Summaries of toxicological data: No-effect levels of three organophosphates in the rat, pig and man

Fd Cosmet. Toxicol. Vol. 2, pp. 311--316. Pergamon Press 1964. Printed in Great Britain

SUMMARIES OF TOXICOLOGICAL DATA

NO-EFFECT LEVELS OF THREE ORGANOPHOSPHATES IN THE RAT, PIG A N D MAN

Summaries* of Jbur unpublished reports of work carried out by Dr. E. F. Edson and colleagues in the Medical Department, Fisons Pest Control Ltd., Saffron Walden, Essex during the

period 1953-1957.

Introduction

Organophosphorus insecticides are believed to act by inhibiting the activity of cholin- esterase (ChE) in the nervous system. Before symptoms or signs of organophosphorus intoxication set in, ChE activity present in the blood is progressively depressed, thus pro- viding a very early and sensitive indicator of toxic action. This criterion was used to deter- mine the maximum no-effect levels of schradan, dimefox and parathion in the rat, pig and man.

1. The effects in the rat and the pig of prolonged administration of schradan at low dosages

(Report dated May 1954)

A summary of the dosage schedules of schradan in the rat and pig is presented in Table 1.

Table I. Dosage schedules of schradan in the rat and pig

Schradan administered No. in Duration of

Experiment group and In diet By injection']" administration no. Species sex (ppm) (mg/kg) (days)

I Rat 4-6M - - 0, 0.007, 0-02, 37 0"1, 0"5, 2.5

1I Rat 10M 0, 0.05, 0.25, - - 14-85 1.0, 5"0

111 Rat 12M+ 12F 0, 0-05, 0.25, - - 112-273 1-0

1V Pig 2F 0, 0.I, 0"5, 2-5 - - 102

M=Males F--Females tlntraperitoneal injection 6 times weekly.

General observations. In experiment I retardation of growth of rats occurred at the 0.5 mg schradan/kg level during days 10-15 but progressed to normal thereafter. Two rats given 0.5 mg schradan/kg also showed slight transient diarrhoea on days 3-5. There was no effect on the gross appearance of organs of survivors at autopsy. In experiment II, III and 1V no effect was exercised by any dietary level of schradan on the general health and condi- tion of the animal, nor on body weight, food consumption, organ weights (experiment II, liver, kidney and spleen; I lI and IV, liver and kidney) and gross appearance of organs at autopsy.

*Prepared by BIBRA and published with the permission of the responsible authorities.

311

312 SUMMARIES OF TOXICOLOGICAL DATA

Changes in ChE. The results of experiment I showed that on day 37 a reduction of ChE was apparent at the 0-1 mg/kg level, mainly in the red cell but possibly also in the plasma. At the highest intraperitoneal dose level of schradan, 0.5 mg/kg, ChE was completely inhibited in the red cell and substantially in the plasma while that of the brain remained unchanged.

A knowledge of the effect of more prolonged dietary administration of schradan on ChE is provided by the results obtained in experiments II and III. Inhibition of enzyme activity was rapid, reaching a maximum by the third week. The suggestion of a lowering of red cell ChE in experiment II at 0.05 ppm was later disproved by the findings of experiment III. The first indication of a significant reduction of red cell ChE occurred at 0.25 ppm, with a further decline at 1 ppm and complete inhibition at 5 ppm. These findings are in good agreement with those obtained in experiment III, where evert more prolonged administra- tion of sehladan at dietary levels of up to 1 ppm brought about only a slight reduction in red c~ll ChE at the 0.25 ppm level, with substantial reduction occurring at I ppm at 16 and 37 weeks. At both these levels, males were more susceptible--as indicated by the enzyme changes--than females. Reduction in plasma ChE occuned only at the 5 ppm level (experi- ment II) and in neither experiment was brain ChE affected.

