Section A6.1.1 Acute Toxicity Oral - ECHA

Sumitomo Chemical Takeda Agro Co., Ltd. TI-435 February 2004

RCC project no. 852224 page 1 of 4

Section A6.1.1 Annex Point IIA VI.6.1.1/01

Acute Toxicity Oral Acute oral toxicity study in rats (LD50)

1 REFERENCE

Officialuse only

1.1 Reference Gardner, J. R. (1997a); TI-435: Acute oral toxicity study in the rat Covance Laboratories, Harrogate, UK unpublished report number 586/120-

1.2 Data protection Yes

1.2.1 Data owner Sumitomo Chemical Takeda Agro Co. Ltd.

1.2.2 Companies with letter of access

None

1.2.3 Criteria for data protection

Data submitted on existing a.s. for its first entry into Annex I.

2 GUIDELINES AND QUALITY ASSURANCE

2.1 Guideline study Yes 92/69/EEC (method B1), EPA FIFRA, Subdivision F, Section 81-1 (1984), Japan MAFF (59 NohSan No. 4200, 1985), OECD No. 401 (1987)

2.2 GLP Yes

2.3 Deviations No

3 MATERIALS AND METHODS

3.1 Test material TI-435

3.1.1 Lot/Batch number 30034708

3.1.2 Specification As given in section 2

3.1.2.1 Description Pale, yellow powder

3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study

3.2 Test Animals 5 male and 5 female fasted rats per group (Crl:CD.BR strain, 6–11 weeks old, body weight range 214–353 g, supplied by Charles River UK Ltd) No control group

3.3 Administration/ Exposure

Oral

3.3.1 Postexposure period

14 days





Oral

3.3.2 Type Gavage

3.3.3 Concentration 1758, 2283, 2965, 3850 and 5000 mg/kg bw

3.3.4 Vehicle 5% w/v aqueous gum arabic

3.3.5 Concentration in vehicle

Prepared according to individual bw and dosing volume

3.3.6 Total volume applied

10 mL/kg bw

3.3.7 Controls -

3.4 Examinations Clinical signs: recorded on at least 5 occasions on the day of treatment and at least once daily thereafter. Body weights were recorded pre-dose and on days 1, 4/5, 8 and 15. Decedents, animals killed in a moribund condition, and all survivors were subjected to necropsy and post mortem examination.

3.5 Method of determination of LD50

Since no group showed mortality of >20%, the data were not analysed statistically.

3.6 Further remarks -

4 RESULTS AND DISCUSSION

4.1 Clinical signs Single females and males died at ≥2965 and at 5000 mg/kg bw, respectively; palpebral closure, decreased activity, lethargy, ataxia, hunched posture, vocalisation, tremor, wasted appearance, hair loss, alopecia at all dose levels (see table A6_1_1/01); additionally discoloration of urine in females ≥2283 mg/kg bw

4.2 Pathology No gross lesions were apparent during post mortem examination of the animals.

4.3 Other Reduced bw development at all dose groups in the first few days after application; at ≥3850 mg/kg bw loss of bw in individual animals also in the 2nd week after application

4.4 LD50 >5000 mg/kg bw (males, females, males and females combined)

5 APPLICANT'S SUMMARY AND CONCLUSION

5.1 Materials and methods

Toxicity evaluation (bw, clinical signs, post mortem examination) after acute oral application to rats (gavage); no relevant deviation from guidelines OECD 401)

5.2 Results and discussion

Acute oral LD50 in rats is >5000 mg/kg bw

5.3 Conclusion No classification for oral toxicity is considered required for TI-435 according to Directive 2001/59/EC (adaptation of 67/548/EEC).

5.3.1 Reliability 1

5.3.2 Deficiencies No





Evaluation by Competent Authorities Use separate "evaluation boxes" to provide transparency as to the

comments and views submitted

EVALUATION BY RAPPORTEUR MEMBER STATE

Date 2004-10-07

Materials and Methods Although testing method 401 has been deleted from OECD guidelines since December 2002 the experiments described are acceptable since they were performed in 1997. Apart from two minor deviations as regards contents such as the time of checking the body weight of two rat groups for the second time (day 5 instead of day 4) or the time of removal of food prior to dosing to main study rats (20 hours rather than 18 hours before administration of the test substance) the study complies with the requirements of the guideline. The deviations from protocol were considered not to have affected the outcome or reliability of the study.

Results and discussion Acute oral LD50 in rats is >5000 mg/kg bw for both sexes. Single females and males died at ≥2965 and at 5000 mg/kg bw, respectively. For clinical signs and pathology see table A6_1_1/01.

Conclusion No classification for oral toxicity is considered required for TI-435 according to Directive 2001/59/EC (adaptation of 67/548/EEC).

Reliability 1

Acceptability Acceptable

Remarks There is an acute oral neurotoxicity study in rats (Cain, D. M., 2000; see also A 6.9/01) the supplement of which is an acute oral dose range-finding study (Sheets, L. P., 2002) originally not presented in this section. Since an LD50 value can be calculated from this study using a different rat strain than above it was included in evaluation at this place by RMS (see A6.1.1/03).

COMMENTS FROM ...

Date Give date of comments submitted

Materials and Methods Discuss additional relevant discrepancies referring to the (sub)heading numbers and to applicant's summary and conclusion. Discuss if deviating from view of rapporteur member state

Results and discussion Discuss if deviating from view of rapporteur member state

Conclusion Discuss if deviating from view of rapporteur member state

Reliability Discuss if deviating from view of rapporteur member state

Acceptability Discuss if deviating from view of rapporteur member state

Remarks





Table A6_1_1/01. Table for Acute Oral Toxicity (rat)

Dose [mg/kg bw]

Number of dead / number of investigated

Time of death (range) Observations

1758 0/5 females 0/5 males

- -

Palpebral closure, decreased activity, lethargy, ataxia, hunched posture, vocalisation, tremor, wasted appearance, stained snout, piloerection, hair loss; all males recovered within day 2, all females (except the hair loss) recovered until end of exposure period reduced bw development in first week after treatment, post mortem examinations revealed no effect of treatment


- -

Palpebral closure, decreased activity, lethargy, ataxia, hunched posture, vocalisation, tremor, wasted appearance, hair loss, alopecia, discoloration of urine; except the hair loss/alopecia all animals recovered until end of exposure period reduced bw development in first week after treatment, post mortem examinations revealed no effect of treatment


Day 2 -

Palpebral closure, decreased activity, ataxia, hunched posture, vocalisation, tremor, hypothermia, wasted appearance, hair loss, alopecia, discoloration of urine, chromodacryorrhoea; except the hair loss/alopecia all survivors recovered until end of exposure period reduced bw development in first week after treatment (survivors), post mortem examinations revealed no effect of treatment


Day 12 -

Palpebral closure, decreased activity, ataxia, arched gait, hunched posture, tremor, hypothermia, wasted appearance, hair loss, discoloration of urine, soft faeces, chromo-dacryorrhoea; except the hair loss all survivors recovered until end of exposure period bw loss in some individuals over the observation period and reduced bw development in first week after treatment in the remaining survivors, post mortem examinations revealed no effect of treatment


Day 4 Day 12

Palpebral closure, decreased activity, lethargy, ataxia, hunched posture, tremor, wasted appearance, stained snout, hair loss, discoloration of urine, chromodacryorrhoea; not all clinical signs disappeared until end of exposure period bw loss in some individuals over the observation period and reduced bw development in first week after treatment in the remaining survivors, post mortem examinations revealed no effect of treatment

LD50 value Males: >5000 mg/kg bw Females: >5000 mg/kg bw Combined: >5000 mg/kg bw




Acute Toxicity Oral Acute oral toxicity study in mice (LD50)

1 REFERENCE

Officialuse only

1.1 Reference Gardner, J. R. (1997b); TI-435: Acute oral toxicity study in the mouse; Covance Laboratories, Harrogate, UK; unpublished report number 586/121-




None


Data submitted on existing a.s. for its first entry into Annex I



2.2 GLP Yes

2.3 Deviations None relevant for the scientific integrity or validity of the study: mice were used as they were considered the more sensitive species than rats






3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study (test-article-vehicle formulations prepared at the day of dosing)

3.2 Test Animals 5 male and 5 female fasted mice per group (Crl:CD-1(ICR)BR strain, 5 – 7 weeks old, weight range 22 – 30g, supplied by Charles River UK Ltd) No control group


Oral


14 days





Oral

3.3.2 Type Gavage

3.3.3 Concentration 304, 380, 475, 594 and 742 mg/kg bw





10 mL/kg bw

3.3.7 Controls -

3.4 Examinations Clinical signs: recorded on at least 5 occasions on the day of treatment and at least once daily thereafter. Body weights were recorded pre-dose and on days 1, 4, 8 and 15. Decedents, animals killed in a moribund condition, and all survivors were subjected to necropsy and post mortem examination.

3.5 Method of determination of LD50

The acute median lethal dose and 95% confidence limits were estimated using moving average interpolation (C. S. Weil, 1952).



4.1 Clinical signs Mortality at ≥380 mg/kg bw; decreased activity, palpebral closure, ataxia and tremors at all dose levels; additionally lethargy, prone posture, twitching and breathing anomalies (dyspnea, hyperpnea or bradypnea) at ≥594 mg/kg bw (see table A6_1_1/02)


4.3 Other -

4.4 LD50



Toxicity evaluation (bw, clinical signs, post mortem examination) after acute oral application to mice (gavage); no relevant deviation from guidelines (92/69/EEC method B1; EPA FIFRA 81-1, Japan MAFF, OECD 401)


Acute oral LD50 in mice (males+females combined): 425 mg/kg bw

5.3 Conclusion Classification as harmful if swallowed (R22) is considered required for TI-435 according to Directive 2001/59/EC (adaptation of 67/548/EEC).

5.3.1 Reliability 1









Date 2004-10-07

Materials and Methods Although testing method 401 has been deleted from OECD guidelines since December 2002 the experiment described is acceptable since it was performed in 1997. Apart from two minor deviations as regards contents such as the time of assigning study groups (three days before dosing instead of one in mice of groups 1 to 6) or the method of calculating the acute median lethal dose level with 95 % confidence limits (moving average interpolation (Weil, C. S., 1952, Biometrics 8, 249-263) instead of probit analysis) the study complies with the requirements of the guideline. The deviations from protocol were considered not to have affected the outcome or reliability of the study.

Results and discussion Acute oral LD50 in mice is 389 mg/kg bw for males, 465 mg/kg bw for females and 425 mg/kg bw for both sexes. For clinical signs and pathology see table A6_1_1/02. TI-435 is approximately one order of magnitude more toxic to mice than to rats by the oral route.

Conclusion Classification as ‘harmful if swallowed’ (R22) is considered required for TI-435 according to Directive 2001/59/EC (adaptation of 67/548/EEC).

Reliability 1


Remarks None

COMMENTS FROM ...







Remarks





Table A6_1_1/02. Table for Acute Oral Toxicity

Dose [mg/kg bw]

Number of dead / number of inves-tigated

Time of death (range) Observations


- -

palpebral closure, decreased activity, ataxia and tremor; all animals normal by day 3; bw development and post mortem examinations revealed no effect of treatment


Day 1 Day 1-2

palpebral closure, decreased activity, ataxia and tremor; all survivors normal by day 3; bw development (survivors) and post mortem examinations revealed no effect of treatment


Day 2 Day 2

palpebral closure, decreased activity, ataxia and tremor; all survivors normal by day 3; bw development (survivors) and post mortem examinations revealed no effect of treatment


Day 1-2 Day 1-2

palpebral closure, decreased activity, ataxia and tremor; lethargy and respiratory impairment (tachypnoe, bradypnoe, dyspnoe or hyperpnoe); all survivors normal by day 3; bw development (survivors) and post mortem examinations revealed no effect of treatment


Day 1 Day 1

palpebral closure, decreased activity, ataxia and tremor lethargy and respiratory impairment (tachypnoe, bradypnoe, dyspnoe or hyperpnoe); post mortem examinations revealed no effect of treatment

LD50 value Males: 389 mg/kg bw (348-433 mg/kg bw) Females: 465 mg/kg bw (384-561 mg/kg bw) Combined: 425 mg/kg bw (380-475 mg/kg bw)

Sumitomo Chemical Takeda Agro Co., Ltd. TI-435 May 2005


Section A6.9

Neurotoxicity studies Acute study in rats Acute oral neurotoxicity study in rats

1 REFERENCE

Officialuse only

1.1 Reference Sheets, L. P. (2000); Original: An acute oral neurotoxicity study with technical grade TI-435 in Fischer 344 rats, Supplemental: An acute oral dose range-finding study with technical grade TI-435 in Fischer 344 rats. Bayer CropScience LP, Toxicology, Kansas 66085-9104, USA unpublished report number 108960 Supplement Submission #2, 30.12.2002, original : 97-912-OH, Supplemental : 97-912-OE




None




2.1 Guideline study No: range finding study was conducted to provide information on acute toxicity needed for the selection of dose levels for a guidelines-conform acute neurotoxicity study.

2.2 GLP Yes

2.3 Deviations No





3.1.2.1 Description Pale yellow powder

3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study (stability, homogenicity and content of test-article/vehicle formulations was analysed)

3.2 Test Animals 5 male and 5 female fasted 9 week old Fischer 344 rats per group (CDF[F-344]/BR strain, SASCO Inc., Michigan, USA)


Oral


4 days

3.3.2 Type Gavage

3.3.3 Concentration 250, 500 and 1000 mg/kg bw

3.3.4 Vehicle aqueous 0.5% methylcellulose/0.4% Tween 80 in deionized water






Section A6.9


3.3.7 Controls -

3.4 Examinations Detailed physical examination for clinical signs of toxicity, mortality: 0.5, 1, 2, 3, 4 and 24 hours after treatment, daily thereafter Body weights: prior to treatment and on day 4 or when they were found dead.

3.5 Sacrifice and pathology

None



4.1 Body weight The surviving males at 1000 mg/kg bw lost weight until day 4 of the study. At 500 mg/kg bw the surviving rats had a lower body weight development than at 250 mg/kg bw.

4.2 Clinical signs At 500 mg/kg bw 1/5 females, at 1000 mg/kg bw all females and 2/5 males died. Treatment-related clinical signs like tremors, decreased activity, locomotor incoordination, nasal discharge, lacrimation as well as nasal and oral stains were seen at ≥500 mg/kg bw. The sings occurred within 3 hours after treatment and were partly reversible after 72 hours. The Animals that died were cool-to-touch and gasping prior to death.

4.3 Pathology -



5.3 Conclusion The time of peak effect was 4 hours after application. Dose levels selected for the main study (acute neurotoxicity study) were 100, 200 and 400 mg/kg bw.

5.3.1

5.3.2 NO(A)EL 250 mg/kg bw (for clinical signs and mortality)

5.3.3 Reliability 1

5.3.4 Deficiencies No (study fulfilled its purpose as a range finding study)






Date 2005-06-06

Materials and Methods Deviations from test method B.1, of directive 92/69/EEC mentioned were: (i) survivors were sacrificed on day 4 instead of day 14, when recovery was evident, and when it was apparent that no additional animals would die as a result from treatment (ii) no gross necropsy examination was performed. Since the study was intended as a range-finding study for an acute neurotoxicity study (determination of time of peak for clinical signs), the deviations are not considered to affect the integrity of the study. Based on analytical results, the actual doses of TI-435 were 290, 523 and 1216 mg/ kg bw for males and females In addition an acute oral study was conducted at a limit dose of 2000 mg/kg bw with young adult Fischer-344 rats (5 males and 5 females) receiving a single dose by gavage according to the methods described above.

Results and discussion 1216 mg/kg bw < LD50 (M) <2000 mg/kg bw 523 mg/kg bw < LD50 (F) <1216 mg/kg bw. All animals treated with 2000 mg/kg bw died on day 1 or 2 after treatment

Conclusion Classification as ‘harmful if swallowed’ (R22) is required for TI-435 according to Directive 2001/59/EC (adaptation of 67/548/EEC).

Reliability

Acceptability

Remarks

COMMENTS FROM ...







Remarks



Table A6_9/04-1: Table for Acute Oral (Neuro)toxicity (rat)

Males Females mg/kg bw 250 500 1000 250 500 1000

Mortality [g] day 1 day 2 day 4

0/5 0/5 0/5

0/5 0/5 0/5

0/5 1/5 1/4

0/5 0/5 0/5

1/5 0/4 0/4

4/5 1/1 -

Cumulative bw gain [g] a day 0-4 25 10 -6 18 5 -2 Clinical signs [affected/total animals] Tremors Decreased activity Locomotor incoordination Nasal staining (red and/or clear) Oral staining (red and/or clear) Nasal discharge, clear Lacrimation, clear or red Lacrimal stain, red Cool-to-touch Gasping Moribund

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

5/5 5/5 0/5 4/5 5/5 0/5 0/5 0/5 0/5 0/5 0/5

5/5 5/5 1/5 5/5 5/5 1/5 1/5 0/5 3/5 0/5 0/5

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

3/5 5/5 3/5 1/5 3/5 0/5 3/5 2/5 3/5 0/5 3/5

5/5 5/5 3/5 2/5 3/5 2/5 3/5 0/5 3/5 1/5 3/5

a : Terminal body weight on day 4 for animals that survived to study termination ot when animals were found dead.

ANNEX Evaluation by Rapporteur Member State, CA-Table CA-Table: Table for Acute Oral Toxicity at 2000 mg/kg bw

Dose [mg/kg bw]

Number of dead / number of investigated

Time of death (range)

Observations


Day 1-2 Day 1-2

Not determined

LD50 value Males: 1216 mg/kg bw < LD50 < 2000 mg/kg bw Females: 523 mg/kg bw) < LD50 < 1216 mg/kg bw




Acute Toxicity Dermal Acute dermal toxicity study in rats (Limit Test)

1 REFERENCE

Officialuse only

1.1 Reference Gardner, J. R. (1997c); TI-435: Acute dermal toxicity study in the rat; Covance Laboratories, Harrogate, UK; unpublished report number 586/122-103




None





2.2 GLP Yes

2.3 Deviations None






3.1.2.2 Purity


3.2 Test Animals 5 male and 5 female, singly-caged rats (Crl:CD.BR strain, 6 – 11 weeks old, weight range 225 – 273 g, supplied by Charles River UK Ltd) No control group



14 days





Dermal

3.3.2 Area covered 10% of body surface (25 cm2)

3.3.3 Occlusion Semiocclusive

3.3.4 Vehicle Distilled water


TI-435 (dry powder) wetted with 0.2 mL distilled water


See 3.3.5 (dose level TI-435: 2000 mg/kg bw)

3.3.7 Duration of exposure

24 h

3.3.8 Removal of test substance

Swabbed with moist cotton wool

3.3.9 Controls -


4.1 Clinical signs No animals died at the limit dose level (2000 mg/kg bw). There were no systemic clinical signs of a reaction to treatment and no overt signs of irritation at the application site in any animal. All animals progressively gained weight during the 2-week observation period.


4.3 Other -

4.4 LD50 No lethal effect at maximal dose dermal LD50 (males, females, males + females) >2000 mg/kg bw




Dermal LD50 in rats (males, females, males + females) >2000 mg/kg bw no mortality at limit dose

5.3 Conclusion No classification required for TI-435 according to Directive 2001/59/EC (adaptation to 67/548/EEC).

5.3.1 Reliability 1









Date 2004-10-07

Materials and Methods Acceptable, no deviations from OECD guideline 402

Results and discussion Acute dermal LD50 in rats is >2000 mg/kg bw for both sexes. No animal died at the limit dose level of 2000 mg/kg bw. Nor were there clinical signs in reaction to the treatment neither pathological findings during post mortem examination of the animals.

Conclusion No classification is needed for TI-435 according to Directive 2001/59/EC (adaptation to 67/548/EEC).

Reliability

Acceptability

Remarks

COMMENTS FROM ...







Remarks




Acute Toxicity Inhalation

Acute inhalation toxicity study in rats (Limit Test)

1 REFERENCE

Officialuse only

1.1 Reference Shepherd, N. M. (1998); TI-435: Single dose inhalation (head-only) toxicity study in the rat; Covance Laboratories, Harrogate, UK; unpublished report number 586/129-D6154, 21.04.1998




None




2.1 Guideline study Yes 92/69/EEC (method B2), EPA FIFRA, Subdivision F, Section 81-3 (1982), OPPTS 870.1330 (1996), Japan MAFF (59 NohSan No. 4200, 1985), OECD No. 403 (1981)

2.2 GLP Yes

2.3 Deviations No



3.1.1 Lot/Batch number 30034708b



3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study (4 h exposure period)

3.2 Test Animals 5 male and 5 female rats group (Crl:CD.BR strain, 7 weeks old, weight range 201 – 278 g, supplied by Charles River UK Ltd)

Control group included


Inhalation


14 days






Inhalation

3.3.2 Concentrations Nominal concentration 10400 mg/m³

Gravimetrical concentration 6141 mg/m³ (concentration over the totally 4.5 h exposure period was 5538 mg/m3 including the first half hour where difficulties with the aerosol generation occurred)

3.3.3 Particle size MMAD (mass median aerodynamic diameter) 2.78 µm + GSD (geometric standard deviation) 2.38 µm

3.3.4 Type or preparation of particles

13% particles <1 µm 68% particles <4 µm

3.3.5 Type of exposure nose/head only

3.3.6 Vehicle -


-


4 h (due to technical difficulties with the aerosol generation in the first 0.5 h of exposure, the exposure period was prolonged to totally 4.5 hours to guarantee a 4 h exposure at the target concentration)

3.3.9 Controls sham exposition


4.1 Clinical signs No deaths occurred in either experimental group. Treatment-related clinical signs comprised ataxia and semi-closed eyes (days 1 and 2), hunched posture and lethargy (days 1 – 3), staining (days 1 – 4). Reduced food and/or water consumption (subjective assessment) occurred on days 2 – 4. All animals were of normal appearance from day 5 until termination. Slight weight loss occurred in both test and control group animals during the exposure period, but a further treatment-related transient weight loss occurred in all test group animals on day 2 (see table A6_1-3/01). Thereafter all treated animals gained weight at a comparable rate to the controls.

4.2 Pathology There were no treatment-related gross or microscopic lesions and the lung weights of test group animals were unaffected by exposure to TI-435.

4.3 Other -

4.4 LC50 LC50 (male, females, males + females) >6141 mg/m3 No lethal effect at the limit dose








Toxicity evaluation (bw, clinical signs, post mortem examination including signs of irritation in the inhalation tract, lung weights) after acute inhalation exposure of rats; no relevant deviation from guidelines

OECD 403)


LC50 of TI-435 in rats (male, females, males + females) >6141 mg/m3 No lethal effect at limit dose

5.3 Conclusion No classification required for TI-435 according to Directive 2001/59/EC (adaptation to 67/548/EEC)

5.3.1 Reliability 1





Date 2004-10-07

Materials and Methods Apart from minor deviations as regards contents the study complies with the requirements of OECD guideline 403. Deviations from protocol were: - Slightly aberrant temperature and relative humidity inside the exposure chamber as well as in the study room on a few occasions, - Prolonged exposure duration of 4 ½ hours instead of 4 hours due to difficulties with the aerosol generator resulting in a period of reduced aerosol concentration and - Missing particle size measurement during the extension of exposuretime. The deviations from protocol were considered not to have affected the outcome or reliability of the study.

Results and discussion Acute inhalation LC50 in rats is >6141 mg/m3 air for both sexes. No animal died at the limit concentration of 6141 mg/m3 air. Treatment-related clinical signs comprised ataxia and semi-closed eyes (days 1 and 2), hunched posture and lethargy (days 1 – 3), staining (days 1 – 4). Reduced food and/or water consumption (subjective assessment) occurred on days 2 – 4. All animals were of normal appearance from day 5 until termination. Slight weight loss occurred in both test and control group animals during the exposure period, but a further treatment-related transient weight loss occurred in all test group animals on day 2 (see table A6_1-3/01). Thereafter all treated animals gained weight at a comparable rate to the controls. There were no treatment-related gross or microscopic lesions apparent during post mortem examination of the animals.

Conclusion No classification required for TI-435 according to Directive 2001/59/EC (adaptation to 67/548/EEC)

Reliability 1


Remarks None






COMMENTS FROM ...







Remarks

Table A6_1-3/01. Table for Acute Inhalation Toxicity: body weights [g]

Dose [mg/ m3 air]

Day

Males Females

1 (pre-dose)

1 (post-dose) 2 8 15

1 (pre-dose)

1 (post-dose) 2 8 15

0 273 260 275 321 363 213 201 211 228 242

6141 254 242 232 269 328 209 198 193 218 234




Acute Toxicity Skin irritation Acute skin irritation study in rabbits

1 REFERENCE

Officialuse only

1.1 Reference Gardner, J. R. (1997d); TI-435: Skin irritation study in the rabbit; Covance Laboratories, Harrogate, UK; unpublished report number 586/124-1032, 25.06.1997




None





2.2 GLP Yes

2.3 Deviations None relevant for the integrity or validity of the study: six rabbits instead of 3 (92/69/EEC B.4) were used since this is a regulatory requirement of EPA-FIFRA and JMAFF






3.1.2.2 Purity


3.2 Test Animals Five male and one female New Zealand White rabbits (Crl:NZW/Kbl.BR strain, 11 to 14 weeks old, body weight 2.42 to 2.85 kg, supplied by Charles River UK Ltd) No control group


Dermal

3.3.1 Application

3.3.1.1 Preparation of test substance

Test substance was used as delivered; the application site was moistened with 0.1 mL distilled water immediately before application of 500 mg TI-435 (powder).

3.3.1.2 Test site and Preparation of Test Site

Dorsal area of the trunk (6 cm2) Shaved skin (clipped)

3.3.2 Occlusion Semiocclusive





3.3.3 Vehicle Distilled water


See 3.3.1.1


See 3.3.1.1

3.3.6 Removal of test substance

Swabbed with moist cotton wool


4 h


3 days

3.3.9 Controls -

3.4 Examinations

3.4.1 Clinical signs Yes

3.4.2 Dermal examination

Yes

3.4.2.1 scoring system Erythema and Eschar: no erythema 0 very slight erythema (barely perceptible) 1 well-defined erythema 2 moderate to severe erythema 3 severe erythema (beet redness) or eschar preventing reading of erythema 4 Oedema: no oedema 0 very slight oedema (barely perceptible) 1 slight oedema (edges of area well-defined by definite raising) 2 moderate oedema (edges raised approximately 1 mm) 3 severe oedema (raised >1 mm and expanding beyond area of exposure) 4

3.4.2.2 Examination time points

60 min, 24 h, 48 h, 72 h

3.4.3 Other examinations -



4.1 Average score

4.1.1 Erythema There were no skin reactions in any rabbit at any of the observation points

4.1.2 Edema There were no skin reactions in any rabbit at any of the observation points.

4.2 Reversibility Not relevant

4.3 Other examinations

-

4.4 Overall result TI-435 did not induce any skin reaction at all after dermal application for 4 hours.





Table A6_1-4S/01. Table for skin irritation study

score (average animals investigated) time Erythema Edema

average score Draize scores (0 to maximum 4)

60 min 0 0

24 h 0 0

48 h 0 0

72 h 0 0

other times State time - -

average score 24h, 48h, 72h 0 0

reversibility: * - -

average time for reversibility - -

* c : completely reversible n c : not completely reversible n : not reversible



Evaluation of skin reactions in rabbits after 4 hour dermal exposure; no relevant deviation from guidelines (92/69/EEC B4, EPA FIFRA 81-5, Japan MAFF, OECD 404)


TI-435 did not induce any skin reaction at all after dermal exposure for 4 hours.

5.3 Conclusion No classification for skin irritation is considered required for TI-435 according to Directive 2001/59/EC (adaptation of 67/548/EEC).

5.3.1 Reliability 1





Date 2004-10-07

Materials and Methods Acceptable, no deviations from OECD guideline 404

Results and discussion There were neither erythema or edema nor any skin reaction in any rabbit during the study. TI-435 is not irritating to the skin.

Conclusion No classification for skin irritation is required for TI-435 according to Directive 2001/59/EC (adaptation of 67/548/EEC).

Reliability 1

Acceptability

Remarks





COMMENTS FROM ...







Remarks



Section 6.1.4 Annex Point IIA VI.6.1.4/02

Acute Toxicity Eye Irritation Acute eye irritation study in rabbits

1 REFERENCE

Officialuse only

1.1 Reference Gardner, J. R. (1997e); TI-435: Eye irritation study in the rabbit; Covance Laboratories, Harrogate, UK; unpublished report number 586/123-1032, 30.07.1997




None





2.2 GLP Yes

2.3 Deviations None relevant for the integrity or validity of the study: six rabbits instead of 3 (92/69/EEC B.5) were used since this is a regulatory requirement of EPA-FIFRA and JMAFF






3.1.2.2 Purity


3.2 Test Animals Six male New Zealand White rabbits (Crl:NZW/Kbl.BR strain, 11 to 14 weeks old, body weight 2.10 to 2.47 kg, supplied by Charles River UK Ltd) No control group


3.3.1 Preparation of test substance

Test substance was used as delivered

3.3.2 Amount of active substance instilled

66 mg corresponding to 0.1mL

3.3.3 Exposure period The eyes remained unwashed


3 days





3.4 Examinations

3.4.1 Ophthalmoscopic examination

yes

3.4.1.1 Scoring system Cornea (degree of opacity: area most dense assessed): no ulceration or opacity 0 scattered or diffuse areas of opacity other than slight dulling of normal lustre, details of iris clearly visible 1 easily discernible translucent area, details of iris slightly obscured 2 nacreous area, no details of iris visible but size of pupil barely discernible 3 opaque cornea, iris not discernible through opacity 4 Cornea (area of corneal opacity): total area of opacity amounts to <25% of corneal area 1 area of opacity amounts to 25 - <50% of corneal area 2 area of opacity amounts to 50 - <75% of corneal area 3 area of opacity amounts to 75 or more of corneal area 4 Iris: no reaction 0 markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia or injection, any of these or any combination thereof; iris still reacting to light 1 no iridial reflex to light, haemorrhage or gross destruction (any or all of these) 2 Conjunctivae, redness: blood vessels normal 0 some blood vessels definitely hyperaemic (injected) 1 diffuse, crimson colour, individual vessels not easily discernible 2 diffuse beefy red appearance 3 Conjunctivae, chemosis: no swelling 0 any swelling above normal 1 obvious swelling with partial eversion of lids 2 swelling with lids about half-closed 3 swelling with lids more than half-closed 4

3.4.1.2 Examination time points

0, 30 min, 60 min, 4 h, 24 h, 48 h, 72 h

3.4.2 Other investigations Initial 'sting response' (immediately after instillation): no response 0 a few blinks only, normal within two minutes 1 rabbit blinks and attempts to open eye but reflex closes it 2 rabbit keeps eye shut and puts pressure on it, may rub eye 3 rabbit holds eye tightly shut, may struggle or squeal 4

3.5 Further remarks As requested by the US-EPA-guideline, conjunctival discharge was also assessed.






4.1 Clinical signs No effects

4.2 Average score (24-72 hours)

4.2.1 Cornea 0

4.2.2 Iris 0

4.2.3 Conjunctiva

4.2.3.1 Redness 0

4.2.3.2 Chemosis 0

4.3 Reversibility Yes Conjunctival redness: 24 h Conjuntival chemosis: 1 h

4.4 Other Slight discharge (grade 1) was observed in 2/6 rabbits immediately after treatment (reversible within 30 minutes).

4.5 Overall result Cornea and iris were unaffected by treatment. Slight conjunctival redness was recorded up to 4 hours and slight chemosis up to 0.5 hours after treatment. TI-435 is considered to be practically non-irritant to the eye.



Evaluation of eye reactions in rabbits after instillation into the eye; no relevant deviation from guidelines (92/69/EEC B5, EPA FIFRA 81-4, Japan MAFF, OECD 405)


Cornea and iris were unaffected by treatment. Slight conjunctival redness was recorded up to 4 hours and slight chemosis up to 0.5 hours after treatment.

5.3 Conclusion No classification for eye irritation is considered required for TI-435 according to Directive 2001/59/EC (adaptation of 67/548/EEC).

5.3.1 Reliability 1









Date 2004-10-07

Materials and Methods Acceptable, no relevant deviations from OECD guideline 405

Results and discussion Cornea and iris were unaffected by treatment. Slight conjunctival redness was recorded up to 4 hours and slight chemosis up to 0.5 hours after treatment. Under the terms of Directive 2001/59/EC (adaptation of 67/548/EEC) TI-435 is not irritating to the eye.

Conclusion No classification for eye irritation is required for TI-435 according to Directive 2001/59/EC (adaptation of 67/548/EEC).

Reliability 1


Remarks None

COMMENTS FROM ...







Remarks





Appendix

Table A6_1_4E/02. Results of eye irritation study

Cornea Iris Conjunctiva

redness chemosis

score (average of animals investigated) 0 to 4 0 to 2 0 to 3 0 to4

60 min 0 0 1.0 0

24 h 0 0 0 0

48 h 0 0 0 0

72 h 0 0 0 0

Average 24h, 48h, 72h 0 0 0 0

Area effected - - - -

Maximum average score (including area affected, max 110) - - - -

Reversibility* - - C C

average time for reversion - - 24 h 1 h

Give method of calculation maximum average score.

* c : completely reversible n c : not completely reversible n : not reversible

- - - -




Acute Toxicity Skin sensitisation Guinea pig maximisation test (GPMT)

1 REFERENCE

Officialuse only

1.1 Reference Denton, S. M. (1997); TI-435: Skin sensitisation study in the guinea pig; Covance Laboratories, Harrogate, UK; unpublished report number 586/125-1032, 23.10.1997




None





2.2 GLP Yes

2.3 Deviations No






3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study (test-article-vehicle formulations prepared at the day of application)

3.1.2.4 Preparation of test substance for application

for induction: intradermal: 1% w/v TI-435 in Alembicol D topical: 55% w/w TI-435 in Alembicol D for challenge: 10% and 20% w/w TI-435 in Alembicol D

3.1.2.5 Pretest performed on irritant effects

Yes Intradermal application: one injection with 0, 0.1, 1, 2.5, 5, 7.5 and 10% w/v TI-435 in Alembicol D Topical application for induction (exposure 48 h): 10, 25, 40 and 55% w/w TI-435 in Alembicol D Topical application for challenge (exposure 24 h): 5, 10, 20 and 30% w/w TI-435 in Alembicol D





3.2 Test Animals 20 female Guinea pigs per treated and negative control group (Dunkin-Hartley strain, 5 – 7 weeks old, body weight 345 – 571g, supplied by D. Hall Ltd., Burton-on-Trent, UK)

concurrent positive control study 10 female Guinea pigs (same strain and supplier) were used per positive and negative control group In addition biannual positive control experiments are performed in the laboratory; about 7 month previous to the study with TI-435 10 male Guinea pigs were treated with 2-mercaptobenzothiazole (MBT) with further 5 males as negative control group (supplied by D. Hall, Newchurch, UK)


Adjuvant

3.3.1 Induction schedule day 1 (intradermal induction) – day 8 (topical induction) – day 21 (topical challenge) see table in appendix

3.3.2 Way of Induction Intradermal and topical

Occlusive (topical)

3.3.3 Concentrations used for induction

Intradermal: 1% w/v TI-435 (causing mild to moderate irritation)

3.3.4 Concentration Freunds Complete Adjuvant (FCA)

50% FCA in water (1:1 v/v)

3.3.5 Challenge schedule

3.3.6 Concentrations used for challenge

10% and 20% w/w TI-435 (usually maximum non-irritant concentration)

3.3.7 Rechallenge No

3.3.8 Scoring schedule 24h, 48h after challenge

3.3.9 Removal of the test substance

24 hours after challenge application treated skin areas were washed with arachis oil

3.3.10 Positive control substance

Concurrent positive control: 2,4-dinitrochlorobenzene (DNCB): Intradermal induction: 0.1% w/v in corn oil (and/or adjuvant) Topical induction: 0.5% w/v in corn oil Topical challenge: 0.2% and 0.05% w/v in corn oil Biannual positive control studies: 2-mercaptobenzothiazole Intradermal induction: 2.5% w/w in Alembicol D (and/or adjuvant) Topical induction: 60% w/w in Alembicol D Topical challenge: 15% and 30% w/w in Alembicol D

3.4 Examinations

3.4.1 Pilot study Yes







4.1 Results of pilot studies

Intradermal application: no skin reactions at 0.1% w/v slight skin reactions (grade 1) in 1/2 Guinea pigs at 24 h Higher concentrations were considered not to be injectable when mixed with FCA (2.5% and 5% w/v) or could not be correctly injected even without mixing with FCA (7.5% and 10% w/v); 1% w/v selected for main part of study Topical induction: slight erythema in 1/2 Guinea pigs at 25-55% w/w; no erythema at 10% w/w 55% w/w selected for main part of study Topical challenge: slight erythema in 1/3 Guinea pigs at 30% w/w; no erythema at 5-20% w/w 10% and 20% selected for main part of study

4.2 Results of test

4.2.1 24h after challenge

4.2.2 48h after challenge

4.2.3 Other findings The fact that skin irritation after the topical induction was observed in negative control but not in treated animals is considered not to impair the validity of the study, as the pilot test has shown that the selected TI-435 concentration (55% w/w) is an irritant concentration.

4.3 Overall result The study director considered the results being negative for skin sensitisation in 19/20 treated animals and inconclusive for 1/20 treated Guinea pig. Therefore the positive sensitisation incidence was 0%.



Evaluation of the skin sensitisation potential according to the maximisation procedure (intradermal and topical induction, topical challenge) in Guinea pigs; no relevant deviation from guidelines (92/69/EEC B6, EPA FIFRA 81-6, Japan MAFF, OECD 406)


The study director considered the results being negative for skin sensitisation in 19/20 treated animals and inconclusive for 1/20 treated Guinea pig. Therefore the positive sensitisation incidence was 0%.

5.3 Conclusion No classification for skin sensitisation is required for TI-435 according to Directive 2001/59/EC (adaptation of 67/548/EEC), as the positive skin reactions were below the threshold for classification (30%) defined in the Directive.

5.3.1 Reliability 1









Date 2004-10-07

Materials and Methods In the above described study induction was performed intradermally (day 1) as well as topically (day 8). The test substance preparations used for injection (0.1 mL per site, two sites per animal) were 1 % TI-435 (w/v) in Alembicol D and 1 % TI-435 (w/v) in Alembicol D/Freund’ s Complete Adjuvant (FCA) emulsion 1:1, in which the concentration of FCA was 50 % (v/v) in water. For topical induction an aliquot (approximately 0.5 mL) of 55 % TI-435 (w/w) in Alembicol D was applied to the skin on a patch of filter paper. Challenge was performed using 0.1 mL each of 10 % as well as 20 % TI-435 (w/w) in Alembicol D. The study partly complies with OECD guideline 406. Treatment of the skin 24 hours prior to topical induction application with an irritant adjuvans such as sodium dodecyl sulphate is missing, although skin reactions in a dose-finding pretest were only slight (grade 1, 24 h reading) and disappeared within 96 hours at the highest concentration applied. According to the study director this concentration (55 % w/w) represented the maximal preparable concentration. No explanation is given for the deviation from guideline.

Results and discussion For detailed results see tables A6_1_5/01-1 and A6_1_5/01-2 in Appendix. After intradermal injection skin reactions were seen at all injection sites, i. e. in test animals as well as in control animals. Even though this is not a normal finding in GPMTs, it has not been commented by the test facility. On the other hand after topical induction in the main study only control animals but not test animals exhibited erythema or skin irritations. No discussion or explanation is provided in respect to different results in screening test and main study. At 24 hours after challenge, 10 % and 15 % of the guinea pigs had erythema following application of 10 % (w/w) or 20 % (w/w) TI-435, respectively. Both values are below the threshold for classification (i. e. 30 %) according to Directive 2001/59/EC. TI-435 is not sensitising to the skin.

Conclusion No classification for skin sensitisation is required for TI-435 according to Directive 2001/59/EC (adaptation of 67/548/EEC), as the positive skin reactions were below the threshold for classification defined in the Directive.

Reliability 2


Remarks None





COMMENTS FROM ...







Remarks

Table A6_1_5/01-1. Detailed information including induction/challenge/scoring schedule for skin sensitisation test

Inductions GPMT Observations/Remarks

day of treatment

application

Induction 1 1 intradermal Treated: well-defined erythema (grade 2, day 2) Negative controls: slight to well-defined erythema (grade 1-2, day 2)

Induction 2 8 topical Treated: no erythema (day 11) Negative Controls: slight to well defined erythema (day 11)

challenge 21 topical scoring 1 23 scoring 2 24

Table A6_1_5/01-2. Result of skin sensitisation test (modify if necessary)

Number of animals with signs of allergic reactions / number of animals in group

TI-435 challenge concentrations (w/w)

Negative control Test group* Positive control

10% 20% 10% 20% concurrent biannual

scored after 24h 1/20 0/20 2/20 3/20 9/10 6/10

scored after 48h 1/20 0/20 0/20 0/20 8/10

* Results considered negative for 19/20 animals and inconclusive for skin sensitisation for the remaining single animal.


Section A6.2 Annex Point IIA VI.6.2

Percutaneous absorption (in vivo) Rhesus monkeys

1 REFERENCE

Officialuse only

1.1 Reference Sebesta, C. A. (2003); A study to determine the dermal absorption of TI 435 FS 600 when administered dermally to male Rhesus monkeys Charles River Laboratories, Worcester, MA 01608, USA unpublished report number 200494, 27.02.2003


1.2.1 Data owner Bayer Crop Science / Sumitomo Chemical Takeda Agro Co., Ltd.


None


Data submitted on existing a.s. for the purpose of its first entry into Annex I.


2.1 Guideline study Yes No applicable EU guideline; US-EPA OPPTS 870.7600, OECD 427

2.2 GLP Yes

2.3 Deviations None relevant for the integrity and validity of the study: Monkeys were used as test animals. Therefore skin samples were not collected as the animals were not sacrificed at the end of the observation period. Almost complete recovery of the applied radioactive dose revealed that no relevant amount of test compound remained associated with the treated skin after washing.


