,782). Applicators who provided incomplete information differed wrt, age, state and education. The symptoms listed in the questionnaire were based on a previously used and established questionnaire ‘Q16’. The responses were used to derive the applicators frequency of experiencing these symptoms at least once in the year before enrolment. “Cases” were categorised as those with a high frequency of

calculated (not shown)


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symptoms i.e. ≥ 10 symptoms

Kamel 2007b US White male private pesticide applicators (mostly farmers) aged 18-75 years taken from the Agricultural Health Study (AHS) cohort in 1993-97 who at the time were applying for new or renewed licenses (due to the Iowa and North Carolina requirement for licenses to be renewed every 3 years). From the approximate 52,400 applicators (82% of those eligible) who completed the first questionnaire at enrolment, 44% completed the second applicator questionnaire, (which included questions on neurological symptoms. The authors noted those who completed the second questionnaire were similar to those who did not in most respects including

“Controls” were categorised as those applicators; who experienced < 10 symptoms (low frequency). The authors noted this comprised 80% of the applicators. See study population column for further details

The authors used two questionnaires at enrolment to collect information from applicators on demographic characteristics, lifestyle, medical history and pesticide use. The second questionnaire (completed a month after the first) included questions on neurological symptoms experienced during the year before enrolment. These questions were based on a previously established symptoms questionnaire (Q16), that was used to evaluate effects of occupational exposure to neurotoxicants. Twenty-three symptom categories were reported. These symptoms were categorised a-priori in groups under several functional domains which included an ‘autonomic’ (comprising of nausea, loss of appetite, excessive sweating and fast heart rate), and ‘other’ group (comprising of dizziness, loss of consciousness, headache, fatigue and insomnia). However, the authors reported that they did not analyse the group of ‘other’ symptoms as it was considered too heterogeneous to provide interpretable results. Group symptom variables were

Various neurological symptoms that were categorised a priori into groups representing several functional domains were evaluated. This included an autonomic domain (as well as affect, cognition, motor and vision groups)

Several measures of pesticide exposure were constructed from questionnaire data: reported duration (years) and frequency (days/year) of use for any pesticide in categories; cumulative days of use (created by multiplying duration times by frequency using midpoints of the reported categories and then categorising the product in quartiles, ranging from 0 to >500 days); cumulative lifetime days of use of pesticides in functional groups (insecticides, herbicides, fungicides and fumigants) or chemical subgroups of insecticides (OPs, organochlorines, carbamates and pyrethroids) were created by multiplying duration by frequency of use for each pesticide in the functional or chemical group,

Age, state, education, cigarette smoking, alcohol use. The authors also screened for medical history

Logistic regression was used to estimate associations of symptom outcomes with pesticide exposure. Results were expressed as odd ratios with 95% CIs


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prevalence of symptoms and pesticide exposure. Only subjects who provided complete information on neurological symptoms (89% of those who returned the second questionnaire) were included (n=18,782). Applicators who provided incomplete information differed wrt, age, state and education. The symptoms listed in the questionnaire were based on a previously used and established questionnaire ‘Q16’. The responses were used to derive the applicators frequency of experiencing these symptoms at least once in the year before enrolment. “Cases” were categorised as those with a high frequency of symptoms i.e. ≥ 10 symptoms. The authors noted this comprised 20% of

created by summing, for each applicator, the number of times per year any symptom in the group was experienced, and then dichotomising the distribution of sums so that the positive category included approximately 10% of the participants

summing across pesticides in the group, and categorising the sum into never use and other categories of use. The authors also classified application methods likely to involve low or high personal exposure i.e. (aerial application, tractor boom, pre-applied to seed) and (airblast, mist blower, backpack sprayer, and hand spraygun) respectively to categorise applicators as using low or high exposure methods


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the applicators Kilburn

1999 US 22 male and female patients (mean age 44.7 years, SD 13.0, range 34 to 77 years) representing 6% of 384 patients being evaluated in a neurotoxicology clinic for possible neurobehavioural effects of exposure to chemicals. Subjects were selected on the basis that they identified chlorpyrifos insecticides as the main chemical of exposure. (NB. The authors noted that other major shared chemicals included diazinon, carbamates, pyrethroids and a phenolic chemical)

[Neurobehavioural function analysis]: 264 male and female referents (mean age 45 years, range 18 to 83 years) unexposed to neurotoxic chemicals were recruited at random from voter registration polls in Arizona and Los Angeles and tested to generate predicted values

Subjects completed a questionnaire which collected frequency data on 35 common health complaints scaled from rare (1) to frequent or daily (11). The assessment included a standard respiratory questionnaire. Complete physical and neurological examinations were also done, and assessments of respiratory flows and vital capacities. Neuropsychological and neurophysiological test batteries were conducted by staff at the clinic.

CNS effects i.e. neurobehavioural impairments (that included assessment of neurophysiological function)

Three different measures were used: (i) detailed schedules of pesticide application for offices (exposed by spraying every 3 to 9 weeks); (ii) analysis of personal clothing or rugs; (iii) case histories of exposure incidents. NB. The authors noted that no air sampling of chlorpyrifos or other OPs were conducted in the premises due to the time elapsed since exposure (9-108 months)

Adjustments were made for age, sex and education, gender, height and other factors with significant coefficients. Authors collected information on possible confounding exposures i.e. other hazardous chemicals including pesticides and herbicides, alcohol, tobacco and drug use (illicit and prescription), unconsciousness, anaesthesia, head trauma and neurologic and medical histories

Comparisons between exposed subjects and unexposed referents were made using the following equation: observed/predicted multiplied by 100 equals the percentage of predicted value. This was done by calculating the predicted values for each test of each patient from stepwise linear regression equations, adjusted for factors with significant coefficients. Also analysis of variance was used to compare mean percentage of predicted neurobehavioural test scores (adjusted) and SD between exposed and unexposed, and to compare symptom frequencies in chlorpyrifos-exposed


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subjects. Statistical significance was defined as p<0.05

Pilkington 2001 UK 612 male and female sheep farmers/farm workers (mean aged 45.1 years, SD 13.4) who had dipped sheep were recruited from databases maintained by the Ministry of Agriculture, Fisheries and Food (MAFF) and the Scottish Office for farms in England, Wales and Scotland. This represented 293 of 335 eligible farms (prior to exclusions based on potential confounding conditions)

Low exposure groups for comparisons comprised of (i) 53 male and female farmers (mean age 39.2 years, SD 12.7) without sheep-dipping experience recruited from MAFF/ Scottish Office database; (ii) 107 ceramics factory workers (brick makers) recruited via the British Ceramics Confederation and selected from two companies based in Scotland and England

Neurological assessments were conducted using a symptoms questionnaire in conjunction with a series of quantitative sensory tests. Symptoms were categorised into three groups that included autonomic symptoms i.e. sweating, fainting and impotence (the other two groups were sensory and muscle weakness). Analysis was based on an overall dichotomous symptom indicator where a positive score was obtained for at least two symptoms in the autonomic group. NB. The authors noted that autonomic symptoms were downweighted (in comparison to sensory and motor symptoms which required at least one symptom to be considered positive) due to Mayo questionnaire criteria and also to increase specificity

Clinically detectable abnormalities of the peripheral and autonomic nervous system

Retrospective exposure information was obtained via use of an exposure history questionnaire administered by a trained interviewer and developed during the first phase of the study. The first study phase consisted of an occupational hygiene study of dipping practice at 20 farms. Cumulative exposure models that were based on a dermal exposure model that utilised the two urinary OP metabolites diethylphosphate (DEP), and diethylthiophosphate (DETP) were derived and were used in subsequent analyses of sensory test data. They identified both concentrated dip handling and splashing dilute dip in a bath as the principal sources of exposure among

Authors adjusted for the following potential confounders of neurological symptoms: age, sex, country and alcohol habit, exposure to vibration via occupation/recreationally, other chemical exposures. Subjects were also screened for education, and having a disease or taking medication known to confound PN diagnosis. NB. Comparative group analyses showed differences in age, gender and alcohol consumption (the latter only between ceramic workers and farmer groups)

Linear logistic regression was used to calculate ORs for reporting symptoms and to test the significance of the differences between occupational groups and the effect of cumulative exposure. Cumulative exposure variables were scaled in the regression analyses by their interquartile range across all subjects. A 5% level was used to determine significance


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dippers

Srivastava 2000 India 59 male quinalphos (QP) manufacturing workers (mean age 30.0 years, SD 6.61). NB. The authors noted that these workers are also exposed to other chemicals e.g. raw materials and by-products of QP, e.g. organic solvents, o-phenylene diamine (OPD), Glyoxalic Methyl Ester Hemiacetal (GMHA), 2-hydroxyquinoxaline (2-HQ), diethyl thiaryl phosphorocyanide (DETCN), and xylene

17 eligible male control subjects (mean age 28.9 years, SD 4.5) not engaged in manufacture or handling of QP but employed as tea vendors, roadside hawkers, etc (sourced on the basis of being situated near the manufacturing unit and having no direct or indirect exposure to OP pesticides)

Detailed clinical history of subjects were recorded via a structured interview. Each subject was clinically evaluated for general health status and possible abnormalities by medically qualified doctors after 5-6 hrs of typical work exposure (in QP manufacturing workers). This included cardiovascular, respiratory, gastrointestinal, genitounrinary, ophthalmic and dermal examinations. Evaluation also included a haematological analysis. Clinical psychological tests were conducted via a battery of neuro-behavioural tests

Various CNS outcomes in particular reflex and neurobehavioural abnormalities

Detailed occupational history of subjects were recorded via a structured interview. AChE levels in blood were measured via a previously published method

Variables investigated included age, sex, height, weight, education, income, smoking and drinking habits, work history duration and previous neurological conditions. Both groups were similar wrt. age, work history, physical activity at work, sex, education, and drinking and smoking habits (p>0.05), although exposed subjects tended to be more educated, smokers and alcohol drinkers

Student’s t-test was used to test the significance of the difference in mean values between exposed and control subjects. Chi-square test was used to test the significance of prevalence of signs and symptoms in exposed and control subjects. Fisher’s exact test was used where the expected cell frequencies were > 5

Steenland

2000 US 193 current and former termiticide applicators (mean age 39.3 years, SD 9.4) who had reported using chlorpyrifos for a year or more in a 12 county area of North Carolina. NB. Some also used chlordane.