In female pigs (experiment IV) red cell ChE declined progressively to reach 45 700 of normal activity as the dose was increased from 0-5 to 2.5 ppm. The slight reduction in red cell ChE at 0.1 ppm was considered insignificant. There was only slight reduction in plasma ChE at 2.5 ppm and the brain enzyme level was unaffected.

Conclusions. The no-effect level of schradan was estimated to be 0.02 mg/kg/day in both the rat and the pig. By extrapolating this figure to mart the conclusion was drawn in 1954 that the consumption of large amounts of foodstuffs containing 3 ppm of schradan residues would present no hazard to public health.

H. The effect of prolonged ingestion of low dosages of schradan in man

(Report dated February 1955)

A knowledge of the no-effect level of schradan in the rat and pig now made it possible to establish a dose safe enough to be used in a study involving mart. A total of 13 male and 12 female adults took part; 6 males and 6 females ingested doses of 1.4 mg schradan for 5 days a week until 44 mg had been consumed. This regime was considered to be toxicologi- cally equivalent to 44 daily doses of 1 mg. One subject took 4.2 mg daily for 74 days, on two occasions as much as 12-6 mg. The remaining 12 adults received no schradan and served as control subjects. ChE of whole blood, red cells and plasma was estimated weekly. No consideration was given to the possible ingestion of schradan residues in food consumed by either control or treated subjects.

General observations and changes in ChE. Clinical symptoms did not develop at any level of schradan dosage. Oral ingestion of 44 mg schradan over 44 days (intake rate 0.013-0.015 mg/kg/day) caused a slow fall in whole blood ChE, reaching 80 ~ of control activity by day 18 and 75 ~o by day 44. Three weeks later enzyme activity had recovered to 90 ~ of control activity. At the higher intake of 4.2 mg daily (0.057 mg/kg/day) for 74 days there was a striking fall in whole blood ChE which was unaccompanied by toxic symptoms. Maximum inhibition of whole blood ChE was attained after 2 months with 33% of control activity in whole blood, 23 ~o in red cells and 5 0 ~ in plasma. Recovery of whole blood ChE com- menced 2 weeks before dosage ceased and in the following 25 schradan-free days the rise

SUMMARIES OF TOXICOLOGICAL DATA 313

had steadily progressed to 77 % of control activity. At both dosage levels the red cells were more sensitive to changes in ChE than the plasma.

Conclusions. The no-effect level of schradan in man was estimated to be 0.01 mg/kg/day, a figure which provides an adequate margin of safety for the human consumption of food- stuffs likely to contain schradan residues in Britain.

HI. The effects of prolonged administration of small daily doses of dimefox in the rat, pig and man

(Report dated December 1956)

A summary of dosage schedules of dimefox in the rat, pig and man is presented in Table 2

Table 2. Dosage schedules of dimefox in the rat, pig and matt

Dimefox administered No. in Durat ion of

Experiment group and In diet Oral dosage administration no. Species sex (ppm) (mg/kg/day) (days)

I Rat 20 F 0, 0.01, 0"05, - - 28-287 0-25, 1.0, 5.0

II Pig 2 F 0, 0.005 #, 0.025, - - 133 0"1, 0.5-[-

III Man 1 M - - 0-0014, then 0"004 14 then 95 IV Man 41: - - 0.0012, 0.002, 70

0"0034

M ~ M a l e s F--Females *This dose level was given to control group for the last 69 days of the experiment. tF i r s t 14 days on 0.25 ppm. ~;Groups were mixed male and female, based on similar body weight.

General observations. At the four lowest dietary levels in the weanling rat (experiment I) and at all levels in the weanling pig (experiment II) there was no effect on growth, food con- sumption, condition and health of the animal nor on gross appearance of organs at autopsy. Similarly, no clinical effects developed in man (experiment III and IV). The only toxic effect was seen at the 5 ppm level in rats where initially a slight impairment of growth, appearance and behaviour was evident but animals showed subsequent improvement.

