Sumitomo Chemical Takeda Agro Co., Ltd. TI-435 February Acute Toxicity. Acute oral toxicity study in rats (LD 50 )

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1 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.1 Annex Point IIA VI.6.1.1/1 Acute Toxicity Oral Acute oral toxicity study in rats (LD 5 ) 1 REFERENCE 1.1 Reference Gardner, J. R. (1997a); TI435: Acute oral toxicity study in the rat Covance Laboratories, Harrogate, UK unpublished report number 586/ Data protection Yes Data owner Sumitomo Chemical Takeda Agro Co. Ltd Companies with letter of access Criteria for data protection None Data submitted on existing a.s. for its first entry into Annex I. Official use only 2 GUIDELINES AND QUALITY ASSURANCE 2.1 Guideline study Yes 92/69/EEC (method B1), EPA FIFRA, Subdivision F, Section 811 (1984), Japan MAFF (59 NohSan No. 42, 1985), OECD No. 41 (1987) 2.2 GLP Yes 2.3 Deviations No 3.1 Test material TI Lot/Batch number Specification As given in section Description Pale, yellow powder Purity 3 MATERIALS AND METHODS 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 g, supplied by Charles River UK Ltd) No control group 3.3 Administration/ Exposure Postexposure period Oral 14 days RCC project no page 1 of 4

2 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.1 Annex Point IIA VI.6.1.1/1 Acute Toxicity Oral Acute oral toxicity study in rats (LD 5 ) Oral Type Gavage Concentration 1758, 2283, 2965, 385 and 5 mg/kg bw Vehicle 5% w/v aqueous gum arabic Concentration in vehicle Total volume applied Controls Prepared according to individual bw and dosing volume 1 ml/kg bw 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 predose 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 Since no group showed mortality of >2%, the data were not analysed statistically. LD Further remarks 4 RESULTS AND DISCUSSION 4.1 Clinical signs Single females and males died at 2965 and at 5 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_1/1); 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 385 mg/kg bw loss of bw in individual animals also in the 2 nd week after application 4.4 LD 5 >5 mg/kg bw (males, females, males and females combined) 5.1 Materials and methods 5.2 Results and discussion 5 APPLICANT'S SUMMARY AND CONCLUSION Toxicity evaluation (bw, clinical signs, post mortem examination) after acute oral application to rats (gavage); no relevant deviation from guidelines OECD 41) Acute oral LD 5 in rats is >5 mg/kg bw 5.3 Conclusion No classification for oral toxicity is considered required for TI435 according to Directive 21/59/EC (adaptation of 67/548/EEC) Reliability Deficiencies No RCC project no page 2 of 4

3 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.1 Annex Point IIA VI.6.1.1/1 Acute Toxicity Oral Acute oral toxicity study in rats (LD 5 ) Evaluation by Competent Authorities Use separate "evaluation boxes" to provide transparency as to the comments and views submitted Date 2417 Materials and Methods Results and discussion Conclusion Reliability 1 Acceptability Remarks EVALUATION BY RAPPORTEUR MEMBER STATE Although testing method 41 has been deleted from OECD guidelines since December 22 the experiments described are acceptable since they were performed in 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 (2 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. Acute oral LD 5 in rats is >5 mg/kg bw for both sexes. Single females and males died at 2965 and at 5 mg/kg bw, respectively. For clinical signs and pathology see table A6_1_1/1. No classification for oral toxicity is considered required for TI435 according to Directive 21/59/EC (adaptation of 67/548/EEC). Acceptable There is an acute oral neurotoxicity study in rats (Cain, D. M., 2; see also A 6.9/1) the supplement of which is an acute oral dose rangefinding study (Sheets, L. P., 22) originally not presented in this section. Since an LD 5 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 A6.1.1/3). COMMENTS FROM... Date Materials and Methods Results and discussion Conclusion Reliability Acceptability Remarks Give date of comments submitted Discuss additional relevant discrepancies referring to the (sub)heading numbers and to applicant's summary and conclusion. RCC project no page 3 of 4