3.1 Test material TI-435 FS 600 formulation

3.1.1 Lot/Batch number 15209/1

3.1.2 Specification

3.1.2.1 Description Red liquid (suspension)

3.1.2.2 Purity

3.1.2.3 Stability 7 days at 40°C, 97 days at ≤-20°C

3.1.2.4 Radiolabelling Nitroimino-14C TI-435:

radiochemical purity 99.3%, 117.2 µCi/mg (4.34 MBq/mg)

3.2 Test Animals

3.2.1 Species Rhesus monkeys

3.2.2 Strain Bred at Charles River Laboratories

3.2.3 Source See 3.2.2

3.2.4 Sex Male




3.2.5 Age/weight at study initiation

3.3-5.5 kg

3.2.6 Number of animals per group

5

3.2.7 Control animals No


Dermal

3.3.1 Preparation of test site

shaved skin

3.3.2 Concentration of test substance

5.70 µg/cm2 (target 6.13 µg/cm2) corresponding to 1.5 mg TI-435 equivalents/mL

3.3.3 Specific activity of test substance

117.2 µCi/mg

3.3.4 Volume applied 100 µL

3.3.5 Size of test site 24 cm²

3.3.6 Exposure period 8 hours

3.3.7 Sampling time 4, 8, 12, 24, 48, 72, 96 and 120 hours after dermal treatment.

3.3.8 Samples Urine, faeces, skin wash, chair wipe, pan wash, cage wash


4.1 Toxic effects, clinical signs

Faeces were not produced for 2, 1 and 5 animals at the 4-, 8- and 12-hour observation points, respectively. One monkey had mildly soft faeces at the 24-hour observation point.

4.2 Dermal irritation None reported

4.3 Recovery of labelled compound

Mean total recovery was 98.0% of applied dose with the majority (97.75%) recovered from the application site (dressing, skin washes).

4.4 Percutaneous absorption

The total of all samples associated with excretion (faeces, urine and combined cage, chair and pan washes) was 0.24% of applied dose. As excretion of applied radioactivity was essentially complete within 24 hours, the result was normalized to 100% total recovery resulting in a normalized dermal absorption of 0.245%.



The dermal absorption of TI-435 was investigated in Rhesus monkeys (no applicable EU guideline; no relevant deviations from US-EPA OPPTS 870.7600, OECD 427).


Percutaneous absorption of TI-435 in Rhesus monkeys was (normalized) 0.245% of applied dose. The used concentration of TI-435 of 1.5 g/L corresponds well with the recommended use concentration as a wood preservative (1 g/L) even if the base formulation used in this study was different than the biocide formulation. Due to the high dilution factor (1:600) of the FS 600 formulation, the inert formulation ingredients are expected not to influence the dermal penetration properties of TI-435. Therefore the data obtained with the (diluted) FS 600 formulation of TI-435 are considered relevant for the biocide use of TI-435.




5.3 Conclusion (Normalized) dermal absorption of TI-435 at a concentration corresponding well its use as wood preservative was 0.245% of applied dose in Rhesus monkeys. The same value can be considered for human risk assessment.

5.3.1 Reliability 1





Date 2006-10-23

Materials and Methods acceptable

Results and discussion In two out of five animals, faecal excretion was observed even during the last time interval (96-120 h). The mean overall total recovery of radioactivity was 98 % (sum of eliminated and residual radioactivity). From the results presented, it cannot be ruled out with certainty, that the 2 % of radioactivity that were not accounted for, were in fact stored in deeper layers of the skin, from where they could have been gradually released into the system.

Conclusion In conclusion, a value of 2 % is derived from this study as a conservative estimate of the absorption of TI 435 FS 600 through the skin of rhesus monkeys in vivo.

Reliability 1

Acceptability acceptable

Remarks Co-formulants of the plant protection product are not identical but are considered sufficiently similar to permit the use of this study in deriving a dermal absorption value for the biocidal product. Both formulations are water-based and contain dispersing agents in similar quantities. The glycerol antifreeze content of the biocidal product is higher and as glycerol has been shown to reduce skin penetration the value derived for the plant protection product may actually overestimate dermal absorption from the biocidal product.

COMMENTS FROM ...







Remarks


Table A6_2_02-1: Excretion and recovery of TI-435 after dermal application to Rhesus monkeys

% of applied dose

Urine 0-120 hours 0.05 Faeces 0-120 hours 0.13 Cage wash/wipes 8-120 hours 0.00 Urine pan/chair wash/wipes 4 + 8 hours 0.06

Total excretion 0.24 Dressing (patch, dome) 2.75 Skin wash first four soap swabs

remaining soap swabs dry swabs alcohol swabs Total

91.24 3.41 0.03 0.09

97.75

5.3.3 Recovery 98.0




Metabolism studies in mammals Absorption, distribution, metabolism and excretion in rats

1 REFERENCE

Officialuse only

1.1 Reference Weber, E. (2000); [Nitroimino-14C]- and [thiazolyl-2-14C] TI-435 toxicokinetic behaviour and metabolism in the rat including whole body autoradiography; Bayer AG., Agrochemical Division, D-51368 Leverkusen-Bayerwerk, Germany; unpublished report number MR 348/




None




2.1 Guideline study Yes eatment regimes selected according to 91/414/EEC as amended by

94/79/EEC, OECD guideline no. 417, and EPA OPPTS 870.7485

2.2 GLP Yes

2.3 Deviations None relevant for the integrity and validity of the study: only one animal per time point was used for investigation of the kinetic of tissue residues (whole body radiography); clinical signs of toxicity had not been recorded (not a relevant endpoint for this type of study)



3.1.1 Lot/Batch number [nitroimino-14C] TI-435: batch nos. 11553/11 and 11553/17 (chemical purity >98%, specific activity 102.2 μCi/mg, radiochemical purity >99%), [thiazolyl-2-14C] TI-435: batch no. 11649/2 (chemical and radiochemical purities >99%, specific activity 103.8 μCi/mg) unlabelled TI-435: batch no. M00157 (purity 99.8%)

3.1.2 Specification Highly pure batches as required for metabolism studies

3.1.2.1 Description Powder

3.1.2.2 Purity


3.1.2.4 Radiolabelling





3.2 Test Animals Groups of male and female 7 - 8 week old Sprague-Dawley rats (Crl:CD BR strain, weight range 196 - 228 g, Charles River, Sulzfeld, Germany); Group distribution see following table.

Group number

No. & sex of animals

oral Dosemg/kg bw

14C label position

Sample collection regime (hours post treatment)

1 4M 2.5 Nitroimino Expired air: 0-4, 4-8, 8-24, 24-48, 48-72 Urineb: 0-4, 4-8, 8-24, 24-48, 48-72 Faecesc: 0-24, 24-48, 48-72 Carcassd: 72

2 4M + 4F 2.5 Nitroimino Urineb: 0-4, 4-8, 8-24, 24-48, 48-72 Faecesc: 0-24, 24-48, 48-72 Organs & tissuesd: 72 Plasma 14C concn as a function of time

3 4M 2.5 Thiazolyl-24 4M 250 Nitroimino 5 4M 25a Nitroimino

6 6M 5 Nitroimino One animal killedd 1, 4, 8, 24, 48 and 72 hours post-treatment for whole body autoradio-graphy (radioluminography)

7 1M 25 Thiazolyl-2 Collection of urine and faeces, additional test for the isolation of metabolites

a pre-treated with 14 oral doses of unlabelled TI-435 b subsequently combined with de-mineralised water wash from collection funnels c subsequently lyophilised and homogenised d animals were anaesthetised by CO2 inhalation and killed by exsanguination


Oral (gavage) Concentration: see table above Vehicle: 0.5% aqueous tragacanth Concentration in vehicle: prepared according to individual bw and dosing volume Total volume applied: 2 mL per rat (∼10 mL/kg bw) Sampling time/samples collected: see table above


4.1 Toxic effects, clinical signs

Not determined





4.2 Absorption and elimination

Single doses of 2.5 mg/kg bw or 250 mg/kg bw, and repeated doses of 25 mg/kg bw TI-435 were rapidly and extensively absorbed from the gastrointestinal tract and rapidly eliminated, predominantly in the urine (see tables below).

Parameter Units 2.5 mg/kg [nitroimino-14C]

a pre-treated for 14 days with unlabelled TI-435

Based on the extent of cumulative urinary elimination, the extent of oral absorption was >89% for all doses and dose regimens and for both 14C labels and sexes.

Time [h] Cumulative excretion (% administered dose) mg/kg bw

sex 2.5b male

2.5b male

2.5b female

250b male

25ab male

2.5c male

Expired air: 24 0.013 - - - - - 48 0.015 - - - - - 72 0.017 - - - - -

Urine: 24 90.60 88.21 93.32 57.17 91.33 87.85 48 91.24 88.80 94.21 88.86 92.57 88.88 72 91.48 89.10 94.56 90.47 92.99 89.25

Faeces: 24 6.56 5.99 2.90 3.49 6.18 7.02 48 6.80 6.18 3.20 7.82 6.35 7.34 72 6.89 6.27 3.29 8.58 6.60 7.79

Total 98.39 95.37 97.85 99.05 99.58 97.04 a pre-treated for 14 days with unlabelled TI-435 b [nitroimino-14C] TI-435; c [thiazolyl-2-14C] TI-435





Maximum plasma concentrations of between 1.27 – 1.82 ppm occurred 1.4 – 2.1 hours after administration of a 2.5 mg/kg bw dose for both 14C labels. The maximum plasma concentration after repeated doses of 25 mg/kg bw was 15.0 ppm. AUC values were approximately proportional to dose for a single dose of 2.5 mg/kg bw (7.3 – 10.3 μg/mL⋅h) and repeated doses of 25 mg/kg bw (116μg/mL⋅h). The maximum plasma concentration following treatment at 250 mg/kg bw was approximately 79.5 ppm, which occurred 6 hours after administration. However, at this high dose level a further peak concentration of 47.8 ppm occurred at 32 hours, indicating saturated absorption.

4.3 Distribution See also the two following tables. Radioactivity was rapidly distributed to all tissues within one hour of oral administration of 5 mg/kg bw [nitroimino-14C] TI-435, at which time most tissues showed their maximum concentration. The exceptions were the ocular vitreous body, kidney medulla and urinary bladder, which showed maximal concentrations after 4 or 8 hours - the latter consistent with extensive urinary elimination. Thereafter tissue concentrations declined rapidly and after 72 hours all tissue concentrations were close to/below the limit of quantification. Residual radioactivity in the carcass at 72 hours post-treatment was 0.093 – 0.327% of applied dose for all groups. Therefore, there was no evidence of accumulation in any tissue or organ examined. Mean disposition residence times (MRTdisp) were 4.3 – 5.9 hours for groups treated with the [nitroimino-14C] label but higher for a similar dose of the [thiazolyl-2-14C] label (9.2 hours), possibly reflecting different residence times for the metabolites traced by the radiolabels.





Total tissue residues [ppm] after administration of 5 mg/kg bw [nitroimino-14C] TI-435, by whole body autoradiography

Organ/tissue 1 h 4 h 8 h 24 h 48 h 72 h Adrenal 2.94 1.93 0.64 0.025 0.010 0.010 Blood 3.00 1.14 0.38 0.010 <LOQ <LOQ Bone marrow 0.91 0.69 0.24 <LOQ <LOD <LOD Brain 0.34 0.25 0.12 <LOD <LOD <LOD Brown fat 1.85 1.33 0.33 <LOQ <LOD <LOD Eye (vitreous body) 0.01 0.55 0.32 0.112 0.048 0.020 Heart 1.99 1.17 0.43 0.007 <LOQ <LOD Kidney (cortex) 4.08 2.47 0.78 0.020 0.009 0.008 Kidney (medulla) 2.89 4.39 0.87 0.019 0.004 <LOQ Kidney (whole) 3.72 3.59 0.86 0.023 0.008 0.006 Liver 3.42 2.23 0.82 0.065 0.026 0.021 Lung 0.49 0.15 0.22 0.006 <LOD <LOD Muscle 1.88 1.13 0.39 <LOQ <LOD <LOD Nasal mucosa 5.64 3.82 4.31 0.072 0.040 0.022 Pituitary 1.88 1.08 0.35 0.006 - - Pineal body 1.35 0.88 0.32 0.007 - - Salivary gland 2.22 1.50 0.54 0.008 <LOD <LOD Skin 2.24 1.34 0.56 0.013 <LOQ <LOQ Spinal marrow 0.28 0.21 0.11 <LOD <LOD <LOD Spleen 1.53 0.96 0.32 0.006 <LOD <LOD Testes 0.66 0.53 0.21 0.004 <LOD <LOD Thymus 1.37 0.86 0.28 <LOQ <LOD <LOD Thyroid 2.34 1.53 0.47 0.006 <LOQ <LOD Tongue 2.23 1.33 0.40 0.007 <LOD <LOD Urinary bladder 6.00 6.17 5.99 - 0.018 <LOQ

LOD limit of detection; LOQ limit of quantitation; - value not measured

Organ/tissue Total radioactive tissue residue [ppm] at 72 hours mg/kg bw

sex2.5b

male 2.5 b

female 250 b male

25a b male

2.5 c male

Erythrocytes 0.0056 0.0044 0.789 0.0537 0.0119 Plasma 0.0033 0.0027 0.361 0.0257 0.0079 Spleen 0.0020 - 0.263 - 0.0047 GI tract 0.0021 0.0031 0.245 - 0.0234 Liver 0.0313 0.0167 2.880 0.2077 0.0329 Kidney 0.0093 0.0070 0.864 0.0693 0.0380 Testes 0.0011 - 0.167 0.0124 0.0031 Skeletal muscle - - 0.235 - 0.0028 Bone (femur) - - - - 0.0041 Heart 0.0022 0.0022 0.318 0.0219 0.0046 Lung 0.0042 0.0037 0.560 0.0400 0.0084 Brain - - - - 0.0018 Skin 0.0035 0.0034 0.317 - 0.0104 Carcass 0.0020 0.0021 0.254 0.0267 0.0037

a pre-treated for 14 days with unlabelled TI-435 b [nitroimino-14C] TI-435; c [thiazolyl-2-14C] TI-435





4.4 Metabolism

The predominant component identified for all groups was the parent compound TI-435 (55-74% of applied dose, see table below). The major metabolites identified (≥10% of applied dose) were TZNG and MNG; MTCA and NTG were found at 1-10% of applied dose. These metabolites occurred predominantly in urine. The main metabolite occurring in faeces was TMG. All other metabolites occurred at less than 2% of administered dose and together accounted for less than 11% of applied dose.

Metabolite % of applied dose

mg/kg bwsex

2.5b male

2.5 b female

250c male

25ac male

2.5 b male

label [nitroimino-14C] [thiazolyl-2-14C] ACT - - - - 1.31 CTCA - - - - 0.94 MTCA - - - - 8.54 Polar HPLC fraction 0.60 0.34 - Urea 0.25 0.13 0.06 0.09 - NTG 3.92 1.42 3.49 1.92 - MNG 13.21 7.75 9.46 8.78 - MG 0.45 0.30 0.30 0.24 - TZG 0.36 0.19 0.54 TMG 1.55 0.73 0.19 0.16 2.37 TZU 0.63 0.56 0.64 0.67 0.42 THMN 0.11 0.12 0.09 0.17 TZMU 0.26 0.13 0.29 0.06 0.19 TZNG 11.39 7.07 12.48 10.18 10.62 TI-435 (parent) 55.55 74.02 59.98 66.53 61.33 Total identified 88.29 92.76 86.99 88.82 86.25 Total excreted 95.49 97.95 99.22 99.68 97.37

a pre-treated for 14 days with unlabelled TI-435; b from urine and faeces samples; c from urine samples







ADME evaluation in the rat (gavage application, single treatment at three dose levels, multiple treatment at one dose; absorption, distribution and elimination kinetics; identification and quantification of metabolites); no relevant deviation from guidelines (91/414/EEC as amended by 94/79/EEC, OECD 417, EPA OPPTS 870.7485)


Single low and high doses (2.5–250 mg/kg bw) and repeated doses (25 mg/kg bw) of TI-435 are rapidly and extensively absorbed (∼90% of applied dose) from the gastrointestinal tract and rapidly and almost completely eliminated, predominantly in the urine. High doses of TI-435 saturate the gastrointestinal absorptive mechanism and show a transient delay in excretion (at 24 h after application: 61% of applied dose at 250 mg/kg bw vs. ≥94% at 2.5/25 mg/kg bw). TI-435 is rapidly and extensively distributed to all tissues and organs but is rapidly and almost completely eliminated from all tissues with no evidence of accumulation. The parent TI-435 is the major fraction in excreta (55-74% of applied dose); . TZNG and MNG are the two major metabolites (≥10% of applied dose); a further metabolite is MTCA (8.5% of applied dose). Other than saturated absorption and transiently delayed elimination of high doses, the biokinetics and metabolism of TI-435 are not markedly influenced by dose level, dose regimen and sex.

5.3 Conclusion Rapid and almost complete absorption and excretion of TI-435 in rats after oral application essentially independent of sex, dose level, pre-treatment and label position (slight delay at high dose levels due to saturation of absorption process). No potential for accumulation. TI-435 (parent) was the major fraction in excreta. metabolites were identified (TZNG and MNG ≥10%, MTCA ∼8.5% of applied dose).

5.3.1 Reliability 1









Date 2004-10-07

Materials and Methods Applicant’s version is acceptable.

Results and discussion Applicant's version is amended by: Female rats were found to have a higher percentage of parent compound in their metabolic profile than males (74% vs. 56%). However, there were no apparent sex differences in the proportions of specific metabolites produced when the metabolised fraction of the dose was considered. The about 10-fold higher radioactive residues in the gastrointestinal tract of males given [thiazolyl-2-14C] TI-435 in conjunction with only a slight increase in fecal excretion and higher plasma levels at 24 and 48 hours post dose suggests biliary excretion and enterohepatic recirculation of thiazole-containing metabolites resulting from the cleavage of the molecule at the nitrogen-carbon bond between the thiazolyl-methyl position and the nitroimino moiety.

Conclusion Other conclusions: Applicant's version is amended by: The main metabolic reactions are oxidative demethylation to form TZNG and cleavage of the nitrogen-carbon bond between the thiazolyl-methyl position and the nitroimino moiety. Due to the difference in metabolic profile, exposure of females to the major metabolites can be expected to amount to approximately 60% of male exposure at low doses.

Reliability 1


Remarks Results and conclusions are supported by published data on tests with low and high doses of [nitroimino-14C]- and [thiazolyl-2-14C] TI-435 in male and female Wistar rats (Yokota, T., Mikata, K., Nagasaki, H., and Ohta, K. (2003). Absorption, tissue distribution, excretion, and metabolism of clothianidin in rats. J Agric Food Chem 51, 7066-7072).

COMMENTS FROM ...







Remarks

Sumitomo Chemical Takeda Agro Co., Ltd. TI-435 March 2004



Metabolism studies in mammals Dermal absorption

JUSTIFICATION FOR NON-SUBMISSION OF DATA Officialuse only

Other existing data [ ] Technically not feasible [ ] Scientifically unjustified [ x ] Limited exposure [ x ] Other justification [ ]

Detailed justification:

Undertaking of intended data submission [ ]




Date

Evaluation of applicant's justification

Conclusion

Remarks

COMMENTS FROM OTHER MEMBER STATE (specify)



Discuss if deviating from view of rapporteur member state


Remarks




Short-term repeated dose toxicity (28 days) Oral 28-day dietary toxicity study in mice

1 REFERENCE

Officialuse only

1.1 Reference Chambers, P. R. (1997a); TI-435: Toxicity to mice by dietary administration for 4 weeks; Huntingdon Life Sciences Ltd., Huntingdon, UK; unpublished report number TDA 180/960497, 19.02.1997




None


Data submitted on existing a.s. its first entry into Annex I.


2.1 Guideline study Yes 92/69/EEC (method B7), OECD no. 407 (1981), EPA FIFRA Subdivision F (1984), Japan MAFF (59 NohSan No. 4200, 1985)

2.2 GLP Yes

2.3 Deviations No






3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study (stability of the compound and its homogenicity and stability in food were tested).

3.2 Test Animals 6 male and 6 female 6 week old mice per group (Crl:CD-1(ICR)BR strain, weight range 19 – 35 g, Charles River Ltd., UK)


Oral (dietary)

3.3.1 Duration of treatment

28 days

3.3.2 Frequency of exposure

daily


none





3.3.4 Oral

3.3.4.1 Type in food

3.3.4.2 Concentration 0, 500, 1000, 2000 and 4000 ppm corresponding to 0, 90, 190, 383 and 683 mg/kg bw/day for males and 0, 122, 248, 491 and 619 mg/kg bw/day for females food consumption per day: ad libitum

3.3.4.3 Vehicle None (TI-435 was mixed with diet directly)

3.3.4.4 Concentration in vehicle

-

3.3.4.5 Total volume applied

-

3.3.4.6 Controls plain diet

3.4 Examinations

3.4.1 Observations

3.4.1.1 Clinical signs yes (daily)

3.4.1.2 Mortality yes (twice daily)

3.4.2 Body weight yes (weekly)

3.4.3 Food consumption yes (weekly)

3.4.4 Water consumption yes (by visual assessment)


no

3.4.6 Haematology yes, all animals during week 4 Parameters: Haematocrit (packed cell volume), haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, reticulocyte count

3.4.7 Clinical Chemistry yes, all animals during week 4 Parameters: sodium, potassium, calcium, inorganic phosphorous, chloride, glucose, total cholesterol, blood urea nitrogen, total bilirubin, creatinine, total protein, albumin, alanine aminotransferase (= GPT), aspartate aminotransferase (= GOT), alkaline phosphatase, triglycerides

3.4.8 Urinalysis yes all animals (paired resulting in 3 samples per group) during week 4 Parameters: volume, specific gravity, pH, protein, glucose, blood, ketones, bile pigments, urobilinogen


3.5.1 Organ Weights yes organs: liver, kidneys, adrenals, testes, ovaries, thymus, spleen, brain, heart, lungs, thyroid (with parathyroid), pituitary

3.5.2 Gross and histopathology

yes gross pathology: all dose groups histopathology: abnormal tissues from animals that died during the studies; any gross lesion in any animal; tissues marked (*) from all surviving control, intermediate high and high dose group animals





organs: brain, spinal cord, pituitary, thyroid, parathyroid, thymus, oesophagus, salivary glands, stomach, small and large intestines, liver*, pancreas, kidneys*, adrenals*, spleen*, heart*, trachea, lungs, aorta, gonads, uterus, female mammary gland, prostate, urinary bladder, gall bladder, lymph nodes, peripheral nerve, bone marrow, skin, eyes, head, larynx, pharynx, tongue


3.5.4 Statistics Where appropriate: Bartlett’s test for heterogeneity of variance between groups followed by one-way analysis of variance (or ANOVA) on homogeneous or transformed data followed by Student’s t test and Williams’ test or a Kruskal-Wallis ranking test, as appropriate.

3.6 Further remarks


4.1 Observations

4.1.1 Clinical signs During week 2, 4 male and 6 female animals treated at 4000 ppm died or were killed in a moribund condition. Prior to death, the animals showed reduced food consumption and weight loss. There were no other treatment-related deaths although 2 males treated at 2000 ppm died as a result of stress associated with anaesthesia and blood sampling. Treatment-related clinical signs were confined to the groups treated at 2000 ppm (thin appearance and hunched posture) and 4000 ppm (lethargy, tremors, hunched posture, piloerection, emaciation, half-closed eyes, unsteady gait, apparent hypothermia and pale extremities).

4.1.2 Mortality

4.2 Body weight gain Animals treated at ≥2000 ppm showed a dose-related decrease in body weight throughout the study. Females treated at 1000ppm also showed weight loss and males treated at 1000 ppm showed decreased weight gain during week 1, but subsequently gained weight (see table A6_3_1/01-1). Thus, there was a treatment-related reduction in the overall weight gains of both sexes at 1000 ppm, although there was no statistical significance.

4.3 Food consumption and compound intake

The cumulative food consumption of both sexes was reduced at all dose levels (see table A6_3_1/01-1). The effect was very marked at dose levels of 2000 and 4000 ppm, particularly during the first week of treatment. Since the slightly lower food consumption at 500 ppm did not result in a significant reduction in weight gain, it is considered to be without toxicological relevance. Compound intake see 3.3.4.2. The test diets were stable for 22 days at ambient temperature and the achieved concentrations in the diets prepared for weeks 1 and 4 were within –3.5% to +7.6% of nominal concentrations.

4.4 Ophtalmoscopic examination

-

4.5 Blood analysis

4.5.1 Haematology Decreased red blood cell count, haemoglobin and packed cell volume in the 2 surviving males at 4000 ppm, and reduced mean cell volume (MCV) and mean cell haemoglobin (MCH) values in these animals and in females at 2000 ppm (see table A6_3_1/01-1).





Neutrophil counts in all male groups and the eosinophil count of the female group at 2000 ppm were slightly elevated, but total white blood cell counts were unaffected by treatment at all dose levels in both sexes. These minor changes in relative white cell numbers are considered to be stress-related and of no toxicological relevance.

4.5.2 Clinical chemistry Treatment-related slight increases in plasma GPT activity in females and in plasma GOT activity in males occurred at 2000 ppm (see table A6_3_1/01-1). The single male at 4000 ppm also showed elevated plasma transaminase activities. Plasma glucose and triglyceride concentrations were reduced in females at 2000 ppm and in the male at 4000 ppm. Electrolyte concentrations (Na+, K+, Cl-) were marginally elevated in males at 2000 ppm (Na+ and Cl- only), in females at 1000 and 2000 ppm and in the male at 4000 ppm.

4.5.3 Urinalysis rinary protein levels were reduced in all treated males and females, and urine specific gravity was slightly increased in the male at 4000 ppm (see table A6_3_1/01-1). Meaningful urine analysis in mice is difficult due to the often small sample size (removal of water during sample collection). Concerning protein levels in females only a single sample could be obtained from the control group and only two samples at 2000 ppm. The variations in urinary protein were not accompanied by any relevant changes in blood protein levels nor any indication of kidney toxicity. Therefore (and due to the small sample size in females), the reduction of urinary protein is considered to be of doubtful toxicological relevance and at least the only moderate reduction at 500 ppm is considered to be not adverse, as it was not accompanied by any other toxicologically relevant effect. In addition there is no apparent reason why - independent of treatment - the urinary protein levels were about 10 times lower in females than in males.


4.6.1 Organ weights There were no effects on organ weights that are considered a direct effect of treatment, although a number of organ weight changes at ≥2000 ppm are considered to reflect impaired weight gain and lower body weights at necropsy.


Relevant macroscopic observations at necropsy were confined to the small size of some organs at dose levels of 2000 and 4000 ppm, notably the thymus and reproductive organs of both sexes and the spleen at 4000 ppm. Gall bladder distension, depressions in the corpus mucosa of the stomach and congestion of the adrenals also occurred in some animals treated at 4000 ppm. There were histomorphological correlates for some of the observed macroscopic lesions in the immune and reproductive systems that are considered to reflect inferred stress and markedly reduced weight gain, respectively (reduced cellularity in the splenic white pulp, atrophy of the red pulp and thymic involutionary; atrophy of testicular seminiferous epithelia, reduced colloid in the seminal vesicles and prostate; reduced thickness of the uterine wall due to inanition and absent ovarian corpora lutea) (see table A6_3_1/01-2). There were no treatment-related histomorphological correlates in the liver and kidneys to account for the observed increases in plasma transaminase activities and electrolyte concentrations. Similarly, there were no histomorphological correlates for the gross lesions noted at





necropsy in the gall bladder, adrenals and stomach.

4.7 Other -



Toxicity evaluation after short-term (28 day) dietary exposure of mice to TI-435; no relevant deviation from guidelines (92/69/EEC B7, OECD 407, EPA FIFRA, Japan MAFF


Reduced food consumption and body weight development at ≥1000 ppm leading to reduced survival at 4000 ppm accompagnied by clinical signs at ≥2000 ppm. In absence of a clear dose-relation ship at 500-1000 ppm and without a relevant effect on bw development, the slightly lower food consumption at 500 ppm was considered to be not adverse. Lower red blood cell parameters in the surviving males at 4000 ppm and in females at 2000 ppm. Slightly increased liver enzyme values in blood among the animals treated with ≥2000 ppm, reduced glucose and triglyceride levels in 2000 ppm treated females and in males at 4000 ppm (considered related to poor nutrition status), marginally elevated electrolytes at ≥1000 ppm (of doubtful toxicological relevance). Slightly higher urine specific gravity at 4000 ppm; lower urinary protein values in all treated groups generally were of doubtful toxicological relevance due to the small sample size (1-3 per group and sex) and in absence of any other indication of kidney toxicity nor any relevant change in blood protein levels. The effect is considered to be not adverse at least at 500 ppm in absence of any other relevant sign of toxicity. No relevant effects on organ weights (slight reductions of absolute or increased relative organ weight due to effect on body weight); histopathological effects in the spleen and ovaries at ≥2000 ppm.

5.3 Conclusion 4000 ppm exceeded the maximum tolerated dose level. Effects of treatment comprised clinical signs of toxicity, weight loss, haematological and blood biochemistry perturbations and histomorphological lesions in the spleen and ovaries.

5.3.1 LO(A)EL LOAEL = 1000 ppm corresponding to 190 and 248 mg/kg bw/day for males and females, respectively due to the reduced bw development and food consumption

5.3.2 NO(A)EL NOAEL = 500 ppm corresponding to 90 and 122 mg/kg bw/day for males and females, respectively

5.3.3 Other -

5.3.4 Reliability 1









Date 2004-10-07

Materials and Methods The applicants version is acceptable

Results and discussion 4 male and 6 female died at the top dose of 4000 ppm in the second week of treatment. 2 males treated at 2000 ppm died as a result of stress associated with anaesthesia and blood sampling. Reduced food consumption and body weight development at ≥1000 ppm leads to reduced survival at 4000 ppm accompagnied by clinical signs at ≥2000 ppm. Lower red blood cell parameters were found in the surviving males at 4000 ppm as well as an increase of neutrophiles in males at ≥1000 ppm. Increased liver enzyme values in blood were observed among the animals treated with ≥2000 ppm. In females at 2000 ppm and in males at 4000 ppm occured reduced glucose and triglyceride levels (considered related to poor nutrition status). Lower urinary protein values in all treated groups generally were of doubtful toxicological relevance due to the small sample size (1-3 per group and sex) and in absence of any other indication of kidney toxicity nor any relevant change in blood protein levels. The effect is considered to be not adverse at least at 500 ppm in absence of any other relevant sign of toxicity. No relevant effects on organ weights were reported (slight reductions of absolute or increased relative organ weight due to effect on body weight); histopathological effects were found in the spleen, thymus and ovaries at ≥2000 ppm.

Conclusion LO(A)EL: 1000 ppm for males and females NO(A)EL: 500 ppm for males and females

Reliability

Acceptability

Remarks

COMMENTS FROM ... (specify)







Remarks



Table A6_3_1/01-1. Bw development, food consumption and results of clinical chemistry, haematology and urinalysis (28-day mouse)

Parameter [Unit] Controls 0 ppm

low dose 500 ppm

medium dose 1000 ppm

interm. high 2000 ppm

high dose 4000 ppm

weeks after start of treatment

0-1 0-4 0-1 0-4 0-1 0-4 0-1 0-4 0-1 0-4

Cumulative bw gain [g] males 3.1 5.8 2.5 5.9 1.9 4.0 -1.6** -4.1** -9.0** -12.6**

females 0.8 2.9 1.3 2.2 -0.2 0.9 -1.9** -4.0** -6.4** -

Total food consumption [% control] males 100 ↓ 79 ↓ 84 ↓ 74 ↓ 49

females 100 80 ↓ 84 ↓ 66 ↓ -

Week 4 ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀

RBC (1012/L) ↓ -

Hb (g/dL) ↓ -

PCV (%) ↓ -

MCV (fL) ↓ ↓ -

MCH (pg) ↓ ↓ -

Neutrophils (109/L) (↑) ↑ ↑ ↑ -

Eosinophils (109/L) ↑ -

GPT (mU/ml) ↑ (↑) -

GOT (mU/ml) ↑ (↑) -

Glucose (mg/dL) ↓ (↓) -

Trig (mg/dL) ↓ (↓) -

Na+ (mEq/L) ↑ +1.3%

↑ +3.3%

↑ +3.3%

(↑) +3.3%

-

K+ (mEq/L) ↑ +12%

↑ +17%

(↑) +11.5%

-

Cl- (mEq/L) ↑ +1.8%

↑ +1.8%

↑ +3.6%

↑ +3.6%

(↑) +3.6%

-

Urine SG ↑ -

Urine Prot (mg/dL) ↓ ↓ ↓ ↓ ↓ ↓ - -

* p <0.05, ** p <0.01 arrows in brackets (↓↑) indicate a trend that was not statistically significant



Table A6_3_1/01-2. Results of repeated dose toxicity study (28-day mouse): histopathology Parameter 0 ppm 500 ppm 1000 ppm 2000 ppm 4000 ppm dose-

response

+/-

ma fa ma fa ma fa ma fa ma fa m f

Spleen reduced cellularity of white pulp atrophy of red pulp

0/6

0/6

0/6

0/6

-/0

-/0

0/1

0/1

-/0

-/0

-/0

-/0

0/4

0/4

1/6

2/6

3/3

3/3

6/6

5/6

Thymus involution -/0 -/0 -/0 -/0 -/0 -/0 1/1 3/3 2/2 -/0

Prostate colloid absent or reduced

-/0 - -/0 - -/0 - -/0 - 2/2 -

Seminal veshicles reduced colloid -/0 - -/0 - -/0 - -/0 - 1/2 -

Testes atrophy -/0 - -/0 - -/0 - -/0 - 2/2 -

Ovaries no corpora lutea - 0/2 - 0/1 - 0/1 - 2/5 - -/0

Uterus reduced mural thickness

- -/0 - 0/2 - -/0 - 5/5 - -/0

a number of animals affected/total number of animals




Short-term repeated dose toxicity (28 days) Oral 28-day dietary toxicity study in dogs

1 REFERENCE

Officialuse only

1.1 Reference Moore, M. R. (2000); 4-week dietary toxicity study with TI-435 in dogs; Covance Laboratories Inc., Virginia 22182-1699, USA; unpublished report number 6155-106, 01.02.2000




None




2.1 Guideline study Yes 88/302/EEC (method B27), EPA FIFRA Subdivision F, section 82-1 (1984)

2.2 GLP Yes

2.3 Deviations



3.1.1 Lot/Batch number

3.1.2 Specification


3.1.2.2 Purity


3.2 Test Animals 3 male and 3 female beagle dogs per group (7 months old, weight range 7.7–11.2 kg, supplied by Covance Research Products Inc., USA)


Oral (dietary)


At least 28 days (the surviving animals in the highest dose group were sacrificed after 3 weeks of treatment as an interim sacrifice group)


Daily


None





3.3.4 Oral


3.3.4.2 Concentration 0, 1250, 2500 and 5000 ppm corresponding to 0, 34.3, 36.9 and 62.4 mg/kg bw/day for males and 0, 35.8, 53.5 and 57.4 mg/kg bw/day for females food consumption per day: ad libitum



-


-


3.4 Examinations

3.4.1 Observations

3.4.1.1 Clinical signs yes (daily), thorough clinical examination was performed weekly



3.4.3 Food consumption yes (daily)

3.4.4 Water consumption No


Yes (all animals at pre-test and on all survivors during week 4)

3.4.6 Haematology yes, all animals prior to treatment and during week 3 and 4 Parameters: Haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, blood cell morphology

3.4.7 Clinical Chemistry yes, all animals prior to treatment and during week 3 and 4 Parameters: sodium, potassium, calcium, inorganic phosphorous, chloride, glucose, cholesterol, blood urea nitrogen, total bilirubin, creatinine, total protein, albumin, globulin, albumin/globulin ratio, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, triglycerides

3.4.8 Urinalysis yes all animals prior to treatment and during week 5 Parameters: appearance, specific gravity, protein, glucose, blood, ketones, total bilirubin, sediment


3.5.1 Organ Weights yes organs: liver (with drained gall bladder), kidneys, adrenals, testes with epididymis, ovaries, brain, thyroid (with parathyroid)






Yes gross pathology: all animals histopathology: all surviving animals (interim and terminal sacrifice) organs: brain, spinal cord, pituitary, thyroid, parathyroid, thymus, oesophagus, mandibular salivary gland, stomach, small and large intestines, liver, pancreas, kidney, adrenals, spleen, heart, trachea, lung, aorta, gonads, uterus, female mammary gland, prostate, urinary bladder, gall bladder, lymph nodes, peripheral nerve, bone marrow, skin, lesions, skeletal muscle, vagina


3.5.4 Statistics Where appropriate: Bartlett’s test for heterogeneity of variance between groups followed by one-way analysis of variance on homogeneous or transformed data followed by Dunnetts’ t-test for multiple comparisons between treated and control groups at 1% and 5% probability

3.6 Further remarks


4.1 Observations

4.1.1 Clinical signs One male treated at 5000 ppm died and one 2500 ppm treated male were killed in a moribund condition on day 18. On day 19, a male and a female at 5000 ppm were moribund and also killed. The remaining 3 animals at 5000 ppm were killed on day 22 due to deteriorating condition (interim sacrifice). A female at 2500 ppm was killed on day 32, one day prior to scheduled necropsy. All other animals survived the designated treatment period. Treatment-related clinical signs were limited to the 2500 and 5000 ppm treated groups. They occurred first on day 18 and progressed rapidly. Prior to death or unscheduled sacrifice, animals showed clinical signs in various combinations of prostration or hunched posture, discoloured and mucoid faeces, salivation, dyspnea or polypnea, sanguinous nasal discharge, thin appearance, pale gums and dehydration.

4.1.2 Mortality

4.2 Body weight gain Males at 2500 ppm and both sexes at 5000 ppm lost weight and females at 2500 ppm showed markedly reduced weight gain, throughout the study or until termination (see Table A6_3_1/02-1). Although the group treated at 1250 ppm showed a slightly reduced weight gain, it was not statistically significant (variation of individual values comparable to controls) and occurred in the absence of other treatment-related effects. Therefore it is considered to be non-adverse.


Food consumption was markedly reduced throughout the study for both sexes treated at 5000 ppm and for males at 2500 ppm (see table A6_3_1/02-1). Females treated at 2500 ppm showed reduced food consumption during the first 2 weeks of treatment only. The food consumption at 1250 ppm was marginally lower than controls but was considered not to be an adverse effect of treatment. It was not accompanied by any relevant effect on bw development. The fact that food consumption was lower than controls already at the first day of the study at ≥2500 ppm and in 1250 ppm treated females indicates that low palatability played a role in the observed reductions in food consumption. Compound intake see 3.3.4.2. Chemical analysis of test diets indicated adequate stability and homogeneity in the range 1000–5000 ppm. After





10 days storage at room temperature, TI-435 concentrations were within 4–5% of nominal concentrations. Homogeneity was within 3.5–3.7% of target concentrations for the dose range 50–5000 ppm. Achieved concentrations for representative diets prepared in weeks 1 and 4 were within 7% of nominal concentrations.


There were no treatment-related ophthalmological findings at any dose level.

4.5 Blood analysis Treatment-related effects on the haematological and blood chemistry profiles were limited to the 2500 and 5000 ppm dose groups.

4.5.1 Haematology Decreased red and white blood cell counts (RBC and WBC), haemoglobin (Hb) concentration, haematocrit, segmented neutrophil, lymphocyte counts and platelet count were apparent at the week 3 and/or week 4 sampling interval (see table A6_3_1/02-1).

4.5.2 Clinical chemistry Reduced plasma concentrations of glucose (5000 ppm males only), alanine aminotransferase (ALT) activity, total protein, albumin, globulin, inorganic phosphorus (I/phos) and chloride ion were apparent at 2500 and/or 5000 ppm, and increased plasma alkaline phosphatase activity, total cholesterol and triglyceride concentrations were also primarily apparent at 5000 ppm (see table A6_3_1/02-1).

4.5.3 Urinalysis There were no effects on urine constituents at any dose level.


4.6.1 Organ weights There were no treatment-related effects on absolute and relative organ weights at any dose level, other than generally lower weights at 5000 ppm reflecting depressed weight gain.


Four of the 5 animals treated at ≥2500 ppm that died showed mottled lungs at necropsy with histological evidence of congestion, haemorrhage or oedema in 3 animals. However, these findings are considered to be a consequence of opportunistic infection that developed in the presence of declining health of the animals and treatment-related depression of the lymphoreticular/hematopoietic system. None of the other macroscopic findings in these animals had histological correlates. There were no treatment-related macroscopic findings in the survivors at ≥2500 ppm or in any animal treated at 1250 ppm. Treatment-related histomorphological changes were confined to animals treated at ≥2500 ppm (see table A6_3_1/02-2). Depression of haematopoietic/ lymphoid tissues occurred at both dose levels and was manifested as diffuse hypocellularity of bone marrow with increased amounts of fat, congestion of marrow sinusoid, haemorrhage in sternal and femoral bone marrow and depletion of lymphoid cells in the thymus, spleen and mesenteric lymph nodes. A subtle increase in the incidence and severity of duodenal crypt gland dilatation accompanied by some necrotic epithelial cells within the gland occurred at ≥2500 ppm.

4.7 Other -



Toxicity evaluation after short-term (28 day) dietary exposure of dogs to TI-435; no relevant deviation from guidelines (88/302/EEC B27, EPA





FIFRA 82-1)


Reduced food consumption and body weight development at ≥2500 ppm leading to reduced survival accompanied by clinical signs. The marginally lower food consumption at 1250 ppm was considered to be not adverse in absence of any relevant effect on bw development or any other treatment-related effect. Lower red blood cell parameters and reduced white blood cell counts among the surviving animals ≥2500 ppm. Slight blood biochemistry perturbations at 2500 and/or 5000 ppm: reduced glucose, protein, albumin, globulin values, (marginally lower chloride and inorganic phosphorus), and increased alkaline phosphatase, cholesterol and triglycerides levels. No relevant effects on organ weights (slight reductions of organ weight due to effect on body weight); histopathological effects in the haematopoietic/lymphoid tissues and duodenum at ≥2500 ppm.

5.3 Conclusion Dose levels of 2500 and 5000 ppm exceeded the maximum tolerated dose level. Effects of treatment comprised clinical signs of toxicity, weight loss, haematological and blood biochemistry perturbations, and histomorphological lesions in haematopoietic/lymphoid tissues and duodenum.

5.3.1 LO(A)EL LOAEL = 2500 ppm corresponding to 36.9 and 53.5 mg/kg bw/day for males and females, respectively Effects at LOAEL: clinical signs of toxicity, weight loss, haematological and blood biochemistry perturbations, and histomorphological lesions in haematopoietic/lymphoid tissues and duodenum.

5.3.2 NO(A)EL NOAEL = 1250 ppm corresponding to 34.3 and 35.8 mg/kg bw/day for males and females, respectively

5.3.3 Other Remark: For males the NOAEL expressed in mg/kg bw/day is only slightly lower than the LOAEL. The latter is due to the markedly reduced food consumption at the LOAEL (2500 ppm) leading to an in total only slightly higher test article intake than at the NOAEL with normal food consumption at 1250 ppm. Nevertheless the NOAEL at 34.3/35.8 mg/kg bw/day for males/females is considered valid.