Two non-exposed groups were used: (i) 106 age (within 5 years) and sex-matched controls (mean age 38.0 years, SD 9.7) who were friends of the exposed subjects. The authors did not clarify whether these friend

A short 24-item self-administered questionnaire (modified version of the Hogstedt symptom questionnaire) was used to determine neurological symptoms experienced in the last month. This included questions on ‘dizziness’ and ‘heart palpitations’. The authors also conducted

Central and peripheral nervous system abnormalities

Subjects were interviewed to obtain information on work history i.e. termiticide use and job history to determine whether subjects used other pesticides, including previous poisoning. Biological samples of urine were

The following demographic variables were checked for potential confounding effects and included in the final models: age, race, education, smoking, BMI,

Symptom questionnaire responses were considered either continuous or dichotomised. Linear regression was used to compare neurological test data between subjects.


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The source population identified from pest control operators/ companies licensed between 1987-1997 comprised of 3605 pest control workers of which 2917 were screened and 688 were unscreened due to the companies either being out of business or refusing to provide information. 383 of the 2917 screened candidates were considered eligible i.e. worked as termiticide applicators for a minimum of one year. However, 239 (61%) of the 383 eligible participants were contactable and 153 of the contactable participants were tested (64%). Reasons for non-participation included refusals, ineligibility, and lack of availability. The authors sourced a further 206 applicators among the previously

controls were screened for their exposure to OPs; (ii) 83 from 856 age, sex and race-matched blue-collar North Carolina State employees (maintenance workers and correction officers) (mean age 42.6 years, SD 8.7) living in central North Carolina who had never worked (or been poisoned) with pesticides

a series of neurological tests for both central and peripheral nerve function – none of which appeared to have assessed ANS function.

collected to measure levels of the metabolite TCP via gas chromatography in applicators who reported current chlorpyrifos exposure (n=105) and in non-exposed subjects (n=52). Buccal swabs were also collected for genotyping participants (all exposed and 55 non-exposed) with regards to paraoxonase polymorphisms at amino acid 54 of chromosome7 i.e. their ability to detoxify chlorpyrifos (as individuals homozygous for methionine exhibit lower serum concentrations of paraoxonase and subsequently at greater risk of chlorpyrifos oxon-induced neurotoxicity). This was done by first isolating the DNA, followed by PCR amplification of the enzyme gene sequences. Exposed subjects were subsequently classified as being either current

Subjects were interviewed to obtain information on other pesticide, solvent use. NB. Subjects were asked to refrain from alcohol consumption on the night before testing. Subjects were tested for this via completion of a saliva test performed on the morning of the test. Individuals who failed (i.e. had an alcohol saliva concentration of > 0.03% returned at a later time)

Significance level was (α = 0.05)


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unscreened group. 43 subjects (21%) were subsequently scheduled for testing – reasons for non-participation included refusals, unavailability and inability to locate

applicators, former applicators, applicators with a susceptible genotype and previously poisoned applicators

6.B.4. Case series studies – none

6.B.5. Case reports – none Table 7. AUTONOMIC NERVOUS SYSTEM EFFECTS RESULTS AND CONCLUSIONS

First Author




7.A.1. Cohort studies Kawana 2001 [VICTIMS OF THE 1995 TOKYO SARIN ATTACK FOLLOWED UP AFTER 2, 3 AND 5 YEARS]

Health Outcome Data: [Questionnaire-based] For symptoms suggestive of abnormalities in the ANS, symptom frequency was highest for ‘tiredness of eyes’, followed by ‘difficulty focussing’. Other, frequently reported symptoms (>10%) included diarrhoea. Reports of most physical symptoms did not change significantly from survey to survey except for abdominal pain which decreased from 7.4% in 1997 to 2.4% in 1998 (P<0.05). NB. The authors noted that most of the reported symptoms were medically unexplained (evidenced by the lack of medical treatment) according to medical diagnostic criteria.

Symptom 1997 No and % of 283

1998 No and % of 205

2000 No and % of 191

Strengths Subjects known to have definite exposure to the OP sarin. Adequate sample size. Limitations Low and inconsistent response rate (declines with subsequent follow-ups). Exposure assessment based solely on hospital admittance as a sarin victim.

This study reports a higher incidence of several symptoms suggestive of abnormalities in the autonomic nervous system in victims of the 1995 Tokyo sarin attack (up to 5 years) compared to referent Matsumoto control group. However, due to the non-specific nature of some symptoms it would be imprudent to

THIS IS A DRAFT PAPER FOR DISCUSSION. IT SHOULD NOT BE QUOTED, CITED OR REPRODUCED TOX/2012/26 Annex 8

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patients patients patients Diarrhoea 35 (12.4) 16 (7.8) 17 (8.9) Dizziness 28 (9.9) 12 (5.8) 14 (7.3) Abdominal pain 21*(7.4) 5 (2.4) 12**(6.3) Nausea 18 (6.4) 8 (3.9) 5 (2.6) Difficulty breathing 15 (5.3) 8 (3.9) 12 (6.3) Appetite loss 14 (4.9) 6 (2.9) 7 (3.7) Sudden palpitation 27 (9.5) 18 (8.7) 12 (6.3) Feeling of squeezing one’s chest

20 (7.1) 10 (4.9) 14 (7.3)

Tiredness of the eyes

106 (37.5) 69 (33.5) 75 (39.3)

Difficulty focussing 60 (21.2) 36 (17.5) 34 (17.8) Fisher’s exact test: *p<0.05; **p<0.1 (comparisons of 1997 vs. 1998 and 1998 vs. 2000) Results of comparing symptom incidence of the current study with those reported by victims of two other groups exposed to sarin (i.e. Tokyo sarin attack studied by a non-governmental organisation (NGO) and the 1994 Matsumoto sarin attack), and a control group from Matsumoto city, showed that for all symptoms suggestive of abnormalities in the ANS shown below, the symptom incidence rate was highest in the NGO Tokyo victim group and lowest in the Matsumoto control group. NB. Many symptoms were non-specific for ANS

Symptom St Luke’s (191 patients)

NGO (655 patients)

Matsumoto (88 patients)

Control (M) (87 patients)

Diarrhoea 8.9 12.7 6.8 1.1 Dizziness 7.3 20.9 13.7 4.6 Abdominal pain 6.3 11.1 5.7 2.3 Nausea 2.6 7.5 9.2 1.1 Difficulty breathing

6.3 13.6 11.4 4.5

Appetite loss 3.7 7.4 2.3 4.6 Sudden palpitation

6.3 19.2 14.9 3.4

Feeling of squeezing one’s chest†

7.3 15.0 5.7 3.4

Tiredness of the eyes

39.3 60.9 25.2 14.9

No objective health assessment used - based on subjective self-reports. The follow-up period for the two other sarin victim groups is not specified in the symptom incidence rate comparisons. No analysis of possible associations between exposure and risk of reported symptoms. Possible confounders not considered. No statistical tests of significance to rule out chance findings when comparing results between different study groups

classify them as neuropathic disorders of autonomic nerves in the absence of more objective tests. For example, although ‘dizziness’ could be caused by orthostatic hypotension/presyncope (which would be suggestive of an ANS abnormality) it could also arise from vertigo, or disequilibrium. With this in mind and the various design limitations this study provides very limited evidence to support the development of autonomic abnormalities in victims of the sarin attack in Japan


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Difficulty focussing

17.8 32.7 12.5 3.4

The authors suggested that the reason for symptom differences between these groups may be due to the cohort receiving multiple interventions, including physical examination, counselling and laboratory work