Changes in ChE. In the rat, pig and man maximum inhibition of ChE was obtained after 4 weeks of dosage. Red cell ChE was again the most sensitive index of enzyme change in all three species. In the rat, significant lowering of red cell ChE first appeared at the 0.25 ppm level proceeding to almost complete loss of enzyme activity at 5 ppm. Red cell ChE in the pig was slightly reduced at 0.025 ppm and moderately affected at 0.1 ppm, with only 13-23 of control activity present at the highest dietary level of 0.5 ppm. Reduction of plasma ChE was first apparent at 1 ppm in the rat and at 0-1-0.5 ppm in the pig. Brain ChE, which was only measured in the rat, was lowered at 5 ppm.

In a preliminary study involving one male adult for 109 days (experiment III) the first 14 days dosage at 0.0014 mg]kg/day caused no ChE change, but increase to 0.004 mg/kg/ day caused whole blood ChE to decline gradually to 60 ~o of normal activity by week 7. This effect was entirely due to the lowering of red cell ChE since plasma ChE was unchanged. Whole blood ChE slowly recovered to 90 Yo of control activity 56 days after administration had ceased. The group study in man lasting 70 days (experiment IV) showed that reduction

314 SUMMARIES OF TOXICOLOGICAL DATA

in whole blood ChE was only evident at the highest level of 0-0034 mg/kg/day and was of the order of 75 % of normal activity.

Conclusions. The no-effect levels of dimefox based on changes in red cell ChE in the rat, pig and man were estimated to be 0.003, 0,006 and 0-002 mg/kg/day respectively. I t was concluded that the no-effect level in man "is in fact about one hundred times less than the daily dose which would probably cause obvious ill-effects or illness", thus providing art adequate margin of safety to the consumer.

IV. The effects of prolonged administration of small daily doses of parathion in the rat, pig and man (Report dated March 1957)

A summary of the dosage schedules of parathion in the rat, pig and man is presented in Table'3.

Table 3, Dosage schedules of parathion in the rat, pig and man

Parathion administered No. in Duration of

Experiment group and In diet Oral dosage* administration no. Species sex (ppm) (mg/day) (days)

I Rat 20 F 0, 0'05, 0'5, 5.0 - - 84 (max.) II Pig 2 F 0, 0-2"1", 1"0~;,. - - 33-122

5.0, 25t, 100~ III Man 4 MF - - 0, 0.6§, 1.2, 2.4, 25-70

4-8§, 7.211

M--Males F--Females (In experiment III, subjects were grouped by body weight, not by sex). #On 5 days each week. "l'Increased to 25 ppm during days 33-72. ~lncreased to 100 ppm during days 73-114. §Increased to 4.8 mg/day during weeks 4-13. IIGiven to 4 F for 6 weeks.

General observations. No effect on growth, food consumption, general condition and health of animals or gross appearance of organs was noted in the weanling rat or weanling pig and no clinical symptoms developed in man.

Changes in ChE. Red cell ChE was again found to be most affected in the rat and pig but in man it appeared that parathion produced a greater effect on plasma ChE than on red cell ChE. Maximum irthibition of ChE was usually attained during weeks 4, 6 and 7 in the rat, man and pig respectively.

In the rat (experiment I) there was no effect on ChE below 0.05 ppm. Increasing the dose level from 0.5 to 5.0 p p m brought about a dramatic lowering of red cell ChE activity from 46~o to 2 0 ~ of control activity by week 12. By comparison the pig (experiment IH) was extremely resistant; lowering of red cell ChE was first seen at 25 ppm, but the slight effect observed at this level became more pronounced at 100 ppm where red cell ChE fell f rom 50 ~o to 20 ~o of control activity during weeks 4-7. Plasma ChE was slightly irthibited at 5 p p m in the rat but not at any level given to the pig; and in neither species did brain ChE change. In mart (experiment III) it was significant that whole blood ChE was only reduced at the highest dose level of 7-2 mg (given 5 days a week), declining to 67 ~o of control activity after 6 weeks. At this time red cell ChE was 84 ~ of control activity and plasma

SUMMARIES OF TOXICOLOGICAL DATA 315

ChE 63 %. Within 28 days of withdrawal of parathion whole blood ChE was restored to about 87 % of control activity.