5.3.4 Reliability 1





Date 2006-10-23

Materials and Methods The applicants version is accepted. The applicant has suggested that the dose levels given in “3.3.4.2. Concentration” should correspond with the mean values for 1-28 days for groups 1-3 and for 1-15 days for group 4 (died or sacrificed prior to termination). Therefore, 0, 36.3, 35.8 and 62.4 mg/kg/day in the males and 0, 35.6, 52.3 and 57.4 mg/kg/day in the females seems to be appropriate.





Results and discussion Applicant’s version is acceptable with the remark that concerning the red blood cell parameters the reported decrease at 5000 ppm bases on only one surviving male dog. The decrease at 2500 ppm in females is not reflected in the female dose group of 5000 ppm. Considering this a substance related effect on red blood cell parameters is doubtful. Also the described effect on glucose and inorganic phosphorus bases on the blood sample of one animal which is not considered to be representative in general.

Conclusion LO(A)EL: 1250 ppm in male and female NO(A)EL: 2500 ppm in male and female

Reliability 1


Remarks








Remarks



Table A6_3_1/02-1. Bw development and food consumption, results of clinical chemistry and haematology (28-day dog)

Parameter [Unit] 0 ppm 1250 ppm 2500 ppm 5000 ppm ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀

Cumulative bw gain [kg] week 1-2 week 1-3 week 1-4 week 1-5

0.7 0.5 0.8 0.8

0.7 0.7 0.7 0.7

0.4 0.3 0.6 0.6

0.5 0.2 0.3 0.2

-0.1* -0.4* -0.7 -0.7

0.1** 0.0* 0.1 0.1

-0.4** -0.8** -1.7

-

-0.2** -0.8** -1.3

-

Total food consumption [kg] week 1 week 1-2 week 1-3 week 1-4

2.46 4.32 6.19 8.01

2.21 3.80 5.40 7.08

2.43 4.20 5.96 7.65

1.74 3.00*4.36 5.79

1.17** 2.21** 3.11** 4.25*

1.52* 2.82* 4.34 5.81

1.02** 1.76**

- -

0.95** 1.63**

- -

week 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4

RBC (106/μL) (↓) (↓) Hb (g/dL) (↓) (↓) (↓) PCV (%) (↓) (↓) (↓) WBC (103/μL) (↓) (↓) (↓) (↓) (↓) (↓) (↓) (↓) Seg neut (103/μL) (↓) (↓) (↓) (↓) (↓) (↓) (↓) (↓) Lympho. (103/μL) (↓) (↓) (↓) (↓) (↓) (↓) (↓) (↓) Platelets (103/μL) (↓) (↓) (↓) (↓) (↓) Glucose (mg/dL) (↓) (↓) ALT (U/L) (↓) (↓) ↓ ↓ (↓) (↓) (↓) (↓) Total prot. (g/dL) ↓ (↓) ↓ I/phos (mg/dL) (↓) Cl- (mEq/L) (↓)

-2.6% (↓) -6.2%

(↓) -2.7%

AP (U/L) (↑) (↑) (↑) Cholest. (mg/dL) (↑) (↑) (↑) Triglyc. (mg/dL) (↑) (↑) Albumin (g/dL) ↓ (↓) (↓) (↓) Globulin (g/dL) (↓) (↓) (↓)

* p <0.05, ** p <0.01; arrows in brackets (↓↑) indicate a trend that was not statistically significant Seg neut = segmented neutrophils, Lympho. = lymphocytes, prot. = protein, Cholest. = cholesterol, Triglyc. = triglycerides



Table A6_3_1/02-2. Results of repeated dose toxicity study (28-day dog)

Parameter 0 ppm 1250 ppm 2500 ppm 5000 ppm dose-response

+/-

ma fa ma fa ma fa ma fa m f

Duodenum glandular epithelial necrosis dilated crypt gland

0/3

1/3

0/3

1/3

1/3

1/3

0/3

0/3

1/3

1/3

0/3

2/3

0/3

2/3

3/3

3/3

Lymph node lymphoid depletion 0/3 0/3 0/3 0/3 0/3 0/3 2/3 2/3

Spleen lymphoid depletion 0/3 0/3 0/3 0/3 1/3 2/3 2/3 3/3

Thymus lymphoid depletion 0/3 0/3 0/3 0/3 1/3 2/3 3/3 2/3

Sternal marrow hypocellular haemorrhage

0/3 0/3

0/3 0/3

0/3 0/3

0/3 0/3

1/3 1/3

2/3 1/3

2/3 2/3

3/3 2/3

Femoral marrow hypocellular haemorrhage

0/3 0/3

0/3 0/3

0/3 0/3

0/3 0/3

2/3 1/3

2/3 1/3

2/3 2/3

3/3 2/3

a number of animals affected/total number of animals




Subchronic dermal toxicity Rat 28-day dermal toxicity study in rats

1 REFERENCE

Officialuse only

1.1 Reference Weiler, M. S. (2000); 28-day dermal toxicity study with TI-435 in rats Covance Laboratories Inc., Wisconsin 53704-2595, USA unpublished report number 6155-120, 13.10.2000




None




2.1 Guideline study Yes 92/69/EEC (method B9), OECD no. 410 (1981), US EPA-OPPTS 870.3200 (August 1998), Japan MAFF (59 NohSan No. 4200, 1985)

2.2 GLP Yes

2.3 Deviations No






3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study (prepared daily)

3.2 Test Animals 10 male and 10 female 8 week old rats per group (Crl:CD[SD]IGS BR strain, weight range 171 – 317 g, Charles River Laboratories Inc., USA)


dermal


28 days


daily


none





3.3.4 Dermal

3.3.4.1 Area covered 10% of body surface (5 x 5 cm2)

3.3.4.2 Occlusion occlusive

3.3.4.3 Vehicle deionised water (reverse osmosis water)


Application site was wetted with deionised water before the solid test material was applied and covered with a gauze pad moistened with another 1.0 mL of deionised water


See 3.3.4.4 Dose levels: 0, 100, 300 and 1000 mg/kg bw/day

3.3.4.6 Duration of exposure

6 h per day

3.3.4.7 Removal of test substance

Water-moistened tissue

3.3.4.8 Controls deionised water

3.4 Examinations

3.4.1 Observations







Yes (all animals at pre-test and during week 4)

3.4.6 Haematology yes, all animals at termination (week 5) Parameters: Haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, prothrombin time, activated partial thromboplastin time, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, blood cell morphology, reticulocyte count

3.4.7 Clinical Chemistry yes, all animals at termination (week 5) Parameters: sodium, potassium, calcium, chloride, inorganic phosphorus, magnesium, glucose, total cholesterol, blood urea nitrogen, total bilirubin, creatinine, total protein, albumin, globulin, albumin/globulin ratio, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transpeptidase, sorbitol dehydrogenase, creatine kinase

3.4.8 Urinalysis no


3.5.1 Organ Weights yes organs: liver, kidneys, adrenals, testes, epididymis, uterus, ovaries, thymus, spleen, brain, heart

3.5.2 Gross and yes





histopathology gross pathology: all dose groups histopathology: all control and high dose animals (treated and untreated skin from the mid-dose groups also); all animals that died during the study; organs: adrenals, aorta, bone and bone marrow (femur and sternum), brain, cecum, colon, duodenum, epididymis, esophagus, eyes with potic nerve, gross lesions, heart, ilium, jejunum, kidneys, larynx, liver, lungs, female maammary gland, lymph nodes, muscle, nasal turbinates, ovaries, pancreas, pharynx, pituitary, prostate, rectum, salivary glands, sciatic nerve, seminal vesicle, skin (treated and untreated), spinal cord, spleen, stomach, testis, thymus, thyroid with parathyroid, trachea, urinary bladder, uterus

3.5.3 Other examinations Observations for neurobehavioural function (hand-held and open-field observations, sensory reactivity to stimuli, grip strength) and motor activity measurement (by photocell recording at 2-minute intervals for 40 minutes) were performed blind, on all animals immediately before dosing during week 4. Dermal irritation reactions were scored daily according to the modified Draize scale.

3.5.4 Statistics Where appropriate: Levene’s test for heterogeneity of variance between groups followed by one-way analysis of variance on homogeneous or transformed data followed by Dunnett’s t test for significant ANOVA.



4.1 Observations

4.1.1 Clinical signs There were no treatment-related deaths during the study although one female at 100 mg/kg bw/day and one female at 1000 mg/kg bw/day died on day 15. The latter animal had the collar caught in the cage and its death is considered not to be treatment-related. Neither decedent showed clinical signs of a reaction to treatment or treatment-related macroscopic and microscopic lesions. All other animals survived the treatment period and none showed clinical signs of a reaction to treatment. There was no treatment-related dermal irritation at the application site at any dose level.

4.1.2 Mortality See above

4.2 Body weight gain The study director discussed that there were no treatment-related effects on weight gain in either sex at any dose level. A reduced weight gain during week 1 was seen in 1000 mg/kg bw/day treated males, but thereafter weight gains were comparable to all other groups (see table 6_3_2/01-1). Therefore the initial lower weight gain was considered incidental to treatment.


There were no consistent treatment-related effects on food consumption at any dose level.


No treatment-related ocular lesions occurred at any dose level.

4.5 Blood analysis There were no treatment-related effects at any dose level on haematological and blood biochemistry profiles.






There were no treatment-related effects at any dose level on macroscopic pathology, organ weights and ratios, and no histomorphological lesions in any tissue at 1000 mg/kg bw/day.

4.7 Other There were no treatment-related effects at any dose level on qualitative or quantitative neurobehavioural function tests and quantitative motor activity.



Toxicity evaluation after 28-day dermal exposure of rats to TI-435; no relevant deviation from guidelines (92/69/EEC B9, OECD 410, US EPA-OPPTS 870.3200, Japan MAFF)


Beside a slight reduction in initial bw gain in 1000 mg/kg bw/day treated males, there was no relevant treatment-related effect. The study author considered the variations in week 1 bw gain to be unrelated to treatment, as the bw gain was comparable to the other groups for the later three weeks of the study and in absence of any similar changes in females.

5.3 Conclusion TI-435 was well-tolerated upon dermal application for 28 days. There were no infications for skin irritation.

5.3.1 LO(A)EL -

5.3.2 NO(A)EL 1000 mg/kg bw/day as discussed above, in absence of a similar effect in females and as bw development was normal for weeks 2-4, the lower bw gain in the 1st week of treatment in males was considered to be unrelated to treatment

5.3.3 Other -

5.3.4 Reliability 1





Date 2004-10-07

Materials and Methods The applicants version is accepted.

Results and discussion Applicant’s version is acceptable with the remark that the pathological findings of the female (100 mg/kg bw/day) which died on day 15 have been enlarged liver with dark brown lobes, dark red ovaries, kidney mineralisation, thyroid cyst and uterus dilatation. These findings are not considered to be substance related. The RMS takes the view of the applicant concerning the reduced weight gain in 1000 mg/kg bw/day in males. The body weight of the male rats in the high dose group after 28 days is about 6% lower than in the control group which is not considered to be relevant.

Conclusion LO(A)EL: not detectable NO(A)EL: > 1000 mg/kg BW Applicant's version is adopted





Reliability 1


Remarks CA-Table A6_3_2/01-1 is added by CA








Remarks

Table A6_3_2/01-1 Bw development (28-day dermal, rat)

Parameter [Unit] Males Females mg/kg bw/day 0 100 300 1000 0 100 300 1000

Bw gain [g] week 1 week 2 week 3 week 4 week 1-4°

18 34 32 17 101

18 35 27 13 92

18 26 28 16 87

7* ↓ 29 28 16

80* ↓

7 17 12 9 45

8 18 13 6 44

9 22 8 6 45

7 20 7 10 42

* p <0.05 ° values may not calculate to sum of weekly values due to rounding and the death of one female each in the low and high dose group



Evaluation by Rapporteur Member State, CA-Tables CA-Table A6_3_2/01-1 Summary of body weight data (g)



Section A6.3.3 Annex Point IIA VI.6.3.3

Short-term repeated dose toxicity (28 days) Inhalation



Detailed justification: relevant inhalation exposure is

considered unlikely for humans.





Date 2006-10-24


Conclusion Applicant's justification is accepted

Remarks






Remarks




Subchronic oral toxicity test (90 days) Dog 90-day dietary toxicity study in dogs

1 REFERENCE

Officialuse only

1.1 Reference Bernier, L. (2000a); 13-week dietary toxicity study with TI-435 in dogs; Covance Laboratories Inc., Virginia 22182-1699, USA; unpublished report number 6155-111, 14.03.2000




None




2.1 Guideline study Yes 2001/59/EC (method B27), EPA FIFRA Subdivision F (section 82-1, 1984), OECD guideline no. 409 (1981), Japan MAFF (59 NohSan No. 4200, 1985), US EPA OPPTS 870.3150

2.2 GLP Yes

2.3 Deviations No





3.1.2.1 Description Paleyellow powder

3.1.2.2 Purity


3.2 Test Animals 4 male and 4 female Beagle dogs per group (5-6 months old, weight range 7.3-11.4 kg, supplied by Covance Research Products Inc., USA)


Oral (dietary)


at least 13 weeks


Daily


None





3.3.4 Oral


3.3.4.2 Concentration 0, 325, 650, 1500 and 2250 ppm corresponding to 0, 9.2, 19.3, 40.9 and 58.2 mg/kg bw/day for males and 0, 9.6, 21.2, 42.1 and 61.8 mg/kg bw/day for females food consumption per day: ad libitum



-


-


3.4 Examinations

3.4.1 Observations








3.4.6 Haematology yes, all animals prior to treatment and during week 5 and 13 (in addition complete blood count with differential was performed in week 9) Parameters: Haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, blood cell morphology, prothombin time, activated partial thromboplastin time, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration

3.4.7 Clinical Chemistry yes, all animals prior to treatment and during week 5 and 13 Parameters: sodium, potassium, calcium, inorganic phosphorous, chloride, glucose, cholesterol, blood urea nitrogen, total bilirubin, creatinine, total protein, albumin, globulin, albumin/globulin ratio, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyltransferase

3.4.8 Urinalysis Yes all animals prior to treatment and during week 5 and 13 Parameters: appearance, volume, pH, specific gravity, protein, glucose, blood, ketones, total bilirubin, sediment


3.5.1 Organ Weights Yes organs: liver (with drained gall bladder), kidneys, adrenals, testes with epididymis, ovaries, brain, thyroid (with parathyroid), lung, heart, uterus, thymus, spleen






Yes gross pathology: all animals histopathology: all animals organs: brain, spinal cord, pituitary, thyroid, parathyroid, thymus, oesophagus, mandibular salivary gland, stomach, small and large intestines, liver, pancreas, kidney, adrenals, spleen, heart, trachea, lung, aorta, gonads, uterus, female mammary gland, prostate, urinary bladder, gall bladder, lymph nodes, peripheral nerve, bone marrow, skin, lesions, skeletal muscle, cervix and vagina, bone, eye


3.5.4 Statistics Where appropriate: Bartlett’s test for heterogeneity of variance between groups followed by one-way analysis of variance on homogeneous or transformed data followed by Dunnetts’ t-test for multiple comparisons between treated and control groups at 1% and 5% probability

3.6 Further remarks Neurological examinations, including observation of gait and tests of reflexes and nociception, were performed pre-test and during weeks 1, 5 and 13.


4.1 Observations

4.1.1 Clinical signs There were no deaths during the study but 3 animals treated at 1500 ppm and 5 animals at 2250 ppm appeared thin during the latter part of the treatment period. All other clinical observations were considered to be unrelated to treatment. One 2250 ppm treated female had a recurrent large swollen mass on the right shoulder leading to non-use of the right forelimb. The popliteal lymph nodes were drained of a purulent material and a clinical diagnosis of non-treatment-related lymphadenitis was made. Antibiotic therapy was administered and by day 68 the animal was returning to normal.

4.1.2 Mortality None

4.2 Body weight gain The overall weight gain of males treated at 2250 ppm showed a treatment-related depression of 72.7%, which correlated with the clinical observation of thinness (see table A6_4_1/01-1). There was no clear effect of treatment on the weight gain of females treated at 2250 ppm and of both sexes treated at lower dose levels. The reduced overall weight gain of males treated at 1500 ppm was due to weight loss during the second half of the treatment period in one animal. It is therefore considered unrelated to treatment.


The food consumption was reduced at 2250 ppm during the first 5 weeks of the study, but approached control levels thereafter (see table A6_4_1/01-1). There was no effect of treatment on the food consumption at ≤1500 ppm. Compound intake see 3.3.4.2. Chemical analysis of diets indicated adequate stability, homogeneity and achieved concentrations. The test diets were stable at room temperature for 14 days and homogeneity analyses showed all samples from all concentrations to be within 10% of nominal concentration. Analysis of all diets prepared for weeks 1 and 13 were within 7.7% of nominal concentrations.


The neurological and ophthalmological examinations did not reveal any treatment-related effects in either sex at any dose level.





4.5 Blood analysis

4.5.1 Haematology Reduced white blood cell counts (WBC) due to reduced neutrophil and lymphocyte counts were recorded at 2250 ppm. A marked reduction in circulating platelets and panleukopenia in a male treated at 1500 ppm and slight anaemia in a male at 2250 ppm are considered to have an equivocal relationship to treatment.

4.5.2 Clinical chemistry Reduced alanine aminotransferase (ALT) activity were seen at ≥1500 ppm, and reduced total protein levels in females at 1500 ppm and both sexes at 2250 ppm. The latter was mainly due to reduced albumin levels at ≥1500 ppm.

4.5.3 Urinalysis Qualitative and quantitative urinalysis revealed no treatment-related effects at any dose level.


There were no treatment-related effects on organ weights and ratios or on the nature and incidence of macroscopic lesions at necropsy in either sex at any dose level. Histomorphological findings were consistent with commonly occurring lesions of spontaneous etiology and none was considered to be treatment-related in either sex at any dose level.



Toxicity evaluation after subchronic (90 day) dietary exposure of dogs to TI-435; no relevant deviation from guidelines (2001/59/EC B27, EPA FIFRA 82-1, OECD 409, Japan MAFF, US EPA OPPTS 870.3150)


There was an increased incidence of thinness, slight decreases in serum albumin and total protein concentrations and reduced alanine aminotransferase activity at ≥1500 ppm. In addition reduced weight gain in males and reduced food consumption and white blood cell counts were recorded in both sexes at 2250 ppm.

5.3 Conclusion Effects of treatment comprised clinical signs of toxicity, reduced bw development, haematological and blood biochemistry perturbations but no histomorphological lesions.

5.3.1 LO(A)EL LO(A)EL = 1500 ppm corresponding to 40.9 and 42.1 mg/kg bw/day for males and females, respectively Effects at LOAEL: clinical signs of toxicity, slight haematological and blood biochemistry perturbations

5.3.2 NO(A)EL NO(A)EL = 650 ppm corresponding to 19.3 and 21.2 mg/kg bw/day for males and females, respectively

5.3.3 Other -

5.3.4 Reliability 1





Date 2004-10-07





Materials and Methods The applicants version is accepted

Results and discussion Increased incidence of thinness, slight decreases in serum albumin and total protein concentrations and reduced alanine aminotransferase activity at ≥1500 ppm were observed. In addition reduced weight gain (males), reduced food consumption and white blood cell counts were recorded in both sexes at 2250 ppm. Lymphoid intestinal hyperplasia was observed in male at 650ppm and above and in female at 1500 ppm and above.

Conclusion LO(A)EL: 1500 ppm (based on decrease of WBC parameters and protein) NO(A)EL: 650 ppm

Reliability 1


Remarks At week 7 some data of body weight, mortality check, environmental conditions, clinical observation and food consumption were lost due to computer malfunction








Remarks



(u/L)

(g/dL)

(g/dL)




Subchronic oral toxicity test (1-year) Dog 1-year dietary toxicity study in dogs

1 REFERENCE

Officialuse only

1.1 Reference Bernier, L. (2000b); 52-week dietary chronic toxicity study with TI-435 in dogs; Covance Laboratories Inc., Virginia 22182-1699, USA; unpublished report number 6155-113, 22.03.2000




None




2.1 Guideline study Yes 87/302/EEC (method B30), EPA FIFRA Subdivision F (section 83-1, 1984), OECD guideline no. 452 (1981), Japan MAFF (59 NohSan No. 4200, 1985), US EPA OPPTS 870.4100 (1998)

2.2 GLP Yes

2.3 Deviations No






3.1.2.2 Purity


3.2 Test Animals 4 male and 4 female beagle dogs per group (6 – 7 months old, weight range 8.5 – 12.8 kg, supplied by Covance Research Products Inc., USA)


Oral (dietary)


52 weeks


Daily


None





3.3.4 Oral


3.3.4.2 Concentration 0, 325, 650, 1500 and 2000 ppm corresponding to 0, 7.8, 16.6, 36.3 and 46.4 mg/kg bw/day for males and 0, 8.5, 15.0, 40.1 and 52.9 mg/kg bw/day for females food consumption per day: ad libitum



-


-


3.4 Examinations

3.4.1 Observations



3.4.2 Body weight yes (weekly for the first 13 weeks, every 4 weeks thereafter and at week 53)

3.4.3 Food consumption Yes (weekly for the first 13 weeks, every 4 weeks thereafter and at week 52)




3.4.6 Haematology yes, all animals prior to treatment and during week 5, 13, 15 (2000 ppm treated male No. G35518 only), 26, 39 and 52 Parameters: Haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, blood cell morphology, prothombin time, activated partial thromboplastin time, reticulocyte count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration

3.4.7 Clinical Chemistry yes, all animals prior to treatment and during week 13, 26, 39 and 52 Parameters: sodium, potassium, calcium, inorganic phosphorous, chloride, glucose, cholesterol, blood urea nitrogen, total bilirubin, creatinine, total protein, albumin, globulin, albumin/globulin ratio, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyltransferase, creatine kinase, triglycerides

3.4.8 Urinalysis Yes all animals prior to treatment and during week 13, 26, 39 and 52 Parameters: appearance, volume, pH, specific gravity, protein, glucose, blood, ketones, bilirubin, sediment, urobilinogen


3.5.1 Organ Weights Yes organs: liver (with drained gall bladder), kidneys, adrenals, testes with





epididymis, ovaries, brain, thyroid (with parathyroid), lung, heart, uterus, spleen, pituitary, prostate


Yes gross pathology: all animals histopathology: all animals organs: adrenals, aorta, brain, caecum, cervix, colon, duodenum, esophagus, eyes with optic nerve, femur with bone marrow, heart, ileum, jejunum, kidney, lesions, liver with gall bladder, lung, lymph nodes, female mammary gland, ovaries, pancreas, peripheral nerve, pituitary, prostate, rectum, mandibular salivary gland, skeletal muscle, skin, spinal cord, spleen, sternum with bone marrow, stomach, testis with epididymis, thymus, thyroid with parathyroid, trachea, urinary bladder, uterus, vagina


3.5.4 Statistics Where appropriate: Levene’s test for homogeneity of variance between groups followed by one-way analysis of variance on homogeneous or transformed data followed by Dunnetts’ t-test for multiple comparisons between treated and control groups at 1% and 5% probability.

3.6 Further remarks Neurological examinations, including observation of posture and gait, changes in autonomic functions, reactivity to handling and the presence of tonic/clonic movements, were performed pre-test and during weeks 26, 38 and 53.


4.1 Observations

4.1.1 Clinical signs Redness of the skin inside the ears occurred in females treated at 1500 ppm and in males and females at 2000 ppm. Although this observation was considered to be treatment-related, it was not associated with any systemic change and did not adversely affect the animals. There were no other treatment-related clinical signs at any dose level, but one male at 2000 ppm was found to have a fractured tibia on day 30, which, after appropriate treatment, had healed by day 89.

4.1.2 Mortality None

4.2 Body weight gain A slight and transient treatment-related weight loss occurred in males at 2000 ppm during the first week of treatment, but thereafter weight gain and terminal group mean body weight were comparable to controls (see table A6_4_1/02-1). Females treated at 2000 ppm showed a slight decrease in overall weight gain during the study such that the group mean terminal body weight was 5.5% lower than controls. There was no treatment-related effect on weight gain at lower dose levels.


The food consumption of females treated at 2000 ppm was significantly reduced by 22.2% during the first week of treatment only (see table A6_4_1/02-1). The food consumption of males at 2000 ppm and of both sexes at lower dose levels was unaffected by treatment. Compound intake see 3.3.4.2. Analysis of test diets for stability and homogeneity had been performed for previous studies. Chemical analysis of diets indicated adequate achieved concentrations. Analysis of all diets prepared for weeks 1, 13, 26, 39 and 52 demonstrated they were within 8% of nominal concentrations.


The neurological and ophthalmological examinations did not reveal any treatment-related effects in either sex at any dose level.





4.5 Blood analysis

4.5.1 Haematology Treatment-related effects were confined to reduced total white blood cell and neutrophil counts at week 5 in animals of both sexes at 2000 ppm and in females only at week 39 (see table A6_4_1/02-1). Lower lymphocyte counts in week 5 in males treated at ≥650 ppm were considered incidental to treatment because lymphocyte counts had already been low prior to treatment.

4.5.2 Clinical chemistry Treatment-related effects on serum chemistry were confined to a dose-related decrease in serum alanine aminotransferase (ALT) activity in both sexes at 1500 and 2000 ppm (see table A6_4_1/02-1). ALT activity was also reduced on one occasion in both sexes at 650 ppm. The biological significance of reduced serum ALT activity is equivocal, and in the absence of correlating histomorphological changes in the liver, the effect on ALT activity is considered not to be an adverse effect.



There were no treatment-related effects on organ weights and ratios or on the nature and incidence of macroscopic lesions at necropsy in either sex at any dose level. Histomorphological findings were consistent with commonly occurring lesions of spontaneous etiology and none was considered to be treatment-related in either sex at any dose level.



Toxicity evaluation after subchronic (1-year) dietary exposure of dogs to TI-435; no relevant deviation from guidelines (87/302/EEC B30, EPA FIFRA 83-1, OECD 452, Japan MAFF, US EPA OPPTS 870.4100)


There was a transient or slightly reduced weight gain and reduced total white blood cell counts and neutrophil counts in both sexes at 2000 ppm. In absence of any other indication for liver damage any other toxicologically relevant effect, the slight decrease in serum ALT activity at ≥650ppm is considered to be non-adverse.

5.3 Conclusion Relevant effects of treatment comprised a reduced bw development and haematological perturbations but no histomorphological lesions.

5.3.1 LO(A)EL LOAEL = 2000 ppm corresponding to 46.4 and 52.9 mg/kg bw/day for males and females, respectively Effects at LOAEL: see 4.3

5.3.2 NO(A)EL NOEL = 650 ppm corresponding to 16.6 and 15.0 mg/kg bw/day for males and females, respectively NOAEL = 1500 ppm corresponding to 36.3 and 40.1 mg/kg bw/day for males and females, respectively

5.3.3 Other -

5.3.4 Reliability 1









Date 2004-10-07

Materials and Methods The applicants version is accepted.

Results and discussion The applicant's version is adopted with some additions. Slight decrease of red blood cell mass (Hct, Hb and RBC count) were observed in female at 2000 ppm. In both sexes at 2000 ppm transient or slightly reduced weight gain combined with reduced total white blood cell counts and neutrophil counts were observed. In absence of any other indication for liver damage any other toxicologically relevant effect, the slight decrease in serum ALT activity at ≥650ppm is considered to be non-adverse.

Conclusion LO(A)EL: 2000 ppm (based on decrease of white blood cells) NO(A)EL: 1500 ppm LOEL: 650 ppm (based on decrease in ALT activity) NOEL: 325 ppm

Reliability 1


Remarks








Remarks



Table A6_4_1/02-1. Bw development and food consumption, results of clinical chemistry and haematology (1-year dog)

Parameter [Unit] Males Females ppm 0 325 650 1500 2000 0 325 650 1500 2000

Body weight [kg] week 0 week 1 week 4 week 13 week 25 week 37 week 52

11.4 11.7 11.8 12.4 12.3 12.6 12.7

10.9 11.2 11.6 12.3 12.7 12.9 12.6

10.3 10.6 11.1 11.6 12.0 12.7 13.5

10.7 10.6 11.0 11.5 12.1 12.2 12.5

11.2 11.0 11.2 11.6 12.0 12.4 12.5

9.4 9.6 9.5 9.9

10.0 10.4 10.9

9.5 9.6 10.0 10.5 10.7 11.4 11.3

9.4 9.6 9.9

10.4 10.3 10.7 11.3

9.3 9.3 9.4

10.0 10.0 10.7 11.0

9.5 9.4 9.5 9.6

10.0 10.2

10.3 (↓)

Cumulative bw gain [kg] week 0-52 1.3 1.7 3.3 1.8 1.2 1.5 1.8 1.9 1.7 0.8 (↓)

Total food consumption [kg] week 1-2 week 1-53

1.8 43.4

2.1 44.6

2.0 47.8

1.9 44.7

1.6 44.0

1.8 43.7

1.8 46.4

1.7 38.2

1.8 40.9

1.4* ↓41.6

WBC (103/µL) week 0° week 5 week 13 week 26 week 39 week 52

10.6 10.9 10.9 10.0 9.0 8.9

11.4 10.5 10.6 10.2 9.8 9.9

9.6 10.4 9.4 9.8 8.7 9.6

10.0 9.8 9.4 10.8 10.0 10.7

10.8 7.2 (↓)

10.0 8.4 8.5 8.1

10.0 11.2 11.4 9.7 9.5 9.5

10.0 9.4 9.0 8.3 8.5 9.1

9.8 8.5 8.6 7.8 7.9

10.8

11.4 9.7 8.9 8.7 9.5 8.1

9.1 6.1* ↓

8.0 7.4

6.9* ↓7.4

Neutrophils (103/µL) week 0° week 5 week 13 week 26 week 39 week 52

6.0 5.8 6.3 5.7 4.9 5.0

7.5 5.9 6.5 6.1 6.3 6.4

5.7 5.9 5.4 5.3 4.6 5.1

6.4 6.4 6.0 7.1 6.7 7.1

6.7 3.9 (↓)

6.4 4.4 5.1 4.8

6.1 6.9 7.6 5.8 6.0 5.9

6.1 5.4 5.3 5.0 5.4 5.9

6.0 4.5 4.9 4.3 4.6 7.1

6.9 5.6 4.9 4.8 5.9 4.4

5.0 2.8 (↓)

4.8 4.0

3.6* ↓4.3

Lymphocytes (103/µL) week 0° week 5 week 13 week 26 week 39 week 52

3.7 4.2 3.4 3.3 3.2 2.8

2.9 3.8 3.2 3.2 2.7 2.8

2.8 3.2* 3.0 3.3 3.1 3.1

2.6 2.4* 2.5 2.8 2.5 2.6

3.0 2.7* 2.8 3.2 2.8 2.5

2.9 3.2 2.8 2.8 2.5 2.6

3.0 3.2 2.7 2.5 2.2 2.3

2.9 3.2 2.8 2.8 2.5 2.5

3.2 3.2 3.0 2.9 2.5 2.6

3.1 2.9 2.7 2.8 2.5 2.4

ALT (U/L) week 0° week 13 week 29 week 37 week 52

41 42 48 43 40

39 34 39 34 38

43 34

34* ↓34 34

37 27* ↓27* ↓25* ↓25* ↓

45 17* ↓18* ↓16* ↓18* ↓

38 38 41 41 37

33 30 35 30 32

40 26* ↓

37 29 28

38 21* ↓ 19* ↓ 20* ↓ 19* ↓

32 14* ↓13* ↓12* ↓10* ↓

* p <0.05, * p <0.01; arrows in brackets (↓↑) indicate a trend that was not statistically significant ° mean of two pre-dose values

Sumitomo Chemical Takeda Agro Co., Ltd. TI-435 June 2004



Subchronic oral toxicity test (90 days) Rat 90-day dietary toxicity study in rats

1 REFERENCE

Officialuse only

1.1 Reference Wahle , B. S. (2000); Technical grade TI-435: A subchronic toxicity testing study in the rat; Toxicology, Bayer Corporation, 17745 South Metcalf, USA unpublished report number 98-172-QO, 22.02.2000




None




2.1 Guideline study Yes 2001/59/EC (method B26), EPA FIFRA Subdivision F (section 82-1, 1984), OECD guideline no. 408, Japan MAFF (59 NohSan No. 4200)

2.2 GLP Yes

2.3 Deviations No






3.1.2.2 Purity


3.2 Test Animals 30 male and 30 female Sprague Dawley rats (8 weeks, supplied by Charles River Laboratories Inc., USA) in the control and high dose group; 15 male and 15 female rats in the low and intermediate dose groups


Oral (dietary)


13 weeks


Daily


for the control and high dose group 15 rats/sex/group were kept for a recovery period of 7 weeks





3.3.4 Oral


3.3.4.2 Concentration 0, 150, 500 and 3000 ppm corresponding to 0, 9.0, 27.9 and 202 mg/kg bw/day for males and 0, 10.9, 34.0 and 254 mg/kg bw/day for females food consumption per day: ad libitum

3.3.4.3 Vehicle Acetone/corn oil (to suspend the test article prior to mixing with diet)


-


-

3.3.4.6 Controls plain diet (with acetone/corn oil as treated diet)

3.4 Examinations

3.4.1 Observations

3.4.1.1 Clinical signs yes (daily), detailed clinical examination was performed weekly

3.4.1.2 Mortality yes (daily)





Yes (all animals at pre-test and prior to termination)

3.4.6 Haematology yes, all surviving animals after 13 weeks±1; in addition recovery animals in week 20±1. Parameters: Haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, blood cell morphology, prothrombin time, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, reticulocyte count, Heinz body count

3.4.7 Clinical Chemistry yes, all surviving animals after 13 weeks±1; in addition recovery animals in week 20±1. Parameters: sodium, potassium, calcium, inorganic phosphorous, chloride, magnesium, glucose, cholesterol, urea nitrogen, uric acid, total and direct bilirubin, creatinine, triglyceride, total protein, albumin, globulin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyltransferase, creatine kinase, lactate dehydrogenase, thyroxine, triiodothyronine, thyroid stimulating Hormone; hepatic enzymes (cytochrome P-450, N-demethylase, O-demethylase, ethoxyresorufin O-deethylase, pentoxyresorufin O-dealkylase)

3.4.8 Urinalysis Yes all surviving animals after 13 weeks±1; in addition recovery animals in week 20±1. Parameters: appearance, colour, clarity, volume, pH, specific gravity, protein, glucose, blood, ketones, bilirubin, urobilinogen, leukocytes, nitrite, sediment






3.5.1 Organ Weights Yes organs: liver, kidneys, adrenals, testes, epididymides, ovaries, brain, thyroid, lungs, heart, uterus, thymus, spleen


Yes gross pathology: all animals histopathology: control and high dose groups (not done for organs marked with *); in case of organs-effects also in low and intermediate dose groups organs: skin, mammary gland, bone, joint, skeletal muscle, skull, lungs, larynx, trachea, nasopharynx, heart, aorta, spleen, bone marrow, lymph nodes, thymus, liver, cecum, colon, rectum, small intenstine (duodenum, ileum, jejunum), stomach, oesophagus, stomach, pancreas, kidney, ovaries, uterus, testes, epididymides, prostate, preputial gland*, seminal vesicles, urinary bladder, cervix, vagina*, clitoral gland*, adrenals, thyroids, parathyroids, pituitary, brain, spinal cord, optic nerve, sciatic nerve, eyes, exorbital lacrimal gland*, harderian glands, Zymbal’s gland, salivary glands, gross lesions (all animals)

3.5.3 Other examinations Estrous cycle staging (females, during 3 weeks prior to sacrifice); females were sacrificed at Day 2 diestrus)

3.5.4 Statistics Where appropriate: Bartlett’s test for heterogeneity of variance between groups (p ≤0.001); for equal variances: one-way analysis of variance on homogeneous or transformed data followed by Dunnett’s test for multiple comparisons between treated and control groups at 1% and 5% probability; for unequal variances: Kruskal-Wallis ANOVA followed by Mann-Whitney-U test. Frequency data: Chi-square and/or Fisher exact test



4.1 Observations

4.1.1 Clinical signs There were no treatment-related clinical signs. No effect of treatment was noted on the mean number of complete oestrus cycles in the 3-week period prior to sacrifice in females.

4.1.2 Mortality The death of one 500 ppm treated male was considered to be unrelated to treatment.

4.2 Body weight gain The cumulative body weight gain during treatment was reduced at 3000 ppm by 22-32% in comparison to controls resulting in 11-12% lower absolute body weights at day 91 (see table A6_4_1/03-1). During the recovery period the high dose rats had a higher body weight gain than controls but the absolute body weights remained below control levels.


Food consumption was reduced in the first week of treatment at 3000 ppm. However no relevant effect of treatment was noted for the mean food consumption during treatment or recovery period (see table A6_4_1/03-1). Compound intake see 3.3.4.2. Chemical analysis of diets indicated adequate stability, homogeneity and achieved concentrations. The test diets were stable at room temperature and at 4°C for at least 7 and 28





days, respectively, and homogeneity analyses showed all samples from all concentrations to be within 10% of nominal concentration. Analysis of all diets prepared for weeks 1, 5, 9 and 14 were within 15% of nominal concentrations.


The ophthalmological examinations did not reveal any treatment-related effects in either sex at any dose level.

4.5 Blood analysis

4.5.1 Haematology There was no treatment-related effect on any haematological parameter.

4.5.2 Clinical chemistry Except a slight increase of liver enzymes (cytochrome P450, aminopyridine N-demethylase, p-niroanisole O demethylase in both sexes and ethoxyresorufin O-deethylase, pentoxyresorufin O-dealkylase in males), which was considered to indicate an adaptive response of the liver and no adverse effect of treatment, there were no treatment-related effects in any blood biochemistry parameter (see table A6_4_1/03-1).



There were no direct effects of treatment on organ weights. The occasionally slightly lower absolute and/or higher relative weight of some organs in the 3000 ppm treated group, was considered to be secondary to the lower body weight development in these animals. There were no treatment-related effects on the nature and incidence of macroscopic lesions at necropsy in either sex at any dose level. Beside increased incidence of pigmentation of the spleen in 3000 ppm treated males, there were no treatment-related histopathological findings (see table A6_4_1/03-1).



Toxicity evaluation after subchronic (90 day) dietary exposure of rats to TI-435; no relevant deviation from guidelines (2001/59/EC B26, EPA FIFRA 82-1, OECD 408, Japan MAFF)


The body weight development was reduced at 3000 ppm in both sexes. Lower food consumption was noted at the same dose in the first week of treatment. Slight increases in liver enzymes after treatment indicated an increased functional load of the liver, which was considered not to be adverse in absence of any indication of liver damage. An increased incidence of spleen pigmentation was noticed in high dose males. During the recovery period high dose animals gain more weight than controls. All other effects of treatment were completely reversible during the recovery period

5.3 Conclusion Effects of treatment comprised reduced bw development, indications for an increased functional load of the liver (in absence of liver damage) and pigmentation of the spleen in males.

5.3.1 LO(A)EL LO(A)EL = 3000 ppm corresponding to 202 and 254.2 mg/kg bw/day for males and females, respectively Effects at LOAEL: reduced body weight development (secondary changes in some absolute and/or relative organs weights), pigmentation of the spleen in males

5.3.2 NO(A)EL NO(A)EL = 500 ppm corresponding to 27.9 and 34.0 mg/kg bw/day for males and females, respectively

5.3.3 Other -





5.3.4 Reliability 1





Date 2004-10-07

Materials and Methods The applicants version is accepted

Results and discussion The applicant's version is adopted with some remarks. In the males at 3000 ppm some slight increase of RBC, Hb and Hct was noted as well as a prolongation of PT. This result was no more evident after recovery. In the report the finding was linked to slight dehydration of the animals. In contrast to the applicant's view the slight increase of N- and O-demethylase activities occurring in male at the top dose is considered to be a mild induction of enzyme activity by the substance in the liver. After recovery the increase was no more significant. The activities of EROD and PROD were increased in tissue homogenate in male in the top dose which let assume a induction of CPY1A1 and CYP2B1 activity in the liver. The CYP450 activities were determined on snap-frozen liver samples which may lead to less optimal results compared to fresh liver tissue.

Conclusion LO(A)EL:3000 ppm (based on body weight, body weight gain, increase of enzyme activity in liver and pigmentation of spleen) NO(A)EL: 500 ppm

Reliability 1


Remarks








Remarks



Table A6_4_1/03-1. Bw development and food consumption, results of clinical pathology and histopathology (90-day rat)

Parameter [Unit] 0 ppm 150 ppm 500 ppm 3000 ppm ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀

Body weight [g] day 0 day 7 day 28 day 91 day 91 (recovery) day 140 (recovery

280.1 330.5 439.7 560.1 593.1 606.2

192.6 212.9 257.6 308.3 329.7 331.0

281.4 337.2 452.3 585.3

- -

190.3 211.7 261.3 303.1

- -

278.5 337.3 443.8 565.9

- -

188.6 209.3 257.4 298.2

- -

280.2 307.8* ↓ 404.4* ↓ 499.8* ↓ 529.3* ↓

576.5

192.5 197.9* ↓234.9* ↓270.8* ↓ 282.4* ↓299.8*

Cumulative bw gain [g] day 0-28 day 0-91 day 0-35 (recovery)

159.6 280.0 13.1

65.0 115.7 1.3

171.0 303.9

-

71.1 112.8

-

165.9 287.4

-

68.7 109.6

-

124.2* ↓ 219.6 ↓ 47.2 ↑

42.4* ↓ 78.3 ↓ 17.4 ↑

Mean food consumption [g/rat/day] week 1 week 1-14 week 15-21 (recovery)

28.62 29.61 29.38

20.28 20.72 21.20

28.98 29.77

-

20.59 20.62

-

29.66 29.37

-

19.95 20.69

-

23.26* ↓ 28.15 30.21

15.95* ↓20.62 20.39

N-demethylase [nM/min/mg protein] week 14 week 21 (recovery)

2.43 2.42

0.75 1.03

2.23 -

0.85 -

2.51 -

0.82 -

3.21* ↑ 2.54

1.06 (↑) 0.86

O-demethylase [nM/min/mg protein] week 14 week 21 (recovery)

0.10 0.13

0.08 0.09

0.10 -

0.09 -

0.13 -

0.09 -

0.15* ↑ 0.13

0.11 0.10

Cytochrome P-450 [mM/mg protein] week 14 week 21 (recovery)

0.34 0.30

0.14 0.22

0.31 -

0.16 -

0.33 -

0.16 -

0.44 (↑) 0.35

0.20 (↑) 0.16

pentoxyresorufin O-dealkylase week 14 week 21 (recovery)

0.036 0.036

0.011 0.023

0.042 -

0.009 -

0.045 -

0.007 -

0.112* ↑ 0.049

0.007 0.012

ethoxyresorufin O-deethylase week 14 week 21 (recovery)

0.15 0.12

0.13 0.10

0.18 -

0.08 -

0.23 -

0.08 -

0.34* ↑ 0.15

0.07 0.05

Spleen pigmentation (grading) week 14 week 21 (recovery)

6/15 (1.0)

12/15 (1.1)

6/15 (1.0) -

8/15 (1.0) -

13/15# (1.3) 11/15 (1.6)

14/15 (1.3) 14/15 (2.1)

13/14 (1.2)

-

14/15 (1.3)

-

15/15 (1.7) 15/15 (2.2)

*# p <0.05




Subchronic oral toxicity test (90 days) Mouse 90-day dietary toxicity study in mice

1 REFERENCE

Officialuse only

1.1 Reference Chambers, P. R. (2000); TI-435 - Toxicity to mice by dietary administration for 13 weeks; Huntingdon Life Sciences Ltd., Huntingdon, Cambridgeshire, UK; unpublished draft-report number TDA 193/962813, 08.12.2000




None




2.1 Guideline study Yes 88/302/EEC B.7 (predecessor of 2001/59/EC B.26), OECD 408

2.2 GLP The study was intended to be performed according to GLP but not finalised as serious GLP-problems were found at the test facility, which were considered to compromise the quality of the data and the validity of results.