Nakajima 1999 [VICTIMS OF THE MATSUMOTO SARIN ATTACK, FOLLOWED UP 3 WEEKS, 4 MONTHS, 1, 2 AND 3 YEARS AFTER THE SARIN INCIDENT]. NB. The authors evaluate symptoms reported after one and three years after the attack; the results of earlier/other surveys are reportedly published elsewhere: Nakajima et al, 1998. J Epidemol, 8:33-41. Health Outcome Data: [Questionnaire-based] {First year survey}: 58 sarin victims (28 men and 30 women) reported symptoms associated with sarin exposure, (mean age 45.9 years, SD ± 19.0); symptoms resolved in 260 sarin victims, (mean age 48.5, SD ± 20.1). For 54 symptomatic subjects, 11 had been admitted, 26 were outpatients and 17 were non-patients. Significant differences in the percentage of symptoms were seen between admitted and outpatient groups

Symptom Admitted patients (n=11)

Outpatients (n=26)

Non-patients (n=17)

Total (n=54)

Blurred vision 7(63.6) 12(46.2) 11(64.7) 30(55.6)

Asthenopia (eye strain)

11(100) 16(61.5) 11(64.7) 38(70.4)

Palpitation 3(27.3) 3(11.5) 0(0) 6(11.1)a*

a Significant differences were noted among the group admitted, out and non-patients * p<0.05 Erythrocyte AChE activity (measured 4 weeks after the incident) was significantly lower in subjects reporting asthenopia one year after exposure (cf. subjects reporting no symptoms):

Symptom na E-AChE(IU/L)b Blurred vision Yes = 10

No = 83 1.40±0.31 1.54±0.33

Asthenopia Yes = 16 No = 77

1.36±0.35 (p<0.05) 1.55±0.32

Palpitation Yes = 4 No = 89

1.28±0.53 1.53±0.32

aNo. of subjects who had (yes) and did not have (no) symptoms

Strengths Subjects received definite exposure to an OP agent. Health examinations were performed (although it appears that this was not a consistently applied). Limitations Lack of unexposed control subjects. Background prevalence of sarin-related symptoms is unclear. Possible information bias of an overestimated risk of later sequelae in victim group due to over-recall of each symptom. Possible underestimation of risk due to lower questionnaire compliance in the victim group (36.1% vs. 54.5% in non-victim group). Clinical assessment of symptoms not fully described. No objective neurological tests used to validate health sequelae. Does not appear to

This study reports an increased prevalence of chronic symptoms (e.g. blurred vision, asthenopia and palpitations) in victims one and three years after acute sarin poisoning. NB. Although ‘blurred vision’ can be a sign of ANS dysfunction (if associated with ciliary muscle defects), it can also be associated with dysfunction/abnormality in the eye (e.g. cornea), optic nerve or brain. In view of the various study limitations, which includes the lack of objective data to verify the symptoms reported, this study provides very weak evidence of autonomic abnormalities in victims of Matsumoto sarin attack


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Limitations Conclusions bmean ±SD (or value when no’s were under 3) Postal code mapping of the location of subjects with symptoms showed that percentage of symptomatic subjects were greatest in the block where sarin was deliberately released (17.5%), followed by blocks north and north east; most victims were located in an elliptical area, with a 400m axis, north east of the site (data represented by illustration). {Three year survey}: The table below shows the number (and percentages) of all victims reporting symptoms suggestive of abnormalities in the autonomic nervous system (incl. for ‘Victims A’ i.e. those with symptoms both one and three years after sarin incident). The percentage of victims reporting blurred vision, asthenopia (and palpitations)† were significantly greater than the percentage of non-victims reporting these symptoms. The likelihood of these symptoms being reported (depending on whether subject was a victim or not) is greater in victims than non-victims. † The authors reported that no differences were noted in palpitation symptoms between non-victims and victims, or for ORs. This conflicts with the data provided in the table below when comparing all victims with non-victims

Symptom Non-victims 669 (%)

All victims 167 (%)

ORs (95% CI)

Victims A ORs (95% CI)

Blurred vision 13 (1.9) 18 (10.8)*** 6.10(2.92-12.72) 10 (6.0)** 3.21(1.44-7.16)

Asthenopia 21 (3.1) 40(24.0)*** 9.72(5.54-17.04) 12(7.2)* 2.39(1.17-4.86)

Palpitation 5 (0.7) 5 (3.0)* 4.10 (1.17-14.33) 1 (0.6) 0.80 (0.09-6.86)

*, **, ***Significant differences were noted between victims and non-victims at P<0.05, p<0.01 and p<0.001, respectively The prevalence of victims with symptoms both one and three years after the sarin incident was also compared to those of non-victims. Significant differences were seen in the prevalence of those with blurred vision and asthenopia between victims A and non-victims. Odd ratios for blurred vision and asthenopia were also significantly greater in the victims than in non-victims.

account for possible exposure to other OP agents or medical conditions that could confound reported symptoms

7.A.3. Cross-sectional studies McCauley 2001 [US TROOPS DEPLOYED IN KHAMISIYAH AND EXPOSED TO SARIN/CYCLOSARIN]

Exposure Data: The authors noted that 50 of the 653 KHAM subjects reported that they had never been in Iraq (which the authors suggested questions the status provided by the US Department of Defence). Furthermore, the authors reported that at least 53 of the 610 N-KHAM subjects claim to have been within 50-km of Khamisiyah to which 15 reported being involved in or watching detonations Health Outcome Data:

Strengths Good sample size. Attempts to stratify exposure level and measure possible associations with reported health symptoms. Highlights US

This study does not present any convincing evidence to suggest the development and persistence of symptoms suggestive of abnormalities in the autonomic nervous system (ANS) in


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[Telephone interview-based survey]: {Symptoms experienced during the first 2 weeks after the Ground War}: No difference was found in reported symptoms between the Khamisiyah (KHAM) and non-Khamisiyah (N-KHAM) deployed groups when adjusted for confounders. Comparison between the two KHAM subgroups showed that KHAM witness subgroup were significantly more likely to report the following symptoms suggestive of abnormalities in the autonomic nervous system compared to KHAM non-witness subgroup: nausea, abdominal cramping, runny nose, increased urination, and sweating of hands or feet. NB. Increased hunger, salivation, heart palpitations and tearing of the eyes did not reach statistical significance.

Symptom Khamisiyah Witness (n=162)

Frequency (%)

Khamisiyah Non-witness (n=405) Frequency (%)

OR (95% CI)*

Vision problems 14.2 6.7 2.4(1.3-4.2) Nausea 26.5 13.6 2.3(1.4-3.6) Abdominal cramping 24.7 11.6 2.4(1.5-3.8) Increased hunger 17.9 12.1 1.6(0.9-2.6) Increased salivation 6.8 3.5 2.0(0.9-4.5) Runny nose 21.6 12.6 1.9(1.2-3.0) Heart palpitation 13.6 9.9 1.4(0.8-2.4) Increased urination 19.1 10.4 2.1(1.3-3.8) Sweating of hands or feet

25.3 14.3 2.0(1.3-3.1)

Coughing 38.3 23.4 2.1(1.4-3.1) Tearing of the eyes 19.8 14.1 1.4(0.9-3.2)

* Adjusted for age and gender. {Current health symptoms}: Reported symptoms in the KHAM group as a whole were not significantly different from the N-KHAM (data not shown below). Deployed veterans (KHAM and N-KHAM) reported significantly higher rates of nearly all health symptoms suggesting abnormalities in the autonomic nervous system (shown below) compared to non-deployed (ND) veterans (except for fainting). However, the KHAM-witness subgroup did not significantly differ from the KHAM non-witness subgroup in their reports of symptoms suggestive of abnormalities in the autonomic nervous system. NB. Many symptoms were non-specific for the ANS

Symptom Deployed (n=1263) vs

Non-deployed (n=516) OR (95% CI)*

Khamisiyah Witness (n=162) vs.

Khamisiyah Non-witness (n=405)

OR (95% CI) † Dizzy spells 2.4(1.7-3.4) 1.5(0.9-2.4) Fainting 2.5(0.9-8.4) 1.1(0.3-3.7) Choking sensation 2.1(1.3-3.7) 1.6(0.9-3.1) Problems breathing, wheezing, coughing, 4.3(2.6-7.6) 1.4(0.8-2.4)

government’s possible misclassification of potential chemical warfare agents (CWA) exposure status in KHAM group subjects. Limitations Exposure assessment based on crude measures of exposure. Lacks objective health assessment. Possible information bias (since all KHAM subjects were informed by the Dept of Defence that they had potentially been exposed to low levels of chemical warfare agents (CWA), and US army training materials refer to symptoms associated with CWA exposure). Possible recall bias (since the Kham witness subgroup would be more interested in the study and differ in their ability to recall symptoms). Possible selection bias (i.e. sample may not be representative of the entire population of troops serving in the GW, since the sample was limited to individuals whose telephone numbers could be tracked using common search mechanisms; extensive tracking measures were

individuals who witnessed/involved in the Khamisiyah detonations. Although this subgroup were almost twice as likely to report experiencing many symptoms suggesting ANS abnormalities during the 2-week period in which the detonations occurred, this was not the case eight years later. Higher prevalence of ‘bloody diarrhoea’ in sarin exposed subjects In view of the possible sources of bias introduced into the study and the limitations associated with the exposure and health assessment, this study does not provide any convincing evidence of ANS abnormalities in sarin exposed troops.