Conclusions. Based on changes in ChE, the no-effect levels of parathion in the rat, pig and man were estimated to be 0.02, 1.0 and 0.05 mg/kg/day. That the pig showed outstanding resistance to changes in ChE from parathion dosage was interesting in view of its lower tolerance, compared with the rat, to the related compound dimefox.

The conclusion was drawn that a 60-kg adult could "consume up to 3 rag. parathion daily, equivalent to 1 kg. foodstuff containing 3 p.p.m, residual parathion (and pro rata), without cholinesterase depression, and with a high margin of safety from toxic effects". The maximum allowable concentration in air of 0.1 mg/m 3 for industrial exposure provides a threefold safety factor from ChE inhibition and hence can be considered to be adequately safe.

Summary and conclusions

A study of the effects of schradan, dimefox and parathion on red cell ChE, the most sensitive indicator of organophosphorus intoxication, has made it possible to establish maximum no-effect levels of the three organophosphates in the rat, pig and man. The estimated maximum no-effect level of each compound in each species studied are given in Table 4 together with those levels found to exercise no-effect, the smallest effect and the greatest effect on red cell ChE. In contrast to the pig, changes irt the activity of the rat en- zyme closely paralleled that of man. With schradan, however, good agreement in the behaviour of red cell ChE was obtained in all three species. Above all, studies in the rat and pig showed that a wide margin of safety existed between the maximum no effect level and that needed to evoke a true toxic effect. Organophosphate residues in food would be likely, at the very most, to cause only slight and fully reversible physiological change in ChE, thus ensuring adequate safety to the consumer.

Table 4. Effect of dietary intake of schradan, dimefox and parathion on red cell Cholinest;rase of the rat, pig and man

0%

R a t P i g M a n

Category of effect on Dietary intake Red Cell ChE Dietary intake Red cell CItE Oral intake Red cell ChE

ChE (Izg/kg/day) (% of control) (Izg/kg/day) (~. of control) (Izg/kg/day) ( ~o of control)

SCHRADAN

No-effect 2.5--4'5" - - 3"9-5"2 - - - - - - (0-05 ppm) (0.1 ppm)

Smallest effect 2.4-22" 70 20-27 75 13-15 75-80 Greatest effect 48-88* 8 90-124 15 57 23 M a x i m u m n o - e f f e c t l e v e l 2 0 - - 2 0 - - 1 0 - -

U~

DIMEFOX

No-effect 0.7-0.9 - - 0' 12 - - 1.2-2 - - 3--4'6 (0"05 ppm)

(0.01, 0.05 ppm) Smallest effect 17-220 51-68 0.8 80 3.4 72 Greatest effect 360--430 0-7 11-I 3 13-23 4 56 M a x i m u m n o - e f f e c t l e v e l 3 - - 0 - 6 - - 2 - -

0

N O g

P A R A T H I O N

No-effect 4-6 - - 8-10 - - 7-3-58 - - (0"05 ppm) 34--43

150-220 (0.2 1, 5 ppm)

Smallest effect 40-60 46 1000 S 78 63t Greatest effect 440-610 20 4000 20 78 63t M a x i m u m n o - e f f e c t l e v e l 2 0 - - 1 0 0 0 - - 5 0 - -

The range of dietary intakes is given for each dietary level. Dietary levels in ppm are included in brackets below the no-effect levels in the rat and pig to permit ready cross-reference to data in Tables 1, 2 and 3.

S Slight but unspecified reduction of red cell ChE. * Results from experiment II, Table 1 are only cited. t Percentage of control plasma ChE.