2.3 Deviations






3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study (homogeneity and stability in food were tested in an earlier study in this laboratory).

3.2 Test Animals 10 male and 10 female Crl: CD-1 (ICR) BR mice per group (about 6 weeks old, weight range 21-36 g, supplied by Charles River UK Ltd, Kent, UK)


Oral (dietary)


13 weeks


Daily


None

3.3.4 Type in food





3.3.5 Concentration 0, 100, 500, 1000 and 1500 ppm corresponding to 0, 16, 82, 160 and 263 mg/kg bw/day for males and 0, 22, 107, 207 and 329 mg/kg bw/day for females food consumption per day: ad libitum

3.3.6 Vehicle None (TI-435 was mixed with diet directly)


-


-

3.3.9 Controls plain diet

3.4 Examinations

3.4.1 Observations

3.4.1.1 Clinical signs yes (at least once daily), detailed clinical examination was performed on 5 days/week during the first 4 weeks of treatment and weekly thereafter






yes (all animals at pre-test and control and high dose animals during week 13)

3.4.6 Haematology yes, all animals during week 12 Parameters: Packed cell volume, haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, reticulocyte count, blood cell morphology, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration

3.4.7 Clinical Chemistry yes, all animals during week 13 Parameters: sodium, potassium, calcium, inorganic phosphorous, chloride, glucose, cholesterol, triglycerides, urea nitrogen, total bilirubin, creatinine, total protein, albumin, globulin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase

3.4.8 Urinalysis yes all animals during week 13 Parameters: volume, pH, specific gravity, protein, glucose, blood, ketones, urobilinogen, bile pigments, microscopic sediment, haem pigments, total reducing substances


3.5.1 Organ Weights yes organs: liver, kidneys, adrenals, testes, epididymides, ovaries, brain, thyroid (with parathyroid), lungs, heart, uterus, thymus, spleen, pituitary, prostate, seminal vesicles






yes gross pathology: all animals histopathology: all animals (organs marked with *) organs: brain*, spinal cord*, pituitary*, thyroid with parathyroid*, thymus*, oesophagus*, salivary gland*, stomach*, small and large intestines* (duodenum, jejunum, ileum, caecum, colon, rctum), liver*, pancreas*, kidneys*, adrenals*, spleen*, heart*, trachea*, lungs*, aorta*, gonads*, uterus*, mammary gland*, prostate*, urinary bladder*, gall bladder, lymph nodes*, peripheral nerve*, bone marrow*, skin*, lesions*, skeletal muscle*, cervix* and vagina*, bone*, eye*, seminal vesicles*, ovaries*, epididymides*, femur (with joint)*, Harderian gland, head, larynx, pharynx, tongue


3.5.4 Statistics Where appropriate: Bartlett’s test for heterogeneity of variance between groups followed by one-way analysis of variance on homogeneous or transformed data followed by Student’s t- test and Williams’ test. In case of heterogeneity of variance a Kruskal-Wallis analysis was performed followed by the non-parametric equivalents of the t test and Williams’ test. Macrospcopic and microscopic data were analysed by Fisher’s exact test where appropriate.



4.1 Observations

4.1.1 Clinical signs Vocalisation was observed at 1500 ppm from week 4 onwards among 4 males and 6 females. There were no treatment-related clinical findings up to 1000 ppm.

4.1.2 Mortality There were no treatment-related deaths during the study. However several animals died in weeks 12/13 due to the stress of blood sampling. In total these were 4 males of the 500 ppm group, 3 males of the 1000 ppm group, 2 males and 3 females of the 1500 ppm group.

4.2 Body weight gain The body weight development was reduced significantly at 1500 ppm (predominantly during the first 2 weeks of the study) (see Table A6_4_1/04-1). A trend for lower body weight gains was also observed at 1000 ppm. A marginally lower body weight development was noted during the first two weeks of the study at lower dose levels among the males. However this was considered unrelated to treatment as the overall body weight gain was comparable to controls (normal body weight gain during weeks 2-13).


Food consumption was slightly reduced in females at ≥500 ppm during the first two weeks of the study (see Table A6_4_1/04-1). No effect of treatment was observed in males at this period nor in any group during weeks 3-13. As the overall food consumption was not reduced for 500 ppm treated females and as it was not accompanied by a lower body weight development, the marginal reduction during the first two weeks is considered not adverse. Efficiency of food utilisation was reduced at ≥1000 ppm (see Table A6_4_1/04-1). Compound intake see 3.3.4.2. Chemical analysis of diets indicated that adequate concentrations were achieved. Diets prepared for weeks 1 and 11 were within 2.2% of nominal concentrations.






The ophthalmological examinations did not reveal any treatment-related effects in either sex.

4.5 Blood analysis

4.5.1 Haematology No treatment-related changes were observed. In absence of a dose-relationship and a change in females the marginally higher eosinophil count in males treated with 100 and 1500 ppm was considered fortuitous and of no toxicological relevance (see Table A6_4_1/04-1).

4.5.2 Clinical chemistry Blood urea nitrogen was decreased at 1500 ppm (see Table A6_4_1/04-1). Higher alkaline phophatase and aspartate aminotransferase values were noted for females at ≥1000 ppm and ≥100 ppm. Due to the only marginal degree of changes, in absence of a clear dose-relationship and as there were no further indications of a liver effect (organ weight, histoapthology), these are considered not to be adverse. Higher electrolyte values were also noted among females: sodium at ≥1000 ppm, phosphorous at ≥500 ppm and potassium at 1500 ppm. In addition protein was reduced at ≥500 ppm for females along with reduced globulin at 1500 ppm. These were considered not to be adverse due to their minor degree and in absence of a similar effect in males. Minor changes in glucose and bilirubin values among the treated males were considered to be without toxicological relevance as the changes were only slight, did not occur in females and were not dose-related.

4.5.3 Urinalysis A slightly lower pH for 1500 ppm treated males was considered to be of no toxicological relevance due to the only marginal degree of change and in absence of a similar effect in females (see Table A6_4_1/04-1).


Lower kidney weights were seen at ≥1000 ppm (see Table A6_4_1/04-2). Reduced kidney weights observed for males at <1000 ppm were considered to be unrelated to treatment as they were well within the range of historical controls (0.44-0.81 g, mean 0.629 g) and in absence of any histopathological changes. The apparent difference to the concurrent control was due to an extraordinary high value for the latter. As relative kidney weights were comparable to controls, the reduction in absolute weight might be secondary to the lower body weight development at ≥1000 ppm. Lung weights for 1500 ppm treated females were decreased in comparison to controls (see Table A6_4_1/04-2). Some further changes – increased relative brain and epididymis weight - achieving statistical significance were considered to be secondary to the reduced body weight development among the ≥1000 ppm treated males. A higher number of females with sparse corpora lutea and follicles in the ovaries were observed at 1500 ppm than in controls (see Table A6_4_1/04-2), which was considered to be related to the lower body weight development in this group. Increased prominence of uterine endometrial glands was also observed at 1500 ppm. The latter finding was considered to represent normal structures within the range of cyclical change in the female mouse reproductive tract with an indication of an exacerbation of the normal cyclical activity.







Toxicity evaluation after subchronic (90 day) dietary exposure of mice to TI-435; no relevant deviation from guidelines (88/302/EEC B.7 [predecessor of 2001/59/EC B.26], OECD 408) apparent. However the quality and validity of data was considered to be compromised due to serious GLP-problems at the test facility.


Body weight development and food consumption (females only) were reduced at ≥1000 ppm. Blood urea nitrogen was increased 1500 ppm. Further minor changes in electrolytes or liver enzymes were considered to be of no toxicological relevance in absence of any further indication of treatment-related changes in liver. Lung weights were reduced in females at 1500 ppm. Lower absolute kidney weights observed at ≥1000 ppm can be considered to be secondary to the reduced body weight development in these groups. A higher number of females with sparse corpora lutea was observed in the 1500 ppm treated females, which was also considered to be related to the reduced body weight development in this group. Occurrence of prominent endometrial glands was associated with an indication for an exacerbation of normal cyclical activity at 1500 ppm.

5.3 Conclusion Due to the GLP-problems in the laboratory, the results of this study are not considered relevant for the risk assessment of TI-435. Effects were mainly a reduced body weight development and food consumption at ≥1000 ppm accompanied by associated findings in organ weights and increased blood urea nitrogen (at 1500 ppm). For females reduced lung weights were seen at 1500 ppm. Also in high dose females there was an indication for exacerbation of normal cyclic activity. (For comparison no treatment-related changes in ovaries and uterus were detected in the 18-month mouse study up to the highest dose level of 1800 ppm.)

5.3.1 LO(A)EL LO(A)EL = 1000 ppm corresponding to 160 and 207 mg/kg bw/day for males and females, respectively Effects at LOAEL: reduced bw development and food consumption (females)

5.3.2 NO(A)EL NO(A)EL = 500 ppm corresponding to 82 and 107 mg/kg bw/day for males and females, respectively

5.3.3 Other -

5.3.4 Reliability

5.3.5 Deficiencies Yes. Due to GLP-problems occurring in the test facility at that time the study was performed, the quality and validity of data was considered to be compromised. Also the findings in the uterus and ovaries of the Huntingdon mouse study could not be reproduced in the 18-month mouse study (also Crl: CD-1 (ICR) mice) in another test facility. In addition a 90-day rat study was performed at the same test facility [Huntingdon] at about the same time as the 90-day mouse study and was not finalised for the same reasons. A repetition of this 90-day rat study in another laboratory with the same strain of rats also revealed that several effects observed in the Huntingdon study could not be reproduced.








Date 2006-11-27

Materials and Methods Achieved doses in males: 0-16-82-160 and 263 mg/kg bw during 90 d achieved doses in females: 0-22-107-207 and 329 mg/kg bw during 90 d.

Results and discussion A review of the individual clinical chemistry values shows that there are no consistent, relevant changes within or between treatment groups (see CA-Figure 1 for inorganic phosphate). Based on body weights and food conversion efficiency clear adverse effects are present in males and females at dose levels ≥ 1000 ppm. A slight effect was observed also at 500 ppm in males only but this is not considered truely adverse. Relative kidney weights in males were reduced at dose levels ≥ 100 ppm also in the absence of effects on body weight. As similar results were obtained in the 78-week study this effect is regarded as substance-related even though a clear dose reponse in the dose range of 100-1000 ppm is lacking. The mean relative kidney weights in treated males differed by 12 - 17 % from values in control males. A comparison of individual data showed that treated male relative kidney weights were at the lower end of the control range in this study. No histologic correlates have been found. The deaths that occurred following blood sampling (0, 0, 4, 3, 2 males; 0, 0, 0, 0, 3 females at 0, 100, 500, 1000 and 1500 ppm, repectively) are considered to be a consequence of the treatment with clothianidine in combination with the exposure to ether anaesthesia, possibly through an interaction at nicotinic acetylcholine receptors (nAChR). There is evidence of such an effect for at least one other insecticide belonging to the neonicotinoid class. It is known that volatile and intravenous anaesthetics can interact with nAChRs and modulate their ion channel activity (Flood et al. , Anaesthesiology 92, 1418-1425, 2000). No mortality related to blood sampling was observed in the 78-week study where anaesthesia was performed with O2/CO2.

Conclusion LO(A)EL: 1000 ppm (160 mg/kg bw for males and 207 mg/kg bw for females) based on reduced body weight gain and food conversion efficiency NOAEL: 500 ppm (82 mg/kg bw for males and 107 mg/kg bw for females) NOEL: 100 ppm (16 mg/kg bw) for males based on mortality and 500 ppm (107 mg/kg bw) for females)

Reliability 2


Remarks In the absence of any detailed description of the alleged GLP problems and how they may have compromised the study it has been decided to accept the study as a non-GLP study. The effects reported are plausible when compared to other studies in mice with the same substance or a structurally/functionally similar compound.












Remarks



Table A6_4_1/04-1: Body weight development and food consumption, clinical pathology (90-day mouse)

Parameter [Unit]

0 ppm 100 ppm 500 ppm 1000 ppm 1500 ppm

♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀

Body weight [g] week 0 week 2 week 7 week 13

32 37 41 44

25 27 31 32

31 35 42 44

25 28 31 33

32 35 41 42

25 28 31 32

31 34 39 39

25 27 30 31

30 32 37 37

24 25 28 29

Cumulative bw gain [g] / [% control] week 0- 2 week 2-13 week 0-13

4.4 7.2 11.7

2.0 4.7 6.7

3.9 8.8

12.7 / 109

2.7 5.4

8.0 / 119

3.6 7.1

10.1 / 86

2.8 4.3

7.1 / 106

3.5 ↓ 5.6

8.8 / 75 ↓

1.6 ↓ 3.6

5.2 / 78 ↓

1.2** ↓ 5.5

6.8**/58↓

0.1** ↓4.1

3.8 / 57 ↓

Total food consumption [g/mouse/week] / [% control] week 1-2 week 3-13 week 1-13

83 489 572

90 480 570

81 488

569 / 99

87 507

595 / 104

85 500

585 / 102

79* 502

581 / 102

79 465

544 / 95

81* 474

556 / 98

84 477

561 / 98

74** 473

548 / 96

Food conversion ratio [g food/g bw gain] week 1-4 week 5-8 week 9-13 week 1-13

29.3 50.3 94.9 49.0

48.855.5

- 85.0

25.8 53.3 82.8 44.8

42.7 78.1 151.8 74.0

27.3 65.9 93.0 57.8

43.9 50.0

- 82.2

29.4 120.4 ↑102.7 61.5 ↑

52.0 48.8

- 106.2 ↑

50.9 ↑ 172.8 ↑ 117.6 ↑ 81.3 ↑

105.9 ↑60.8

- 144.5 ↑

Clinical pathology Eos. [109/L] 0.08 0.06 0.12 0.06 0.08 0.08 0.08 0.08 0.13* 0.07

Glu [mg/dL] Bili [mg/dL]

247 0.1

199 0.2

217 0.1

203 0.2

220 0.2

203 0.2

191** 0.1

183 0.1

199** 0.2*

175 0.2

UN [mg/dL] AP [U/L] GOT [mU/ml] Prot [g/dL] Glob [g/dL] Na [mEq/L] K [mEq/L] P [mEq/L]

26 78

105 5.3 2.9 152 5.4 3.4

25 98 53 5.5 2.5 150 4.6 3.3

26 108 90 5.3 2.8 152 5.1 3.2

27 118 78* 5.3 2.4 150 4.5 3.4

28 83 118 5.4 2.8 153 5.0 3.5

26 119

97** 5.2* 2.4 150 5.1

3.8**

26 71 115 5.4 2.8 152 5.5 3.6

23 150* 106** 5.2* 2.4

151* 5.1

3.9**

20* 106 87 5.5 2.8 152 5.7 3.7

20* 145* 87** 5.2* 2.3*

152** 5.2* 3.9**

pH (urine) 6.6 6.8 6.5 6.6 6.4 7.0 6.6 6.7 6.3* 7.1

* p <0.05, ** p <0.01 Eos. = eosinophils, Glu = glucose, Bili = bilirubin, UN = urea nitrogen, AP = alkaline phosphatase, GOT = aspartate aminotransferase, Prot = protein, Glob = globulin



Table A6_4_1/04-2: Organ weights and pathology results (90-day mouse)

Parameter [Unit]


♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀

Terminal body weight [g] 43.1 31.6 43.1 32.9 41.1 31.6 38.6 29.6 36.7 28.4

Kidney weight, absolute/adjusted [g], relative [‰] absolute adjusted relative [% control]

0.791 0.763 18.5

0.4440.44214.1

0.689 0.662** 16.0**

86

0.456 0.450 14.3 101

0.666 0.662**16.2*

88

0.430 0.428 13.7 97

0.625 0.652**16.2**

88

0.388 0.393* 13.2 94

0.564 0.614** ↓ 15.4** ↓

83

0.384 0.392* ↓

13.6 96

Lung weight, absolute/adjusted [g], relative [‰] absolute relative

0.264 6.2

0.2678.4

0.317 7.3

0.266 8.2

0.316 7.7

0.293 9.4

0.242 6.2

0.324 11.0

0.256 7.0

0.177**6.3*

Histopathology Ovaries: sparse c l

- 1/10 - 1/10 - 2/10 - 1/10 - 6/10

Uterus: prom endom glands

- 1/10 - 2/10 - 3/10 - 2/10 - 7/10

* p <0.05, ** p <0.01 c l = corpora lutea, prom endom = prominent endometrial

Evaluation by Rapporteur Member State, CA-Tables




Subchronic dermal toxicity test


Other existing data [ ] Technically not feasible [ ] Scientifically unjustified [ x ] Limited exposure [ ] Other justification [ ]


he available short-term dermal and subchronic/chronic oral toxicity studies with TI-435 are considered sufficient for human risk assessment of TI-435 and accordingly no subchronic dermal toxicity study is required.


-




Date 2004-10-07


Conclusion Applicant's justification is acceptable

Remarks






Remarks




Subchronic inhalation toxicity test




a subchronic inhalation toxicity study is considered not to be required for human risk assessment of TI-435 as a biocide.





Date 2006-10-26


Conclusion The applicant's justification is acceptable

Remarks






Remarks



Section A6.5/6.7 Annex Point IIA VI.6.5 and 6.7/01

Carcinogenicity study (2-year) Rat 2-year dietary chronic toxicity/carcinogenicity study in rats

1 REFERENCE

Officialuse only

1.1 Reference Biegel, L.B. (2000a); 104-week dietary combined chronic toxicity and carcinogenicity study with TI-435 in rats Covance Laboratories Inc, Wisconsin 53704-2595, USA unpublished report No. 6155-108, 11.04.2000




None


Data submitted on existing a.s. for the purpose of its first entry into Annex I


2.1 Guideline study Yes 87/302/EEC B.33 (May 1988); Japan MAFF (59 NohSan no. 4200, 1985); EPA-FIFRA section 83-5; OECD no. 453 (1981) and EPA-OPPTS 870.4300 (June 1998)

2.2 GLP Yes

2.3 Deviations None






3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study (stability of the compound and its homogeneity and stability in food were tested).

3.2 Test Animals 80 male and 80 female albino rats per group (Crl:CD (SD)BR VAF/Plus

strain, age 34-40 days old, bodyweight range 105-194 g, Charles River Laboratories Inc., USA): 20 animals per sex and group for interim sacrifice after 52 weeks of treatment


Oral (dietary)


104 weeks


Daily


None





3.3.4 Oral


3.3.4.2 Concentration 0, 150, 500, 1500 and 3000 ppm corresponding to 0, 8.1, 27.4, 82 and 157 mg/kg bw/day for males and 0, 9.7, 32.5, 97.8 and 193 mg/kg bw/day for females food consumption per day: ad libitum



-


-


3.4 Examinations

3.4.1 Observations


3.4.1.2 Mortality yes

3.4.2 Body weight yes

3.4.3 Food consumption yes

3.4.4 Water consumption yes


yes

3.4.6 Haematology yes, 10 animals/sex/group during weeks 13, 27, 53, 79 and prior to the terminal sacrifice (weeks 103-105) Parameters: Haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, blood cell morphology, prothombin time, activated partial thromboplastin time, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration

3.4.7 Clinical Chemisty yes, 10 animals/sex/group during weeks 13, 27, 53, 79 and prior to the terminal sacrifice (weeks 103-105) Parameters: sodium, potassium, calcium, inorganic phosphorous, chloride, glucose, cholesterol, blood urea nitrogen, total bilirubin, creatinine, total protein, albumin, globulin, albumin/globulin ratio, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyltransferase, creatine kinase, triglycerides

3.4.8 Urinalysis Yes 10 animals/sex/group during weeks 13, 27, 53, 79 and prior to the terminal sacrifice (weeks 103-105) Parameters: appearance, volume, pH, specific gravity, protein, glucose, blood, ketones, bilirubin, sediment, urobilinogen


3.5.1 Organ Weights Yes (all animals surviving to scheduled interim or terminal sacrifice) organs: liver, kidneys, adrenals, testis, epididymis, ovaries, brain,





thyroid (with parathyroid), lung, heart, uterus with cervix, spleen, pituitary, prostate, seminal vesicle, thymus


Yes gross pathology: all animals histopathology: all animals (* tissues were not examined microscopically) organs: adrenals, aorta, brain, caecum, cervix, colon, duodenum, epididymis, esophagus, eye, femur with bone marrow, harderian gland*, head*, heart, ileum, jejunum, kidney, larynx*, lesions, liver, lung, lymph nodes, female mammary gland, masses and associated tissues, muxcle (tigh), optic nerve, ovaries, pancreas, pituitary, prostate, rectum, mandibular salivary gland, sciatic nerve, seminal vesicle, skin, spinal cord, spleen, sternum with bone marrow, stifle joint, stomach, testis, thymus, thyroid with parathyroid, trachea, urinary bladder, uterus, vagina


3.5.4 Statistics Body weights, bw changes, food consumption, food efficiency, organ weights, organ-body weight ratios and organ-brain weight ratios: one-way analysis of variance (ANOVA). Levene’s test was performed to test for heterogeneity, and, where appropriate, transformations were performed to stabilise the variances. ANOVA was then performed on the transformed data. If ANOVA was significant, Dunnett’s multiple comparison t-test was used for pairwise comparisons between treated and control groups. Survival data. log-rank test at the 5%, two-tailed probability level for group comparisons. Dose-response relationship of mortality: logistic regression method at the 5% one-tailed probability level. Fatal, non-fatal and incidental tumours: Peto’s mortality-prevalence test.

3.6 Further remarks During week 66 or 67, 10 animals/sex from each group were subjected to a functional observation battery (FOB) of neurological tests.


4.1 Observations

4.1.1 Clinical signs There were no treatment-related effects at any dose level on clinical signs, ophthalmological findings and FOB observations.

4.1.2 Mortality There were no adverse effects of treatment on survival at any dose level. Probably related to the reduced bw development, high dose animals had highest survival (55% and 50% for males and females, respectively, at termination) (see also table A6_7/01-1). Overall the survival incidences were sufficient to evaluate the carcinogenic potential of TI-435, even if the group survival was <50% at termination at 0-1500 ppm. In case of a carcinogenic test compound increased tumour incidences would have occurred with the highest probability at the highest dose level - for TI-435 no relevant increase of tumour incidence was observed with a survival ≥50% at 3000 ppm. Survival in the other groups fell below 50% only a few weeks before termination (weeks 91 and 88 onwards for males and females, respectively). Therefore a sufficient number of rats survived long enough to allow tumour formation - in case of a carcinogenic test compound - and thus a meaningful evaluation of carcinogenicity.

4.2 Body weight gain A reduced bw development was noted for 1500 ppm treated females and





at 3000 ppm (both sexes) (see table A6_7/01-1). Cumulative bw gain in the first 50 weeks of the study were 13% and 28% below controls for 1500 and 3000 ppm treated females, respectively, and 13% below controls for males at 3000 pmm. Lower body weights were occasionally recorded for males at 1500 ppm and females at 500 ppm mostly in the first 14 weeks of the treatment period. As these reductions did not exceed 5% in comparison to controls and as the overall weight gains were not significantly decreased, they are considered to be not-adverse.

4.3 Food/water consumption and compound intake

The food consumption was reduced at 3000 ppm and slightly lower values were also noted for 1500 ppm treated females mainly in the first part of the study (see table A6_7/01-1). No relevant effect on food consumption was seen for the other groups. Compound intake see 3.3.4.2 Analysis of diet samples demonstrated adequate homogeneity, stability and achieved concentration of TI-435 in diet. Mean values of the homogeneity were 95.9-96.9% and 96.0-100% of the theoretical concentrations of 100 and 3000 ppm, respectively. Stability analysis revealed TI-435 was stable in diet at concentrations of 25 or 5000 ppm for 29 days at room temperature and between –10 and –30°C. The mean achieved concentrations of all diets ranged from 88.7 to 109% of nominal concentrations. Water consumption was unaffected by treatment at all dose levels.


4.5 Blood analysis

4.5.1 Haematology No treatment-related effects were observed.

4.5.2 Clinical chemistry A slight but consistent elevation of serum inorganic phosphorus concentration at all sampling intervals in male rats treated at 3000 ppm was shown, which might have been associated with the increased incidence of mineralisation of renal pelvic observed in these animals.



4.6.1 Organ weights There were no effects on organ weights that were directly attributable to treatment, although some minor fluctuations occurred at 3000 ppm that were considered to be associated with reduced body weight gain.


No treatment-related macroscopic lesions were evident at any dose level. Treatment-related non-neoplastic histomorphological alterations occurred in the glandular stomach, kidneys and ovaries (see table 6_7/01-2). Indication for irritation were seen at 3000 ppm in the glandular stomach: increased incidences of erosion (females), hemorrhage (males) and edema of the stomach wall (both sexes). Incidences of pelvic mineralisation and hyperplasia of the transitional epithelium in the kidney were slightly increased in 3000 ppm treated males. Increased incidences of interstitial hyperplasia of the ovaries occurred in females at ≥500 ppm. However the severity of this finding was only marginally increased at 3000 ppm in correspondence to controls. It was





seen in ovaries undergoing atrophy with little or no active follicular development and was considered to be an age-related change of dubious toxicological significance (in average it was found in rats sacrificed later than about 701 days of the study). The increased incidence of this finding is considered to be at least partly due to the longer survival for females at ≥1500 ppm. No treatment-related effects occurred at any dose level on the nature and incidence of neoplastic lesions. Although the incidences of thyroid C-cell adenoma were higher in the female groups treated at ≥1500 ppm, the combined incidence of C-cell adenoma and carcinoma of 3000 ppm treated females was not significantly greater than the control incidence. All other neoplastic changes and incidences were consistent with spontaneously occurring lesions expected in Sprague-Dawley-derived rats.



Toxicity evaluation after chronic (2-year) dietary exposure of rats to TI-435; no relevant deviation from guidelines (87/302/EEC; Japan MAFF; EPA-FIFRA 83-5; OECD 453 and EPA-OPPTS 870.4300)


Food consumption and bw development was reduced at 3000 ppm (both sexes) and at 1500 ppm in females. Slightly lower body weights were also recorded for 500 ppm treated females and 1500 ppm treated males during the first part of the study. Clinical pathology revealed a slight increase in inorganic phosphorus for males at 3000 ppm. Histopathological changes were seen in the glandular stomach (irritation, both sexes at 3000 ppm) and the kidney (males at 3000 ppm). Increased incidences of interstitial cell hyperplasia of the ovaries were recorded at ≥500 ppm in females representing an age-related change and therefore of dubious toxicological relevance. It was considered to be at least partly due to the longer survival in the ≥1500 ppm treated females.

5.3 Conclusion Chronic treatment with up to 3000 ppm TI-435 for two years was tolerated well in rats and the high dose represented the MTD. Main effects of treatment were reduced food consumption and bw development leading to a better survival, and some histopathological changes in the stomach (slight irritation), kidney and ovaries (age-related, of dubious toxicological relevance). Treatment with TI-435 did not result in any indication for a carcinogenic response.

5.3.1 LO(A)EL LO(A)EL = 1500 and 500 ppm in males and females, respectively, corresponding to 82 and 32.5 mg/kg bw/day for males and females, respectively males: reduced food consumption and bw gain (slight increase in blood phosphorus levels, histopathological changes in glandular stomach and kidney) females (interstitial cell hyperplasia in ovaries, considered to be of only dubious toxicological relevance but was used as the basis for the LOAEL/NOAEL as a conservative assumption)

5.3.2 NO(A)EL NOAEL = 500 and 150 ppm for males and females, respectively, corresponding to 27.4 and 9.7 mg/kg bw/day for males and females, respectively

5.3.3 Other -





5.3.4 Reliability 1





Date 2004-10-07


Results and discussion Applicant’s version is acceptable with exception of the opinion regarding effects on the ovary. The increased incidences of interstitial cell hyperplasia of the ovaries at ≥500 ppm are considered to be toxicologically relevant. The finding is age-related insofar as it occured only in females older that 630 days (>600 days on study). However, there was a clear relationship to treatment and dose, especially when only the subpopulations at risk (animals with more than 600 days on study) were compared (see CA-Table 1).

Conclusion LO(A)EL: 500 ppm (32.5 mg/kg bw/day) for females based on interstitial cell hyperplasia of the ovaries 1500 ppm (82 mg/kg bw/day) for males based on reduced food consumption, reduced bw gain, clinical chemistry findings and histopathological changes in glandular stomach and kidney NO(A)EL: 150 ppm (9.7 mg/kg bw/day) for females 500 ppm (27.4 mg/kg bw/day) for males

Reliability 1


Remarks CA-table 1 is added by the RMS








Remarks



Table A6_7/01-1. Survival, bw development and food consumption and results of clinical chemistry (2-year rat)

Parameter Males Females ppm 0 150 500 1500 3000 0 150 500 1500 3000

Survival [%] week 78 week 96 (= 24 x 4) termination (week 103-105)

78 43

28

62 40

30

73 45

30

73 52

40

93 67

55

72 35

25

70 37

27

72 37

28

73 50

38

83 62

50


152 543 778 759

150 534 766 784

151 532 743 799

150 522* 741 754

149 491** ↓691** ↓

725

137 287 392 447

136 282 408 500

134 276* 387 456

135 267** ↓ 355** ↓

424

137 249** ↓320** ↓356** ↓

Body weight gain [g] week 1-13 week 13-50 week 1-50 week 1-104

391 235 626 607

384 232 616 637

381 211 592 655

372 219 591 603

343** ↓200** ↓ 543** ↓

574

150 105 254 313

146 126 272 370

142 111 253 321

132**↓ 89 (↓)

221**↓ 289

112** ↓72** ↓ 183** ↓220** ↓

Food consumption [% of control] week 1-13 week 17-101

100 100

98 99

101 100

96 98

87 ↓ 94 ↓

100 100

95 102

94 98

89 ↓ 95

81 ↓ 90 ↓

Inorganic phosphorus [mg/dL] week 13 week 27 week 53 week 79 week 105

7.7 7.5 6.5 6.0 5.9

7.8 7.7 6.8 6.2 6.2

7.7 7.4 6.8 6.0 5.9

8.0 7.8 6.5 6.6 7.4

8.7* ↑8.2 (↑)7.5* ↑7.4* ↑7.1* ↑

6.0 6.1 5.5 5.6 4.7

6.0 5.5 6.4 5.8 4.8

6.0 6.1 5.4 5.8 5.1

6.6 6.6 5.7 5.8 5.3

6.6 6.7 5.6 6.4 5.4

* p <0.05, ** p <0.01; arrows in brackets (↓↑) indicate a trend that was not statistically significant



Table A6_7/01-2. Results of chronic toxicity/carcinogenicity study (2-year rat): histopathology and tumour incidences Parameter 0 ppm 150 ppm 500 ppm 1500 ppm 3000 ppm

ma fa ma fa ma fa ma fa ma fa

Glandular Stomach erosion hemorrhage edema

5/80 0/80 1/80

2/80 0/80 0/80

6/80 0/80 1/80

0/80 0/80 2/80

3/80 0/80 0/80

2/80 0/80 2/80

5/80 1/80 1/80

7/80 1/80 2/80

8/80 5/80 8/80

11/80 1/80 6/80

Kidney pelvic mineralisation transitional cell hyperplasia

34/80 33/80

77/80 60/80

30/80 30/80

76/80 58/80

34/80 29/80

74/80 59/80

43/80 29/80

75/80 65/80

52/80 51/80

79/80 65/80

Ovaries interstitial cell hyperplasia (grade°)

- 5/80 (2.6)

- 10/80 (2.3)

- 13/80 (2.3)

- 20/80 (2.4)

- 33/80 (3.0)

Thyroid C-cell adenoma C-cell carcinoma

8/80 5/80

7/80 2/80

13/801/80

13/802/80

17/801/80

9/80 1/80

16/801/80

17/80 1/80

5/80 3/80

16/801/80

total 13/80 9/80 14/80 15/80 18/80 10/80 17/80 18/80 8/80 17/80

No of animals interim sacrifice unscheduled death terminal sacrifice

18 45 17

20 45 15

18 44 18

19 45 16

19 43 18

20 48 12

19 37 38

19 38 23

20 27 33

20 30 30

total 80 80 80 80 80 80 80 80 80 80

Benign neoplasms interim sacrifice unscheduled death terminal sacrifice

7 56 25

14 57 21

8 57 29

8 63 31

9 50 28

7 68 24

8 45 41

9 53 41

5 34 40

10 38 60

total 88 92 94 102 87 99 94 103 79 108

Malign neoplasms interim sacrifice unscheduled death terminal sacrifice

2 18 5

3 25 6

0 4 4

1 25 10

0 20 4

3 30 9

1 5 8

3 18 11

0 15 6

1 16 9

total 25 34 8 36 24 42 14 32 21 26

Benign+malign neoplasms interim sacrifice unscheduled death terminal sacrifice

9 74 30

17 82 27

8 61 33

9 88 41

9 70 32

10 98 33

9 50 49

12 71 52

5 49 46

11 54 69

total 113 126 102 138 111 141 108 135 100 134 a number of animals affected/total number of animals ° minimal - grade 1 slight grade 2 moderate grade 3 moderate severe grade 4 severe grade 5



Evaluation by Rapporteur Member State, CA-Table CA-Table 1. Results of chronic toxicity/carcinogenicity study (2-year rat): Ovary interstitial cell hyperplasia Parameter 0 ppm 150 ppm 500 ppm 1500 ppm 3000 ppm

Interim sacrifice N examined N affected % affected

20 0 0

19 0 0

20 0 0

19 0 0

20 0 0

Unscheduled deaths N examined N affected % affected

45 2

4.4

45 4

8.9

48 7

14.6

38 7

18.4

30 11

37.9

Terminal sacrifice N examined N affected % affected

15 3 20

16 6

37.5

12 6 50

23 13

56.5

30 22 73

Survivors >600 days N examined N affected % affected

34 5

14.5

35 10

28.6

34 13

38.2

36 20

55.6

48 33

68.8



Section A6.6.1

Annex Point IIA

VI.6.6.1/01

Genotoxicity In vitro gene mutation in bacteria

Ames test (+/- S9) using S. typhimurium and E. coli

1 REFERENCE

Official

use only

1.1 Reference Thompson, P. W. (2000); TI-435: reverse mutation assay “Ames test” using Salmonella typhimurium and Escherichia coli; Safepharm Laboratories Ltd., Derby, UK. unpublished report number 178/110, 08.03.2000




None




2.1 Guideline study Yes 2000/32/EC (successor of 92/69/EEC) B13/14, OECD no. 471 (1983), EPA FIFRA section 84-2 (1984), Japan MAFF (59 NohSan no. 4200, 1985)

2.2 GLP Yes

2.3 Deviations No (study was performed previous to current EU-guideline but was checked for compliance with the above)






3.1.2.2 Purity

3.1.2.3 Stability Stable under conditions of this study (storage for 24 hours at RT)

3.2 Study Type Bacterial reverse mutation test

3.2.1 Organism/cell type S. typhimurium: TA 1535, TA 1537, TA 98, TA 100 E. coli: WP2 uvr A

3.2.2 Deficiencies / Proficiencies

Histidine deficient (S. typhimurium) Tryptophan deficient (E. coli)

3.2.3 Metabolic activation system

S9 mix Prepared from livers of male Sprague Dawley rats (bw about 200 g) treated with a single i.p. injection of 500 mg/kg bw Arochlor 1254 5 days prior to sacrifice.



Section A6.6.1

Annex Point IIA

VI.6.6.1/01



3.2.4 Positive control In absence of S9: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) at 2 µg/plate for WP2uvrA 3 µg/plate for TA 100 5 µg/plate for TA 1535 9-Aminoacridine (9AA) at 80 µg/plate for TA 1537 4-Nitroquinoline-1-oxide (4NQO) at 0.2 µg/plate for TA 98 In presence of S9: 2-Aminoantracene (2AA)at 1 µg/plate for TA 100 2 µg/plate for TA 1535 and TA 1537 10 µg/plate for WP2uvrA 0.5 µg/plate for TA 98

3.3 Administration /

Exposure;

Application of test

substance

3.3.1 Concentrations 0, 50, 150, 500, 1500, 5000 µg/plate

3.3.2 Way of application Stock soluations: TI-435 was dissolved in DMSO. The bacterial suspensions (0.1 mL) were mixed with top agar (2.0 mL, supplemented with histidine/tryptophan), 0.1 mL of TI-435 stock solutions or vehicle control, 0.5 mL S9 mix or phosphate buffer before pured onto minimal agar plates. The plates were then incubated for 48 hours at 37°C.

3.3.3 Pre-incubation time None (plate incorporation assay)

3.3.4 Other modifications -

3.4 Examinations Cytotoxicity (pre-test with TA 100 and WP2uvrA): frequency of revertant colonies and thinning of background lawn Mutagenicity (main test): frequency of revertant colonies Criteria for a positive response: The test was considered to be positive if there was a dose-related, reproducible and statistically significant increase in mutation frequency of at least twice the spontaneous reversion frequency in one or more strains with or without metabolic activation in both experiments at sub-toxic dose levels.


4.1 Genotoxicity Non-entry field

4.1.1 without metabolic activation

None of the observed results fulfilled the criteria of a positive response (see Table A6_6_1/01-1). Reproducible, statistically significant increases in the number of revertant colonies were seen at 1500 µg/plate in TA 1535 which however did not reach twice the spontaneous or concurrent control mutant frequencies and therefore did not fulfill the criteria of aposition response.



Section A6.6.1

Annex Point IIA

VI.6.6.1/01



4.1.2 with metabolic activation

Yes: Reproducible, statistically significant increases in the number of revertant colonies were seen at 5000 µg/plate in TA 1535, which just barely reached more than twice the concurrent control mutant frequencies (2.5 and 2.2 times of concurrent control; however less than double of the given spontaneous frequencies) (see Table A6_6_1/01-1). No: for all other concentrations and strains

4.2 Cytotoxicity No (no indications for growth inhibition)


5.1 Materials and

methods

Evaluation of the in vitro gene mutation potential in S. typhimurium and E. coli strains; no relevant deviation from guidelines (2000/32/EC B13/14, OECD 471, EPA FIFRA 84-2, Japan Maff)

5.2 Results and

discussion

Reproducible, statistically significant increases in revertant numbers in TA 1535 at 1500 ppm in absence of S9 and at 5000 ppm in presence of S9, which however only for the latter just barely fulfilled the criteria of a positive response (double mutant frequencies than concurrent negative controls). No relevant effect on the number of revertant colonies of TI-435 in the other strains and no indication for growth inhibition were noted.

5.3 Conclusion Due to the (barely) positive response in TA 1535, a mutagenic potential might not be excluded for TI-435.

5.3.1 Reliability 1





Date 2006-10-26


Results and discussion Applicant's version is adopted.

Conclusion Due to the weak but reproducible positive response in TA 1535 (base pair substitution), a mutagenic potential can not be excluded for TI-435.

Reliability 1


Remarks



Section A6.6.1

Annex Point IIA

VI.6.6.1/01



COMMENTS FROM ...


Materials and Methods Discuss additional relevant discrepancies referring to the (sub)heading numbers

and to applicant's summary and conclusion.






Remarks



Section A6.6.1

Annex Point IIA

VI.6.6.1/01



Table A6_6_1/01-1. Table for Gene Mutation Assay (Ames test, Thompson 2000)

Strain/concen-tration [µg/plate]

Number of mutant cells Comments Experiment 1 Experiment 2

— S9 + S9 — S9 + S9

TA 100: 0 50 150 500 1500 5000 Positive control

91 83 94 83 97

104 384

119 116 96

107 115 123 771

111 105 109 104 114 115 608

111 114 99 97 97

121 838

No precipitation of TI-435, no growth inhibition


20 27 21 26

30* 32* 190

11 12 15 13

20* 28** 189

19 19 25 24 29

37** 385

12 13 15 12 12

26** 221


WP2uvrA: 0 50 150 500 1500 5000 Positive control

24 19 18 22 12 11

587

18 21 18 19 17 14

861

19 15 14 14 16 12

1046

18 20 21 19 15 17

739



18 16 16 20 18 20

175

30 32 30 26 26 28

407

24 20 20 21 15 23

162

27 22 25 20 22 27

462



12 11 13 12 11 10

605

10 13 11 9 11 8

279

9 8 8 6 6 6

879

10 9 7 8 8 7

269


** p<0.01




Genotoxicity In vitro gene mutation in bacteria Ames test (+/- S9) using S. typhimurium and E. coli

1 REFERENCE

Officialuse only

1.1 Reference Otsuka, M. (1990b); Bacterial reverse mutation test of TIR-435 Hita Research Laboratories, Chemical Biotesting Center, Chemicals Inspection & Testing Institute, Oita, Japan unpublished report number T-2276, 23.04.1990




None




2.1 Guideline study Yes 2000/32/EC (method B13/14), Japan MAFF (59 NohSan no. 4200, 1985)

2.2 GLP Yes






3.1.2.1 Description White crystall

3.1.2.2 Purity ≥99%

3.1.2.3 Stability Stable under conditions of this study (tested; storage at RT in the dark)


3.2.1 Organism/cell type S. typhimurium: TA 1535, TA 1537, TA 98, TA 100 E. coli: WP2 uvr A


Histidine deficient (S. typhimurium) Tryptophan deficient (E. coli)


S9 mix Prepared from livers of 7 week old male Sprague Dawley, which were induced once with 30 mg/kg bw phenobarbital, three times with 60 mg/kg bw phenobarbital and once with 80 mg/kg bw 5,6-benzo-flavone by i.p. injection.