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shortness of breath Bloody diarrhoea 2.3(1.4-4.0) 3.1(1.6-6.0) Continuous eye irritation or sensitivity 3.5(2.4-5.1) 1.2(0.8-1.8)

* Adjusted for age, gender and region of residence †Adjusted for age and gender.

not used). Potential for the differences in health symptoms reported during the detonations and those currently experienced to be associated with differences in work exposures and not specifically the detonations themselves (due to differences in GW job codes). Non-specific exposures to chemical agents among deployed veterans


2002 [US DESERT STORM VETERANS DEPLOYED IN KHAMISIYAH AND EXPOSED TO SARIN/CYCLOSARIN] Health Outcome Data : [Telephone interview-based survey]: The authors calculated the likelihood (ORs) for reported medical conditions diagnosed by a physician since the Gulf War. Deployed troops [Khamisiyah subgroup and Non-Khamisiyah subgroup] were significantly more likely to report a diagnosis of several medical conditions that included high blood pressure (OR=1.7, 95%CI[1.3-2.4]) compared to non-deployed troops. NB. The authors considered that the reliability of this diagnosis was high (as indicated by the test-retest reliability coefficient (k=0.74)) – no explanation was provided re: how this coefficient was generated). However, this did not differ significantly between Khamisiyah vs. Non-Khamisiyah group (OR=0.9, 95%CI[0.6-1.3]). Hospitalisation rates among deployed and non-deployed troops did not differ.) No further evaluations of symptoms suggestive of abnormalities in the ANS were conducted.

Strengths 76.7%% participation rate. Addressed possible selection bias by comparing veterans with contactable telephone no’s with those who were unavailable or untraceable. Evaluated the reliability of self-reported medical diagnoses Limitations Sample not representative of the entire population of troops serving in the GW due to sample limited to individuals whose telephone numbers could be tracked using common

This study does not provide any evidence of an increased prevalence of symptoms suggestive of abnormalities in the ANS in Khamisiyah-based veterans exposed to sarin


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search mechanisms. Exposure assessment based on subject’s deployment status, which was later shown to be subject to possible misclassification. Lack of precise characterisation of exposure. Symptoms recalled eight years after the event No validation of self-reported medical diagnoses

Ohtani 2004 [VICTIMS OF THE 1995 TOKYO SARIN ATTACK FOLLOWED UP AFTER 5 YEARS] [Questionnaire based] The mean total score of the self-rating questionnaire was 36.5 (SD 29.9). Those suggestive of abnormalities in the ANS are listed below:

Somatic symptom No. of victims None Mild Severe

Tightness in the chest 19 15 0 Palpitation 20 14 0 Nausea 29 4 1 Diarrhoea 28 5 1 Abdominal pain 29 5 0 Loss of appetite 29 3 2 Dizziness 23 10 1 Eyes tend to become easily tired 15 16 3 Blurred vision 14 15 5 Difficulty in focussing 11 17 6

NB. Most of these symptoms are non-specific/general, particularly in the absence of specific information. For e.g. it is not known whether the nausea is postprandial, which would be more indicative of an autonomic insufficiency. The authors noted that the following symptoms were rated at a high rate: blurred vision was found in 63% of the subjects; difficulty focusing in 60%; eyes tend to become easily tired 56.7%. No further information was provided.

Strengths Long follow-up period. Subjects known to have received sarin exposure Limitations Small sample Low response and participation rate (30% and 11% respectively) The authors did not attempt to control for possible selection bias Lack of comparison group Health assessment based on self-rating questionnaire there being no objective tests conducted Lack of significance testing Inadequate control of confounding exposures/conditions

This study describes the presence of several chronic symptoms suggestive of abnormalities in the ANS in a select group of victims of the sarin Toyko attack. However, the reliability of this finding is compromised by lack of attempts to control for selection and recall bias, and confounding exposures/ conditions. Furthermore, the lack of an objective assessment, information on background prevalence, and tests to rule out chance limits the validity of these findings


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Spencer 2001 [US DESERT STORM VETERANS DEPLOYED IN KHAMISIYAH AND EXPOSED TO SARIN/CYCLOSARIN] Health Outcome Data : [Telephone-based interview survey]: {Acute symptoms} During the time period of the Khamisiyah detonations no symptom differences were evident between participants in the US Dept of Defence’s broadly defined Khamisiyah group and troops outside the defined area. However, differences were found between the small no. of individuals who witnessed detonations and others in the larger Khamisiyah group: 16 symptoms were reported in excess and were mostly mild immediate responses to low doses of OPs. The authors did not provide further information but refer readers to McCauley et al 2000. NB. No articles were published by McCauley in 2000 - it is likely that this was an editorial error and Spencer 2001 intended to cite an appended manuscript as explained below. {Current symptoms} The following information in parentheses was abstracted from’ McCauley et al, under review’ – which was attached to the current study. A subsequent Pubmed search revealed that this manuscript has been published as ‘McCauley et al (2002). Illness Experience of Gulf War Veterans Possibly Exposed to Chemical Warfare Agents. Am J Prev Med; 23(3):200-6’: (Nine years following the Gulf War, deployed troops [Khamisiyah subgroup and Non-Khamisiyah subgroup] were significantly more likely to report a diagnosis of several medical conditions that included high blood pressure (OR=1.7, 95%CI[1.3-2.4]) compared to non-deployed troops. However, this did not differ significantly between Khamisiyah vs. Non-Khamisiyah group (OR=0.9, 95%CI[0.6-1.3]). Hospitalisation rates among deployed and non-deployed troops did not differ.) No further evaluations of symptoms suggestive of abnormalities in the ANS were conducted.

Strengths 76.7% participation rate. Addressed possible selection bias by comparing veterans with contactable telephone no’s with those who were unavailable or untraceable. Used intensive telephone tracking systems to minimise possible selection bias. Limitations Exposure assessment based on subject’s deployment status. Two tiered clinical examination did not include any tests of the ANS. Lack of precise characterisation of exposure. Symptoms recalled nine years after the event which may question its accuracy

The positive finding that deployed troops were more likely to report being medically diagnosed with ‘high blood pressure’ cf. non-deployed troops is limited by the fact that this was not specific to Khamisiyah-based troops.

Stallones & Beseler

2002b [FARM RESIDENTS EXPOSED TO OPS AND OTHER PESTICIDES] Health Outcome Data: [Questionnaire-based] Analyses of the odds for reporting re-occurring neurological symptoms in those who experienced a

Strengths Addressed possible selection bias with regards to type of farming operation used by those who refused and those who took

The finding that farm operators who reported having experienced a pesticide-related illness had a 2.8-fold increased likelihood of reporting ‘heart palpitations


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pesticide-related illness showed a significantly increased association between previous poisoning and current ‘heart palpitations without exertion’ (OR=2.83[95% CI:1.22-6.54). ‘Difficulty driving due to feeling dizzy or tired’ failed to reach statistical significance (OR=2.18, 95% CI[0.79-6.05]). The χ2 test for trend showed that ‘difficulty driving due to feeling dizzy or tired’ was significantly associated with having a pesticide related illness (χ2 4.36, P = 0.0368). In the remaining analyses, the authors did not provide data for associations with specific symptoms suggestive of abnormalities in the ANS, but rather presented data on the odds of reporting an acute pesticide-related illness with regards to the type of pesticide used and the method of pesticide application

part in the study. Limitations 57% response rate. Exposure assessment based on self-reports. Exposure and health outcome analyses is not specific for OP exposure. Lacks objective measures of neurological symptoms (health outcome based on self reports). Lack of verification from physicians of pesticide poisonings. Authors noted that time sequence of exposure and outcome was flawed: pesticide poisoning reflected cumulative lifetime incidence while exposure information came from the previous year; the time frame for the neurological symptom assessment did not match that used to assess current exposure. Co-exposures to other pesticides

without exertion’ should be considered with caution. This is because the above finding is not specific for OP exposure as subjects had co-exposure to other non-OP pesticides. Furthermore, the study failed to use objective measures of neurological assessment; lacks any physician-verified pesticide diagnosis; used crude/subjective measures of OP exposure, and had flawed time frames for both exposure and neurological symptom assessment. Therefore, this study provides very limited evidence for the development of chronic peripheral neuropathy in acutely OP exposed individuals. NB. Although dizziness could have an ANS aetiology (e.g. presyncope/ orthostatic hypotension), it could also arise from vertigo, or disequilibrium. Furthermore, aside from the fact that ‘difficulty driving due to feeling dizzy or tired’ is too vague a symptom to be considered within the ANS category, presyncope/orthostatic hypotension is typically associated with


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changes from supine position to an upright position which questions whether this would be suggestive of an ANS abnormality.