4 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.1 Annex Point IIA VI.6.1.1/1 Table A6_1_1/1. Acute Toxicity Oral Acute oral toxicity study in rats (LD 5 ) Table for Acute Oral Toxicity (rat) Dose [mg/kg bw] Number of dead / number of investigated 1758 /5 females /5 males 2283 /5 females /5 males /5 females /5 males 385 1/5 females /5 males 5 1/5 females 1/5 males Time of death (range) Day 2 Day 12 Day 4 Day 12 LD 5 value Males: >5 mg/kg bw Females: >5 mg/kg bw Combined: >5 mg/kg bw Observations 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 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 Palpebral closure, decreased activity, ataxia, arched gait, hunched posture, tremor, hypothermia, wasted appearance, hair loss, discoloration of urine, soft faeces, chromodacryorrhoea; 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 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 RCC project no page 4 of 4

5 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.1 Annex Point IIA VI.6.1.1/2 Acute Toxicity Oral Acute oral toxicity study in mice (LD 5 ) 1 REFERENCE 1.1 Reference Gardner, J. R. (1997b); TI435: Acute oral toxicity study in the mouse; Covance Laboratories, Harrogate, UK; unpublished report number 586/ Data protection Yes Data owner Sumitomo Chemical Takeda Agro Co. Ltd Companies with letter of access Criteria for data protection None Data submitted on existing a.s. for its first entry into Annex I Official use only 2 GUIDELINES AND QUALITY ASSURANCE 2.1 Guideline study Yes 92/69/EEC (method B1), EPA FIFRA, Subdivision F, Section 811 (1984), Japan MAFF (59 NohSan No. 42, 1985), OECD No. 41 (1987) 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 Test material TI Lot/Batch number Specification As given in section Description Pale, yellow powder Purity 3 MATERIALS AND METHODS Stability Considered stable under conditions of this study (testarticlevehicle formulations prepared at the day of dosing) 3.2 Test Animals 5 male and 5 female fasted mice per group (Crl:CD1(ICR)BR strain, 5 7 weeks old, weight range 22 3g, supplied by Charles River UK Ltd) No control group 3.3 Administration/ Exposure Postexposure period Oral 14 days RCC project no page 1 of 4

6 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.1 Annex Point IIA VI.6.1.1/2 Acute Toxicity Oral Acute oral toxicity study in mice (LD 5 ) Oral Type Gavage Concentration 34, 38, 475, 594 and 742 mg/kg bw Vehicle 5% w/v aqueous gum arabic Concentration in vehicle Total volume applied Controls Prepared according to individual bw and dosing volume 1 ml/kg bw 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 predose 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 The acute median lethal dose and 95% confidence limits were estimated using moving average interpolation (C. S. Weil, 1952). LD Further remarks 4 RESULTS AND DISCUSSION 4.1 Clinical signs Mortality at 38 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_1/2) 4.2 Pathology No gross lesions were apparent during post mortem examination of the animals. 4.3 Other 4.4 LD Materials and methods 5.2 Results and discussion 5 APPLICANT'S SUMMARY AND CONCLUSION 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 811, Japan MAFF, OECD 41) Acute oral LD 5 in mice (males+females combined): 425 mg/kg bw 5.3 Conclusion Classification as harmful if swallowed (R22) is considered required for TI435 according to Directive 21/59/EC (adaptation of 67/548/EEC) Reliability Deficiencies No RCC project no page 2 of 4

7 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.1 Annex Point IIA VI.6.1.1/2 Acute Toxicity Oral Acute oral toxicity study in mice (LD 5 ) Date 2417 Materials and Methods Results and discussion Conclusion Reliability 1 Acceptability Remarks Evaluation by Competent Authorities Use separate "evaluation boxes" to provide transparency as to the comments and views submitted EVALUATION BY RAPPORTEUR MEMBER STATE Although testing method 41 has been deleted from OECD guidelines since December 22 the experiment described is acceptable since it was performed in 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, ) 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. Acute oral LD 5 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_1/2. TI435 is approximately one order of magnitude more toxic to mice than to rats by the oral route. Classification as harmful if swallowed (R22) is considered required for TI435 according to Directive 21/59/EC (adaptation of 67/548/EEC). Acceptable None Date Materials and Methods Results and discussion Conclusion Reliability Acceptability Remarks COMMENTS FROM... Give date of comments submitted Discuss additional relevant discrepancies referring to the (sub)heading numbers and to applicant's summary and conclusion. RCC project no page 3 of 4