3.2.4 Positive control In absence of S9: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) at 0.01 µg/plate for TA 100 and WP2uvrA 0.1 µg/plate for TA 98 sodium azide (NaN3) at 0.5 µg/plate for TA 1535 2-methoxy-6-chloro-9-[3-(2-chlrorethyl)-aminopropylamino]acridine ⋅2 HCl (ICR-191) at 1 µg/plate for TA 1537 In presence of S9: 2-Aminoantracene (2AA) at 1 µg/plate for TA 100 2 µg/plate for TA 1535 and TA 1537 20 µg/plate for WP2uvrA 0.5 µg/plate for TA 98

3.3 Administration / Exposure; Application of test substance

3.3.1 Concentrations 0, 100, 500, 1000, 2000, 5000 µg/plate (1st experiment) 0, 313, 625, 1250, 2500, 5000 µg/plate (2nd experiment)

3.3.2 Way of application Stock solutions: TI-435 was dissolved in DMSO. The bacterial suspensions (0.1 mL) were mixed with top agar (2.0 mL), 0.1 mL of TI-435 stock solutions or vehicle control, 0.5 mL S9 mix or phosphate buffer before pured onto minimal glucose agar plates. The plates were then incubated for 48 hours at 37°C.

3.3.3 Pre-incubation time None (plate incorporation assay)


3.4 Examinations Cytotoxicity (pre-test): frequency of revertant colonies, growth inhibition and precibitation of the test article Mutagenicity (main test): frequency of revertant colonies Criteria for a positive response: The test was considered to be positive if there was a dose-related and reproducible increase in mutation frequency of at least twice the solvent control reversion frequency, in one or more strains.


4.1 Genotoxicity


No (see Table A6_6_1/02-1)


No (see Table A6_6_1/02-1)

4.2 Cytotoxicity No (no indications for growth inhibition)







Evaluation of the in vitro gene mutation potential in S. typhimurium and E. coli strains; no relevant deviation from guidelines (2000/32/EC B13/14, Japan Maff)


TI-435 did not increase the number of revertant colonies in any strain at any concentration in presence or in absence of metabolic activation.

5.3 Conclusion No mutagenic potential was detected for TI-435 in this test system. The barely positive response in the study by Thompson (2000) in strain TA 1535 could not be reproduced in this study also using similar concentrations. Therefore TI-435 is considered not to have a relevant potential for bacterial gene mutation.

5.3.1 Reliability 1





Date 2004-10-07



Conclusion Other conclusions: No mutagenic potential was detected for TI-435 in this test with high purity material.

Reliability 1


Remarks

COMMENTS FROM ...







Remarks





Table A6_6_1/02-1. Table for Gene Mutation Assay (Ames test, Otsuka 1990b)


Number of mutant cells Comments Experiment 1 (pre-test) Experiment 2 (main test)

— S9 + S9 µg/plate — S9 + S9


81 81 88 97 89 96 399

104 99 96 98 99 106

3408

0 313 625

1250 2500 5000

pos. ctr

79 74 82 90 86 81 253

83 73 97 79 85 87 954

No precipitation of TI-435, no growth inhibition in the pre-test


7 6 7 6 6 4

309

7 7 5 7 7 8

464

0 313 625

1250 2500 5000

pos. ctr

8 5 7 7 4 6

353

12 10 7

10 11 8

415


WP2uvrA: 0 100 500 1000 2000 5000 Positive control

50 45 38 40 39 36 252

44 47 55 57 50 48

1358

0 313 625

1250 2500 5000

pos. ctr

36 46 38 42 32 32 184

48 51 48 60 36 50

1375



26 33 26 25 31 26 557

31 34 32 34 29 32 555

0 313 625

1250 2500 5000

pos. ctr

27 25 25 27 24 27 377

26 29 28 39 29 28 251



5 8 4 4 5 4

639

6 6 8 8 6 4

638

0 313 625

1250 2500 5000

pos. ctr

6 4 8 4 4 4

1006

9 7

10 9 6 7

180






1 REFERENCE

Officialuse only

1.1 Reference Herbold, B. (1999a); TI 435: Salmonella/microsome test, plate incorporation and preincubation method Bayer AG, Wuppertal, Germany unpublished report number 28849, 16.06.1999 (revised version of Bayer report no. 26584 - first version)




None





2.2 GLP Yes






3.1.2.1 Description yellowish powder

3.1.2.2 Purity

3.1.2.3 Stability Stable under conditions of this study (stability in solvent was tested)


3.2.1 Organism/cell type S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 102


Histidine deficient


S9 mix Prepared from livers of male Sprague Dawley rats (200-300 g bw) treated with a single i.p. injection of 500 mg/kg bw Arochlor 1254 in corn oil 5 days prior to sacrifice. Livers were washed, homogenised and centrifuged (9000 g for 10 min at 4°C). The supernatant was supplemented with co-factors.





3.2.4 Positive control In absence of S9: 4-nitro-1,2-phenylenediamine (NP) at 0.5 µg/plate for TA 98 10 µg/plate for TA 1537 Nitrofurantoin (NF) at 0.2 µg/plate for TA 100 Cumene hydroperoxide (CH) at 50 µg/plate for TA 102 Sodium azide (SA) at 10 µg/plate for TA 1535 In presence of S9: 2-Aminoantracene (2AA) at 3 µg/plate for all strains


3.3.1 Concentrations 0, 16, 50, 158, 500, 1581 and 5000 µg/plate for experiments 1 (plate incorporation assay) and 2 (pre-incubation assay) 0, 16, 32, 48, 64, 80, 96 and 112 µg/plate for an additional 3rd experiment with TA 102 (pre-incubation assay)

3.3.2 Way of application Stock solutions: TI-435 was dissolved in DMSO. Plate incorporation assay: The bacterial suspensions (0.1 mL) were mixed with soft agar (2.0 mL), 0.1 mL of TI-435 stock solutions or vehicle control, 0.5 mL S9 mix or phosphate buffer before pured onto minimal agar plates. Pre-incubation assay: The bacterial suspensions (0.1 mL) were mixed with 0.1 mL of TI-435 stock solutions or vehicle control, 0.5 mL S9 mix or phosphate buffer and incubated for 20 minutes at 37°C. Then 2.0 mL of soft agar were added and pured onto minimal agar plates. For both assay types the plates were then incubated for 48 hours at 37°C.

3.3.3 Pre-incubation time None for the plate incorporation assay 20 min at 37°C for the pre-incubation assay


3.4 Examinations Cytotoxicity: reduction of background lawn and total bacterial counts (titer), reductions of revertant colonies Mutagenicity: frequency of revertant colonies Criteria for a positive response: The assay was considered positive if there was a dose-related increase in revertants and a reproducible doubling of the revertants/plate at any concentration for one or

more of strains TA 98, TA 100 and TA 1535 and/or a threefold increase of the number of revertants/plate at any

concentration for strain TA 1537 and/or an increase of approximately 150 mutants at any

concentration for strain TA 102.


4.1 Genotoxicity


None of the observed results fulfilled the criteria of a positive response (see Table A6_6_1/03-1).






None of the observed results fulfilled the criteria of a positive response (see Table A6_6_1/03-1). The slight increase in revertant number at 50 µg/plate in TA 102 by about 100 colonies over vehicle control was further investigated in the 3rd experiment with a narrow dose spacing around 50 µg/plate, where no relevant increase of revertant colonies was detected.

4.2 Cytotoxicity Plate incorporation assay (experiment 1): no indication for inhibition of bacterial growth Pre-incubation assay (experiments 2 and 3): inhibition of bacterial growth at concentrations >158 µg/plate; reduction in bacterial titres at 5000 µg/plate for TA 100, TA 102 and TA 98.



Evaluation of the in vitro gene mutation potential in S. typhimurium strains; no relevant deviation from guidelines (2000/32/EC B13/14, OECD 471, EPA FIFRA 84-2, Japan Maff)


TI-435 did not increase the number of mutant colonies in any strain at any concentration up to 5000 µg/plate both in the plate incorporation and the pre-incubation assay.

5.3 Conclusion TI-435 did not induce gene mutations in this test system. The barely positive result in the study by Thompson (2000) in strain TA 1535 could not be reproduced in this study using similar concentrations of TI-435. Therefore no relevant potential for bacterial gene mutation is considered for TI-435.

5.3.1 Reliability 1





Date 2004-10-07



Conclusion No mutagenic potential was detected for TI-435 in this test.

Reliability 1


Remarks





COMMENTS FROM ...


Materials and Methods






Remarks





Table A6_6_1/03-1 Table for Gene Mutation Assay (Ames test, Herbold 1999a)

Strain/conc. [µg/plate]

Number of mutant cells Cytotoxicity: Bacterial titres [x 108/mL]

Experiment 1 2 3 1 2 3 -S9 +S9 -S9 +S9 µg/plate +S9 +S9 +S9 +S9

TA 100: 0 16 50 158 500 1581 5000 Positive ctr

60 65 70 67 70 57 81 203

101 92 94 95 90

104 110 1267

102 89 88 91 91 80 -

237

100 107 101 101 127 102 128

1211

- - 1.3 1.2 1.6 1.5 1.9 1.4 0.7

1.1 / 0.9°

3.7 4.5 4.4 4.1 4.6 3.1

0.9* 4.0 / 4.0°

-

TA 1535: 0 16 50 158 500 1581 5000 Positive ctr

9 8 8 6 6 9

12 820

11 9 9 6

10 8

11 80

6 5 5 5 9 9 8

912

10 11 8 7 7

11 16 137

- - 6.4 7.0 7.0 8.8 8.1 7.7 7.4

11.2 / 5.1°

11.6 11.7 12.3 12.4 11.7 9.6

2.9* 11.2 / 9.6°

-

TA 102: 0 16 50 158 500 1581 5000 Positive ctr

215 195 217 204 169 200 193

371

254 271 266 224 179 172 163

411

126 135 113 115 140 137 120

494

148 186 251 228 146 155 168

474

0163248648096

112 pos. ctr

195 203 226 233 187 178 197 201 467

15.6 16.3 17.1 20.9 18.1 16.1 16.6

19.0 / 19.1°

16.0 19.5 20.5 18.7 14.0 19.1 21.8

22.1 / 22.4°

11.6 13.7 14.7 15.2 16.3 14.9 14.1 13.9 14.9

TA 98: 0 16 50 158 500 1581 5000 Positive ctr

27 24 22 23 28 29 26 155

25 19 29 23 22 25 21

1146

16 16 17 28 19 20 23

151

22 19 25 27 24 22 28

1755

- - 16.7 18.0 14.8 14.9 19.1 18.3 16.7

16.0 / 16.1

18.8 19.7 21.3 20.5 22.4 18.2

11.3* 21.1 / 19.9°

-

TA 1537: 0 16 50 158 500 1581 5000 Positive ctr

7 8 6 7 6 4 8

85

7 7 7 6 7 6 7

235

6 4 4 3 2 2 3

63

6 4 3 3 4 2 4

28

- - 5.5 4.8 5.5 6.5 6.2 6.2 5.6

6.6 / 5.6°

0.3 0.5 0.2 0.3 0.4 0.2 0.1

0.5 / 0.6°

-

*cytotoxic ° two values for the two positive control in absence and presence of S9, respectively conc. = concentration, ctr = control



Section A6.6.1

Annex Point IIA

VI.6.6.1/04


Ames test (+/- S9) using S. typhimurium TA 1535

1 REFERENCE

Official

use only

1.1 Reference Herbold, B. (1999b); TI 435: Salmonella/microsome test using Salmonella typhimurium TA 1535, plate incorporation and preincubation method Bayer AG, Wuppertal, Germany unpublished report number 25739, 31.05.1999 (revised version of Bayer report no. 25739A - first revision)




None





2.2 GLP Yes

2.3 Deviations None relevant for the integrity and validity of the study (study was performed previous to current EU-guideline but was checked for compliance with the above): only strain TA 1535 was used in this study to clarify the borderline response seen in this strain in an earlier study at 5000 µg/plate.



3.1.1 Lot/Batch number 30034708 NLL 6100-3


3.1.2.1 Description beige powder

3.1.2.2 Purity

3.1.2.3 Stability Stable under conditions of this study (stability in solvent was tested)


3.2.1 Organism/cell type S. typhimurium TA 1535



Section A6.6.1

Annex Point IIA

VI.6.6.1/04




Histidine deficient


S9 mix Prepared from livers of male Sprague Dawley rats (200-300 g bw) treated with a single i.p. injection of 500 mg/kg bw Arochlor 1254 in corn oil 5 days prior to sacrifice. Livers were washed, homogenised and centrifuged (9000 g for 10 min at 4°C). The supernatant was supplemented with co-factors.

3.2.4 Positive control In absence of S9: Sodium azide (SA) at 10 µg/plate In presence of S9: 2-Aminoantracene (2AA) at 3 µg/plate


Exposure;

Application of test

substance

3.3.1 Concentrations Plate incorporation assay (+/- S9): 0, 1000, 2000, 3000, 4000 and 5000 g/plate (batch NLL 6100-3) and 0, 3000, 5000 and 7000 µg/plate (batch 30034708) Pre-incubation assay (+/- S9): 0, 1000, 2000, 4000, 6000 and 8000 µg/plate (both batches)

3.3.2 Way of application Stock solutions: TI-435 was dissolved in DMSO. Plate incorporation assay: The bacterial suspensions (0.1 mL) were mixed with soft agar (2.0 mL), 0.1 mL of TI-435 stock solutions or vehicle control, 0.5 mL S9 mix or phosphate buffer before pured onto minimal agar plates. Pre-incubation assay: The bacterial suspensions (0.1 mL) were mixed with 0.1 mL of TI-435 stock solutions or vehicle control, 0.5 mL S9 mix or phosphate buffer and incubated for 20 minutes at 37°C. Then 2.0 mL of soft agar were added and pured onto minimal agar plates. For both assay types the plates were then incubated for 48 hours at 37°C.

3.3.3 Pre-incubation time None for the plate incorporation assay 20 min at 37°C for the pre-incubation assay


3.4 Examinations Cytotoxicity: reduction of background lawn and total bacterial counts (titer), reductions of revertant colonies Mutagenicity: frequency of revertant colonies Criteria for a positive response: The assay was considered positive if there was a dose-related increase in revertants and a reproducible doubling of the revertants/plate at any concentration for TA 1535


4.1 Genotoxicity


No increase in the number of revertant colonies was observed results in any of the two assay types with two batches of TI-435 (see Table A6_6_1/04-1).


No increase in the number of revertant colonies was observed results in any of the two assay types with two batches of TI-435 (see Table A6_6_1/04-1).



Section A6.6.1

Annex Point IIA

VI.6.6.1/04



4.2 Cytotoxicity Reduction in bacterial titres at 5000 µg/plate


5.1 Materials and

methods

Evaluation of the in vitro gene mutation potential in S. typhimurium strain TA 1535; no relevant deviation from guidelines (2000/32/EC B13/14, OECD 471, EPA FIFRA 84-2, Japan Maff)

5.2 Results and

discussion

TI-435 did not increase the number of mutant colonies in TA 1535 at any concentration up to 8000 µg/plate both in the plate incorporation and the pre-incubation assay using two batches of TI-435.

5.3 Conclusion TI-435 did not induce gene mutations in this test system. The barely positive result in the study by Thompson (2000) in strain TA 1535 could not be reproduced in this study using the same and another batch of TI-435 and similar or higher concentrations of TI-435. Therefore no relevant potential for bacterial gene mutation is considered for TI-435.

5.3.1 Reliability 1





Date 2004-10-07

Materials and Methods Ad 2.2: The study is not compliant to GLP insofar as no stability analysis for the test substance in solution and no QAU inspections were conducted.


Conclusion Applicant's version is adopted.

Reliability 1


Remarks Stability data for the test substance for this type of test have been provided in earlier studies.

COMMENTS FROM ...








Remarks



Section A6.6.1

Annex Point IIA

VI.6.6.1/04



Table A6_6_1/04-1 Table for Gene Mutation Assay (Ames test, Herbold 1999b)

Strain/concentration

[µg/plate]

Number of mutant cells

Assay plate incorporation pre-incubation

Batch NLL 6100-3 30034708 NLL 6100-3 30034708

-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9

TA 1535: 0 1000 2000 3000 4000 5000 6000 7000 8000# Positive control

13 9

11 12 11 11 - - -

652

11 10 13 9

12 13 - - -

209

13 - -

13 -

11 -

16 -

652

11 - -

17 -

14 -

18 -

209

9 9

12 -

10 - 8 - 9

626

10 10 11 -

14 - 9 - 7

302

11 11 8 - 9 - 8 - 9

672

10 12 13 -

10 -

11 -

12 296

Cytotoxicity: Bacterial titres [x 108/mL]

TA 1535: 0 1000 2000 3000 4000 5000 6000 7000 8000# Positive control°

14.3 13.0 13.5 13.4 13.0

11.6* - - -

12.8 / 9.7*

14.3 - -

13.2 -

11.4* -

9.4* -

12.8 / 9.7*

15.6 16.7 17.6

- 15.5

- 10.1*

- 6.5*

18.0 / 16.0

15.8 15.5 16.1

- 15.2

- 6.6*

- 5.4*

17.1 / 14.3

*cytotoxic ° two values for the two positive control in absence and presence of S9, respectively # precipitation




Genotoxicity In vitro cytogenicity in mammalian cells Chromosome aberration study in Chinese hamster lung cells

1 REFERENCE

Officialuse only

1.1 Reference Wright, N. P. (2000); TI-435: Chromosome aberration test in CHL cells in vitro Safepharm Laboratories Ltd., Derby, UK unpublished report number 178/111, 08.03.2000




None




2.1 Guideline study Yes 2000/32/EC (successor of 92/69/EEC) B10, OECD no. 473 (1983), EPA FIFRA section 84-2 (1984), Japan MAFF (59 NohSan no. 4200, 1985)

2.2 GLP Yes







3.1.2.2 Purity


3.2 Study Type In Vitro mammalian chromosome aberration test:

3.2.1 Organism/cell type Chinese hamster lung (CHL) cells


-


S9 mix Prepared from livers of male Sprague Dawley rats (bw about 200 g) induced with a single i.p. injection of 500 mg/kg bw Arochlor 1254 up to 5 days prior to sacrifice.

3.2.4 Positive control 0.075, 0.05 and 0.025 µg/mL mitomycin C (MMC) was used in absence of metabolic activation for the 12, 24 and 48 hour harvests; 10 µg/mL cyclophosphamide (CP) was used as positive control in the experiments in presence of metabolic activation and for the 6 (18) hour harvest in absence of metabolic activation






3.3.1 Concentrations 2500 µg/mL corresponds to about 10 mM of TI-435 (limit concentration). Cytotoxicity pre-test: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL Mutagenicity experiments:

Exposure [h] Recovery [h] Concentrations [µg/mL] 1st experiment

12 - - S9 0, 156.25, 312.5, 625, 937.5, 1250, 1875

4 8 + S9 0, 156.25, 312.5, 625, 937.5, 1250, 1875

2nd experiment 12 - - S9 0, 78.1, 156.3, 312.5, 625, 937.5, 1250 6 18 - S9 0, 312.5, 625, 937.5, 1250

24 - - S9 0, 78.1, 156.3, 312.5, 625, 937.5, 1250 48 - - S9 0, 39, 78.1, 156.3, 312.5, 625, 937.5 4 8 + S9 0, 312.5, 625, 937.5, 1250, 1562.5,

1875 6 18 + S9 0, 312.5, 625, 937.5, 1250

3.3.2 Way of application TI-435 was dissolved in DMSO (further diluted to result in the selected concentrations). Cytotoxicity pre-test: Cell cultures (0.3–0.45⋅106 cells for exposures up to 12 hours, 0.1⋅106 cells for 48 hours) were exposed continuously for 12, 24 and 48 hours without metabolic activation, and for 4 and 6 hours followed by 8 and 18-hour recovery periods, respectively, with metabolic activation. Growth inhibition was estimated by electronic counting of cells and comparison with solvent control values. Mutagenicity experiments: Cell cultures (number of cells as in pre-test) were exposed to TI-435 according to the table above. Two hours before harvest mitosis was arrested by addition of 0.1 µg/mL colcemid. At harvest a sample of cells was counted to determine growth inhibition. After hypotonic treatment, fixation and staining 100 metaphases from each culture were counted for gaps, chromosome and chromatid-type breaks and rearrangements, and number of chromosomes (23-27).

3.3.3 Pre-incubation time -


3.4 Examinations Cytotoxicity: growth inhibition Mitotic index: number of metaphases in a total of 1000 cells Cells with structural chromosome aberrations (breaks, exchanges with and without gaps) Cells with 28-31 and >31 chromosomes were classified as aneuploid and polyploid, respectively. A positive result is recorded if the % cells with aberrations (including gaps) is ≥10%. An equivocal response is recorded for values between 5 and 10%, and a negative response for values less than 5%. An incidence of polyploidy >10% is generally recorded as positive.





3.4.1 Number of cells evaluated

Where possible: 100 metaphases per culture (totally 200 metaphases per concentration)


4.1 Genotoxicity


Yes (2nd experiment): the number of aberrant cells (including gaps) fulfilled the criteria of a positive response at 625 µg/mL after exposure for 48 h (the highest concentrations with scorable metaphases). An equivocal response was observed at 1250 µg/mL after 6 h exposure (+ 18 h recovery) and at 625 µg/mL after 24 h exposure but no relevant increase at the next higher dose level of 937.5 µg/mL. No (both experiments): no relevant increase in the number of aberrant metaphases were seen after 12 h treatment at concentrations up to 937.5 µg/mL.


Yes (2nd experiment): the number of aberrant cells (including gaps) fulfilled the criteria of a positive response at 1250 µg/mL after exposure for 6 h (+ 18 h recovery) (highest concentrations with scorable metaphases). An equivocal response was observed at 1563 µg/mL after 4 h exposure (+ 8 h recovery) (highest concentrations with scorable metaphases). No (1st experiment): no increase in the number of aberrant metaphases were seen after 4 h treatment (+ 8 h recovery) at concentrations up to 1875 µg/mL.

4.2 Cytotoxicity Yes: Markedly reduced cell counts and/or mitotic indices were observed at concentrations of ≥625 µg/mL for treatment without S9 for 12 and 48 hours ≥1250 µg/mL for treatment without S9 for 24 hours and with S9 for 6 hours (+ 18 h recovery) ≥1562.5 µg/mL for treatment with S9 for 4 hours (+ 8 h recovery) ≥2500 µg/mL for treatment without S9 for 6 hours (+ 18 h recovery)



Evaluation of the in vitro cytogenetic potential in mammalian cells; no relevant deviation from guidelines (2000/32/EC B10, OECD 473, EPA FIFRA 84-2, Japan MAFF)


Relevant increases in the number of aberrant metaphases were seen at the highest dose levels with scorable metaphases after treatment for 48 hours in absence of metabolic activation and for 6 hours (+18 h recovery) in presence of metabolic activation. Marked cytotoxicity was also seen at these concentrations (mitotic index were 25% and 39% of the respective controls).

5.3 Conclusion TI-435 induced chromosome aberrations at cytotoxic concentrations in vitro in Chinese hamster lung cells. No relevant effect was detected at lower concentrations inducing less or no cytotoxicity.

5.3.1 Reliability 1









Date 2004-10-07



Conclusion Applicant's version is adopted.

Reliability 1


Remarks

COMMENTS FROM ...







Remarks





Table A6_6_2/01-1. Table for Cytogenetic In-Vitro-Test: Cytotoxicity

Concentration [µg/mL] Treatment / Recovery Number of cells x 105/mL % of controls -S9 +S9 -S9 +S9 Cytotoxicity pre-test 0 9.8 19.5 39.1 78.1 156.3 312.5 625 1250 2500

-S9 12 h / -

+S9 4 h / 8 h

2.07 2.00 2.25 2.03 1.84 1.71 1.80 1.70 1.28 1.97

1.44 1.36 1.42 1.31 1.58 1.29 1.49 1.32 1.75 1.10

100 97 109 98 89 82 87 82 62

95°#

100 95 99 91

110 90

104 92

121 77°#

0 9.8 19.5 39.1 78.1 156.3 312.5 625 1250 2500

-S9 6 h / 18 h

+S9 6 h / 18 h

1.59 1.55 1.59 1.37 1.34 1.63 1.27 1.13 1.11 0.27

1.20 1.27 1.49 1.37 1.33 1.22 1.31 1.29 1.27 1.07

100 97 100 86 85 103 80 71 70

17°#

100 106 124 114 110 102 109 108 105 89°#

0 9.8 19.5 39.1 78.1 156.3 312.5 625 1250 2500

-S9 24 h / -

-S9 48 h / -

24 h: 1.92 1.88 1.90 1.91 2.03 1.75 1.71 1.18 0.96 1.44

48 h: 1.36 1.18 1.44 1.28 1.32 1.05 0.54 0.36 0.37 0.80

24 h: 100 98 99 100 106 91 89 62

50°° 75°#

48 h: 100 87

106 94 97 77 40 27 27° 59°#

# precipitate ° no scorable metaphases, °° few scorable metaphases





Section A6.6.3

Annex Point IIA VI.6.6.3/01

Genotoxicity In vitro gene mutation in mammalian cells

In vitro gene mutation in L5178Y mouse lymphoma cells

1 REFERENCE

Officialuse only

1.1 Reference 2000a);


1.2.1 Data owner




2.1 Guideline study Yes 2000/32/EC B17 (successor of 87/303/EEC B14), OECD no. 476 (1984), EPA FIFRA section 84-2 (1984), Japan MAFF (59 NohSan no. 4200, 1985)

2.2 GLP Yes

2.3 Deviations No




3.1.2 Specification


3.1.2.2 Purity


3.2 Study Type In vitro mammalian cell gene mutation test

3.2.1 Organism/cell type Mouse lymphoma L5178Y TK+/- cells


Thymidine kinase proficient (heterozygous)


S9 mix

3.2.4 Positive control 800 µg/mL ethylmethansulphonate (EMS) and 7.5 µg/mL cyclophos-phamide (CP) were used as positive controls in the experiments absence and presence of metabolic activation, respectively



Section A6.6.3





3.3.1 Concentrations

3.3.2 Way of application

3.3.3 Pre-incubation time


3.4 Examinations

significant increase in mutant frequency a greater tha



Section A6.6.3





4.1 Genotoxicity



4.2 Cytotoxicity



In vitro evaluation of gene mutation in mammalian cells; no relevant deviation from test guidelines (2000/32/EC B17, OECD 476, EPA FIFRA 84-2, Japan MAFF)


5.3 Conclusion TI-435 and/or metabolites increased the frequency of mutation at the TK +/- locus of the L5178Y cells indicating a mutagenic effect at cytotoxic dose levels in presence of S9. No positive response was detected in absence of S9.

5.3.1 Reliability 1





Date 2006-10-27




Section A6.6.3




Results and discussion

Conclusion

Reliability 1


Remarks

COMMENTS FROM ...







Remarks



Section A6.6.3








Section A6.6.3



In vitro gene mutation in Chinese hamster lung V79 cells

1 REFERENCE

Officialuse only

1.1 Reference (1999a);


1.2.1 Data owner




2.1 Guideline study Yes 2000/32/EC B17 (successor of 88/302/EEC), OECD no. 476 (1984), EPA FIFRA section 84-2 (1984)

2.2 GLP Yes

2.3 Deviations No




3.1.2 Specification

3.1.2.1 Description Beige powder

3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study (stability in vehicle tested)

3.2 Study Type In vitro mammalian cell gene mutation test

3.2.1 Organism/cell type Chinese hamster lung V79 cells


Hypoxanthine-guanine phosphoribosyl transferase (HRPT) proficient


S9 mix

3.2.4 Positive control 900 µg/mL ethylmethansulphonate (EMS) and 20 µg/mL dimethylbenz-anthracene (DMBA) were used as positive controls in the experiments absence and presence of metabolic activation, respectively



Section A6.6.3





3.3.1 Concentrations

3.3.2 Way of application

3.3.3 Pre-incubation time -


3.4 Examinations



Section A6.6.3





4.1 Genotoxicity



4.2 Cytotoxicity



In vitro evaluation of gene mutation in mammalian cells; no relevant deviation from test guidelines (2000/32/EC B17, OECD 476, EPA FIFRA 84-2, Japan MAFF)


No mutagenic response after treatment of Chinese hamster V79 cells with TI-435 was detected in presence or absence of S9 at concentrations up to twice the limit concentration of 10 mM. Slight to moderate cytotoxicity was seen at ≥2500 µg/mL.

5.3 Conclusion TI-435 and/or metabolites did not indicate any potential for mutagenic effects in this test. TI-435 was considered to be non-mutagenic.

5.3.1 Reliability 1





Date 2004-10-07


Results and discussion Applicant’s version is adopted.

Conclusion Applicant’s version is adopted.

Reliability 1


Remarks



Section A6.6.3




COMMENTS FROM ...







Remarks



Section A6.6.3








Section A6.6.4


Genotoxicity 1st in vivo mutagenicity study

Mouse micronucleus test

1 REFERENCE

Officialuse only

1.1 Reference (2000b);


1.2.1 Data owner




2.1 Guideline study Yes 2000/32/EC (successor of 92/69/EEC) B12, OECD guideline no. 474 (1983), EPA section 84-2 (1984), Japan MAFF, 59 NohSan no. 4200 (1985)

2.2 GLP Yes

2.3 Deviations No




3.1.2 Specification


3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study; analysis of TI-435 formulations prepared for the main study demonstrated adequate stability, homogeneity and achieved concentrations.

3.1.2.4 Maximum tolerable dose

100 mg/kg bw were chosen as maximum

dose level for this study, which is considered to represent the MTD

3.2 Test Animals male and female 5–7 week old mice per group body weight range 20–26 g, including a positive and negative (vehicle) control group


Oral

3.3.1 Number of applications

1

3.3.2 Interval between applications

-



Section A6.6.4





24, 48, and 72 h after treatment

Oral

3.3.4 Type gavage

3.3.5 Concentration 0, 25, 50 and 100 mg/kg bw

3.3.6 Vehicle Arachis oil


2.5-10 mg/mL (for 25-100 mg/kg bw)


10 mL/kg bw

3.3.9 Controls Negative control: vehicle Positive control: 50 mg/kg bw cyclophosphamide (CP) in distilled water (sacrifice 24 hours after application)

3.4 Examinations


3.4.2 Tissue



4.1 Clinical signs

Main study: three 100 mg/kg bw treated female mice assigned for the 48 hours sacrifice died prematurely within 24 hours after application. Clinical signs comprising hunched posture, decreased respiratory rate, laboured respiration, ptosis and lethargy were seen in all treated groups.

4.2 Haematology / Tissue examination

There was no indication for cytotoxicity.

4.3 Genotoxicity No



Section A6.6.4




4.4 Other The number of PCE with micronuclei were significantly increased in the positive controls.



Genotoxicity evaluation in vivo in the bone marrow of mice after a single oral application o TI-435; no relevant deviations from guidelines (2000/32/EC B12, OECD 474, EPA 84-2, Japan MAFF)


TI-435 did not increase the incidence of micronucleated PCE after single oral application of mice at dose levels of 25-100 mg/kg bw at three sacrifice time points (24, 48, 72 h).

5.3 Conclusion TI-435 was not genotoxic in this in vivo test system.

5.3.1 Reliability 1





Date 2004-10-07




Reliability 1


Remarks

COMMENTS FROM ...







Remarks



Section A6.6.4




Table A6_6_4/01-1. Table for Micronucleus Test In Vivo



Section A6.6.5


Genotoxicity 2nd in vivo mutagenicity study

UDS in vivo in rats (hepatocytes)

1 REFERENCE

Officialuse only

1.1 Reference (1999b);


1.2.1 Data owner




2.1 Guideline study Yes 2000/32/EC B.39 (successor of 88/302/EEC), OECD 486

2.2 GLP Yes

2.3 Deviations None relevant for the validity or integrity of the study




3.1.2 Specification

3.1.2.1 Description yellow powder

3.1.2.2 Purity


3.1.2.4 Maximum tolerable dose

A pre-test resulted in no mortality at dose levels of 4000 and 5000 mg/kg bw (single gavage application); therefore 5000 mg/kg bw was chosen as maximum dose level for this study, which is considered to represent the MTD

3.2 Test Animals male young adult (10–14 week old) rats 120–140 g bw, per group

No relevant sex-difference in toxicity is expected


Oral

3.3.1 Number of applications

1



Section A6.6.5




3.3.2 Interval between applications

-


4, 16 h after treatment

Oral

3.3.4 Type Gavage

3.3.5 Concentration 0, 2500 and 5000 mg/kg bw

3.3.6 Vehicle 0.5% aqueous Cremophor


-


20 mL/kg bw for vehicle control and TI-435 treated animals 10 mL/kg bw for positive control groups (corn oil and DMSO were used as vehicles for 2-AAF and DMH, respectively)

3.3.9 Controls Negative control: vehicle (0.5% aqueous Cremophor) Positive control: 100 mg/kg bw 2-acetylaminofluorene (2-AAF) for the 16-h sacrifice 40 mg/kg bw N,N'-dimethylhydrazine (DMH) for the 4-h sacrifice

3.4 Examinations


3.4.2 Tissue liver

Number of animals:

all animals

Number of cells:

150 per animal

Time points:

Type of cells hepatocytes

Parameters: Cytotoxicity (viability after perfusion)

3.5 Further remarks


4.1 Clinical signs



Section A6.6.5




4.2 Haematology / Tissue examination

4.3 Genotoxicity

4.4 Other



Genotoxicity evaluation (DNA repair activity) after single oral application of TI-435 in rats (hepatoytes); no relevant deviation to guidelines (2000/32/EC B.39, OECD 486)


TI-435 and/or metabolites did not induce unscheduled DNA synthesis in rat hepatocytes following in vivo exposure.

5.3 Conclusion TI-435 did not indicate any genotoxic potential in vivo.

5.3.1 Reliability 1





Date 2004-10-07




Reliability 1


Remarks



Section A6.6.5




COMMENTS FROM ...







Remarks





1 REFERENCE

Officialuse only

1.1 Reference Biegel, L.B. (2000a); 104-week dietary combined chronic toxicity and carcinogenicity study with TI-435 in rats Covance Laboratories Inc, Wisconsin 53704-2595, USA unpublished report No. 6155-108, 11.04.2000




None




2.1 Guideline study Yes 87/302/EEC B.33 (May 1988); Japan MAFF (59 NohSan no. 4200, 1985); EPA-FIFRA section 83-5; OECD no. 453 (1981) and EPA-OPPTS 870.4300 (June 1998)

2.2 GLP Yes

2.3 Deviations None






3.1.2.2 Purity


3.2 Test Animals 80 male and 80 female albino rats per group (Crl:CD (SD)BR VAF/Plus

strain, age 34-40 days old, bodyweight range 105-194 g, Charles River Laboratories Inc., USA): 20 animals per sex and group for interim sacrifice after 52 weeks of treatment


Oral (dietary)


104 weeks


Daily


None





3.3.4 Oral


3.3.4.2 Concentration 0, 150, 500, 1500 and 3000 ppm corresponding to 0, 8.1, 27.4, 82 and 157 mg/kg bw/day for males and 0, 9.7, 32.5, 97.8 and 193 mg/kg bw/day for females food consumption per day: ad libitum



-


-


3.4 Examinations

3.4.1 Observations



3.4.2 Body weight yes (weekly for the first 14 weeks, every 4 weeks thereafter)

3.4.3 Food consumption yes (weekly for the first 13 weeks, every 4 weeks thereafter)

3.4.4 Water consumption yes (interim sacrifice groups at weeks 12, 25 and 51)


yes (all surviving animals at pre-test, prior to the interim and terminal sacrifice)

3.4.6 Haematology yes, 10 animals/sex/group during weeks 13, 27, 53, 79 and prior to the terminal sacrifice (weeks 103-105) Parameters: Haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, blood cell morphology, prothombin time, activated partial thromboplastin time, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration

3.4.7 Clinical Chemisty yes, 10 animals/sex/group during weeks 13, 27, 53, 79 and prior to the terminal sacrifice (weeks 103-105) Parameters: sodium, potassium, calcium, inorganic phosphorous, chloride, glucose, cholesterol, blood urea nitrogen, total bilirubin, creatinine, total protein, albumin, globulin, albumin/globulin ratio, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyltransferase, creatine kinase, triglycerides

3.4.8 Urinalysis Yes 10 animals/sex/group during weeks 13, 27, 53, 79 and prior to the terminal sacrifice (weeks 103-105) Parameters: appearance, volume, pH, specific gravity, protein, glucose, blood, ketones, bilirubin, sediment, urobilinogen


3.5.1 Organ Weights Yes (all animals surviving to scheduled interim or terminal sacrifice) organs: liver, kidneys, adrenals, testis, epididymis, ovaries, brain,





thyroid (with parathyroid), lung, heart, uterus with cervix, spleen, pituitary, prostate, seminal vesicle, thymus


Yes gross pathology: all animals histopathology: all animals (* tissues were not examined microscopically) organs: adrenals, aorta, brain, caecum, cervix, colon, duodenum, epididymis, esophagus, eye, femur with bone marrow, harderian gland*, head*, heart, ileum, jejunum, kidney, larynx*, lesions, liver, lung, lymph nodes, female mammary gland, masses and associated tissues, muxcle (tigh), optic nerve, ovaries, pancreas, pituitary, prostate, rectum, mandibular salivary gland, sciatic nerve, seminal vesicle, skin, spinal cord, spleen, sternum with bone marrow, stifle joint, stomach, testis, thymus, thyroid with parathyroid, trachea, urinary bladder, uterus, vagina


3.5.4 Statistics Body weights, bw changes, food consumption, food efficiency, organ weights, organ-body weight ratios and organ-brain weight ratios: one-way analysis of variance (ANOVA). Levene’s test was performed to test for heterogeneity, and, where appropriate, transformations were performed to stabilise the variances. ANOVA was then performed on the transformed data. If ANOVA was significant, Dunnett’s multiple comparison t-test was used for pairwise comparisons between treated and control groups. Survival data. log-rank test at the 5%, two-tailed probability level for group comparisons. Dose-response relationship of mortality: logistic regression method at the 5% one-tailed probability level. Fatal, non-fatal and incidental tumours: Peto’s mortality-prevalence test.

3.6 Further remarks During week 66 or 67, 10 animals/sex from each group were subjected to a functional observation battery (FOB) of neurological tests.


4.1 Observations

4.1.1 Clinical signs There were no treatment-related effects at any dose level on clinical signs, ophthalmological findings and FOB observations.

4.1.2 Mortality There were no adverse effects of treatment on survival at any dose level. Probably related to the reduced bw development, high dose animals had highest survival (55% and 50% for males and females, respectively, at termination) (see also table A6_7/01-1). Overall the survival incidences were sufficient to evaluate the carcinogenic potential of TI-435, even if the group survival was <50% at termination at 0-1500 ppm. In case of a carcinogenic test compound increased tumour incidences would have occurred with the highest probability at the highest dose level - for TI-435 no relevant increase of tumour incidence was observed with a survival ≥50% at 3000 ppm. Survival in the other groups fell below 50% only a few weeks before termination (weeks 91 and 88 onwards for males and females, respectively). Therefore a sufficient number of rats survived long enough to allow tumour formation - in case of a carcinogenic test compound - and thus a meaningful evaluation of carcinogenicity.

4.2 Body weight gain A reduced bw development was noted for 1500 ppm treated females and





at 3000 ppm (both sexes) (see table A6_7/01-1). Cumulative bw gain in the first 50 weeks of the study were 13% and 28% below controls for 1500 and 3000 ppm treated females, respectively, and 13% below controls for males at 3000 pmm. Lower body weights were occasionally recorded for males at 1500 ppm and females at 500 ppm mostly in the first 14 weeks of the treatment period. As these reductions did not exceed 5% in comparison to controls and as the overall weight gains were not significantly decreased, they are considered to be not-adverse.

4.3 Food/water consumption and compound intake

The food consumption was reduced at 3000 ppm and slightly lower values were also noted for 1500 ppm treated females mainly in the first part of the study (see table A6_7/01-1). No relevant effect on food consumption was seen for the other groups. Compound intake see 3.3.4.2 Analysis of diet samples demonstrated adequate homogeneity, stability and achieved concentration of TI-435 in diet. Mean values of the homogeneity were 95.9-96.9% and 96.0-100% of the theoretical concentrations of 100 and 3000 ppm, respectively. Stability analysis revealed TI-435 was stable in diet at concentrations of 25 or 5000 ppm for 29 days at room temperature and between –10 and –30°C. The mean achieved concentrations of all diets ranged from 88.7 to 109% of nominal concentrations. Water consumption was unaffected by treatment at all dose levels.


See 4.1.1

4.5 Blood analysis

4.5.1 Haematology No treatment-related effects were observed.

4.5.2 Clinical chemistry A slight but consistent elevation of serum inorganic phosphorus concentration at all sampling intervals in male rats treated at 3000 ppm was shown, which might have been associated with the increased incidence of mineralisation of renal pelvic observed in these animals.



4.6.1 Organ weights There were no effects on organ weights that were directly attributable to treatment, although some minor fluctuations occurred at 3000 ppm that were considered to be associated with reduced body weight gain.


No treatment-related macroscopic lesions were evident at any dose level. Treatment-related non-neoplastic histomorphological alterations occurred in the glandular stomach, kidneys and ovaries (see table 6_7/01-2). Indication for irritation were seen at 3000 ppm in the glandular stomach: increased incidences of erosion (females), hemorrhage (males) and edema of the stomach wall (both sexes). Incidences of pelvic mineralisation and hyperplasia of the transitional epithelium in the kidney were slightly increased in 3000 ppm treated males. Increased incidences of interstitial hyperplasia of the ovaries occurred in females at ≥500 ppm. However the severity of this finding was only marginally increased at 3000 ppm in correspondence to controls. It was





seen in ovaries undergoing atrophy with little or no active follicular development and was considered to be an age-related change of dubious toxicological significance (in average it was found in rats sacrificed later than about 701 days of the study). The increased incidence of this finding is considered to be at least partly due to the longer survival for females at ≥1500 ppm. No treatment-related effects occurred at any dose level on the nature and incidence of neoplastic lesions. Although the incidences of thyroid C-cell adenoma were higher in the female groups treated at ≥1500 ppm, the combined incidence of C-cell adenoma and carcinoma of 3000 ppm treated females was not significantly greater than the control incidence. All other neoplastic changes and incidences were consistent with spontaneously occurring lesions expected in Sprague-Dawley-derived rats.