7.A.4. Case series studies Dahlgren 2004 [FAMILY ACCIDENTALLY EXPOSED TO DIAZINON]

Exposure Data: A urine sample test was negative for diazinon parent compound. Wipe samples analysed ranged from 0.0048-0.9932 µg/cm2 in June 1999 and 0.2-1.1 µg/cm2 in Jul 1999 to Feb 2000. Air sampling data detected diazinon at concentrations of 1.9 µg/cm3 in the living room, 10 µg/cm3 in the playroom, and 2.7 µg/cm3 in the kitchen in Jul 1999 and 0.0037-0.0183 mg/m3 in Jul 1999 to Feb 2000. Bulk carpet samples ranged from 255 mg/kg (ppm) 24 days after exposure and 1.18-4.72 mg/kg (ppm) 6.5 months after exposure. Follow-up testing was positive for diazonin in two of 11 air, bulk and wipe samples (0.0004645 µg/cm2) for up to 8 months after exposure. Health Outcome Data: [Clinical examination] In addition to acute symptoms experienced soon after exposure, patients also experienced chronic symptoms evident after a 3-year clinical follow-up. Symptoms suggestive of abnormalities in the autonomic system are shown below: Chronic ANS symptoms (clinical evaluation conducted 3 years later)

Patient 1 (Mother)

Patient 2 (Father)





Patient 7 (Son in-utero at 5m gestation at exposure)

CNS Blurred* vision

Blurred vision

Fainting spell

- - - -

Muscular-skeletal

- - - - - - -

Respiratory - - - - - - - GI - - - - - - - Eyes - - - - - - Constant

lacrimation Skin - - - - - - -

* The authors categorised blurred vision as a CNS outcome

Strengths Relatively long follow-up period. Comprehensive assessment of external exposure levels. Limitations Biological measures of exposure based on testing diazinon parent compound in urine. No testing for diazinon metabolites or cholinesterase levels soon after exposure.

This study observed chronic symptoms suggestive of abnormalities in the ANS in adults accidentally exposed to diazinon in the home. However, although ‘blurred vision’ can be a sign of ANS dysfunction (if associated with ciliary muscle defects), it can also be associated with dysfunction/abnormality in the eye (e.g. cornea), optic nerve or brain


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No symptoms indicated by (-)


7.B.2. Case-control studies Jamal 2002a [OP-EXPOSED SHEEP FARMERS/DIPPERS WITH DEFINITE, PROBABLE OR POSSIBLE

NEUROPATHY] Exposure Data: Sheep farmers categorised with definite or probable neuropathy had a mean cumulative exposure of 1758 OPEXP, SD 1486, range 12-6088; Farmers categorised as having possible neuropathy had the highest mean exposure based on the exposure index (OPEXP) derived in the second study (i.e. 4364, SD 8011, range 68-44010). Controls had a mean cumulative OP exposure of 1349 OPEXP, SD 1638, range 10-6534. Health Outcome Data: [Questionnaire] The authors reported (in their discussion) that autonomic nervous system symptoms were commonly reported by the no-neuropathy and possible neuropathy group, but less commonly by the probable neuropathy group. There is no further description of what these symptoms were. The authors suggested that because these symptoms were reported more commonly than peripheral nervous system symptoms this indicates that the autonomic nervous system is possibly more vulnerable than peripheral nerves. The data presented in the paper (shown below) shows that autonomic symptoms were more frequently reported by the probable group followed by the possible and then no-neuropathy group. Symptoms (n(%)): hospital based clinical study

Subject group n Autonomic symptoms*

No neuropathy 15 3 (20)

Possible neuropathy 34 9 (26)

Probable or definite neuropathy 23 14 (61)

Total 72 26 (36)

* Autonomic symptom scores of 0.5 or above There was no further evaluation of reported autonomic symptoms.

Strengths Authors adopted comprehensive measures to minimise observer/ investigator bias. Questionnaires were screened to ensure consistency of recording. Limitations Health outcomes not analysed in relation to increasing OP exposure Reports incidence of abnormalities and not measures of association. Small number of subjects in each group. Low response rate in subjects from probable or definite neuropathy group and the possible neuropathy group

This study was not designed to investigate associations between OP exposure and neurological abnormalities but rather was designed to aid the clinical interpretation of neuropathic outcomes in farmers exposed to OPs. The lack of detailed information on autonomic symptoms, and absence of objective verification suggests caution should be used to interpret the suggestion that the ANS of farmers chronically exposed to OPs may be be more vulnerable


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7.B.3. Cross-sectional studies Abdel-Rasoul 2008 [CHILDREN AGED 16-18 YEARS WORKING AS PESTICIDE APPLICATORS SPRAYING PESTICIDES

ON COTTON CROP] Exposure Data: Children reported working for a mean of 7.2 years (SD 1.5 years), working for a mean of 21.4 days (SD 6.7) during the current application season. Mean AChE activity levels were significantly lower in applicators compared to control group (239.8, SD 60.0 IU/L and 283.1, SD 61.6 IU/L respectively; t =3.6; p< 0.05). Health Outcome Data: [Clinical neurological examination] The neurological examination revealed an increase in neurological symptoms in the applicator group compared to the control group. Significantly more participants in the applicator group had blurred vision (28%) and dizziness (24%) compared to control participants (8 %) (p < 0.05). The authors did not elaborate on the methods used to assess these symptoms. No further analyses specific to symptoms suggestive of abnormalities in the ANS were conducted (blurred vision and dizziness were grouped along with other symptoms as neurological).

Strengths Performed biological assessment of OP exposure. Reported neurological symptoms were verified by specialist. Limitations Small sample. Exposure assessment based on self-reported data. Exposure was not OP specific (other pesticides and growth enhancers were applied during the application period). AChE measurement was performed at one time point after the spraying season (this fails to provide a measure of the AChE variability of individuals). Inadequate account of confounders. Does not fully describe neurological examinations performed (only the results)

The primary aim of this study was to determine whether increasing exposure to OP pesticides was associated with larger neurobehavioural deficits in children seasonally exposed to OP pesticides. This study was not designed to evaluate the development of autonomic nerve related symptoms in exposed subjects. Despite this, data from a sub-analysis of neurological symptoms revealed a significantly higher number of exposed subjects with ‘blurred vision’ compared to controls. However, the strength of this finding is limited by the fact that these children were exposed to other non-OP pesticides during the application season. NB. Although ‘dizziness’ was also increased in exposed subjects it would be imprudent to classify it as a potential ANS abnormality without further information on tests used. This is because dizziness can also result from dysfunction arising outside the ANS i.e.


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vertigo, disequilibrium

Bazylewicz Walczak

1999 [GREENHOUSE WORKERS ASSESSED BEFORE AND AFTER OP/PESTICIDE APPLICATION] Exposure Data: Plant workers had a mean duration of exposure to OPs of 11.9 years (SD 6.8, range 1-24) and the daily cumulative exposure for combined dermal and inhalatory routes ranged from 0.006-1.386 mg (mean 0.177) equivalent to 0.001-0.009% of toxic dose (mean 0.001), which the authors noted is classified as a low a exposure level based on WHO recommendations. NB. The authors only report data on exposure assessment for the greenhouse workers and not for control subjects. Health Outcome Data: [Questionnaire] The only findings of possible relevance reported in the subjective symptom questionnaire results (which compare the first and second examinations in exposed and control subjects) refer to gastrointestinal symptoms, it is assumed that the mean differences were significant as they were included in further analyses (which was conducted for significant findings only). Mean values and SD for NCTB-tests from the 1st and 2nd examinations of exposed and control groups

Name of test variable Exposed group N=26 Control group N=25 Examination 1 Examination II Examination 1 Examination II Mean SD Mean SD Mean SD Mean SD

Subjective Symptoms Questionnaire Gastrointestinal symptoms*

0.65 0.80 1.08 1.13 1.04 0.73 0.88 0.73

* it is assumed these refer to questions relating to poor appetite, diarrhoea, constipation and stomach ache The analysis of the effects of single spraying seasons (season) or long-term exposure (group) on gastrointestinal symptoms reported in the subjective symptoms questionnaire yielded non-significant results. However, interactions between the two factors was apparent (as indicated by F value of 5.63 (p=0.02)). The authors noted that the exposed subjects reported more complaints in the second than in the first examination while this was the reverse for the controls.

Strengths Deployed a comprehensive assessment of external exposure levels. Limitations Small study group. Exposure assessment appears to be conducted on plant workers only and not in controls. Provides no information on the subject selection procedure and response rates. No objective tests used to measure/verify symptoms suggestive of ANS abnormalities. Inadequate control of confounders for autonomic based symptoms Failed to account for plant workers co-exposures to other pesticide compounds

The aim of this study was to assess the behavioural effects of chronic exposure to OP pesticides. The evaluation of disorders suggestive of abnormalities in the autonomic nervous system comprised a minor part of a subjective symptom questionnaire, which itself was a component of a battery of tests used in the psychological assessment. The authors observed possible interaction between the effects of both short and long term exposure for gastrointestinal symptoms, there being more complaints in the exposed subjects after intensive spraying compared to controls.