8 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.1 Annex Point IIA VI.6.1.1/2 Table A6_1_1/2. Acute Toxicity Oral Acute oral toxicity study in mice (LD 5 ) Table for Acute Oral Toxicity Dose [mg/kg bw] Number of dead / number of investigated 34 /5 females /5 males 38 2/5 females 2/5 males 475 3/5 females 5/5 males 594 3/5 females 5/5 males 742 5/5 females 5/5 males Time of death (range) Day 1 Day 12 Day 2 Day 2 Day 12 Day 12 Day 1 Day 1 Observations LD 5 value Males: 389 mg/kg bw ( mg/kg bw) Females: 465 mg/kg bw ( mg/kg bw) Combined: 425 mg/kg bw (38475 mg/kg bw) palpebral closure, decreased activity, ataxia and tremor; all animals normal by day 3; bw development and post mortem examinations revealed no effect of treatment 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 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 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 palpebral closure, decreased activity, ataxia and tremor lethargy and respiratory impairment (tachypnoe, bradypnoe, dyspnoe or hyperpnoe); post mortem examinations revealed no effect of treatment RCC project no page 4 of 4

9 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 May 25 Section A6.9 Neurotoxicity studies Acute study in rats Acute oral neurotoxicity study in rats 1 REFERENCE 1.1 Reference Sheets, L. P. (2); Original: An acute oral neurotoxicity study with technical grade TI435 in Fischer 344 rats, Supplemental: An acute oral dose rangefinding study with technical grade TI435 in Fischer 344 rats. Bayer CropScience LP, Toxicology, Kansas , USA unpublished report number 1896 Supplement Submission #2, , original : 97912OH, Supplemental : 97912OE 1.2 Data protection Yes Data owner Sumitomo Chemical Takeda Agro Co. Ltd Companies with letter of access Criteria for data protection None Data submitted on existing a.s. for its first entry into Annex I Official use only 2 GUIDELINES AND QUALITY ASSURANCE 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 guidelinesconform acute neurotoxicity study. 2.2 GLP Yes 2.3 Deviations No 3.1 Test material TI Lot/Batch number Specification As given in section Description Pale yellow powder Purity 3 MATERIALS AND METHODS Stability Considered stable under conditions of this study (stability, homogenicity and content of testarticle/vehicle formulations was analysed) 3.2 Test Animals 5 male and 5 female fasted 9 week old Fischer 344 rats per group (CDF[F344]/BR strain, SASCO Inc., Michigan, USA) 3.3 Administration/ Exposure Postexposure period Oral 4 days Type Gavage Concentration 25, 5 and 1 mg/kg bw Vehicle aqueous.5% methylcellulose/.4% Tween 8 in deionized water Concentration in vehicle Total volume applied Prepared according to individual bw and dosing volume RCC project no page 1 of 4

10 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 May 25 Section A6.9 Neurotoxicity studies Acute study in rats Acute oral neurotoxicity study in rats Controls 3.4 Examinations Detailed physical examination for clinical signs of toxicity, mortality:.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 3.6 Further remarks None 4 RESULTS AND DISCUSSION 4.1 Body weight The surviving males at 1 mg/kg bw lost weight until day 4 of the study. At 5 mg/kg bw the surviving rats had a lower body weight development than at 25 mg/kg bw. 4.2 Clinical signs At 5 mg/kg bw 1/5 females, at 1 mg/kg bw all females and 2/5 males died. Treatmentrelated clinical signs like tremors, decreased activity, locomotor incoordination, nasal discharge, lacrimation as well as nasal and oral stains were seen at 5 mg/kg bw. The sings occurred within 3 hours after treatment and were partly reversible after 72 hours. The Animals that died were cooltotouch and gasping prior to death. 4.3 Pathology 5.1 Materials and methods 5 APPLICANT'S SUMMARY AND CONCLUSION 5.3 Conclusion The time of peak effect was 4 hours after application. Dose levels selected for the main study (acute neurotoxicity study) were 1, 2 and 4 mg/kg bw NO(A)EL 25 mg/kg bw (for clinical signs and mortality) Reliability Deficiencies No (study fulfilled its purpose as a range finding study) RCC project no page 2 of 4