Toxicity evaluation after chronic (2-year) dietary exposure of rats to TI-435; no relevant deviation from guidelines (87/302/EEC; Japan MAFF; EPA-FIFRA 83-5; OECD 453 and EPA-OPPTS 870.4300)


Food consumption and bw development was reduced at 3000 ppm (both sexes) and at 1500 ppm in females. Slightly lower body weights were also recorded for 500 ppm treated females and 1500 ppm treated males during the first part of the study. Clinical pathology revealed a slight increase in inorganic phosphorus for males at 3000 ppm. Histopathological changes were seen in the glandular stomach (irritation, both sexes at 3000 ppm) and the kidney (males at 3000 ppm). Increased incidences of interstitial cell hyperplasia of the ovaries were recorded at ≥500 ppm in females representing an age-related change and therefore of dubious toxicological relevance. It was considered to be at least partly due to the longer survival in the ≥1500 ppm treated females.

5.3 Conclusion Chronic treatment with up to 3000 ppm TI-435 for two years was tolerated well in rats and the high dose represented the MTD. Main effects of treatment were reduced food consumption and bw development leading to a better survival, and some histopathological changes in the stomach (slight irritation), kidney and ovaries (age-related, of dubious toxicological relevance). Treatment with TI-435 did not result in any indication for a carcinogenic response.

5.3.1 LO(A)EL LO(A)EL = 1500 and 500 ppm in males and females, respectively, corresponding to 82 and 32.5 mg/kg bw/day for males and females, respectively males: reduced food consumption and bw gain (slight increase in blood phosphorus levels, histopathological changes in glandular stomach and kidney) females (interstitial cell hyperplasia in ovaries, considered to be of only dubious toxicological relevance but was used as the basis for the LOAEL/NOAEL as a conservative assumption)

5.3.2 NO(A)EL NOAEL = 500 and 150 ppm for males and females, respectively, corresponding to 27.4 and 9.7 mg/kg bw/day for males and females, respectively

5.3.3 Other -





5.3.4 Reliability 1





Date 2004-10-07


Results and discussion Applicant’s version is acceptable with exception of the opinion regarding effects on the ovary. The increased incidences of interstitial cell hyperplasia of the ovaries at ≥500 ppm are considered to be toxicologically relevant. The finding is age-related insofar as it occured only in females older that 630 days (>600 days on study). However, there was a clear relationship to treatment and dose, especially when only the subpopulations at risk (animals with more than 600 days on study) were compared (see CA-Table 1). Although not statistically significant, an effect appears to be present also at the lowest dose (150 ppm) where a doubling in incidence is observed in all subgroups older that 52 months.

Conclusion LO(A)EL: 500 ppm (32.5 mg/kg bw/day) for females based on interstitial cell hyperplasia of the ovaries 1500 ppm (82 mg/kg bw/day) for males based on reduced food consumption, reduced bw gain, clinical chemistry findings and histopathological changes in glandular stomach and kidney NO(A)EL: 150 ppm (9.7 mg/kg bw/day) for females 500 ppm (27.4 mg/kg bw/day) for males No carcinogenic potential

Reliability 1


Remarks CA-table 1 is added by the RMS.








Remarks



Table A6_7/01-1. Survival, bw development and food consumption and results of clinical chemistry (2-year rat)

Parameter Males Females ppm 0 150 500 1500 3000 0 150 500 1500 3000

Survival [%] week 78 week 96 (= 24 x 4) termination (week 103-105)

78 43

28

62 40

30

73 45

30

73 52

40

93 67

55

72 35

25

70 37

27

72 37

28

73 50

38

83 62

50


152 543 778 759

150 534 766 784

151 532 743 799

150 522* 741 754

149 491** ↓691** ↓

725

137 287 392 447

136 282 408 500

134 276* 387 456

135 267** ↓ 355** ↓

424

137 249** ↓320** ↓356** ↓

Body weight gain [g] week 1-13 week 13-50 week 1-50 week 1-104

391 235 626 607

384 232 616 637

381 211 592 655

372 219 591 603

343** ↓200** ↓ 543** ↓

574

150 105 254 313

146 126 272 370

142 111 253 321

132**↓ 89 (↓)

221**↓ 289

112** ↓72** ↓ 183** ↓220** ↓

Food consumption [% of control] week 1-13 week 17-101

100 100

98 99

101 100

96 98

87 ↓ 94 ↓

100 100

95 102

94 98

89 ↓ 95

81 ↓ 90 ↓

Inorganic phosphorus [mg/dL] week 13 week 27 week 53 week 79 week 105

7.7 7.5 6.5 6.0 5.9

7.8 7.7 6.8 6.2 6.2

7.7 7.4 6.8 6.0 5.9

8.0 7.8 6.5 6.6 7.4

8.7* ↑8.2 (↑)7.5* ↑7.4* ↑7.1* ↑

6.0 6.1 5.5 5.6 4.7

6.0 5.5 6.4 5.8 4.8

6.0 6.1 5.4 5.8 5.1

6.6 6.6 5.7 5.8 5.3

6.6 6.7 5.6 6.4 5.4

* p <0.05, ** p <0.01; arrows in brackets (↓↑) indicate a trend that was not statistically significant



Table A6_7/01-2. Results of chronic toxicity/carcinogenicity study (2-year rat): histopathology and tumour incidences Parameter 0 ppm 150 ppm 500 ppm 1500 ppm 3000 ppm

ma fa ma fa ma fa ma fa ma fa

Glandular Stomach erosion hemorrhage edema

5/80 0/80 1/80

2/80 0/80 0/80

6/80 0/80 1/80

0/80 0/80 2/80

3/80 0/80 0/80

2/80 0/80 2/80

5/80 1/80 1/80

7/80 1/80 2/80

8/80 5/80 8/80

11/80 1/80 6/80

Kidney pelvic mineralisation transitional cell hyperplasia

34/80 33/80

77/80 60/80

30/80 30/80

76/80 58/80

34/80 29/80

74/80 59/80

43/80 29/80

75/80 65/80

52/80 51/80

79/80 65/80

Ovaries interstitial cell hyperplasia (grade°)

- 5/80 (2.6)

- 10/80 (2.3)

- 13/80 (2.3)

- 20/80 (2.4)

- 33/80 (3.0)

Thyroid C-cell adenoma C-cell carcinoma

8/80 5/80

7/80 2/80

13/801/80

13/802/80

17/801/80

9/80 1/80

16/801/80

17/80 1/80

5/80 3/80

16/801/80

total 13/80 9/80 14/80 15/80 18/80 10/80 17/80 18/80 8/80 17/80

No of animals interim sacrifice unscheduled death terminal sacrifice

18 45 17

20 45 15

18 44 18

19 45 16

19 43 18

20 48 12

19 37 38

19 38 23

20 27 33

20 30 30

total 80 80 80 80 80 80 80 80 80 80

Benign neoplasms interim sacrifice unscheduled death terminal sacrifice

7 56 25

14 57 21

8 57 29

8 63 31

9 50 28

7 68 24

8 45 41

9 53 41

5 34 40

10 38 60

total 88 92 94 102 87 99 94 103 79 108

Malign neoplasms interim sacrifice unscheduled death terminal sacrifice

2 18 5

3 25 6

0 4 4

1 25 10

0 20 4

3 30 9

1 5 8

3 18 11

0 15 6

1 16 9

total 25 34 8 36 24 42 14 32 21 26

Benign+malign neoplasms interim sacrifice unscheduled death terminal sacrifice

9 74 30

17 82 27

8 61 33

9 88 41

9 70 32

10 98 33

9 50 49

12 71 52

5 49 46

11 54 69

total 113 126 102 138 111 141 108 135 100 134 a number of animals affected/total number of animals ° minimal - grade 1 slight grade 2 moderate grade 3 moderate severe grade 4 severe grade 5



Evaluation by Rapporteur Member State, CA-Table CA-Table 1. Results of chronic toxicity/carcinogenicity study (2-year rat): Ovary interstitial cell hyperplasia Parameter Ovary interstitial cell hyperplasia


Interim sacrifice N examined N affected % affected

20 0 0

19 0 0

20 0 0

19 0 0

20 0 0

Unscheduled deaths N examined N affected % affected

45 2

4.4

45 4

8.9

48 7

14.6

38 7

18.4

30 11

37.9

Terminal sacrifice N examined N affected % affected

15 3 20

16 6

37.5

12 6 50

23 13

56.5

30 22 73

Survivors >600 days N examined N affected % affected

34 5

14.5

35 10

28.6

34 13

38.2

36 20

55.6

48 33

68.8



Section A6.7 Annex Point IIA VI.6.7/02

Carcinogenicity study (18-month) Mouse 18-month dietary carcinogenicity study in mice

1 REFERENCE

Officialuse only

1.1 Reference Biegel, L.B. (2000b); 78-week dietary carcinogenicity study with TI-435 in mice Covance Laboratories Inc, Wisconsin 53704-2595, USA unpublished report No. 6155-109, 27.03.2000




None




2.1 Guideline study Yes 87/302/EEC (May 1988); Japan MAFF (59 NohSan no. 4200, 1985); EPA-FIFRA section 83-2; OECD no. 451 (1981), EPA-OPPTS 870.4200 (June 1996)

2.2 GLP Yes

2.3 Deviations None






3.1.2.2 Purity


3.2 Test Animals 50 male and 50 female 5 week old albino mice per group (Crl:CD-1

(ICR)BR VAF/Plus strain, bodyweight range 15.8–33.8 g, Charles River Laboratories Inc., USA); no interim sacrifice


Oral (dietary)


18 month (at least 78 weeks)

3.3.2 Interim sacrifice(s) -

3.3.3 Final sacrifice after 78 weeks


Daily


None





Oral

3.3.6 Type in food

3.3.7 Concentration 0, 100, 350, 1250 and 1800/2000 ppm (treatment with 700 ppm for weeks 1-4, increased to 2000/2500 ppm for weeks 5-10/11-34, reduced to 1800/2000 ppm for females/males from week 35 onwards; the average over 78 weeks was 2087 and 1974 ppm for males and females, respectively) corresponding to 0, 13.5, 47.2, 171.4 and 251.9 mg/kg bw/day for males and 0, 17.0, 65.1, 215.9 and 281.1 mg/kg bw/day for females food consumption per day: ad libitum

3.3.8 Vehicle None (TI-435 was mixed with diet directly)


-


-


3.4 Examinations

3.4.1 Body weight yes (weekly for the first 14 weeks, every 4 weeks thereafter)

3.4.2 Food consumption Yes (weekly for the first 14 weeks, every 4 weeks thereafter)


3.4.4 Clinical signs yes (daily), detailed clinical examination was performed weekly mortality: twice daily

3.4.5 Macroskopic investigations

Masses: yes (weekly)


No

3.4.7 Haematology yes, control and high dose animals during weeks 52 and prior to sacrifice (week 79-80) and for unscheduled deaths of all groups Parameters: total and differential leukocyte count, blood cell morphology

3.4.8 Clinical Chemisty No

3.4.9 Urinalysis No

3.4.10 Pathology





3.4.10.1 Organ Weights Yes (all animals surviving to terminal sacrifice) organs: liver with drained gall bladder, kidneys, adrenals, testis, epididymis, ovaries, brain, lung, heart, spleen

3.4.10.2 Gross and histopathology

Yes gross pathology: all animals histopathology: all animals (* tissues were not examined microscopically) organs: adrenals, aorta, brain, caecum, cervix, colon, duodenum, epididymis, esophagus, eye, femur with bone marrow, harderian gland*, head*, heart, ileum, jejunum, kidney, larynx*, lesions, liver with gall-bladder, lung, lymph nodes, female mammary gland, masses and associated tissues, muxcle (tigh), optic nerve, ovaries, pancreas, pituitary, prostate, rectum, mandibular salivary gland, sciatic nerve, seminal vesicle, skin, spinal cord, spleen, sternum with bone marrow, stifle joint, stomach, testis, thymus, thyroid with parathyroid, trachea, urinary bladder, uterus, vagina


3.5 Statistics Body weights, body weight changes, food consumption, food efficiency, organ weights, organ-to-body weight ratios and organ-brain weight ratios: one-way analysis of variance (ANOVA). Levene’s test was performed to test for heterogeneity, and, where appropriate, transformations were performed to stabilise the variances. ANOVA was then performed on the transformed data. If ANOVA was significant, Dunnett’s multiple comparison t-test was used for pairwise comparisons between treated and control groups. Survival data: log-rank test at the 5%, two-tailed probability level for group comparisons; dose-response relationship of mortality: logistic regression method at the 5% one-tailed probability level. Fatal, non-fatal and incidental tumours were analysed by Peto’s mortality-prevalence test.



4.1 Body weight The overall body weight gain was decreased in females treated at 1250 ppm (by 26.4%), in males treated at 2000 ppm (by 23.6%) and in females at 1800 ppm (by 35.2%) (see table 6_7/02-1).


The food consumption of both sexes treated at 2000/1800ppm was reduced throughout the study compared to controls (79-92.5% for males and 75.9-88.9% for females) (see table 6_7/02-1). Compound intake see 3.3.4.2 Analysis of diet samples demonstrated adequate homogeneity, stability and achieved concentration of TI-435 in the diet. Mean values for homogeneity in all diets analysed were 92-99.6% of nominal concentrations. Stability analysis demonstrated TI-435 was stable in diet at concentrations of 25 and 5000 ppm for 29 days at room temperature and at between –10 and –30°C. The mean achieved concentrations in diet were 86.0-107% of nominal concentrations.

4.3 Water consumption

-

4.4 Clinical signs The only clinical sign considered to be treatment-related was vocalisation in animals treated at ≥1250 ppm. There were no statistically significant adverse effects of treatment on survival at any dose level (see also table A6_7/02-1). However slightly





lower survival was noted for the high dose females. The survival incidences were sufficient to evaluate the carcinogenic potential of TI-435.

4.5 Macroscopic investigations

There was no effect of treatment on the occurrence of masses during the study.


-

4.7 Haematology There was no effect of treatment on differential white cell count or cell morphology after 52 and 78 weeks of treatment.

4.8 Clinical Chemistry -

4.9 Urinalysis -

4.10 Pathology No treatment-related macroscopic lesions were evident at any dose level.

4.11 Organ weights There were no treatment-related effects on organ weights and ratios other than minor changes attributable to lower terminal body weights in females treated at 1250 ppm and in males and females treated at 2000 or 1800 ppm. In absence of a clear dose-relationship and in absence of any histopathological evidence for kidney effects, the marginally lower kidney weights seen in treated males are considered to be incidental and unrelated to treatment (or at least not adverse). Relative kidney weight (to bw) variations were 7% at 150 and 300 ppm in comparison to controls, and between 9-10% at 1250/2000 ppm.

4.12 Gross and histopathology

Treatment-related non-neoplastic histomorphological alterations were confined to minimal to moderate centrilobular to panlobular hepatocellular hypertrophy in both sexes treated at ≥1250 ppm (see table 6_7/02-2). Although the incidence of this common finding was also increased in 100 ppm treated males, a similar effect was not apparent in males treated at 350 ppm. The slight increase in 100 ppm treated males is therefore considered to be unrelated to treatment. No treatment-related effects occurred at any dose level on the nature and incidence of neoplastic lesions. All neoplastic changes were consistent with spontaneously occurring lesions expected in the CD-1 strain mouse.

4.13 Other examinations

-

4.14 Time to tumours -

4.15 Other -



Carcinogenicity evaluation after chronic (18-month) dietary exposure of mice to TI-435; no relevant deviation from guidelines (87/302/EEC; Japan MAFF; EPA-FIFRA 83-2; OECD 451, EPA-OPPTS 870.4200)


Body weight development was reduced at ≥1250 ppm and lower food consumption was recorded at 1800/2000 ppm. Increased incidence of hepatocyte hypertrophy occurred at ≥1250 ppm.

5.3 Conclusion Chronic treatment with up to 1800/2000 ppm TI-435 for 18 month was tolerated well in mice and the high dose represented the MTD. Main effects of treatment were reduced bw development and food consumption, and slight to moderate hepatocellular hypertrophy





indicated the liver as a target organ. Treatment with TI-435 did not result in any indication for a carcinogenic response. LOAEL = 1250 ppm corresponding to 171 and 216 mg/kg bw/day for males and females, respectively due to reduced bw development (females) and hepatocellular hypertrophy NOAEL = 350 ppm corresponding to 47.2 and 65.1 mg/kg bw/day for males and females, respectively

5.3.1 Reliability 1





Date 2004-10-07

Materials and Methods Ad 3.3.7: Average substance intake over 78 weeks in the high dose group amounted to 254 mg/kg bw/day for males and 322 mg/kg bw/day for females. The values given by the applicant are based on the intake from week 35 onwards.

Results and discussion Ad 4.1.2: The investigators who conducted the study considered hepatocellular hypertrophy in males at 100 ppm to be a test substance-related, non-adverse effect despite the lack of a similar response at 350 ppm. The RMS agrees with this opinion because the incidence at 100 ppm was clearly elevated (see table 6_7/02-2).

Conclusion LO(A)EL: 1250 ppm (171 mg/kg bw/day for males, 216 mg/kg bw/day for females) based on reduced bw development (females), behavioral changes typical for this class of compounds in mice (vocalisation) and hepatocellular hypertrophy NO(A)EL: 350 ppm (47.2 mg/kg bw/day for males, 65.1 mg/kg bw/day for females) No carcinogenic potential

Reliability 1


Remarks












Remarks

Table A6_7/02-1. Survival, clinical signs, bw development and food consumption (18-month mouse)

Parameter Males Females ppm 0 100 350 1250 2000° 0 100 350 1250 1800°

Survival [%] at termination (week 78-79)

68 84 60 80 68 80 86 58 76 46

Non-traumatic vocalisationAffected/total animals

0/50 1/50 2/50 22/50 45/50 0/50 0/50 3/50 46/50 42/50

Body weight [% of controls] week 1 week 4 week 10 week 34 week 78

100 100 100 100 100

98 98

95 ↓ 96 96

100 98 96 99 96

100 98 96 96 96

98 98

94 ↓ 84 ↓ 88 ↓

100 100 100 100 100

99 103 100 99 98

100 99 98 97 93

99 100 96

93 ↓ 87 ↓

99 101 95 ↓ 83 ↓ 84 ↓

Cumulative body weight gain [g] week 1- 4 week 5-10 week 11-33 week 37-77 week 1-78

6.5 3.6 4.8 0.9 16.1

5.8 2.9 5.1 1.6

15.0

5.7 3.8 4.6 0.9

14.5

5.4** ↓3.3 4.6 0.9

14.6

7.2 0.8** ↓0.7** ↓3.4** ↑12.3**↓

5.5 3.0 6.6 4.0 19.3

5.9 2.9 6.2 3.7

19.0

5.1 3.0 5.8 2.8

16.7

5.5 2.2* ↓ 4.9* ↓ 2.0 (↓)

14.2**↓

5.8 1.6** ↓1.8** ↓

4.2 12.5**↓

Mean food consumption [g/week] week 1- 4 week 5-10 week 11-33 week 37-77

37.0 36.4 36.1 36.1

37.0 35.9 34.7 35.3

37.0 36.6 35.5 35.3

36.7 35.9 35.3 35.8

38.3 33.5 (↓)30.1 (↓)33.9 (↓)

37.8 39.4 38.9 39.4

37.3 39.3 38.9 40.0

39.7 40.7 41.2 41.9

35.8 39.2 37.3 38.5

39.1 37.2 (↓)31.6 (↓)36.3 (↓)

* p <0.05, ** p <0.01; arrows in brackets (↓↑) indicate a trend that was not statistically significant ° from week 35 onwards; before that treatment with 700, 2000 and 2500 ppm for weeks 1-4, 5-10 and 11-34, respectively



Table A6_7/02-2 Results of carcinogenicity study (18-month mouse): histopathology and tumour incidences

Parameter 0 ppm 100 ppm 350 ppm 1250 ppm 2000/1800° ppm ma fa ma fa ma fa ma fa ma fa

Liver Hepatocellular hypertrophy

17/50 5/50 34/50 5/50 15/50 3/50 27/50 11/50 40/50 15/50

No of animals unscheduled death terminal sacrifice

16 34

10 40

8 42

7 43

20 30

21 29

10 40

12 38

16 34

27 23

total 50 50 50 50 50 50 50 50 50 50

Benign neoplasms unscheduled death terminal sacrifice

0 10

1 14

2 10

2 13

1 9

4 9

0 7

0 11

0 8

1 5

total 10 15 12 15 10 13 7 11 8 6

Malign neoplasms unscheduled death terminal sacrifice

2 2

6 7

3 6

2 12

4 5

9 11

0 3

3 5

2 1

2 2

total 4 13 9 14 9 20 3 8 3 4

Benign+malign neoplasms unscheduled death terminal sacrifice

2 12

7 21

5 16

4 25

5 14

13 20

0 10

3 16

2 9

3 7

total 14 28 21 29 19 33 10 19 11 10 a number of animals affected/total number of animals ° from week 35 onwards; before that treatment with 700, 2000 and 2500 ppm for weeks 1-4, 5-10 and 11-34, respectively




Teratogenicity Study Rat Developmental toxicity study (gavage application) in the rat

1 REFERENCE

Officialuse only

1.1 Reference York, R. G. (1998a); Oral (gavage) developmental toxicity study of TI-435 in rats Argus Research Laboratories Inc., Horsham, PA 19044, USA unpublished report number 1120-001, 14.04.1998




None




2.1 Guideline study Yes 87/302/EEC B.31, OECD draft guideline no. 414 (1999), US-EPA OPPTS 870.3700 (1996), Japan MAFF (59 NohSan no. 4200, 1985)

2.2 GLP Yes

2.3 Deviations No






3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study: . The stability and homogeneity of TI-435 in the vehicle was confirmed in a previous study; and all test article-vehicle formulations were analysed for achieved concentration twice during the study

3.2 Test Animals 25 naturally mated female Sprague-Dawley rats per group ] VAF/Plus strain, body weight range 201 – 231 g, Charles River Laboratories Inc., USA); including a control group


Oral


rat day 6-19 post mating


-





Oral

3.3.3 Type Gavage

3.3.4 Concentration 0, 10, 40 and 125 mg/kg bw/day (food consumption per day: ad libitum)

3.3.5 Vehicle 0.5% aqueous methyl-cellulose


0, 1, 4 and 12.5 mg/mL


10 mL/kg bw

3.3.8 Controls Vehicle

3.4 Examinations

3.4.1 Body weight Yes (on day 0 of gestation, daily during the treatment period and on the day of sacrifice)

3.4.2 Food consumption Yes (for days 0-6, 6-9, 9-12, 12-15, 15-18 and 18-20 of gestation)

3.4.3 Clinical signs Yes (before and about one hour after dosing on days 6 to 19, and on day 20); mortality (twice daily)

3.4.4 Examination of uterine content

Gravid uterine weight

Number of corpora lutea

Number of implantations

early and late resorptions

3.4.5 Examination of foetuses

3.4.5.1 General Litter Size, No. of dead Foetuses, Foetal Weight, Sex Ratio, External alternations

3.4.5.2 Skelet Yes (about half of foetuses)

3.4.5.3 Soft tissue Yes (about half of foetuses)



4.1 Maternal toxic Effects

Reduced food consumption and body weight development during treatment (bw loss during the first days of treatment) at 125 mg/kg bw/day (see table A6_8_1/01-1). In addition a transient slight reduction in food consumption and reduction in bw gain was observed at the beginning of treatment with 40 mg/kg bw/day. However as the overall bw development was comparable to controls in this group, this effect is considered to be not adverse.

4.2 Teratogenic / embryotoxic effects

There were no treatment-related effects at any dose level on the litter parameters (see table A6_8_1/01-2). There were no treatment-related effects at any dose level on the nature and incidence of external, soft tissue and skeletal malformations and variations (see table A6_8_1/01-3).

4.3 Other effects -







Evaluation of the embryotoxic and teratogenic potential after oral application to pregnant rats (on days 6-19 of gestation); no relevant deviations to guidelines 87/302/EEC B.31, OECD 414, US-EPA OPPTS 870.3700, Japan MAFF)


A dose level of 125 mg/kg bw/day TI-435 during day 6-19 (gavage) of gestation lead to a reduced food consumption and bw development in dams. A slight transient decrease in food consumption and bw gain at 40 mg/kg bw/day TI-435 in dams is considered to be not-adverse as the overall bw development and food consumption during treatment was comparable to controls. There were no effect on pregnancy rate, litter parameters or at the external, skeletal and visceral examinations of foetuses.

5.3 Conclusion TI-435 did not indicate any embryotoxic or teratogenic potential in this study in rats. Reduced food consumption and bw development were seen in dams.

5.3.1 LOAEL maternal toxic effects

125 mg/kg bw/day reduced food consumption and bw gain during treatment

5.3.2 NO(A)EL maternal toxic effects

NOEL 10 mg/kg bw/day NOAEL 40 mg/kg bw/day

5.3.3 LO(A)EL embryotoxic / teratogenic effects

- no effect of treatment at the highest dose level (125 mg/kg bw/day)

5.3.4 NO(A)EL embryotoxic / teratogenic effects

>125 mg/kg bw/day

5.3.5 Reliability 1









Date 2004-10-07

Materials and Methods Applicants version is acceptable.

Results and discussion A dose level of 125 mg/kg bw/day TI-435 (gavage) during day 6-19 of pregnancy lead to a reduced food consumption and bw development in dams. A slight transient decrease in food consumption and bw gain at 40 mg/kg bw/day TI-435 in dams is considered to be not adverse as the overall bw development and food consumption during treatment was comparable to controls. There were no effects on pregnancy rate, litter parameters or external, skeletal and visceral findings in foetuses. Data from a range-finding study indicate that embryo- or foetotoxic effects (reduced foetal weight) occur at dose levels of 250 mg/kg bw/day and higher (see CA-Table 1).

Conclusion This study did not indicate any embryo/foetotoxic or teratogenic potential of TI-435 in rats. Reduced food consumption and bw development were seen in dams.

Reliability 1


Remarks

COMMENTS FROM ...







Remarks





Table A6_8_1/01-1. Table for Teratogenic effects Maternal effects

Parameter control data 10 mg/kg bw/day

40 mg/kg bw/day

125 mg/kg bw/day

dose-response

+ / - historical study

Number of dams examined (pregnant) 1665 (1542)

25 (23) 25 (22) 25 (24) 25 (25)

Mortality of dams [%] 0.06 0 0 0 0 Abortions 0 0 0 0 0

Body weight gain [g] day 0-6 day 6-9 day 9-12 day 12-15 day 15-18 day 18-20 day 6-20 day 0-20 day 6-20° gravid uterus weight [g]

25.5 11.8 17.1 17.6 37.8 36.6 120.9 146.4 46.7 74.2

26.7 10.2 18.1 18.0 38.3 38.0

122.6 149.3 46.4 76.2

27.7

6.8** ↓ 16.6 16.9 38.8 38.0 117.0 144.7 37.7 79.3

26.1

-6.1** ↓ 17.6 16.8 34.0 38.6

100.8**↓ 127.0**↓ 25.8** ↓

75.1

Food consumption [g/day] day 0-6 day 6-9 day 9-12 day 12-15 day 15-18 day 18-20 day 6-20 day 0-20

19.6 20.9 22.6 24.0 25.9 24.9 23.6 22.4

20.0 20.5 22.2 24.0 26.2 25.4 23.6 22.5

19.7

19.0* ↓ 22.0 23.0 25.6 24.4 22.7 21.8

20.2

11.1** ↓ 18.8** ↓ 21.3** ↓ 23.3** ↓

24.0 19.5** ↓ 19.7** ↓

Pregnancies [%] 93.2 (64-100)

92 88 96 100

° body weight gain minus gravid uterus weight



Table A6_8_1/01-2. Table for Teratogenic effects Litter response (Caesarean section data)


40 mg/kg bw/day

125 mg/kg bw/day

dose-response


Corpora lutea total/number of dams mean

13.8-19.4

365/2315.9

347/2215.8

381/24 15.9

400/25 16.0

Implantations total/number of dams mean

12.9-18.0

314/2313.6

314/2214.3

340/24 14.2

357/25 14.3

Resorptions total/number of dams mean

0-1.6

15/23 0.6

20/22 0.9

10/24 0.4

17/25 0.7

total number of fetuses 299 294 330 340 pre-implantation loss total % per group / % per animal

51 14.0/14.0

33 9.5/8.6

41 10.8/10.6

43 10.8/10.0

post-implantation loss total % per group / % per animal

15 4.8/4.9

20 6.4/6.3

10 2.9/2.8

17 4.8/4.7

total number of litters 23 22 24 25 fetuses / litter 13.0 13.4 13.8 13.6 live fetuses / litter 11.8-17.0 13.0 13.4 13.8 13.6 dead fetuses / litter 0-0.9 0 0 0 0 fetus weight (mean) [g] 3.10-3.78 3.60 3.57 3.63 3.40 Fetal sex ratio m/f % males per litter

42.1-57.0

147/15249.2

144/15049.0

172/158 52.1

162/178 47.6

Table A6_8_1/01-3. Table for Teratogenic effects Examination of the fetuses


40 mg/kg bw/day

125 mg/kg bw/day

dose-response


External malformations* total % litter / foetal incidence

2 8.7 / 0.7

1 4.5 / 0.3

1 4.2 / 0.3

0 0 / 0

External anomalies* total % litter / foetal incidence

0 0 / 0

0 0 / 0

0 0 / 0

0 0 / 0

Skeletal malformations* total % litter / foetal incidence

1 4.3 / 0.6

1 4.5 / 0.6

0 0 / 0

1 4.0 / 0.6

Skeletal variations*° total % litter / foetal incidence

13 30.4/7.1

10 40.9/5.9

16 37.5/6.5

21 40.0/9.1

Visceral malformations* total % litter / foetal incidence

0 0 / 0

0 0 / 0

1 4.2 / 0.6

0 0 / 0

Visceral variations*° total % litter / foetal incidence

2 8.7 / 1.4

1 4.5 / 0.7

1 4.2 / 0.6

3 4.0 / 0.6

° findings were only differentiated into malformation and variations in the report



CA-Table 1 Results of range-finding study Parameter 0

mg/kg bw/day

125 mg/kg bw/day

250 mg/kg bw/day

500 mg/kg bw/day

1000 mg/kg bw/day

Number of dams in study 8 8 8 8 8 Number of dams evaluated (pregnant) 8 7 6 8 - Mortality of dams 0 0 0 0 8 Corpora lutea mean 18.1 18.1 17.4 16.6 - Implantations mean 15.5 16.0 15.8 14.1 - Total litter loss n 0 0 1* 6 - Live litter size mean 14.5 14.7 14.0 1.5 - Gravid uterus weight mean [g] 82 76 61 4 - Live litter size mean 14.5 14.7 14.0 1.5 - Foetal weight mean [g] 3.50 3.22 2.57 1.04 - % males per litter 54 53 51 0 -

* single implantation site (not adverse)




Teratogenicity Study Rabbit Developmental toxicity study (gavage application) in the rabbit

1 REFERENCE

Officialuse only

1.1 Reference York, R. G. (1998b); Oral (stomach tube) developmental toxicity study of TI-435 in rabbits Argus Research Laboratories Inc., Horsham, PA 19044, USA unpublished report number 1120-002, 16.04.1998




None




2.1 Guideline study Yes 87/302/EEC B.31, OECD draft guideline no. 414 (1999), US-EPA OPPTS 870.3700 (1996), Japan MAFF (59 NohSan no. 4200, 1985)

2.2 GLP Yes

2.3 Deviations No






3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study: The stability and homogeneity of TI-435 in the vehicle was confirmed in a previous study; and all test article-vehicle formulations were analysed for achieved concentration twice during the study

3.2 Test Animals 23 naturally mated female New Zealand White rabbits per group (Hra:[NZW]SPF strain, body weight range 3.3 – 4.6 kg, Covance Research Products Inc., USA) including a control group


Oral


rabbit day 6-28 post mating


- (sacrifice on day 29)

Oral

3.3.3 Type Gavage





3.3.4 Concentration 0, 10, 25, 75 and 100 mg/kg bw/day (food consumption per day: ad libitum)

3.3.5 Vehicle 0.5% aqueous methyl-cellulose


0, 1, 2.5, 7.5 and 10 mg/mL


10 mL/kg bw


3.4 Examinations

3.4.1 Body weight Yes (on day 0 of gestation, daily during the treatment period and on the day of sacrifice)

3.4.2 Food consumption Yes (daily)

3.4.3 Clinical signs Yes (before and about one hour after dosing on days 6 to 28, and on day 29); mortality (twice daily)

3.4.4 Examination of uterine content

Gravid uterine weight

Number of corpora lutea

Number of implantations

early and late resorptions

3.4.5 Examination of foetuses

3.4.5.1 General Litter Size, No. of dead Foetuses, Foetal Weight, Sex Ratio, External alternations

3.4.5.2 Skelet Yes

3.4.5.3 Soft tissue Yes



4.1 Maternal toxic Effects

Treatment-related deaths or sacrifice occurred at ≥75 mg/kg bw/day. In addition increased numbers of does aborted or had prematurely deliveries at 100 mg/kg bw/day (see table A6_8_1/02-1). Death and abortion were preceded by weight loss, reduced food consumption, reduced faecal output, orange urine or decreased activity and loss of righting reflex. There were no consistent necropsy findings in the animals that died or aborted. Treatment-related clinical signs in surviving animals were confined to reduced faecal output and orange coloured urine at dose levels of ≥25mg/kg bw/day. In absence of any other indication of a treatment-related effect at 25 mg/kg bw/day, these findings are considered not to be adverse at that dose level - especially as they were seen in only small numbers of does (1-4 of 23). Reduced food consumption and body weight development during treatment were also seen in the survivors at ≥75 mg/kg bw/day.

4.2 Teratogenic / embryotoxic effects

Treatment-related effects on litter parameters were confined to increased post-implantation loss and reduced live foetal weight at 100 mg/kg bw/day (see table A6_8_1/02-2). There was no relevant treatment-related effect on the nature and





incidence of foetal alterations (malformation and variations) at any dose level (see table A6_8_1/02-3). While the incidence of foetuses with variations and malformations was increased at 100 mg/kg bw/day in comparison to controls, all alterations were within the historical control ranges and none of them occurred in conceptuses from animals that died or aborted. They are therefore considered to be incidental and unrelated to treatment. Although lower litter averages for ossified sternal centra and hindlimb phalanges per foetus occurred at 100 mg/kg bw/day, they were considered to be transient variations associated with slightly retarded intrauterine growth.

4.3 Other effects Comment: The study author did not discuss the adverse or not adverse nature of the findings and assigned the NOAEL at the dose level where no effects were observed. This evaluation includes a differentiation between adverse and not-adverse effects, and therefore suggested a higher maternal NOAEL than the study report.



Evaluation of the embryotoxic and teratogenic potential after oral application to pregnant rabbits (on days 6-28 of gestation); no relevant deviations to guidelines (87/302/EEC B.31, OECD 414, US-EPA OPPTS 870.3700, Japan MAFF)


Dose levels of ≥75 mg/kg bw/day TI-435 applied via gavage during day 6-28 of gestation to pregnant rabbits lead to a reduced food consumption and bw development. The high concentration of 100 mg/kg bw/day caused mortality and increased incidences of abortion and/or premature delivery. Mostly in the animals that died or had to be sacrificed, clinical signs like decreased activity, loss of righting reflex or reduced faecal output, orange urine were seen. The latter two was recorded for single or a few does also at 25 mg/kg bw/day. However in absence of any other indication of a treatment-related effect at that dose level, the low incidences of these findings were considered to be non-adverse. Probably due to the severe maternal toxicity/mortality, increased post-implantation loss and reduced live foetal weight was observed at 100 mg/kg bw/day. Associated to the latter, a slight delay in ossification was noted in the foetuses at the same dose level.

5.3 Conclusion TI-435 did not indicate a teratogenic potential in this study in rabbits. Probably related to maternal toxicity (reduced food consumption, bw development even mortality) increased post-implantation loss and lower fetal weights associated with a slight delay in ossification were seen at the highest dose level of 100 mg/kg bw/day.


75 mg/kg bw/day reduced food consumption and bw gain during treatment


NOEL 10 mg/kg bw/day NOAEL 25 mg/kg bw/day

5.3.3 LO(A)EL embryotoxic / teratogenic effects

100 mg/kg bw/day increased post-implantation loss, reduced foetal weight

5.3.4 NO(A)EL embryotoxic / teratogenic effects

75 mg/kg bw/day

5.3.5 Reliability 1









Date 2004-10-07


Results and discussion Ad 4.1: The observation of orange coloured urine at dose levels of ≥25mg/kg bw/day is considered to be due to the renal excretion of the coloured test substance. For all other aspects the applicant’s description of maternal toxic effects is accepted. Ad 4.2: In contrast to the applicant’s view, the observation of a reduced number of lung lobes in 3 foetuses from 3 litters at 75 mg/kg bw/day and 8 foetuses from 5 litters at 100 mg/kg bw/day appears to indicate a dose-related effect on lung branching morphogenesis at high, maternally toxic doses. The incidence is above the historical control range for litters at 75 and 100 mg/kg bw/day and for foetuses at 100 mg/kg bw/day.

Conclusion TI-435 appears to have a teratogenic potential in rabbits at doses which also induced foetal growth retardation and severe maternal toxicity. No classification for reproductive toxicity is considered to be required for TI-435 according to Directive 2001/59/EC (adaptation of 67/548/EEC).

Reliability 1


Remarks

COMMENTS FROM ...







Remarks





Table A6_8_1/02-1 Table for Teratogenic effects Maternal effects


25 mg/kg bw/day

75 mg/kg bw/day

100 mg/kg bw/day historical study

Number of dams examined (pregnant) 700 (664) 23 (21) 23 (23) 23 (22) 23 (22) 23 (23) Clinical findings during application of test substance: No. of observation (does) scant faeces in pan no faeces in pan orange urine red substance in cage pan decreased motor activity lost righting reflex soft/liquid faeces localised alopecia (total)

1 (1) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

45 (4)

4 (3) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

18 (2)

10 (4) 0 (0) 4 (2) 1 (1) 0 (0) 0 (0) 0 (0)

18 (3)

40 (10)**° 1 (1)

38 (9)** 0 (0) 0 (0) 0 (0) 0 (0)

41 (3)

79 (16)**°22 (11)**°64 (9)**8 (4)**°

1 (1)# 1 (1)# 5 (1)

61 (8) Mortality of dams [%] 1.0 0 0 0 8.7 13.0 Abortions°° [%] 1.6 13.0 0 0 13.0 34.8* ↑

Body weight gain [g] day 0- 6 day 6- 9 day 9-15 day 15-21 day 21-29 day 6-29 day 0-29 gravid uterus weight [g]

50 10 90 90 50 320 390

517.8

70 10

120 110 90

320 390

524.5

80 50 90 90 60 300 380

516.5

80 20 30 30 30 170 260

461.6

60

-40** ↓ -160 60 -70

-200** ↓50** ↓ 420.1

Food consumption [g/day] day 6- 9 day 9-15 day 15-21 day 21-24 day 24-29 day 6-29

141.3135.4153.8143.590.0

142.8

154.2 160.5 167.0 143.1 94.2 145.2

168.7 149.4 159.3 137.4 85.4

139.5

142.9 119.0 123.5

101.2* ↓ 89.4

118.8* ↓

90.9* ↓

60.3** ↓86.4** ↓104.7* 64.2

89.6** ↓ Pregnancies [%] 75.0-100 91.3 100 95.6 95.6 100 Necropsy findings in dams dead before end of test§: Uterus: vascularisation Stomach: trichobezoar part of cannibalised pup friable white mucus on mucosa cecum/colon: black substance lungs: spongy, hemorrhagic areas spleen: accessory splenic tissue parovarian cyst yellow perioral/perianal substance

1 1 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

(1) 0

0 (1) 0 0 0 0 0 0

(2) 0

0 1 2 1 0 0 0 0 0 0

0 2 0 0 1 1 1 1 2 1

* p ≤0.01, ° mostly seen in animals that later died/were sacrificed/aborted/prematurely delivered # animal was sacrificed day 19 of gestation, °° including prematurely delivered § number is brackets are from animal with necropsy at the scheduled sacrifice on day 29 of gestation



Table A6_8_1/02-2 Table for Teratogenic effects Litter response (Caesarean section data)


25 mg/kg bw/day°

75 mg/kg bw/day

100 mg/kg bw/day°° historical study

Corpora lutea total/number of dams mean

7.8-11.8

169/189.4

220/23 9.6

198/20 9.9

148/17 8.7

108/11 9.8

Implantations total/number of dams mean

3.8-10.6

155/188.6

204/23 8.9

183/20 9.2

137/17 8.0

97/11 8.8

Resorptions total/number of dams mean

0-3.2

5/18 0.3

3/23 0.1

7/20 0.4

4/17 0.2

15/11 1.4

total number of fetuses 150 201 176 133 82 pre-implantation loss total % per group / % per animal

14 8.3/9.1

16 7.3/7.5

15 7.6/7.5

11 7.4/8.0

11 10.2/10.2

post-implantation loss total % per group / % per animal

5 3.2/3.1

3 1.5/1.4

7 3.8/3.6

4 2.9/3.4

15 15.5/20.6

total number of litters 18 23 20 17 11 fetuses / litter 8.3 8.7 8.8 7.8 7.4 live fetuses / litter 3.2-10.4 8.3 8.7 8.8 7.8 7.4 dead fetuses / litter 0-0.1 0 0 0 0 0 fetus weight (mean) [g] 31.9-55.7 43.85 42.91 40.37 40.59 36.66** Fetal sex ratio m/f % males per litter

32.9-61.8

78/72 52.0

94/107 46.8

89/87 50.7

63/70 47.4

31/51 37.8

° excluding 2 does with only 1 and 2 conceptuses, respectively; including these animals the values would have been: Corpora lutea (total 209, mean 9.5), implantations (total 183, mean 8.5), resorptions (total 7, mean 0.3), fetuses (total 179, mean 8.1), pre-implantation loss (total 23, % group/animal 11.0/10.4), post-implantation loss (total 7, % group/animal 3.8/3.3), litters (22), dead fetuses (0) °°excluding 1 doe with only 3 conceptuses; including this animal the values would have been: Corpora lutea (total 114, mean 9.5), implantations (total 100, mean 8.3), resorptions (total 15, mean 1.3), fetuses (total 85, mean 7.1), pre-implantation loss (total 14, % group/animal 14.0/13.5), post-implantation loss (total 15, % group/animal 15.0/18.9), litters (12), dead fetuses (0)



Table A6_8_1/02-3 Table for Teratogenic effects (including single malformations) Examination of the fetuses

Parameter Control data 10 mg/kg bw/day

25 mg/kg bw/day°

75 mg/kg bw/day

100 mg/kg bw/day°° Historical study

External malformations* total % litter / foetal incidence total / % litter/fetal incidence: Snout: short Eyes, bulge depressed Body: meningocele edema umbical hernia Fore/hindpaws: digits absent Hindpaws: nail absent Tail: short kinked

2 /0-5.9/0-0.71 /0-5.3/0-0.61 /0-5.3/0-0.51 /0-5.3/0-0.66 /0-50/0-12

- 1 /0-5.9/0-0.65 /0-5.6/0-0.7

-

0 0 / 0

0 0 0 0 0 0 0 0 0

1 4.3 / 0.5

0 0 0 0

1 / 4.3/0.50 0 0 0

3 4.5 / 1.1

0 0

1 / 4.5/0.6 0 0 0 0

2 / 4.5/1.1 0

1 5.9 / 0.8

1 / 5.9/0.8

0 0 0 0 0 0 0 0

6 18.2 / 3.5

0

1/ 9.1/1.20

1 /9.1/1.20

1 / 9.1/1.21 / 9.1/1.21 / 9.1/1.21 / 9.1/1.2

External variations*° total % litter / foetal incidence

0 0 / 0

0 0 / 0

4 4.5 / 2.2

0 0 / 0

0 0 / 0

Skeletal malformations* total % litter / foetal incidence total / % litter/fetal incidence: Eye socket: small Thoracic verteb.: hemivertebrae fused centra Sacral vertebr.: open arch Caudal vertebr.: misaligned fused small arch open 16 present 15 present Ribs split extra ossification fused Metacarpals/forepaws digits: 4 pres. Forepaws phalanges: absent Metatarsals/handpaw digits: 2 pres. Hindpaw phalanges: absent

-

7 /0-5.9/0-0.65 /0-5.9/0-0.61 /0-5.3/0-0.521/0-11.8/0-1.49 /0-5.9/0-1.9

- 1/0-5.0/0-0.6#

- -

4 /0-6.2/0-0.8-

4 /0-5.9/0-0.6-

1 /0-5.6/0-0.7-

1 /0-5.9/0-0.6

0 0 / 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

7 17.4 / 2.5

0

1 / 4.3/0.51 / 4.3/0.5

0 2 /8.7/1.0

0 0 0 0 0

1 / 4.3/0.51 / 4.3/0.51 /4.3/0.5

0 0 0 0

6 4.5 / 1.1

0 0 0

1 / 4.5/0.6 0

2 / 4.5/1.1 0

1 / 4.5/0.6 1 / 4.5/0.6 1 / 4.5/0.6

0 0 0 0 0 0 0

0 0 / 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

11 27.3 / 3.5

1 / 9.1/1.2

0 0 0 0

2 /18.2/2.41 /9.1/1.2

0 0 0 0 0 0

1 /9.1/1.21 /9.1/1.21 /9.1/1.2

2 /18.2/2.4Skeletal variations*° total % litter / foetal incidence

5 27.8 /2.7

7 21.7 / 2.5

18 40.9 / 10.5

9 23.5 / 4.5

9 36.4 / 8.2

Visceral malformations* total % litter / foetal incidence total / % litter/fetal incidence: Brain: extreme dilated 3rd/lateral ventricles Nasal passage: small Eyes: small Vessels: persistent truncus arteriosis Lungs: several lobes absent Kidney: small

1 /0-6.2/0-0.8- -

3 /0-6.2/0-0.864 /0-28/0-4.4§

-

0 0 / 0

0 0 0 0 0 0

0 0 / 0

0 0 0 0 0 0

1 4.5 / 0.6

1 / 4.5/0.6 0 0 0 0 0

3 11.8 / 1.5

1 / 5.9/0.8 1 / 5.9/0.8

0 1 / 5.9/0.8

0 0

6 18.2 / 4.7

0 0

1 /9.1/1.20

1 /9.1/1.23 /9.1/3.5

Visceral variations*° total % litter / foetal incidence

0 0 / 0

3 13.0 / 1.5

0 0 / 0

3 17.6 / 2.2

8 36.4 / 8.2

° findings were only differentiated into malformation and variations in the report # irregularly shaped, § one or more lobes, partial or complete agenesis statistically significant incidences marked bold

• Sumitomo Chemical Takeda Agro Co., Ltd.