Cox 2005 [RESIDENTS FROM HOMES ILLEGALLY SPRAYED WITH METHYL PARATHION] Exposure Data: There was no difference in average cholinesterase levels between the low- and high-exposure groups assessed in plasma, red blood cell or whole blood (data presented graphically). Health Outcome Data: [Questionnaire]

Strengths High participation rate. Attempts to control for possible measurement (investigator) bias. Exposure assessment of external and biological exposure. Included objective

This study did not find any significant differences in any health outcome between exposed (high MP levels) and control (low MP level) groups. However, it is likely that the extent of bias


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The authors reported that there were no significant differences in symptom reporting between groups and present data for neuropsychiatric, respiratory and gastrointestinal symptom categories:

Symptom category Control group mean (CI)† Exposure group mean (CI) P Subjects 170 121 Neuropsychiatric 1.4(1.0-1.8) 1.8(1.2-2.3) NS Respiratory 1.4(0.98-1.8) 1.3(0.87-1.7) NS Gastrointestinal 0.62(0.43-0.81) 0.62(0.42-0.82) NS Total 3.7 (2.9-4.5) 3.9(3.0-4.8) NS

† values represent the number of symptoms reported by residents per category The authors noted that when examined individually there were no significant differences for any of the 53 symptoms evaluated between the groups. The most frequently reported symptoms suggestive of abnormalities in the ANS were as follows (although most of these are nonspecific):

Symptom Exposure group (%) Control group (%) Nausea 23.0 17.3 Runny nose 16.4 20.4 Stomach cramps 19.8 16.5 Diarrhoea 15.1 18.9 Blurred vision 17.1 11.3 Dizziness 16.3 11.7 Vomiting 12.7 11.6 Heat intolerance 12.3 10.8

p-value >0.05 for all comparisons [Clinical assessment] There were also no significant differences in the physician’s assessment of subacute or chronic toxicity between subjects in each group. No further relevant evaluations were performed.

assessment of reported symptoms Limitations External exposure assessment based on flat surface wipes that was used to classify subjects does not account for subjects variable cleaning habits and may lead to exposure misclassification bias Sampling did not include carpets and upholstery MP levels were averaged for several rooms which makes the assumption individuals spent an equal amount of time in each room Possible recall and selection bias as subjects knew the results of their home MP sampling prior to enrolment to study Children included in the study group: 39.2% exposed and 43.4% control Failure to screen for potential pesticide exposures outside the home

introduced prevented any detection of effects

Farahat 2003 [AGRICULTURAL ENGINEERS, MIXERS AND MECHANICS WORKING IN PESTICIDE APPLICATION DEPARTMENTS] Exposure Data: Exposed subjects had a mean work duration of 18.04 years (SD 8.29) compared to controls who had a mean work duration of 17.08 years (SD 4.37). Mean level of serum AChE in exposed subjects was 87.34, U/ml which was significantly lower than controls 108.25 U/ml; p=0.0001.

Strengths 79% and 81% response rates among controls and study subjects respectively. Attempts to evaluate

This study did not find any significantly increased incidences of symptoms or signs suggestive of abnormalities in the


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Health Outcome Data: [Questionnaire/clinical exam] Of the two symptoms evaluated that could be suggestive of abnormalities in the ANS, only ‘dizziness’ was significantly higher in the exposed than the control group:

Neurological symptoms† and signs††

Exposed (n=52) n(%)

Control (n=50) n(%)

OR 95% CI Holm adjusted p value*

Blurred vision 15 (29) 5 (10) 3.64 1.28 – 12.07 0.102 Dizziness 14 (27) 2 (4) 8.84 22.29 –

58.52 0.008

* Significant after using Holm’s modification of Bonferroni correction (6 symptoms and 5 signs) †Have you ever had a history of any of the following symptoms? ††Detected by clinical examination The authors noted that most of the reported neurological symptoms and signs were significantly higher in older workers, which they suggest is due to their longer working duration in comparison to younger workers (data not shown). Serum AChE showed no significant differences between those with positive and negative symptoms or signs, however the authors reported a significant trend of lower AChE levels with increasing exposure duration (β= -1.18, SE 0.19, 95% CI -1.55 to -0.81)

dose related effects Adequate control of confounders, which included assessment of tea/coffee consumption Limitations Small sample size. Subjects exposed to other pesticide agents including carbamates and pyrethroids. Inadequate description of clinical neurological tests performed. Use of AChE limited by the fact that carbamates also inhibit AChE.

ANS in Egyptian OP pesticide applicators cf. control subjects. Although dizziness was significantly higher in the exposed group, it would be imprudent to classify it as an ANS outcome in the absence of more specific tests (as it can also arise from cranial nerve dysfunction and inner ear abnormalities). The limitations of this study such as the pesticide treatment protocol’s use of other non-OP pesticides and failure to account for the effect of these multiple co-exposures may have prevented detection of effects. Furthermore, the authors considered the use of AChE as a measure of OP exposure but given that the applicators also used carbamate, which is known to inhibit AChE, makes this assessment non-specific for OP exposure

Kamel 2005 [PRIVATE PESTICIDE APPLICATORS/FARMERS CATEGORISED WITH A HIGH FREQUENCY OF SYMPTOMS (CASES)] Exposure Data: Both cases and controls were exposed to OPs. The authors used frequency of symptoms (rather than duration of exposure) to classify subjects as either cases or controls. The following describes the % of subjects in each group that fall into median, lowest and highest exposure quartiles respectively: 29% of cases (highest number of subjects) had between 51-500 cumulative lifetime days of OP exposure, 9 % with 0 days, and 6% with > 500 days. This compares with 27% of controls with 51-500 cumulative

Strengths Very large sample size. Evaluated the effect of other non-OP pesticide groups on health outcome. Addressed the potential for selection bias.

This study made internal comparisons of exposed individuals from the same population and observed positive associations in applicators with a high


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lifetime days of OP exposure, 14 % with 0 days, and 4% with > 500 days. Health Outcome Data: [Questionnaire] Table below shows the odd ratio of experiencing the following symptoms suggestive of abnormalities in the ANS with high frequency compared with low frequency. These estimates were for the highest category of lifetime days of use of organophosphates. However, it should be noted that these symptoms were also found to be associated with other pesticide groups.

Symptom OR* Dizziness 1.77 Nausea 1.88 Loss of appetite 1.76 Excessive sweating 1.84 Fast heart rate 1.95 Poor night vision 1.85 Loss of consciousness 1.26

* Estimates for which the 95% CI excluded 1.00 The authors also reported that for any particular exposure measure, there was little variation in the magnitude of the associations among symptoms grouped in categories defined a priori e.g. for symptoms reflecting autonomic function (data not shown). In the remaining analyses, symptoms are dichotomised into either “many” or “no” symptoms, which thereby precludes any further evaluation of specific symptoms suggestive of abnormalities in the ANS

Adequate control of confounders (although not specific for evaluation of ANS dysfunction) Limitations All information on exposure and disease states was taken from self reports. No p-values reported. Used an internal control population for comparison (who had both symptoms and exposure to OPs)

frequency of reporting several symptoms suggestive of abnormalities in the ANS compared to those with a low frequency of symptoms. However, the fact that both the exposure assessment and health outcome were based on subjective measures (i.e. self-reports) suggests these findings should be interpreted with caution

Kamel 2007b [PRIVATE PESTICIDE APPLICATORS/FARMERS CATEGORISED WITH A HIGH FREQUENCY OF SYMPTOMS] Exposure Data: 3306 cases (91%) had ever used OPs compared to 13184 (87%) controls. Health Outcome Data: [Questionnaire] Data specific to OP exposure and symptoms suggestive of abnormalities in the ANS were apparent only for the analysis of possible associations between symptoms groups (defined a priori into several neurologic functional domains) and cumulative use of pesticides among applicators. NB. This data was presented in a graph. Use of OPs was associated with an approximate 1.75-fold increased risk of experiencing symptoms related to abnormalities in the ANS i.e. nausea, loss of appetite, excessive sweating and fast heart rate (represented graphically). This effect was not specific for OPs i.e. was apparent for other types of insecticide e.g. organochlorines.

Strengths Very large sample size. Evaluated the effect of other non-OP pesticide groups on health outcome. Adequate control of confounders (although not specific for evaluation of ANS dysfunction) Limitations All information on exposure and disease states was taken from

The finding of an association between ANS system outcomes and use of OPs is limited by the lack of specificity of effect given that other pesticides generated similar findings. However, the subjective nature of the exposure and health assessment is counterbalanced by the very large sample size of the study


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NB. The authors did not consider ‘dizziness’ and ‘loss of consciousness’ to be autonomic symptoms as these were categorised under an “Other” domain along with other symptoms

self-reports. Used an internal control population for comparison (who had both symptoms and exposure to OPs). OR for relevant analyses presented graphically. No p-values reported.