• TI-435 • February 2004


Section A6.8.2 Annex Point IIA6.8.2/01

Multigeneration Reproduction Toxicity Study Two generation dietary reproduction toxicity study in rats

1 REFERENCE

Officialuse only

1.1 Reference (2000)


1.2.1 Data owner




2.1 Guideline study Yes 87/302/EEC B.35, OECD guideline no. 416 (1983), EPA-OPPTS 870.3800 (1998), EPA-TSCA 798.4700; Japan MAFF (59, NohSan no.4200, 1985)

2.2 GLP Yes

2.3 Deviations No






3.1.2.2 Purity


3.2 Test Animals

3.2.1-3.2.6

male and female 7-8 week old Sprague-Dawley rats per group

3.2.7 Mating

3.2.8 Duration of mating 2 weeks

3.2.9 Deviations from standard protocol

In agreement with the test guidelines litters were culled to 8 pups on day 4 by random selection

3.2.10 Control animals Yes


Oral

3.3.1 Animal assignment to dosage groups






3.3.2 Duration of exposure before mating

10 weeks (70 days)

3.3.3 Duration of exposure in general P, F1, F2 males, females

From beginning of the study until sacrifice of parent, F1, F2-generation

Oral

3.3.4 Type in food

3.3.5 Concentration 0, 10.2, 32.7 and 179.6 mg/kg bw/day for males, and 0, 11.8, 37.9 and 212.9 mg/kg bw/day for females (pre-mating phase)

X

3.3.6 Vehicle


-


-


3.4 Examinations

3.4.1 Clinical signs

3.4.2 Body weight

3.4.3 Food/water consumption

3.4.4 Oestrus cycle

3.4.5 Sperm parameters

3.4.6 Offspring

3.4.7 Organ weights P and F1






3.4.8 Histopathology P and F1

3.4.9 Histopathology F1 not selected for mating, F2

3.5 Further remarks Data were analysed statistically using ANOVA for parametric data, followed by Dunnett’s test where appropriate. Non-parametric data were analysed by the Kruskal-Wallis test followed by Dunn’s test.


4.1 Effects






4.1.1 Adults

X






4.1.2 Pups

X

4.2 Other



Evaluation of the potential for reproductive toxicity in a 2-generation study in rats; no relevant deviation from guidelines (87/302/EEC B.35, OECD 416, EPA-OPPTS 870.3800, EPA-TSCA 798.4700; Japan MAFF)







5.3 Conclusion There was no relevant effect of treatment on reproduction at dose levels up to 2500 ppm corresponding to 179.6 and 212.9 mg/kg bw/day for males and females, respectively. Treatment-related changes comprised increases in food consumption and reductions in bw development with further findings considered to be secondary to the latter (changes in organ weight, slightly redcued sperm motility in adult males, slight delay in reaching developmental landmarks in F1 pups). In absence of a similar effect in F1 females and as the final bw was comparable to controls in 500 ppm treated P females, the slightly lower bw development during pre-treatment and the lower bw at day14 of lactation are considered to be not adverse. Also in absence of a similar effect in F2 pups and as the bw development of the F1 generation at 500 ppm during pre-mating was comparable to controls, the marginally lower bw gain of F1 pups at 500 ppm during day 7-14 of lactation was considered to be not adverse. Therefore overall NOAEL of the study is considered to be 500 ppm corresponding to 32.7 and 37.9 mg/kg bw/day in males and females, respectively.






5.3.1 LOAEL

5.3.1.1 Parent males 2500 ppm corresponding to 163.4 mg/kg bw/day increased food consumption, reduced bw development and findings considered secondary to the latter

5.3.1.2 Parent females 2500 ppm corresponding to 188.8 mg/kg bw/day increased food consumption, reduced bw development and findings considered secondary to the latter; as discussed before, the marginal changes in bw in P females at 500 ppm were considered to be not adverse

5.3.1.3 F1 males Adults and pups: 2500 ppm corresponding to 195.7 mg/kg bw/day reduced bw gain and related changes, increased food consumption in adults

5.3.1.4 F1 females Adults and pups: 2500 ppm corresponding to 237.0 mg/kg bw/day reduced bw gain and related changes, increased food consumption in adults

5.3.1.5 F2 males 2500 ppm reduced bw gain

5.3.1.6 F2 females 2500 ppm reduced bw gain

5.3.2 NO(A)EL

5.3.2.1 Parent males NOEL = NOAEL = 500 ppm corresponding to 31.2 mg/kg bw/day

5.3.2.2 Parent females NOEL = 150 ppm corresponding to 11.5 mg/kg bw/day NOAEL = 500 ppm corresponding to 36.8 mg/kg bw/day

5.3.2.3 F1 males Pups: NOEL = 150 ppm, NOAEL = 500 ppm Adults NOEL = NOAEL = 500 ppm corresponding to 34.3 mg/kg bw/day

5.3.2.4 F1 females Pups: NOEL = 150 ppm, NOAEL = 500 ppm Adults NOEL = NOAEL = 500 ppm corresponding to 39.0 mg/kg bw/day

5.3.2.5 F2 males NOEL = NOAEL = 500 ppm

5.3.2.6 F2 females NOEL = NOAEL = 500 ppm

5.3.3 Reliability 1








Date 2006-11-01



Conclusion




COMMENTS FROM ...







Remarks







F F











1 REFERENCE

Officialuse only

1.1 Reference Cain, D. M., Sheets, L. P. and Stuart, B. P. (2000); An acute oral neurotoxicity screening study with technical grade TI-435 in Fischer 344 rats Bayer Corporation, Agriculture Division, Toxicology, Kansas 66085-9104, USA unpublished report number 108960, 12.10.2000




None




2.1 Guideline study Yes No EU guideline available, OECD 424 corresponding EPA-FIFRA Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, Guideline Addendum 10, Neurotoxicity; NTIS, 1991, EPA 540/09-91-123, PB 91-154617, EPA OPPTS 870.6200 (August 1998)

2.2 GLP Yes

2.3 Deviations No






3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study (stability, homogenicity and content of test-article/vehicle formulations was analysed)

3.2 Test Animals 12 male and 12 female fasted 9 week old Fischer 344 rats per group (CDF[F-344]/BR strain, body weight range 103–186 g, SASCO Inc., Michigan, USA); including a control group


Oral


14 days





Oral

3.3.2 Type Gavage


3.3.4 Vehicle aqueous 0.5% methylcellulose/0.4% Tween 80




10 mL/kg bw

3.3.7 Controls vehicle

3.4 Examinations Detailed physical examination for clinical signs of toxicity, mortality: daily Body weights: weekly Functional observation battery (FOB): (all animals 1 week prior to application, 4 hours after application, day 7 and 14) neurological examinations: sensorimotor functions (approach, touch, audition, pain), autonomic functions (pupillary size and reflex, righting reflex, body temperature), sensorimotor coordination (grip strength, landing foot splay); Observations included (cage, handling): posture/gait, salivation, lacrimation, nasal discharge, stains, gait abnormalities, muscle tone, piloerection, involuntary motor movements, vocalisations, palpebral closure, respiratory abnormalities, stereotypy, bizarre behaviour, rearing, defecation, urination, decreased or increased activity, head bobbing, alopecia, other (e.g. bite marks, broken teeth, dehydration, emaciation, exophthalmia, missing toe nail, cool to touch) Motor activity (figure-eight maze): motor activity, locomotor activity, habituation


Gross pathology (all animals) Brain weights (6 animals/sex/group) Histopathology: gross lesions; the following tissues were collected from 6 animals/sex/group (microscopic examination in control and high dose group): brain, spinal cord, eyes (with optic nerve), peripheral nerves (sciatic, tibal, sural), dorsal root ganglia, gasserian ganglion, gastrocnemius muscle

3.6 Further remarks Continuous data were analysed statistically using ANOVA followed by Dunnett’s test in the case of significant F-values. Continuous FOB data and motor/locomotor activity data were analysed using repeated-measures ANOVA followed by one-way ANOVA for significant interactions, and Dunnett’s test. Discrete FOB data were analysed in a similar manner using general linear modelling and categorical modelling followed by Dunnett’s test and analysis of contrasts, respectively. Terminal body and organ weights were analysed for homogeneity of variances using Bartlett's test (p ≤0.001). Homogeneous data were furter analysed using ANOVA and Dunnett's test for pair-wise comparisons. Non-homogeneous data were analysed using the non-parametric Kruskal-Wallis test followed by a Mann-Whitney U test for pair-wise comparisons.






4.1 Body weight The body weight gain was reduced in the first week after application at 400 mg/kg bw (see table A6_9/01-1). While the overall bw gain during the 14 days after application was comparable to controls for 400 mg/kg bw treated males, it remained slightly below control values for females at that dose level.

4.2 Clinical signs, FOB, motor activity

There were no deaths. Treatment-related clinical signs like tremors, decreased activity and ataxia occurred in both sexes at 400 mg/kg bw on the day of dosing with recovery within 2 days. At that dose level an increased incidence of staining by urine was noted in females with one female also exhibiting nasal and oral staining. Treatment-related effects in the FOB and motor activity assessments were evident in males at all 3 dose levels and in females at ≥200 mg/kg bw (see table A6_9/01-1) but were limited to evaluations performed on the day of treatment (approximately 4 hours after dosing); no effects at day 7 or 14. Decreased activity, constriction of the pupils, uncoordinated righting response and decreased body temperature were seen at ≥200 mg/kg bw. Tremors and uncoordinated gait were also evident in animals dosed at 400 mg/kg bw. A single exposure in 100 mg/kg bw treated male revealed hypoactivity during open field observation. Other end-points including forelimb and hindlimb grip strength and landing foot splay were not affected by treatment at any dose level. On the day of treatment the early intervals of the session evaluating motor and locomotor activity revealed lower levels of activity in males at all 3 dose levels and in females at ≥200 mg/kg bw. The generally lower motor activity values recorded for all animals at the day of treatment in comparison to the pre-treatment (and day 7 or 14) were considered due to fasting of the animals before treatment.

4.3 Pathology Post mortem examination revealed no treatment-related gross lesions. There were no treatment-related effects on brain at any dose level. Microscopic examination of tissues from perfused animals revealed no treatment-related histomorphological alterations in the tissues of the central and peripheral nervous systems or muscle tissue. The transient functional effects observed were considered to be neurobehavioural evidence of toxicity and/or signs of pharmacological over-stimulation. There was no evidence of neurohistopathological alterations or persistent functional changes.



Neurotoxicity evaluation after acute oral application of the TI-435 to rats (gavage); no relevant deviation from guidelines (OECD 424, EPA OPPTS 870.6200)


Transient clinical signs (tremors, decreased activity, sluggish arousal) and functional effects (lower motor activity, effects on pupil size, righting reflex and lowered body temperature) were observed on the day of treatment at ≥100 and ≥200 mg/kg bw in males and females, respectively. Lower bw gains were observed at 400 mg/kg bw during the 1st week after application. There was no evidence of neurohistopathological alterations or persistent functional changes.





5.3 Conclusion With exception of the bw development, the observed transient functional effects were considered to be neurobehavioural evidence of toxicity and/or signs of pharmacological over-stimulation but not indications for specific neurotoxicity. Therefore TI-435 is considered to be not neurotoxic in rats after acute exposure.

5.3.1 LOAEL 100 mg/kg bw for males (transient effect on motor activity) 200 mg/kg bw for females (transient effects on FOB and motor activity)

5.3.2 NO(A)EL <100 mg/kg bw for males 100 mg/kg bw for females

5.3.3 Reliability 1







Date 2004-10-07




Reliability 1


Remarks

COMMENTS FROM ...







Remarks



Table A6_9/01-1 Table for Acute Oral Neurotoxicity (rat)

Males Females mg/kg bw 0 100 200 400 0 100 200 400

Cumulative bw gain [g] day 0- 7 day 0-14

39 58

37 55

37 58

31 ↓ 55

27 35

27 36

26 34

21 ↓ 31 ↓

Clinical signs [affected/total animals] Tremors Decreased activity Ataxia Nasal staining Oral staining Urine staining

0/12 0/12 0/12 0/12 0/12 0/12

0/12 0/12 0/12 0/12 0/12 0/12

0/12 0/12 0/12 0/12 0/12 0/12

12/12 10/12 2/12 0/12 0/12 0/12

0/12 0/12 0/12 0/12 0/12 1/12

0/12 0/12 0/12 0/12 0/12 1/12

0/12 0/12 0/12 0/12 0/12 0/12

11/12 11/12 1/12 1/12 1/12 3/12

FOB# Homecage: Tremors Decreased activity Ataxia

0/12 0/12 0/12

0/12 0/12 0/12

0/12 1/12 0/12

8/12* 8/12* 2/12

0/12 0/12 0/12

0/12 0/12 0/12

0/12 0/12 0/12

11/12*11/12*

0/12 Open field: Tremors Posture Incoordinated gait Sluggish arousal

0/12 0/12 0/12 1/12

0/12 0/12 0/12 4/12

0/12 0/12 0/12 5/12

11/12*

5/12 1/12

9/12*

0/12 0/12 0/12 0/12

0/12 0/12 0/12 0/12

0/12 0/12 0/12 3/12

11/12*

5/12 0/12

11/12* Reflex/physiologic observ. Constricted pupil responseRighting reflex - uncoordinated - landing on side Body temperature [°C]

0/12

0/12 0/12 36.5

0/12

0/12 0/12 36.1

1/12

2/12 0/12 34.8*

8/12*

2/12 0/12 32.8*

0/12

1/12 0/12 36.4

0/12

1/12 0/12 36.3

0/12

0/12 0/12 35.1*

9/12*

3/12 1/12 32.7*

Motor activity° pre-treatment day 0# day 7 day 14

473 256 507 566

434

196 ↓ 484 573

505

106 ↓ 580 611

389 48 ↓ 437 540

844 332 798 943

941 358 930 932

867

188 ↓ 893 973

843 94 ↓ 914 908

Locomotor activity° pre-treatment day 0# day 7 day 14

200 86

187 206

170 54 ↓ 175 204

189 33 ↓ 192 209

168 10 ↓ 166 189

305 95

294 334

337 94

335 314

326 52 ↓ 322 324

314 16 ↓ 335 319

* p <0.05, # at time of maximum effect 4 hours after treatment ° number of movements from nine 10-minutes intervals obsv. = observations





1 REFERENCE

Officialuse only

1.1 Reference Sheets, L. P. and Gilmore, R. G. (2000); A special acute oral neurotoxicity study to establish a no-observed-effect-level with technical grade TI-435 in Fischer 344 rats Bayer Corporation, Agriculture Division, Toxicology, Kansas 66085-9104, USA unpublished report number 108960-1, 08.10.2000




None




2.1 Guideline study Yes No EU guideline available, OECD 424 corresponding EPA-FIFRA Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, Guideline Addendum 10, Neurotoxicity; NTIS, 1991, EPA 540/09-91-123, PB 91-154617, EPA OPPTS 870.6200 (August 1998)

2.2 GLP Yes

2.3 Deviations None relevant for the integrity and validity of the study: the study was limited to obtain a NOEL for males as an earlier study already demonstrated a NOEL for females; the transient effects in the earlier study occurred on the day of application only - therefore the post-exposure period in the present study could be limited to 1-2 days after application; in absence of any macro- or histopathological changes or in brain weight in the earlier study, these examinations could be dismissed in the present study.






3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study (stability, homogeneity and content of test-article/vehicle formulations was analysed)

3.2 Test Animals 12 male fasted 9 week old Fischer 344 rats per group (CDF[F-344]/BR strain, body weight range 151–195 g, Charles River Laboratories Inc., USA); including a control group


Oral


1-2 days





Oral

3.3.2 Type Gavage


3.3.4 Vehicle aqueous 0.5% methylcellulose/0.4% Tween 80




10 mL/kg bw

3.3.7 Controls vehicle

3.4 Examinations Detailed physical examination for clinical signs of toxicity, mortality: daily Body weights: on the day of application and during FOB Functional observation battery (FOB): (all animals one week prior to treatment and 4 hours after application) neurological examinations: sensorimotor functions (approach, touch, audition, pain), autonomic functions (pupillary size and reflex, righting reflex, body temperature), sensorimotor coordination (grip strength, landing foot splay); Observations included (cage, handling): posture/gait, salivation, lacrimation, nasal discharge, stains, gait abnormalities, muscle tone, piloerection, involuntary motor movements, vocalisations, palpebral closure, respiratory abnormalities, stereotypy, bizarre behaviour, rearing, defecation, urination, decreased or increased activity, head bobbing, alopecia, other (e.g. bite marks, broken teeth, dehydration, emaciation, exophthalmia, missing toe nail, cool to touch) Motor activity (figure-eight maze): motor activity, locomotor activity, habituation


All animals were sacrificed and discarded without necropsy and tissue preservation 1–2 days after the final assessments.

3.6 Further remarks Continuous data were analysed statistically using ANOVA followed by Dunnett’s test in the case of significant F-values. Continuous FOB data and motor/locomotor activity data were analysed using repeated-measures ANOVA followed by one-way ANOVA for significant interactions, and Dunnett’s test. Discrete FOB data were analysed in a similar manner using general linear modelling and categorical modelling followed by Dunnett’s test and analysis of contrasts, respectively. The report also includes results of another study with male Fischer 344 rats from another supplier using dose levels of 0, 60 and 80 mg/kg bw but an otherwise similar protocol. In absence of any other treatment-related effect, marginally lower motor activity values were seen in the treated males four hours after application. However their biological relevance was considered as the effect did not reach statistical significance and as there was no clear dose-relationship. For further clarification the current study was performed.






4.1 Body weight No effect of treatment.

4.2 Clinical signs, FOB, motor activity

There were no deaths or treatment-related clinical signs. FOB and motor activity assessments did not indicate any effect of treatment. Motor/locomotor activity was comparable to controls in all treated groups (see table A6_9/02-1).

4.3 Pathology -



Neurotoxicity evaluation (assignment of a NOEL) after acute oral application of the TI-435 to male rats (gavage); no relevant deviation from guidelines (OECD 424, EPA OPPTS 870.6200)


No effect of treatment with up to 60 mg/kg bw was noted in male rats. Parameters affected at higher dose level in the previous studies (clinical signs, body temperature, motor/locomotor activity) were comparable to controls in this study.

5.3 Conclusion No indication for neurotoxicity or other effects of treatment were noted in this study after acute oral application of up to 60 mg/kg bw in male rats. The dose level of 60 mg/kg bw was a clear NOEL in this study.

5.3.1 LOAEL >60 mg/kg bw (males)

5.3.2 NO(A)EL 60 mg/kg bw (males)

5.3.3 Reliability 1







Date 2004-10-07




Reliability 1


Remarks

COMMENTS FROM ...







Remarks



Table A6_9/02-1 Table for Acute Oral Neurotoxicity (rat)

Males mg/kg bw 0 20 40 60

FOB# Body temperature [°C] 36.9 36.9 36.9 36.8 Motor activity° pre-treatment day 0#

282 109

224 119

239 119

252 104

Locomotor activity° pre-treatment day 0#

162 54

135 63

131 55

141 49

# at time of maximum effect 4 hours after treatment ° number of movements from nine 10-minutes intervals



Section A6.9

Annex Point IIA VI.6.9/03

Neurotoxicity studies Subchronic study in rats

90-day oral (feeding) neurotoxicity study in rats

1 REFERENCE

Official

use only

1.1 Reference Sheets, L. P. and Cain, D. M. (2000); A subchronic neurotoxicity screening study with technical grade TI-435 in Fischer 344 rats Bayer Corporation, Agriculture Division, Toxicology, Kansas 66085-9104, USA unpublished report number 109400, 12.10.2000




None




2.1 Guideline study Yes The study fulfills the requirements on FOB (including sensory reactivity, motor activity) parameters requested in 2001/59/EC B.26; OECD 424 corresponding EPA-FIFRA Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, Guideline Addendum 10, Neurotoxicity; NTIS, 1991, EPA 540/09-91-123, PB 91-154617, EPA OPPTS 870.6200 (August 1998)

2.2 GLP Yes

2.3 Deviations No






3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study (stability of the compound and its homogeneity and stability in food were tested)

3.2 Test Animals 12 male and 12 female fasted 9 week old Fischer 344 rats per group (CDF[F-344]/BR strain, body weight range 103–186 g, SASCO Inc., Michigan, USA); including a control group

3.3 Administration/

Exposure

Oral


13 weeks


daily


-



Section A6.9




3.3.4 Oral


3.3.4.2 Concentration 0, 150, 1000 and 3000 ppm corresponding to 0, 9.2, 60 and 177 mg/kg bw/day for males and 0, 10.6, 71 and 200 mg/kg bw/day for females food consumption per day: ad libitum

3.3.4.3 Vehicle Corn oil (resulting in a final concentration of 1% corn oil in the diet)


-


-

3.3.4.6 Controls Diet with 1% corn oil

3.4 Examinations

3.4.1 Observations


3.4.1.2 Mortality yes (twice daily except on weekends and holidays [daily])






3.4.6 Clinical pathology No (haematology, clinical chemistry, urine analysis)

3.4.7 Functional observation battery (FOB)

Yes (all animals prior to application, in weeks 4, 8 and 13) neurological examinations: sensorimotor functions (approach, touch, audition, pain), autonomic functions (pupillary size and reflex, righting reflex, body temperature), sensorimotor coordination (grip strength, landing foot splay); Observations included (cage, handling): posture/gait, salivation, lacrimation, nasal discharge, stains, gait abnormalities, muscle tone, piloerection, involuntary motor movements, vocalisations, palpebral closure, respiratory abnormalities, stereotypy, bizarre behaviour, rearing, defecation, urination, decreased or increased activity, head bobbing, alopecia, other (e.g. bite marks, broken teeth, dehydration, emaciation, exophthalmia, missing toe nail, cool to touch)

3.4.8 Motor activity Yes (all animals prior to application, in weeks 4, 8 and 13) (figure-eight maze): motor activity, locomotor activity, habituation

3.5 Sacrifice and

pathology

3.5.1 Organ Weights Yes; limited to brain weight from 6 animals/sex/group


Yes gross pathology: all animals histopathology: gross lesions; the following tissues were collected from 6 animals/sex/group (microscopic examination in control and high dose group): brain, spinal cord, eyes (with optic nerve), peripheral nerves (sciatic, tibal, sural), dorsal root ganglia, gasserian ganglion, gastrocnemius muscle




Section A6.9




3.5.4 Statistics Continuous data were analysed statistically using ANOVA followed by Dunnett’s test in the case of significant F-values. Continuous FOB data and motor/locomotor activity data were analysed using repeated-measures ANOVA followed by one-way ANOVA for significant interactions, and Dunnett’s test. Discrete FOB data were analysed in a similar manner using general linear modelling and categorical modelling followed by Dunnett’s test and analysis of contrasts, respectively.


4.1 Observations There were no treatment-related deaths or clinical signs at any dose level, although one male treated with 3000 ppm died on day 4 having exhibited decreased activity, ataxia and convulsions. Since there were no gross findings at necropsy to suggest a cause of death and because no other animals showed similar clinical symptoms, the death of this animal is considered to be incidental to treatment with TI-435.

4.2 Body weight gain The body weight development was reduced at 3000 ppm by 11-13% in comparison to controls with absolute body weights values reaching statistical significance occasionally during the second half of treatment for both sexes (see Table A6_9/03-1).

4.3 Food consumption

and compound

intake

At 3000 ppm food consumption was lower than controls over the whole study period (see Table A6_9/03-1). Average food consumption was reduced by 7% and 8% for 3000 ppm treated males and females, respectively. Compound intake see 3.3.4.2. The homogeneity, stability and achieved concentrations of TI-435 in the diets were found to be satisfactory. The diets were stable for at least 7 and 28 days at room temperature and refrigerated, respectively. Achieved concentrations were within 89.0-89.7% of nominal concentrations.

4.4 Ophtalmoscopic

examination

There were no treatment-related ophthalmological findings at any dose level.

4.5 FOB, motor

activity

There were no treatment-related effects on any qualitative or quantitative component of the FOB at 4, 8 and 13 weeks post-treatment at any dose level. A measure of inherent variability of motor and locomotor activity results was determined by reference to the pre-treatment variability. The pre-treatment motor activity of the groups designated for treatment was –12 to +20% of the control value, and for locomotor activity was –8 to +14% of the control value (see Table A6_9/03-1). Therefore, differences from the controls of 20% or less are considered not to be biologically significant. Although occasional values in the treated groups exceeded this 20% threshold, there was no consistent pattern to indicate a treatment-related effect, and none of the 90-minute session activity counts were significantly different from the control values (see Table A6_9/03-1). Thus, there was no treatment-related effect at any dose level on motor and locomotor activity at any testing interval.

4.6 Pathology No treatment-related gross lesions were evident at necropsy at any dose level. The absolute brain weights of all groups were unaffected by treatment, but the relative brain weights at 3000 ppm were slightly higher than control values due to the lower terminal body weight. There were no treatment-related histomorphological alterations at



Section A6.9




3000 ppm in any of the central and peripheral nervous system and muscle tissues examined.


5.1 Materials and

methods

Neurotoxicity evaluation after suchronic (90-day) oral application of TI-435 to rats (feeding); no relevant deviation from guidelines (OECD 424, EPA OPPTS 870.6200)

5.2 Results and

discussion

Treatment with 3000 ppm slightly reduced the food consumption and bw development. Due to the latter relative brain weights were slightly increased at that dose level. No relevant treatment-related effect was seen on the FOB, motor activity assessments or histopathological examinations of nervous or muscle tissues.

5.3 Conclusion TI-435 is considered to be not neurotoxic in rats after dietary exposure for 90 days.

5.3.1 LOAEL Systemic: 3000 ppm corresponding to 177 and 200 mg/kg bw/day for males and females, respectively based on the reduced food consumption and bw development Neurotoxicity: -

5.3.2 NO(A)EL Systemic: 1000 ppm corresponding to 60 and 71 mg/kg bw/day for males and females, respectively Neurotoxicity: >3000 ppm corresponding to 177 and 200 mg/kg bw/day for males and females, respectively

5.3.3 Reliability 1







Date 2004-10-07




Reliability 1


Remarks

COMMENTS FROM ...









Remarks



Males Females

ppm 0 150 1000 3000 0 150 1000 3000

Cumulative bw gain [% control] week 0- 1 week 0- 4 week 0- 8 week 0-13

100 100 100 100

96 100 102 105

105 102 101 98

76 93 93

89

100 100 100 100

100 105 106 108

91 96

101 101

64 91

86 87

Body weights [g/animal] week 0 week 13

187.0 335.2

185.3 341.3

191.2 336.1

188.6 320.9

126.7 186.7

126.5 191.0

126.0 186.5

127.0 179.1*

Food consumption [g/animal/day] week 1 week 4 week 8 week 13

18.1 18.7 19.1 19.1

17.4* 18.3 18.3 19.0

18.0 18.8 18.7 18.2

15.0* 17.7* 18.0* 17.9*

12.4 13.0 12.8 12.8

12.1 12.7 12.5 13.0

11.9 12.5 12.7 12.9

9.9* 11.5* 11.8* 11.8*

Motor activity° (% difference to control) pre-treatment week 4 week 8 week 13

290 311 312 255

302 (+4) 375 (+21) 391 (+25) 311 (+22)

333 (+15) 341 (+10) 346 (+11) 328 (+29)

348 (+20) 325 (+5) 302 (-3) 278 (+9)

393 501 477 487

361 (-8) 487 (-3)

529 (+11) 531 (+9)

346 (-12) 527 (+5)

589 (+23) 563 (+16)

450 (+15) 442 (-12) 562 (+18) 541 (+11)

Locomotor activity° (% difference to control) pre-treatment week 4 week 8 week 13

183 174 167 137

191 (+4) 198 (+14) 201 (+20) 162 (+18)

208 (+14) 198 (+14) 182 (+9)

173 (+26)

207 (+13) 182 (+5) 164 (-2)

154 (+12)

242 282 256 261

239 (-1) 301 (+7)

296 (+16) 304 (+16)

223 (-8) 315 (+12) 313 (+22) 301 (+15)

275 (+14) 252 (-11) 310 (+21) 296 (+13)

* p <0.05 ° number of movements from nine 10-minutes intervals


Section A6.9

Developmental neurotoxicity study Rat Developmental neurotoxicity study (orally via diet) in the rat

1 REFERENCE

Officialuse only

1.1 Reference (2000)


1.2.1 Data owner




2.1 Guideline study Yes No applicable EU guideline; US-EPA Health Effects Test Guidelines: Developmental Neurotoxicity Study, OPPTS 870.6300 and OECD 426 (2003) (study was performed previous to the final OECD guideline but was checked for compliance)

2.2 GLP Yes

2.3 Deviations No






3.1.2.2 Purity

3.1.2.3 Stability

3.2 Test Animals naturally mated female Sprague-Dawley rats per group


Section A6.9



Oral


Day 0 of gestation to day 22 post partum (dams)


3.3.3 Type Feeding

3.3.4 Concentration 0, 12.9, 42.9, 142.0 mg/kg bw/day during gestation and 27.3, 90.0 and 299.0 mg/kg bw/day during lactation.

3.3.5 Vehicle None


-


-

3.3.8 Controls Plain diet

3.4 Examinations

3.4.1 Body weight Yes

3.4.2 Food consumption Yes


3.4.4 Physical Development

3.4.5 Sexual Maturation

3.4.6 Brain weights

3.4.7 Neurohistological examination


Section A6.9


3.4.8 Tests

3.4.8.1 Passive Avoidance (learning, memory)

3.4.8.2 Water maize (learning, memory)

3.4.8.3 Motor activity

3.4.8.4 Auditory startle habituation (motor/ sensory function)


3.5 Further remarks Statistical evaluation of results was performed.


4.1 Observations

4.1.1 Clinical signs, pregnancy parameters

4.1.2 Mortality Dams: no deaths

4.2 Body weight gain


Section A6.9



4.4 Physical development / sexual maturation

4.5 Brain weight

4.6 Neurohistology


Section A6.9


4.7 Tests

4.7.1 Passive avoidance

4.7.2 Watermaze

4.7.3 Motor activity

4.7.4 Auditory startle




Evaluation of the developmental neurotoxic potential in the F1 generation after oral application to pregnant rats (during gestation and lactation); no relevant deviations to guidelines OECD 426, US-EPA OPPTS 870.6300


Section A6.9



Dams: no deaths and no clinical signs. The body weight development (gestation) and food consumption (gestation, lactation) were reduced at 1750 ppm. F1-pups: reduced body weight development in both sexes at 1750 ppm and marginally in females of the 500 ppm group. As the latter was only slight and as no toxicologically relevant effect on body weight development had been observed in the 2-generation study at the same dose level in the same strain of rats, the marginal effect at 500 ppm is considered to be not adverse. F1-young adults: There were no treatment-related clinical signs In total 5 animals of the 1750 ppm group died in the first days post-weaning, which was considered due to their low body weight development earlier. The surviving rats of those groups with a reduced body weight after lactation compensated for this after weaning resulting in similar body weights than controls at sacrifice. Reflex and physical development as well as sexual maturation were unaffected by treatment. No biologically relevant effect of treatment on learning and memory as was motor activity and motor/sensory function on all occasions except days 22/23 were recorded. At days 22/23 post partum the motor activity (both sexes) and the auditory startle response (females only) were slightly lower in F1 rats of the 1750 ppm group in comparison to controls. The latter was considered to be related/secondary to the reduced body weight development of these animals during lactation and therefore not to indicate developmental neurotoxicity. Brain weight, morphology and neuropathology were unaffected by treatment. At sacrifice there were no treatment related changes in dams and their offspring at any dose level.

5.3 Conclusion No indication of selective developmental neurotoxicity of TI-435. Systemic toxicity was noted at 1750 ppm in dams and pups; a marginally lower body weight development was also noted in females of the 500 ppm group, which is considered to be not adverse.


1750 ppm corresponding to 142 and 299 mg/kg bw/day during gestation and lactation, respectively


500 ppm corresponding to 42.9 and 90.0 mg/kg bw/day during gestation and lactation, respectively

5.3.3 LO(A)EL developmental neurotoxicity

none

5.3.4 NO(A)EL developmental neurotoxicity

>1750 ppm

5.3.5 LOAEL developmental toxicity

1750 ppm

5.3.6 NOAEL developmental toxicity

500 ppm (= NOEL for males; NOEL for females = 150 ppm)

5.3.7 Reliability 1



Section A6.9





Date




Section A6.9



(continued)

-

Conclusion

Reliability 1


Remarks

COMMENTS FROM ...






Section A6.9




Remarks


Section A6.9









Section A6.10

Annex Point IIA

VI.6.10.1/01

Toxicity studies with compounds other than the a.s.

Ames test (+/- S9) using S. typhimurium with TI-435 metabolite MNG

1 REFERENCE

Official

use only

1.1 Reference Herbold, B. (2001); N-Methylnitroguanidin: salmonella/microsome test – plate incorporation and preincubation method PH-PD Toxicology, Bayer AG, Wuppertal, Germany unpublished report number PH 30755, 21.02.2001


1.2.1 Data owner Sumitomo Chemical Takeda Agro Co. Ltd. and Bayer AG


None




2.1 Guideline study Yes 2000/32/EC (method B13/14), OECD no. 471 (1997), OPPTS 870.5100 (1998)

2.2 GLP Yes

2.3 Deviations None


3.1 Test material N-methylnitrogianidin (MNG)

3.1.1 Lot/Batch number VE96026 Pt. 17004/98

3.1.2 Specification Not applicable

3.1.2.1 Description White powder

3.1.2.2 Purity

3.1.2.3 Stability Stable under conditions of this study (test-article/vehicle solutions were used within 4 hours after preparation)


3.2.1 Organism/cell type S. typhimurium: TA 1535, TA 1537, TA 98, TA 100, TA 102


Histidine deficient


S9 mix Prepared from livers of male Sprague Dawley rats (about 200-300 g body weight) induced with Arochlor 1254 (500 mg/kg bw in corn oil) 5 days prior to sacrifice; supplemented with co-factors

3.2.4 Positive control In absence of S9: Nitrofurantoin (NF) at 0.2 µg/plate for TA 100 Sodium azide (NaN3) at 10 µg/plate for TA 1535 4-nitro-1,2-phenylene diamine (4-NPDA) at 0.5 µg/plate for TA 98 10 µg/plate for TA 1537 Cumenehydroperoxide (Cumene) at 50 µg/plate for TA 102 In presence of S9: 2-Aminoantracene (2-AA) at 3 µg/plate for all strains



Section A6.10

Annex Point IIA

VI.6.10.1/01




Exposure;

Application of test

substance

3.3.1 Concentrations 0, 50, 158, 500, 1581, 5000 µg/plate (+/- S9) for both the plate incorporation and pre-incubation assay

3.3.2 Way of application Stock solutions: MNG was dissolved in DMSO. Plate incorporation assay: The bacterial suspensions (0.1 mL) were mixed with soft agar (2 mL), 0.1 mL of MNG stock solutions or vehicle or positive controls, 0.5 mL S9 mix or buffer before poured onto solid agar plates. Pre-incubation assay: The bacterial suspensions (0.1 mL) were mixed with 0.1 mL of MNG stock solutions or vehicle or positive control, 0.5 mL S9 mix or buffer and incubated for 20 minutes at 37°C. Then 2 mL of soft agar were added and poured onto solid agar plates. For both assay types the plates were then incubated for 48 hours at 37°C.

3.3.3 Pre-incubation time None (plate incorporation assay) 20 min (pre-incubation assay)


3.4 Examinations Cytotoxicity: thinning of background lawn and/or reduction of revertant colonies Mutagenicity: frequency of revertant colonies Criteria for a positive response: The test substance was considered positive if there was a reproducible and dose-related increase in the number of revertants relative to controls. The increase should be

by a factor of 2 for strains TA 1535, TA 100 and TA 98 by a factor of 3 for strain TA 1537 about 100 mutant colonies more than the solvent control


4.1 Genotoxicity





4.2 Cytotoxicity No indication for a growth inhibition.



Section A6.10

Annex Point IIA

VI.6.10.1/01




5.1 Materials and

methods

Evaluation of the in vitro gene mutation potential in S. typhimurium strains; no relevant deviation from guidelines (2000/32/EC B13/14, OECD 471, OPPTS 870.5100)

5.2 Results and

discussion

No effect of treatment with up to 5000 µg/plate of TI-435 metabolite MNG on the number of revertant colonies on 5 S. typhimurium strains was seen. There were also no indications for cytotoxicity (thinning of background lawn).

5.3 Conclusion MNG and/or its metabolites were considered to be not mutagenic in this in vitro test system.

5.3.1 Reliability 1







Date 2004-10-07




Reliability 1


Remarks

COMMENTS FROM ...







Remarks



Table A6_10_1/01-1 Table for Gene Mutation Assay with MNG (Ames test)


Number of mutant cells Comments Plate incorporation assay Preincubation assay

- S9 + S9 - S9 + S9


38 40 40 37 44 44

155

43 37 41 43 40 41

1322

33 31 30 29 26 33

175

36 47 41 40 49 37

1462

No indication for cytotoxicity, no precipitation of the test substance


107 97

100 84

103 101 248

140 122 129 115 137 120

1436

88 91 78 83 82 76

300

98 125 100 110 101 93

1681



179 168 161 153 160 172 309

226 244 257 264 239 210 425

219 235 229 224 227 218 438

293 261 272 261 271 234 404



8 8 7 9 6 6

574

10 8 9 9 8 7

235

10 10 10 9 8 12

701

12 11 10 14 13 9

292



5 5 6 6 6 6 87

8 6 6 8 8 7

291

6 6 6 8 7 6 98

9 9 8 10 10 8

210




Section A6.10

Annex Point IIA

VI.6.10.1/02


Acute oral toxicity study in rats (LD50) with the TI-435 metabolite TZNG

1 REFERENCE

Official

use only

1.1 Reference Ruddock, W. D. (2000c); TZNG: Acute oral toxicity study in the rat Covance Laboratories, Harrogate, UK unpublished report number 586/163-D6144, 20.01.2000




None




2.1 Guideline study Yes 92/69/EEC (method B1), OECD guideline no. 401 (February 1987), EPA OPPTS 870.1100 (August 1998), Japan MAFF (draft guidelines, 1998)

2.2 GLP Yes

2.3 Deviations No


3.1 Test material TZNG




3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study (test-article/vehicle formulations prepared at the day of dosing)

3.2 Test Animals 5 female fasted rats per group (Crl:CD.BR strain, 8–11 weeks old, body weight range 218–246 g, Charles River UK Ltd); in addition 5 male fasted rats from the same supplier, of the same approximate age and weighing 326–366 g were treated at the approximate median lethal dose to demonstrate that males were not more susceptible to the toxic effects of TZNG; No control group

3.3 Administration/

Exposure

Oral


14 days



Section A6.10

Annex Point IIA

VI.6.10.1/02



3.3.2 Type Gavage

3.3.3 Concentration 1125, 1350 and 1620 mg/kg bw (females) 1450 mg/kg bw (males)





20 mL/kg bw

3.3.7 Controls -

3.4 Examinations Clinical signs: recorded on at least 5 occasions on the day of treatment and at least once daily thereafter. Body weights were recorded pre-dose and on days 1, 8 and 15. Decedents, animals killed in a moribund condition, and all survivors were subjected to necropsy and post mortem examination.

3.5 Method of

determination of

LD50

The acute median lethal dose and 95% confidence limits were estimated using probit analysis (Finney, D. J., 1971, Probit analysis, 3rd Ed., Cambridge University Press).



4.1 Clinical signs Mortality occurred at dose levels 1350 mg/kg bw for females; Clinical signs included lethargy and palpebral closure (all animals), arched gait and hunched posture (females), wasted appearance and unkempt appearance in individual males and females (see table A6_10_1/02-1).

4.2 Pathology No relevant treatment-related effects in surviving females. Small or soft testis was seen in males (might be related to the low bw development).

4.3 Other Reduced bw gain or bw loss in all animals during the 1st week after application; the bw gain of the female survivor at 1620 mg/kg bw was also reduced during the 2nd week after application

4.4 LD50 1481 mg/kg bw (females) >1450 mg/kg bw (males)


5.1 Materials and

methods

Toxicity evaluation (bw, clinical signs, post mortem examination) after acute oral application of the TI-435 metabolite TZNG to rats (gavage); no relevant deviation from guidelines (92/69/EEC B1; EPA OPPTS 870.1100, Japan MAFF, OECD 401)

5.2 Results and

discussion

Acute oral LD50 in rats was 1481 mg/kg bw.

5.3 Conclusion Classification as harmful if swallowed (R22) is considered required for TI-453 metabolite TZNG according to Directive 2001/59/EC (adaptation of 67/548/EEC). The same classification had been proposed for the parent TI-435.

5.3.1 Reliability 1




Section A6.10

Annex Point IIA

VI.6.10.1/02






Date 2004-10-07




Reliability 1


Remarks

COMMENTS FROM ...









Remarks



Section A6.10

Annex Point IIA

VI.6.10.1/02



Table A6_10_1/02-1 Table for Acute Oral Toxicity (rat), metabolite TZNG

Dose

[mg/kg

bw]

Number of dead /

number of

investigated

Time of

death

(range) Observations

1125 0/5 females - Lethary, palpebral closure, hunched posture, arched gait, wasted appearance, stained snout, unkempt, anogenital soiling, pink staining around eyes were seen; all animals were normal by day 12; slight bw loss during the 1st week after application with bw gain during the 2nd week; no effect of treatment was noted at gross necropsy

1350 1/5 females Day 5 Lethary, palpebral closure, hunched posture, arched gait, piloerection, wasted appearance, stained snout, bradypnoea were seen; all survivors were normal by day 4; reduced bw gain during the 1st week after; no effect of treatment was noted at gross necropsy in survivors; small spleen and pale surface of stomach mucosa were seen in the female that died

1620 4/5 females Day 3-4 Lethary, palpebral closure, treamors, hunched posture, ataxia, arched gait, piloerection, straub tail, hypothermia were seen; the surviving female was normal by day 4; reduced bw gain over the observation period; no effect of treatment was noted at gross necropsy in the survivor; red and/or dark lungs, discoloration of the surface of stomach mucosa and stmach distension were seen in the females that died

1450 0/5 males - Lethary, palpebral closure, ataxia, piloerection, unkempt were seen; all animals were normal by day 3 bw loss during the 1st week after application with bw gain during the 2nd week; small testis were seen in all males (soft testis in two animals) and one rat had slight colon distension at gross necropsy

LD50 value Males: >1450 mg/kg bw Females: 1481 mg/kg bw (1257 – 1882) mg/kg bw)



Section A6.10

Annex Point IIA

VI.6.10.1/03


Ames test (+/- S9) using S. typhimurium with TI-435 metabolite TZNG

1 REFERENCE

Official

use only

1.1 Reference Dawkes, N. (1999c); TZNG: Reverse mutation in five histidine-requiring strains of Salmonella typhimurium Covance Laboratories, Harrogate, UK unpublished report number 586/165-D5140, 03.06.1999




None




2.1 Guideline study Yes 2000/32/EC (method B13/14), OECD no. 471 (1983), EPA OPPTS 870.5265 (1998)

2.2 GLP Yes

2.3 Deviations None relevant for the integrity and validity of the study (study was performed previous to current EU-guideline but was checked for compliance with the above): no positive control was included for the experiments in presence of S9 for strains TA 1537 and TA 102; however the sensitivity of the strains were shown with positive control compounds in absence of S9 and in three strains in presence of S9


3.1 Test material TZNG




3.1.2.2 Purity





Histidine deficient


S9 mix Prepared from livers of male Sprague Dawley rats induced with Arochlor 1254; supplemented with co-factors



Section A6.10

Annex Point IIA

VI.6.10.1/03



3.2.4 Positive control In absence of S9: 2-Nitrofluorene (2NF) at 5 µg/plate for TA 98 Sodium azide (NaN3) at 2 µg/plate for TA 100 and TA 1535 9-Aminoacridine (ACA) at 50 µg/plate for TA 1537 Glutaraldehyde (GLU) at 25 µg/plate for TA 102 In presence of S9: 2-Aminoantracene (AAN) at 5 µg/plate for TA 98, TA 100 and TA 1535


Exposure;

Application of test

substance

3.3.1 Concentrations Pre-test: 0, 8, 40, 200, 1000, 5000 µg/plate Main test: 0, 8, 40, 200, 1000, 5000 µg/plate in experiment 1 (plate incorporation assay, +/- S9) 0, 156.3, 312.5, 625, 1250, 2500, 5000 µg/plate in experiment 2 (pre-incubation assay, +/- S9)

3.3.2 Way of application Stock solutions: TZNG was dissolved in DMSO. Pre-test and plate incorporation assay: The bacterial suspensions (0.1 mL) were mixed with molten agar (2.5 mL), 0.1 mL of TZNG stock solutions or vehicle control, 0.5 mL S9 mix or phosphate buffer before pured onto minimal agar plates. Pre-incubation assay: The bacterial suspensions (0.1 mL) were mixed with 0.1 mL of TZNG stock solutions or vehicle control, 0.5 mL S9 mix or phosphate buffer and incubated for 20 minutes at 37°C. Then 2.5 mL of molten agar were added and pured onto minimal agar plates. For both assay types the plates were then incubated for 3 days at 37°C.



3.4 Examinations Cytotoxicity (pre-test with TA 100): thinning of background lawn and when possible frequency of revertant colonies Mutagenicity (main test): frequency of revertant colonies Criteria for a positive response: The test substance was considered positive if there was a reproducible increase in the number of revertants relative to controls that was statistically significant by Dunnett’s test (p <0.01) and the data showed a significant dose correlation.




None of the observed results fulfilled the criteria of a positive response (see Table A6_10_1/03-1). In absence of a dose-related effect and as it was not reproducible in the 2nd experiment, the slight but statistically significant increase in revertant colonies at 5000 µg/plate in strain TA 1535 in experiment 1 was considered not to indicate a mutagenic potential.





Section A6.10

Annex Point IIA

VI.6.10.1/03



4.2 Cytotoxicity Yes: Indications for growth inhibition at 5000 µg/plate in all strains except TA 1535.


5.1 Materials and

methods


5.2 Results and

discussion

No relevant effect of treatment with up to 5000 µg/plate of TI-435 metabolite TZNG on the number of revertant colonies on 5 S.

typhimurium strains was seen. Indications for cytotoxicity (thinning of background lawn) were noted at 5000 µg/plate for all strains except TA 1535.

5.3 Conclusion TZNG and/or its metabolites were considered to be not mutagenic in this in vitro test system.

5.3.1 Reliability 1







Date 2004-10-07

Materials and Methods Ad 3.2.4: No positive control in presence of S9 was included in Experiment 1 for strains TA 1537 and TA 102 and in Experiment 2 for strains TA 1535, TA 1537 and TA 102. Ad 3.3.1: In Experiment 2 preincubation was only performed in presence of S9. Plate incorporation was used in cultures without S9.



Reliability 1


Remarks

COMMENTS FROM ...







Remarks



Table A6_10_1/03-1 Table for Gene Mutation Assay with TZNG (Ames test)



- S9 + S9 µg/plate - S9 + S9


34 37 30 26 41 20

1103

34 35 35 35 28 27

1912

0 156.3 312.5 625

1250 2500 5000

Pos. ctr

37 42 36 39 26 33 35

954

46 47 38 35 38 32 27

1466

Thinning of background lawn indicating cytotoxicity was observed at 5000 µg/plate in absence of S9 (both experiments) and in presence of S9 (experiment 2); no precipitation of the test substance


95 107 102 95 99

113

574

120 113 125 113 125 135

1635

0 156.3 312.5 625

1250 2500 5000

Pos. ctr

141 144 138 145 138 145 153 663

157 167 182 165 158 163 145

1582

Thinning of background lawn indicating cytotoxicity was observed at 5000 µg/plate in experiment 2 (+/- S9), no precipitation of the test substance


328 332 331 313 326 248

658

316 343 319 339 262 214

0 156.3 312.5 625

1250 2500 5000

Pos. ctr

316 308 290 282 268 274 225 795

354 396 307 339 283 267 170



15 19 20 20 23

28**

463

21 20 20 17 20 16

163

0 156.3 312.5 625

1250 2500 5000

Pos. ctr

23 21 23 22 21 24 24

450

29 30 22 26 34 18 23



13 18 13 16 16 13

337

14 14 12 14 15 13

0 156.3 312.5 625

1250 2500 5000

Pos. ctr

16 19 15 12 14 14 14

522

18 15 17 16 19 15 10


** p 0.005



Section A6.10

Annex Point IIA

VI.6.10.1/04


Acute oral toxicity study in rats (LD50) with the TI-435 metabolite TMG

1 REFERENCE

Official

use only

1.1 Reference Ruddock, W. D. (1999c); TMG: Acute oral toxicity study in the rat Covance Laboratories, Harrogate, UK unpublished report number 586/164-D6144, 05.07.1999




None




2.1 Guideline study Yes 92/69/EEC (method B1), OECD guideline no. 401 (February 1987), EPA OPPTS 870.1100 (August 1998), Japan MAFF (59 NohSan No. 4200, 1985)

2.2 GLP Yes

2.3 Deviations No


3.1 Test material TMG



3.1.2.1 Description Yellow solid

3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study (test-article/vehicle formulations prepared at the day of dosing)

3.2 Test Animals 5 female fasted rats per group (Crl:CD.BR strain, 8–11 weeks old, body weight range 212–259 g, Charles River UK Ltd); in addition 5 male fasted rats from the same supplier, of the same approximate age and weighing 323–360 g were treated at the approximate median lethal dose to demonstrate that males were not more susceptible to the toxic effects of TMG; No control group

3.3 Administration/

Exposure

Oral


14 days



Section A6.10

Annex Point IIA

VI.6.10.1/04



3.3.2 Type Gavage

3.3.3 Concentration 225, 650 and 1100 mg/kg bw (females) 550 mg/kg bw (males)





20 mL/kg bw

3.3.7 Controls -

3.4 Examinations Clinical signs: recorded on at least 5 occasions on the day of treatment and at least once daily thereafter. Body weights were recorded pre-dose and on days 1, 8 and 15. Decedents, animals killed in a moribund condition, and all survivors were subjected to necropsy and post mortem examination.

3.5 Method of

determination of

LD50

The acute median lethal dose and 95% confidence limits were estimated using probit analysis (Finney, D. J., 1971, Probit analysis, 3rd Ed., Cambridge University Press).



4.1 Clinical signs Mortality occurred at dose levels 650 and at 550 mg/kg bw for females and males, respectively (see table A6_10_1/04-1); Clinical signs included lethargy and palpebral closure (almost all treated rats), tremors, twitiching, tachypnoea, pronation and dyspnoea among individual animals of all dose levels. All survivors appeared normal by day 2.

4.2 Pathology No relevant treatment-related effects in survivors; dark lungs and livers and distended jejunum was seen among the animals that died

4.3 Other No relevant effect on bw development in survivors

4.4 LD50 567 mg/kg bw (females); treatment of males at a dose level of 550 mg/kg bw (with mortality in 3/5 animals) revealed that there is no relevant sex difference in susceptibility to toxic effects of TMG


5.1 Materials and

methods

Toxicity evaluation (bw, clinical signs, post mortem examination) after acute oral application of the TI-435 metabolite TMG to rats (gavage); no relevant deviation from guidelines (92/69/EEC B1; EPA OPPTS 870.1100, Japan MAFF, OECD 401)

5.2 Results and

discussion

Acute oral LD50 of TI-435 metabolite TMG in rats was 567 mg/kg bw.

5.3 Conclusion Classification as harmful if swallowed (R22) is considered required for TI-453 metabolite TMG according to Directive 2001/59/EC (adaptation of 67/548/EEC). The same classification had been proposed for the parent TI-435.

5.3.1 Reliability 1




Section A6.10

Annex Point IIA

VI.6.10.1/04






Date 2004-10-07




Reliability 1


Remarks

COMMENTS FROM ...









Remarks



Table A6_10_1/04-1 Table for Acute Oral Toxicity (rat), metabolite TMG

Dose

[mg/kg

bw]

Number of dead /

number of

investigated

Time of

death

(range) Observations

225 0/5 females - Lethargy, palpebral closure, tremor, piloerection, hunched posture; all animals recovered by day 2 bw development and post mortem examinations revealed no effect of treatment

650 4/5 females Day 1-2 Lethargy, palpebral closure, tremor, prone, twitching, dyspnoea, ataxia, salivation, hunched posture, hypothermia; the surviving female recovered by day 2 the surviving animal gained weight and no effect of treatment was seen at the post mortem examination; dark and/or inflated lungs, dark (and mottled) liver, distended (and discoloured) jejunum were seen in three females that died

1100 5/5 females Day 1 Prior to death: lethargy, palpebral closure, tremor, prone, vasoconstriction, twitching, dyspnoea, tachypnoea, ataxia, piloerection, hunched posture; post mortem examinations revealed dark (and inflated) lung lobes, dark liver and distended jejunum in the animals that died

550 3/5 males Day 1-2 Lethargy, palpebral closure, tremor, twitching, tachypnoea, piloerection; all survivors recovered by day 2 no relevant effect of treatment was seen on bw development of survivors, post mortem examinations revealed no relevant effect of treatment in survivors (one male had slightly mottled kidney), one of the animals that died had dark and inflated lungs (and a partially cannibalised hind leg)

LD50 value Males: 550 mg/kg bw Females: 567 mg/kg bw



Section A6.10

Annex Point IIA

VI.6.10.1/05


Ames test (+/- S9) using S. typhimurium with TI-435 metabolite TMG

1 REFERENCE

Official

use only

1.1 Reference Dawkes, N. (1999d); TMG: Reverse mutation in five histidine-requiring strains of Salmonella typhimurium Covance Laboratories, Harrogate, UK unpublished report number 586/166-D5140, 03.06.1999




None




2.1 Guideline study Yes 2000/32/EC (method B13/14), OECD no. 471 (1983), EPA OPPTS 870.5265 (1998)

2.2 GLP Yes

2.3 Deviations None relevant for the integrity and validity of the study (study was performed previous to current EU-guideline but was checked for compliance with the above): no positive control was included for the experiments in presence of S9 for strains TA 1537 and TA 102; however the sensitivity of the strains were shown with positive control compounds in absence of S9 and in three strains in presence of S9


3.1 Test material TMG



3.1.2.1 Description Yellow solid

3.1.2.2 Purity





Histidine deficient


S9 mix Prepared from livers of male Sprague Dawley rats induced with Arochlor 1254; supplemented with co-factors



Section A6.10

Annex Point IIA

VI.6.10.1/05



3.2.4 Positive control In absence of S9: Nitrofluorene (2NF) at 5 µg/plate for TA 98

Sodium azide (NaN3) at 2 µg/plate for TA 100 and TA 1535 9-Aminoacridine (ACA) at 50 µg/plate for TA 1537 Glutaraldehyde (GLU) at 25 µg/plate for TA 102 In presence of S9: 2-Aminoantracene (AAN) at 5 µg/plate for TA 98, TA 100 and TA 1535


Exposure;

Application of test

substance

3.3.1 Concentrations Pre-test: 0, 8, 40, 200, 1000, 5000 µg/plate Main test: 0, 8, 40, 200, 1000, 5000 µg/plate in experiment 1 (plate incorporation assay, +/- S9) 0, 156.3, 312.5, 625, 1250, 2500, 5000 µg/plate in experiment 2 (pre-incubation assay, +/- S9)

3.3.2 Way of application Stock solutions: TMG was dissolved in dimethylformamide (DMF). Pre-test and plate incorporation assay: The bacterial suspensions (0.1 mL) were mixed with molten agar (2.5 mL), 0.1 mL of TMG stock solutions or vehicle control, 0.5 mL S9 mix or phosphate buffer before pured onto minimal agar plates. Pre-incubation assay: The bacterial suspensions (0.1 mL) were mixed with 0.1 mL of TMG stock solutions or vehicle control, 0.5 mL S9 mix or phosphate buffer and incubated for 20 minutes at 37°C. Then 2.5 mL of molten agar were added and pured onto minimal agar plates. For both assay types the plates were then incubated for 3 days at 37°C.



3.4 Examinations Cytotoxicity (pre-test with TA 100): thinning of background lawn and when possible frequency of revertant colonies Mutagenicity (main test): frequency of revertant colonies Criteria for a positive response: The test substance was considered positive if there was a reproducible increase in the number of revertants relative to controls that was statistically significant by Dunnett’s test (p <0.01) and the data showed a significant dose correlation







4.2 Cytotoxicity Yes: Indications for growth inhibition at 5000 µg/plate in presence of S9 for strains TA 98, TA 100 and TA 1537.



Section A6.10

Annex Point IIA

VI.6.10.1/05




5.1 Materials and

methods


5.2 Results and

discussion

No effect of treatment with up to 5000 µg/plate of TI-435 metabolite TMG on the number of revertant colonies on 5 S. typhimurium strains was seen. There were indications for cytotoxicity (thinning of background lawn) at 5000 µg/plate for strains TA 98, TA 100 and TA 1537 in presence of metabolic activation.

5.3 Conclusion TMG and/or its metabolites were considered to be not mutagenic in this in vitro test system.

5.3.1 Reliability 1







Date 2004-10-07

Materials and Methods Ad 3.2.4: No positive control in presence of S9 was included in Experiment 1 for strains TA 1537 and TA 102 and in Experiment 2 for strains TA 1535, TA 1537 and TA 102. Ad 3.3.1: In Experiment 2 preincubation was only performed in presence of S9. Plate incorporation was used in cultures without S9.



Reliability 1


Remarks

COMMENTS FROM ...







Remarks



Table A6_10_1/05-1 Table for Gene Mutation Assay with TMG (Ames test)



- S9 + S9 µg/plate - S9 + S9


33 28 23 40 30 40

995

32 36 33 30 36 35

1812

0 156.3 312.5 625

1250 2500 5000

Pos. ctr

40 37 33 39 34 38 34

897

48 47 41 40 41 47 36

1615

Thinning of background lawn indicating cytotoxicity was observed at 5000 µg/plate in experiment 2 (+ S9); no precipitation of the test substance


101 93

101 96 99 96

583

105 120 106 110 96

100

1742

0 156.3 312.5 625

1250 2500 5000

Pos. ctr

133 127 119 111 117 137 117 613

149 152 150 139 155 145 144

1820



317 334 303 295 326 297

616

345 235 239 299 292 255

0 156.3 312.5 625

1250 2500 5000

Pos. ctr

304 303 301 311 300 311 270 799

395 426 374 356 364 289 308



16 17 17 17 16 20

441

20 18 16 11 21 18

163

0 156.3 312.5 625

1250 2500 5000

Pos. ctr

24 22 14 25 13 19 21

566

26 30 24 21 19 18 18



13 15 11 14 19 18

379

13 12 11 14 10 13

0 156.3 312.5 625

1250 2500 5000

Pos. ctr

13 8 13 17 17 15 14

655

20 14 13 12 10 12 15




Section A6.10

Annex Point IIA

VI.6.10.2/01

Further study available with the a.s.

Pharmacological study with TI-435 in rats and mice

1 REFERENCE

Official

use only

1.1 Reference Unakami, S. (2000); Pharmacological studies on TI-435 Kashima Laboratory, Mitsubishi Chemical Safety Institute, Tokyo, Japan unpublished report number 9L668, 20.01.2000




None




2.1 Guideline study No No applicable EU guideline, Japan MAFF (59 NohSan 4200, 1985, revision of part 1997)

2.2 GLP Yes

2.3 Deviations -






3.1.2.2 Purity

3.1.2.3 Stability Considered stable under conditions of this study (test-article vehicle formulations were applied on the day of preparation)

3.2 Test Animals The rats used were CD (SD) strain (5-7 weeks old, weight range 136–319 g, Charles River Japan Inc.), the mice used were CD-1 (ICR) strain (5 weeks old, weight range 22.7–34.2 g, Charles River Japan Inc.) and the guinea pigs were Hartley strain (6–8 weeks old, weight range 422–448 g, Japan Slc Co. Ltd). Please see the following table for the number of animals used for which examination.



Section A6.10

Annex Point IIA

VI.6.10.2/01



Test Species Animals/group

Irwin screen – 1 Mouse 3 Anaesthetic effects – 2 Mouse 8 Synergistic effects on convulsion – 3.1 Synergistic effects on convulsion – 3.2

Mouse Mouse

10 10

Effect on body temperature – 4 Rat 6 Effect on cardiovascular parameters – 5 Rat 4 Effect on intestinal transport – 6 Mouse 8 Effect on muscle strength – 7 Mouse 8 Effect on blood coagulation – 8 Rat 6 In vitro: contraction of isolated ilium - 9 ilium derived from Guinea pigs

3.3 Administration/

Exposure

Oral (for in vivo experiments)


See point 3.4

3.3.2 Type Gavage

3.3.3 Concentration Test 1: 0, 12.5, 25, 50, 100, 200, 400 mg/kg bw Test 2: 0, 25, 75, 225 mg/kg bw Test 3.1: 0, 6.25, 12.5, 25, 75, 225 mg/kg bw Test 3.2: 0, 25, 75, 225 mg/kg bw Test 4: 0, 30, 100, 300, 1000, 3000 mg/kg bw Test 5: 0, 100, 300, 1000, 3000 mg/kg bw Test 6: 0, 25, 75, 225 mg/kg bw Test 7: 0, 25, 75, 225 mg/kg bw Test 8: 0, 300, 1000, 3000 mg/kg bw Test 9: 0, 10-6, 10-5 and 10-4 mol/L

3.3.4 Vehicle 5% w/v aqueous gum arabic (in vivo experiments) DMSO (in vitro experiment)




10 mL/kg bw




Section A6.10

Annex Point IIA

VI.6.10.2/01



3.4 Examinations Test 1: general condition and behaviour were observed pre-dose and at 0.5, 1, 3, 6 and 24 hours after treatment of mice with TI-435. Each clinical sign was graded on a scale from 1 to 5. Test 2: 80 mg/kg bw hexobarbital was administered intraperitoneally to each mouse one hour after treatment with TI-435. The anaesthetised animals were placed on a warming pad at 37 C and the time from disappearance to reappearance of the righting reflex was recorded. Test 3.1: one hour after treatment of mice with TI-435, electrodes were attached to the cornea and an electroshock sub-threshold for the induction of convulsions (current 8 mA, pulse duration 5 msec, stimulation interval 10 msec) was delivered for 0.6 sec. The appearance of tonic flexor and extensor convulsions were monitored. Test 3.2: one hour after treatment of mice with TI-435, 55 mg/kg bw pentylenetetrazol was administered subcutaneously. The appearance of clonic and tonic extensor convulsions were monitored for 30 minutes. Test 4: body temperature of rats was recorded via a rectal probe connected to a digital electronic thermometer pre-dose and 0.5, 1, 3 and 6 hours after treatment. Test 5: a tail cuff attached to an automatic blood pressure meter was used in unanaesthetised rats to determine the systolic and mean blood pressures by plethysmography pre-dose and 0.5, 1, 3 and 6 hours after treatment. The heart rate was determined from the pulse wave. Test 6: mice were deprived of food for 19 hours before treatment. A 5% charcoal suspension in 5% gum arabic (0.2mL/animal) was administered orally one hour after treatment with TI-435. The animals were killed 30 minutes after charcoal administration, the stomach and small intestine excised, and the distance from the pylorus to the furthest point travelled was measured. The total length of the small intestine was also recorded. Test 7: mice were checked pre-dose for the ability to grasp a horizontal stretched wire with the paws within 5 seconds of placement. The animals selected were re-tested for grasping ability with the hind-paws 1, 3 and 6 hours after treatment. Test 8: The prothrombin and activated thromboplastin times were determined in rats one hour after treatment with TI-435. Test 9: 2–3 cm strips of isolated ileum, under a static tensive load of 0.5 g, were suspended in a Magnus bath containing Krebs solution at 32 C. Contractions of the preparations were recorded on a recorder via

an isotonic transducer. Contractile agents - 10-6mol/L acetylcholine, 3 10-6mol/L histamine and 10-3mol/L barium - were applied to confirm contractile reactivity. DMSO vehicle and final concentrations of TI-435 at 10-6, 10-5 and 10-4mol/L were applied for 5 minutes each, in succession, interspersed with applications of the 3 contractile agonists. The contractile responses were recorded and measured.

3.5 Further remarks Quantitative data were analysed using Bartlett’s test for homogeneity of variance followed by ANOVA for homogeneous data, or by Dunnett’s mean multiple comparison test. Data with non-homogeneous variances were analysed by the Kruskal-Wallace test followed by Dunnett’s mean multiple comparison test where appropriate. Inter-group comparisons were made using the F test for homogeneity of variance followed by Student’s the test or the Aspin-Welch the test.



Section A6.10

Annex Point IIA

VI.6.10.2/01




4.1 Results

4.1.1 Test 1 (Effects on the CNS – Irwin screen)

There were no clinical signs of a reaction to treatment at dose levels of 12.5 and 25 mg/kg bw. At 50 mg/kg bw transient reduced spontaneous activity occurred for up to 3 hours, and one animal showed tremors and deep respiration. At 100 mg/kg bw decreased grooming and reactivity, reduced locomotor activity, body tone and rectal temperature occurred together with tremor, prone posture, staggering gait, mydriasis and deep respiration. The signs were graded slight to moderate and persisted for up to 6 hours. At 200 mg/kg bw the animals also displayed decreased touch response and grip strength. One animal died 3 hours after treatment at 400 mg/kg bw. In addition to the signs observed at 200 mg/kg bw the remaining animals displayed decreased corneal and pinna reflexes, suppression of the ipsilateral flexor reflex, reduced limb tone, straub tail and cyanosis. The severity of signs was graded as slight to marked.

4.1.2 Test 2 (anaesthetic effects)

The hexobarbital-induced sleeping time was significantly increased at a dose level of 225 mg/kg bw, but not at lower dose levels (25, 75 mg/kg bw). Group mean sleeping times were 27.7, 39.7 and 51.3 minutes in order of ascending dose level of TI-435, compared with a mean control value of 31.4 minutes.

4.1.3 Test 3.1 (synergistic effects on convulsions)

Dose levels 25 mg/kg bw potentiated the sub-threshold electroshock to produce tonic flexor and extensor convulsions. The incidences of a convulsive response were 10, 30, 20, 80, 100 and 100% at dose levels of 0, 6.25, 12.5, 25, 75 and 225 mg/kg bw, respectively. The increased incidences at 25 mg/kg bw were statistically significant.

4.1.4 Test 3.2 (synergistic effects on convulsions)

None of the dose levels of TI-435 potentiated the sub-threshold convulsive dose of pentylenetetrazol. The incidences of induced clonic convulsions were 20, 0 and 0% at dose levels of 25, 75 and 225 mg/kg bw, respectively.

4.1.5 Test 4 (effects on body temperature)

Body temperature was decreased at dose levels of 300 mg/kg bw. At 300 mg/kg bw temperature was reduced by 0.8 C one hour after treatment. At dose levels of 1000 and 3000 mg/kg bw very marked decreases of 4.1 and 6.4 C, respectively, were apparent 6 hours after treatment. Dose levels up to 100 mg/kg bw did not influence body temperature.

4.1.6 Test 5 (effects on cardiovascular parameters)

No effects on blood pressure and heart rate occurred at a dose level of 100 mg/kg bw. Heart rate was significantly increased by 11–12% within 0.5 hours of treatment at 300 and 3000 mg/kg bw. Thereafter heart rates decreased slightly. Systolic and mean blood pressure was unaffected by treatment at 300 mg/kg bw, but significantly reduced by up to 14.6% at dose levels 1000 mg/kg bw.

4.1.7 Test 6 (effects on intestinal transport)

Treatment with TI-435 at dose levels of 75 and 225 mg/kg bw produced a suppression of intestinal transport of 40.2 and 52.0%, respectively. No effect occurred at 25 mg/kg bw.

4.1.8 Test 7 (effects on skeletal muscle)

Muscle strength, assessed by the ability to grasp a horizontal wire, was reduced at 225 mg/kg bw, since 50% of animals were unable to grasp the wire 1 and 3 hours after treatment.



Section A6.10

Annex Point IIA

VI.6.10.2/01



No effects occurred at dose levels of 25 and 75 mg/kg bw.

4.1.9 Test 8 (effects on blood coagulation)

Dose levels of up to 3000 mg/kg bw had no effect on the prothrombin and activated thromboplastin times. Prothrombin times were 14.7, 14.5, 14.6 and 14.5 sec and activated thromboplastin times were 14.6, 13.9, 15.2 and 14.7 sec at dose levels of 0, 300, 1000 and 3000 mg/kg, respectively.

4.1.10 Test 9 (effects on the autonomic nervous system and smooth muscles)

TI-435 applied at 10-4mol/L inhibited the contractile response to barium by 12.2%, but there was no effect on the agonist-induced contractile response to acetylcholine or histamine. TI-435 concentrations of 10-5 and 10-6mol/L had no effect on the contractile response to any of the agonists.


5.1 Materials and

methods

Evaluation of several parameters potentially interessant concenring a compounds pharmacological properties after single acute oral application of the TI-435 to rats or mice (gavage) or in an in vitro experiment (single exposure) with ilium from Guinea pigs; specific Japanese requirement - no EU guideline available

5.2 Results and

discussion

NOELs (species, examined effect) Test 1 25 mg/kg bw (mice, clinical signs) Test 2 75 mg/kg bw (mice, anaesthetic effects) Test 3.1 12.5 mg/kg bw (mice, synergistic effects on convulsions ) this endpoint is not considered relevant for human risk assessment for biocides as synergistic action with electro- schocks is considered an unlikely human exposure situation Test 3.2 >225 mg/kg bw (mice, synergistic effects on convulsions ) Test 4 100 mg/kg bw (rats, body temperature) Test 5 100 mg/kg bw (rats, cardiovascular parameter) Test 6 25 mg/kg bw (mice, intestinal transport) Test 7 75 mg/kg bw (mice, skeletal musle) Test 8 >3000 mg/kg bw (rats, blood coagulation) Test 9 10-5 mol/L (in vitro, contraction of ilium); considered not to be a relevant endpoint for human risk assessment for biocides

5.3 Conclusion The overall NOELs of effects where a potential relevance for human risk assessment for biocides might not be excluded was 25 mg/kg bw for mice and 100 mg/kg bw for rats.

5.3.1 Reliability 1




Section A6.10

Annex Point IIA

VI.6.10.2/01






Date 2006-10-30


Results and discussion Ad 4.1.7: Treatment with TI-435 at a dose level of 25 mg/kg bw produced a very slight suppression of intestinal transport of 61.5% vs. 66.5% in controls. This finding is based on a single animal in this group with a reduced transport (37%). Ad 5.2: The endpoint studied in Test 3.1 (mice, synergistic effects on convulsions after subthreshold electro-shock) and thus the NOEL of 12.5 mg/kg bw could be regarded as relevant for human risk assessment for biocides insofar as this model may be representative for other physicalic (non-pharmacologic) means of eliciting convulsions. However, since the induction of convulsions by electro-shock is not part of the standard repertoire in acute toxicity testing the NOAEL for mice (25 mg/kg bw) will be based on reduction of activity in Test 1 which corresponds to what can also be observed in a standard acute toxicity study.

Conclusion Other conclusions: The overall NOEL of effects 25 mg/kg bw for mice, based clinical signs and decreased intestinal transport, and 100 mg/kg bw for rats, based on body temperature and cardiovascular parameters.

Reliability 1


Remarks

COMMENTS FROM ...









Remarks




Medical surveillance data on manufacturing plant personnel


Other existing data [ ] Technically not feasible [ ] Scientifically unjustified [ ] Limited exposure [ ] Other justification [ X ]



-




Date


Conclusion

Remarks






Remarks




Direct observation, e.g. clinical cases, poisoning incidents





-




Date


Conclusion

Remarks






Remarks




Health records, both from industry and any other available sources





-




Date


Conclusion

Remarks






Remarks




Epidemiological studies on the general population





-




Date


Conclusion

Remarks






Remarks




Diagnosis of poisoning including specific signs of poisoning and clinical tests





-




Date


Conclusion

Remarks






Remarks




Sensitisation/allergenicity observations





-




Date


Conclusion

Remarks






Remarks




Specific treatment in case of an accident or poisoning: first aid measures, antidotes and medical treatment





-




Date


Conclusion

Remarks






Remarks




Prognosis following poisoning





-




Date


Conclusion

Remarks






Remarks

Sumitomo Chemical Takeda Agro Co., Ltd. Clothianidin August 2006

Section 6.14 Annex Point IIIA-XI.2

Other test(s) related to the exposure of humans


Other existing data [ ] Technically not feasible [ ] Scientifically unjustified [ ]

Limited exposure [ ] Other justification [ X ]



-




Date


Conclusion

Remarks






Remarks

RCC project no. 852224


Section 6.15.1 Annex Point IIIA-XI.1

Identification of the residues (identity and concentrations), degradation and reaction products and of metabolites of the active substance in contaminated foods or feeding stuffs



Limited exposure [ X ] Other justification [ ]



-




Date


Conclusion

Remarks






Remarks




Behaviour of the residues of the active substance, its degradation and reaction products and, where relevant, its metabolites on the treated or contaminated food or feeding stuffs including the kinetics of disappearance






-




Date


Conclusion

Remarks






Remarks




Estimation of potential or actual exposure of the active substance to humans or animals through food and feeding stuffs and other means






-




Date


Conclusion

Remarks







Estimation of potential or actual exposure of the active substance to humans or animals through food and feeding stuffs and other means




Remarks

Sumitomo Chemical Takeda Agro Co., Ltd. Clothiandin August 2006

Section 6.15.4 Annex Point IIIA-XI.1.7

Proposed acceptable residues and the justification of their acceptability






-




Date


Conclusion

Remarks






Remarks




Any other available information that is relevant






-




Date


Conclusion

Remarks






Remarks


Reference List geordnet nach Section Numbers Section No / Reference No

Author(s) Year Title. Source (where different from company) Company, Report No. GLP (where relevant) / (Un)Published

Data Protection Claimed (Yes/No)

Owner

A6.1.1/01 1997a

GLP, unpublished

yes

A6.1.1/02* 1997b

GLP, unpublished

yes

A6.1.1/03* 2002

GLP, unpublished

yes

A6.1.2/01* 1997c

GLP, unpublished

yes

A6.1.3/01* 1998

GLP, unpublished

yes

A6.1.4/01* 1997d

GLP, unpublished

yes

Section No / Reference No



Owner

A6.1.4/02* 1997e

GLP, unpublished

yes

A6.1.5/01* 1997

GLP, unpublished

yes

A6.2* 2000d

GLP, unpublished

yes

A6.2/02 Yokota, T. et al. (2003), J Agric Food Chem 51, 7066-7072

2003 Yokota, T. et al. (2003). Absorption, tissue distribution, excretion, and metabolism of clothianidin in rats. J Agric Food Chem 51, 7066-7072

no published

A6.3.1/01* 1997a

GLP, unpublished

yes

A6.3.1/02* 2000

GLP, unpublished

yes

A6.3.1/03 1997b

GLP, unpublished

yes

A6.3.1/04 1998

non-GLP, unpublished

yes




Owner

A6.3.2/01* 2000

GLP, unpublished

yes

A6.4.1/01* 2000a

GLP, unpublished

yes

A6.4.1/02 2000b

GLP, unpublished

yes

A6.4.1/03* 2000

GLP, unpublished

yes

A6.4.1/04 2000a


yes

A6.4.1/05* 2000b


yes

A6.5*

A6.6.1/01* 2000

GLP, unpublished

yes




Owner

A6.6.1/02* 1990b

GLP, unpublished

yes

A6.6.1/03* 1999a

GLP, unpublished

yes

A6.6.1/04* 1999b

GLP, unpublished

yes

A6.6.2/01* 2000

GLP, unpublished

yes

A6.6.3/01* 2000a

GLP, unpublished

yes

A6.6.3/02* 1999a

GLP, unpublished

yes




Owner

A6.6.4/01* 2000b

GLP, unpublished

yes

A6.6.5/01* (1999b) 2001

GLP, unpublished

yes

A6.7/01* 2000a

GLP, unpublished

yes

A6.7/02* 2000b

GLP, unpublished

yes

A6.8.1/01* 1998a

GLP, unpublished

yes

A6.8.1/02* 1998b

GLP, unpublished

yes




Owner

A6.8.2*

2000

GLP, unpublished

yes

A6.9/01*

2000

GLP, unpublished

yes

A6.9/02*

2000

GLP, unpublished

yes

A6.9/03* 2000

GLP, unpublished

yes

A6.9/04* 2000

GLP, unpublished

yes




Owner

A6.10.1/01 2001

GLP, unpublished

yes

A6.10.1/02 2000c

GLP, unpublished

yes

A6.10.1/03 1999c

GLP, unpublished

yes

A6.10.1/04 1999c

GLP, unpublished

yes

A6.10.1/05 1999d

GLP, unpublished

yes

A6.10.1/06 1999b

GLP, unpublished

yes

A6.10.1/07 1999c

yes




Owner

GLP, unpublished A6.10.1/08 1999c

GLP, unpublished

yes

A6.10.1/09 1999d

GLP, unpublished

yes

A6.10.2/01* 2000

GLP, unpublished

yes

Reference List geordnet nach Autoren Author(s) Section No /

Reference No

Year Title. Source (where different from company) Company, Report No. GLP (where relevant) / (Un)Published


Owner

A6.5* see A6.7 A6.4.1/01* 2000a

GLP, unpublished

yes

A6.4.1/02 2000b

GLP, unpublished

yes

A6.7/01* 2000a

GLP, unpublished

yes

A6.7/02* 2000b

GLP, unpublished

yes

A6.6.3/02* 1999a

GLP, unpublished

yes

A6.6.5/01* (1999b) 2001

GLP, unpublished

yes

Author(s) Section No / Reference No



Owner

A6.9/01* 2000

GLP, unpublished

yes

A6.3.1/01* 1997a

GLP, unpublished

yes

A6.3.1/03 1997b

GLP, unpublished

yes

A6.4.1/04 2000a


yes

A6.4.1/05* 2000b


yes

A6.10.1/03 1999c

GLP, unpublished

yes

A6.10.1/05 1999d

GLP, unpublished

yes




Owner

A6.10.1/07 1999c

GLP, unpublished

yes

A6.10.1/09 1999d

GLP, unpublished

yes

A6.1.5/01* 1997

GLP, unpublished

yes

A6.6.3/01* 2000a

GLP, unpublished

yes

A6.6.4/01* 2000b

GLP, unpublished

yes

A6.8.2* 2000

GLP, unpublished

yes

A6.1.1/01 1997a

yes




Owner

GLP, unpublished A6.1.1/02* 1997b

GLP, unpublished

yes

A6.1.2/01* 1997c

GLP, unpublished

yes

A6.1.4/01* 1997d

GLP, unpublished

yes

A6.1.4/02* 1997e

GLP, unpublished

yes

A6.6.1/03* 1999a

GLP, unpublished

yes

A6.6.1/04* 1999b

GLP, unpublished

yes

A6.10.1/01 2001

yes




Owner

GLP, unpublished A6.9/04* 2000

GLP, unpublished

yes

A6.3.1/02* 2000

GLP, unpublished

yes

A6.3.1/04 1998

unpublished

yes

A6.6.1/02* 1990b

GLP, unpublished

yes

A6.10.1/02 2000c

GLP, unpublished

yes

A6.10.1/04 1999c

GLP, unpublished

yes

A6.10.1/06 1999b

GLP, unpublished

yes

A6.10.1/08 1999c yes




Owner

GLP, unpublished

A6.1.1/03* 2002

GLP, unpublished

yes

A6.9/02* 2000

GLP, unpublished

yes

A6.9/03* 2000

GLP, unpublished

yes

A6.1.3/01* 1998

GLP, unpublished

yes

A6.6.1/01* 2000

yes




Owner

GLP, unpublished A6.10.2/01* 2000

GLP, unpublished

yes

A6.4.1/03* 2000

GLP, unpublished

yes

A6.2* 2000d

GLP, unpublished

yes

A6.3.2/01* 2000

GLP, unpublished

yes

A6.6.2/01* 2000

GLP, unpublished

yes

Yokota, T. et al. (2003), J Agric Food Chem 51, 7066-7072

A6.2/02 2003 Yokota, T. et al. (2003). Absorption, tissue distribution, excretion, and metabolism of clothianidin in rats. J Agric Food Chem 51, 7066-7072

no published

A6.8.1/01* 1998a

GLP, unpublished

yes

A6.8.1/02* 1998b

yes




Owner

GLP, unpublished

Documents

Section A6.1.1 Acute Toxicity Oral - ECHA