Kilburn 1999 [PATIENTS EVALAUTED AT A NEUROTOXICOLOGY CLINIC FOLLOWING EXPOSURE TO CHLORPYRIFOS] Exposure Data: Patients were exposed either: outdoors via jobs as a agricultural worker or pesticide applicator (for a duration ranging from 1 day to between 5 to 6 years); indoors (in offices or homes where chlorpyrifos containing pesticides had been applied either repeatedly over days, months and up to 2 years, or were applied only once. Concentrations of chlorpyrifos applied in offices ranged from 0.25 to 24.1% (50% concentrate was used to make dilutions). Chlorpyrifos concentrations in clothing ranged from 0.21 to 0.40 µg/ft2 and in rugs ranged from 23.7 to 125 µg/ft2. Health Outcome Data: [Questionnaire] Comparison of symptom frequencies (on a ten-point scale) showed significant elevations for 33 of 35 symptoms in exposed subjects versus unexposed referents. The most elevated symptoms suggestive of abnormalities in the ANS are shown below (although, many of these symptoms are rather non-specific) :

Symptom Exposed (22) [mean ±SD]


p value

Chest tightness 5.5 ± 3.0 2.2±1.8 0.0001 Palpitations 5.6±3.2 2.1±1.8 0.0001 Burning in chest 4.2±2.5 2.0±1.7 0.0001 Shortness of breath 4.5±2.7 2.5±2.0 0.0001 Dry mouth 6.0±2.9 3.2±2.5 0.0001 Nausea 5.3±3.2 2.4±1.9 0.0001 Dizziness 6.2±2.1 2.1±1.7 0.0001 Lightheadedness 6.4±2.7 2.5±1.9 0.0001 Loss of consciousness 2.2±2.2 1.3±1.0 0.002 Loss of libido 6.5±3.4 3.2±2.7 0.0001 Indigestion 7.1±2.9 3.2±2.3 0.0001

Strengths Performed both subjective and objective assessment of reported neurological symptoms. Adequate control of confounders (although not specific for evaluation of ANS dysfunction) Shortness of breath is further evaluated to help distinguish possible ANS involvement Limitations Small number of study subjects. Acute and chronic-exposed subjects grouped into one category (i.e. range of exposure duration). Exposure assessment based on residues on clothes, rugs, estimates and personal history with no account of possible degradative effects.

This study observed increased frequencies of symptoms suggestive of abnormalities in the ANS in individuals exposed acutely and chronically to chlorpyrifos sprayed inside homes and offices. However, this was based on subjective data only (collected via questionnaires), which suggests that these findings should be interpreted with caution


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Loss of appetite 5.2±3.1 2.6±2.1 0.0001 Swollen stomach 6.3±3.7 2.7±2.3 0.0001

[Respiratory symptoms questionnaire]: The authors noted that complaints of shortness of breath were elevated in exposed cf. unexposed subjects

Percentage of group (%) P values* Exposed Unexposed

Shortness of breath At rest 26 4 0.0001

While walking 47 8 0.0001

Climbing stairs 68 37 0.008

*As percentage predicted [Clinical examination] The authors did not report on the outcomes of physical and neurological examinations in relation to signs suggestive of abnormalities in the ANS.

Pilkington 2001 [SHEEP FARMERS/WORKERS WHO HAD DIPPED SHEEP] Health Outcome Data: NB. Only data relating to symptoms suggestive of abnormalities in the ANS are presented below: [Questionnaire] With regards to prevalence of reported symptoms, autonomic symptoms (i.e. sweating, fainting and impotence) were most often reported within all groups followed by sensory symptoms (highest 11.6% sheep dippers), then muscle weakness symptoms (highest 7.8% in sheep dippers) of the upper and lower limbs.

Symptom group %

Autonomic Ceramics 10.3 Farmers not sheep dippers 20.8 Sheep dippers 28.4

The remaining analyses focussed on all symptoms together or sensory (i.e. thermal and vibration threshold) testing data.

Strengths Large study population. Several exposure measures used i.e. cumulative exposure and exposure intensity. Adequate participation rates 88% and 62% among sheep and pig farmers respectively; 60% and 80% among ceramic workers. Adequate control of confounders (although not specific for evaluation of ANS dysfunction) Limitations Cumulative exposure to OP dips based on self-

This study observed increased prevalence of autonomic symptoms in all groups compared to sensory and motor symptoms. However this is based on a questionnaire. Subsequent analyses of symptom prevalence were based on an overall symptom indicator that combined peripheral and autonomic symptoms together thereby precluding further evaluation of possible associations between OP exposure and prevalence of symptoms suggestive of


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In the discussion the authors noted that the effect of concentrate handling on the presence of specific symptom groups showed that although all types of symptoms were more commonly reported among concentrate handlers than non-handlers the effect was not significant for autonomic symptoms (OR=2.2) compared to sensory symptoms which were nearly 2.5 times higher and significant. No further information was provided.

reports of exposure and exposure modelling. No verification of reported ANS symptoms

abnormalities in the ANS

Srivastava 2000 [QUINALPHOS MANUFACTURING WORKERS] Exposure Data: Mean whole blood AChE levels in QP workers were not statistically different from controls i.e. 24.27 ± SD 11.21 µmol/min/100 ml (range 1.8 to 45.2 µmol/min/100 ml) vs. 24.41 ± SD 12.60 µmol/min/ 100 ml (range 6.6 to 40.6 µmol/min/ 100 ml). Study subject’s work history spanned 5.7 years (SD 4.4) compared to 3.35 years (SD 3.9) in control subjects. Health Outcome Data: [Clinical neurological exam] The authors reported the following as complaints within the clinical findings (those suggestive of possible abnormalities in the ANS shown although they are presented on a system-wide basis). None were significantly higher in subjects engaged in QP manufacture.

Variables Exposed (n=59)

Control (n=17)

P value

Respiratory 4 (7.5%) 2 (11.7%) >0.05 Skin 2 (3.8%) 0 (0%) >0.05 GIT 2 (3.8%) 1 (5.8%) >0.05

Strengths Biological measure of exposure to OP. Employed objective clinical health assessment that was conducted by trained individuals. Adequate control of confounders (although not specific for evaluation of ANS dysfunction) Limitations Small sample size. Questionable referent population/selection process. Exposure assessment based solely on AChE levels in blood with no evaluation of external/ambient levels of OP exposure. Lack of analysis of the effect of co-exposures to other chemicals in the manufacturing process. Authors did not elaborate on the clinical tests performed

This study does not provide any evidence of an increased prevalence of symptoms suggestive of abnormalities in the ANS. This is partly due to the fact that the clinical assessment is only described on a system-wide basis which yielded non-significant results compared to unexposed controls. However, the study is further limited by its use of an questionable control population, inadequate exposure assessment and lack of account of subjects co-exposures to other potentially neurotoxic chemicals during the manufacturing process

Steenland 2000 [TERMITICIDE APPLICATORS EXPOSED TO CHLORPYRIFOS AND CHLORDANE] Strengths This study provides


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Exposure Outcome Data: Applicators (n=191) applied termiticides for a mean of 2.4 years (SD, 2.2), however 34 % had also applied chlordane for a mean of 7.0 years, SD (6.3). 90% of applicators (173) applied other pesticides for a mean of 2.5 years (SD 2.5). 76 and 126 applicators reported having used termiticides within the last week and current year respectively. Mean urinary TCP levels in 65 applicators who applied termiticide in the last week was higher than in 40 applicators who did not (629 ug/L, SD1536.6; and 119 ug/L, SD 196 respectively). This contrasts with the low-level detected in 52 nonexposed subjects): urinary mean TCP level = 6.2 ug/L, SD 6.1) which the authors noted is within the normal range of US adult population (two orders of magnitude lower than that of the termiticide workers). Eight applicators reported having experienced previous poisoning episodes. Health Outcome Data: The paroxonase gene was genotyped in 184 applicators, 10% (18) of whom were homozygous for the gene responsible for potentially producing low levels of paraoxonase and thus had an increased susceptibility to the effects of chlorpyrifos. A similar proportion of non-exposed subjects also had this genotype. [Symptom questionnaire] Exposed subjects reported significantly more symptoms experienced in the last month than nonexposed subjects, which included ‘dizziness’ (p <0.05). The poisoned subgroup had significant associations for ‘dizziness’ (among others) compared to nonexposed group, although the association with ‘heart palpitations’ was borderline (0.05 < p< 0.10). However, the authors noted that ‘heart palpitations’ was the only symptom showing a significantly increased trend with duration of chlorpyrifos exposure (p=0.01). NB. Although ‘dizziness’ could be caused by orthostatic hypotension/presyncope (which would be suggestive of an ANS abnormality) it could also arise from vertigo, or disequilibrium. The objective assessments did not include evaluation of ANS dysfunction

Adequate sample size Used two non-exposed groups that included friend controls. Used extensive methods to locate uncontactable candidates. Adequate account of potential confounders (although not specific for evaluation of ANS dysfunction). Exposure assessment included internal evaluation Limitations Possible selection bias due to low participation rate among contactable eligible participants; there being no evaluation of whether non-participants differed from subjects who participated. Friend controls may represent a select group of more sociable subjects. Age of 90% applicators was young (> 50) which may preclude detection of delayed neurotoxic effects. Applicators co-exposure to other pesticides including chlordane which preceded chlorpyrifos as the main pesticide used against termites. The neurological tests

weak evidence of abnormal ANS effects in termiticide applicators exposed to chlorpyrifos. The data was subjective i.e. based on a symptom questionnaire and due to the non-specific nature of ‘dizziness’ it would be imprudent to classify it as a neuropathic disorder of autonomic nerves in the absence of more objective tests. Furthermore, the study was also limited by the fact subjects had substantial co-exposure to chlordane and other pesticides, and were not representative of all the exposed workers.


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did not include assessment autonomic nerve system abnormalities


TOX/2012/26 Annex 8

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Table 8. EXPOSURE SCENARIO AND AUTONOMIC SYSTEM EFFECTS Section 8.A. ACUTE EXPOSURE WITH CHRONIC EFFECTS Table 8.A.1. Cohort studies

Test Type First Author and

Date

Kawana 2001

Nakajima 1999

Questionnaire + + Table 8.A.3. Cross-sectional studies

Test Type

First Author and Date McCauley

2001 Spencer

2001 Stallones

& Beseler 2002b

Ohtani 2004

Questionnaire - - + + Table 8.A.4. Case series study

Test Type First Author

and Date Dahlgren 2004

Clinical examination + SECTION 8.B. CHRONIC LOW-LEVEL EXPOSURE Table 8.B.2. Case-control study

Test Type First Author

and Date Jamal 2002a

Questionnaire +


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Table 8.B.3. Cross-sectional studies

Test Type First Author and Date

Abdel-Rasoul 2008

Bazylewicz-Walczak

1999

Cox 2005

Farahat 2003

Kamel 2005

Kamel 2007b

Kilburn 1999

Pilkington 2001

Srivastava 2000

Steenland 2000

Questionnaire + - - + + + + + Clinical examination + - - -

Table 9. OVERVIEW OF TESTS FOR AUTONOMIC NERVOUS SYSTEM ABNORMALITIES Clinical Neurological Evaluation 1. A neurological evaluation is a complete clinical assessment of the nervous system, which seeks to detect possible abnormalities by determining the function of relevant components of the nervous system. Autonomic nervous system (ANS) abnormalities are detected via the use of a limited range of standard and well-validated tests as shown below. Subject’s history 2. Prior to any tests being conducted, a subjective assessment of possible health effects is conducted by taking the subject‘s history. This is necessary to help determine the type of neurological disorder and possible causes. Subject history involves collecting data on symptoms (to identify patterns and its development and progression over time), family history of ANS disorders, and any existing medical conditions, viral diseases or medications being taken. There are many symptoms that could suggest autonomic dysfunction that vary depending on the nerves/system affected, and can be non-specific (See table below). Clinical examination


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3. The next stage of the evaluation is an objective assessment of possible ANS disorders. This involves conducting a clinical (physical) neurological examination followed by more specific diagnostic tests. NB. Blood tests and urine screening can also be conducted to help rule out/identify potential causes and measure levels of toxic substances respectively. 4. The physician performs a physical assessment of ANS function by taking the subjects blood pressure and measuring their heart rate to see if levels are normal or suggest autonomic insufficiency. An eye exam will also be performed to identify any lesions. Finally a test for reflexes will be performed as any insufficiencies may suggest issues with the ANS. If the subject displays signs/symptoms suggestive of autonomic insufficiency (See table below) more specific tests are conducted that can help determine the severity and distribution of the insufficiency. NB. ANS disorders that are harmful to health are known as dysautonomias, and are very complex due to their multi-disciplinary nature. Testing for dysautonomia is limited by the lack of speciality training. Common Signs and Symptoms of ANS Abnormalities Body function Symptoms

Signs

Secretomotor Dry eyes and mouth Excessive saliva

Orthostatic (upright posture) Dizzinessa (when standing from rest) Postural hypotension (decrease in blood pressure upon standing) Absence of heart rate variability upon standing

Enteric Constipation, diarrhoea (nocturnal or intermittent)

Abnormal abdominal sounds Swollen abdomen

Postprandial (occuring after a meal) Bloating, fullness, nausea, vomiting (undigested food), dizziness, sweating, orthostatic hypotension

Urological Urinary retention Difficulty with initiation Incomplete emptying

Swollen bladder Anal wink reflex


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Body function Symptoms

Signs

Incontinence Sexual Erectile failure

Ejaculatory dysfunction Retrograde ejaculation into the bladder Vaginal dryness Female orgasm difficulties

Cremasteric reflex Bulbocavernosus reflex

Visual Tiredness of eyes/asthenopiab Difficulty focussingc Blurred visiond Sensitive to light/glare Reduced night visione

Sluggish pupil reaction in the eye: miosis (excessive pupil constriction) and mild ptosis (upper eyelid droop) suggests a sympathetic lesion; a dilated unreactive pupil suggests a parasympathetic lesion

Sudomotor Abnormal sweating Heat intolerance (exercise induced)

Vasomotor Distal colour changes Persistenly cold extremities Raynaud phenomenon Loss of skin wrinkling in water

Other Unexplained weight loss Unexplained syncope Shortness of breath (upon exercise)

Rapid or slow heart rate Irregular heart rhythms High blood pressure Absence of sinus arrythmia

aDizziness is a non-specific symptom that can also result from cranial nerve dysfunction as occurs in vertigo and disequilibrium. An autonomic insufficiency is indicated when dizziness arises from presyncope (i.e. lightheadness/ faintness resulting from orthostatic hypotension) bAssociated with continuous focussing and tightening of the ciliary muscles (innervated by ANS) cCiliary muscle involvement (parasympathetic fibres carried by the occulomotor cranial nerve) dCan be associated with dysfunction/abnormality in the eye (e.g. cornea, ciliary muscle), optic nerve or brain eCould result from dysfunction in the eye (rods), or sympathetic innervation of pupil (carried by the oculomotor nerve) Note: All the symptoms listed above could be related to ANS dysfunction, however the sensitivity and specificity for detecting ANS dysfunction with a single symptom would be low.


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Autonomic testing 5. Laboratories that test autonomic function are increasingly available partly due to reliable non-invasive techniques. However, unlike other systems, autonomic function is not typically directly assessed. Instead, testing relies on the responses of complex overlapping reflex loops that are measured after controlled perturbations. The most common responses assessed are heart rate (HR), blood pressure (BP) and sweating. 6. There are many techniques used to evaluate autonomic function, but only a limited number are considered suitable for routine clinical application. Tests of (i) cardiovagal function (informs on parasympathetic nerve function); adrenergic (sympathetic) nerve function; and (iii) sudomotor (sweating) function are the most commonly performed and are recognised, standard clinical measures with established procedures (See table below). NB. Detailed assessment of components of the ANS is difficult in diagnosing neuropathy, and it is usually sufficient to determine whether there is some autonomic involvement. 7. To minimise confounding factors, subjects are asked to avoid consuming autonomically-active agents prior to testing (i.e. for a minimum of three hours before testing). These include: caffeine, nicotine, large meals and heavy exercise on the day of testing. Subjects are also required to ensure that they have recovered from any acute illness or dehydration. Selected Well-Established Tests of Autonomic Function Type of test Description

Examples

Cardiovagal These tests evaluate the HR response (via ECG rhythm strip) to deep breathing or standing. If the ANS is intact, the heart rate varies with these manoeuvers. The measure used for the Valsava ratio is beat-to-beat BP, which is recorded using a device placed on the subject’s finger. NB. Normal responses to deep breathing and the Valsalva ratio vary by age.

HR variability to cyclic deep breathing HR response to the Valsalva maneuver (Valsalva ratio) HR response to standing (30:15 ratio)

Adrenergic These tests evaluate the response of beat-to-beat BP to

orthostatic stresses and Valsalva maneuvers. The pattern of reflex responses in BP and HR provide an index of adrenergic function.

BP response to the Valsalva maneuver (Phases IV and late phase II)

BP response to orthostatic stress - Head-up tilt - Standing


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Type of test Description

Examples

Sudomotor These tests evaluate either pre- or postganglionic or sudomotor

function by stimulating sweat glands in specific sites or the whole body (either via use of pharmacoactive agents, heat, or electrical shock). The amount of sweat produced is quantified directly or indirectly.

Sympathetic skin response (SSR) Quantitative sudomotor axon reflex test (QSART) Thermoregulatory sweat test (TST) Silastic sweat imprint testing

Other Pharmacological challenges

Vasomotor testing Pupillary tests (pharmacological) Urodynamics GI motility studies Salivary testing Penile plethysmography, Papaverine injection Neuroendocrine tests Cardiac fluorodopamine PET scanning

Bibliography URLs 1. MedilinePlus – Autonomic Nervous System Disorders (Dysautonomia): www.nlm.nih.gov/medlineplus

2. The Merck Manual. Neurologic disorders: www.merckmanuals.com/professional/neurologic_disorders.html 3. The Mayo Clinic. The Utility of Autonomic Testing: www.mayoclinic.org/publications-md/

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