11 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 May 25 Date 2566 Materials and Methods Evaluation by Competent Authorities Use separate "evaluation boxes" to provide transparency as to the comments and views submitted EVALUATION BY RAPPORTEUR MEMBER STATE 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 rangefinding 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 TI435 were 29, 523 and 1216 mg/ kg bw for males and females In addition an acute oral study was conducted at a limit dose of 2 mg/kg bw with young adult Fischer344 rats (5 males and 5 females) receiving a single dose by gavage according to the methods described above. Results and discussion Conclusion Reliability Acceptability Remarks Date Materials and Methods Results and discussion Conclusion Reliability Acceptability Remarks 1216 mg/kg bw < LD 5 (M) <2 mg/kg bw 523 mg/kg bw < LD 5 (F) <1216 mg/kg bw. All animals treated with 2 mg/kg bw died on day 1 or 2 after treatment Classification as harmful if swallowed (R22) is required for TI435 according to Directive 21/59/EC (adaptation of 67/548/EEC). COMMENTS FROM... Give date of comments submitted Discuss additional relevant discrepancies referring to the (sub)heading numbers and to applicant's summary and conclusion. RCC project no page 3 of 4

12 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 May 25 Table A6_9/41: Mortality [g] day 1 day 2 day 4 Cumulative bw gain [g] a Table for Acute Oral (Neuro)toxicity (rat) Males Females mg/kg bw /5 /5 /5 /5 /5 /5 day 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 Cooltotouch Gasping Moribund /5 /5 /5 /5 /5 /5 /5 /5 /5 /5 /5 5/5 5/5 /5 4/5 5/5 /5 /5 /5 /5 /5 /5 a : Terminal body weight on day 4 for animals that survived to study termination ot when animals were found dead. /5 1/5 1/4 5/5 5/5 1/5 5/5 5/5 1/5 1/5 /5 3/5 /5 /5 /5 /5 /5 /5 /5 /5 /5 /5 /5 /5 /5 /5 /5 /5 1/5 /4 /4 3/5 5/5 3/5 1/5 3/5 /5 3/5 2/5 3/5 /5 3/5 4/5 1/1 5/5 5/5 3/5 2/5 3/5 2/5 3/5 /5 3/5 1/5 3/5 ANNEX Evaluation by Rapporteur Member State, CATable CATable: Table for Acute Oral Toxicity at 2 mg/kg bw Dose [mg/kg bw] Number of dead / number of investigated 2 5/5 females 5/5 males Time of death (range) Day 12 Day 12 Observations Not determined LD 5 value Males: 1216 mg/kg bw < LD 5 < 2 mg/kg bw Females: 523 mg/kg bw) < LD 5 < 1216 mg/kg bw RCC project no page 4 of 4

13 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.2 Annex Point IIA VI.6.1.2/1 Acute Toxicity Dermal Acute dermal toxicity study in rats (Limit Test) 1 REFERENCE 1.1 Reference Gardner, J. R. (1997c); TI435: Acute dermal toxicity study in the rat; Covance Laboratories, Harrogate, UK; unpublished report number 586/ Data protection Yes Data owner Sumitomo Chemical Takeda Agro Co. Ltd Companies with letter of access Criteria for data protection None Data submitted on existing a.s. for its first entry into Annex I Official use only 2 GUIDELINES AND QUALITY ASSURANCE 2.1 Guideline study Yes 92/69/EEC (method B3), EPA FIFRA, Subdivision F, Section 812 (1984), Japan MAFF (59 NohSan No. 42, 1985), OECD No. 42 (1987) 2.2 GLP Yes 2.3 Deviations None 3.1 Test material TI Lot/Batch number Specification As given in section Description Pale, yellow powder Purity 3 MATERIALS AND METHODS Stability Considered stable under conditions of this study (testarticlevehicle formulations prepared at the day of dosing) 3.2 Test Animals 5 male and 5 female, singlycaged rats (Crl:CD.BR strain, 6 11 weeks old, weight range g, supplied by Charles River UK Ltd) No control group 3.3 Administration/ Exposure Postexposure period 14 days RCC project no page 1 of 3

14 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.2 Annex Point IIA VI.6.1.2/1 Acute Toxicity Dermal Acute dermal toxicity study in rats (Limit Test) Dermal Area covered 1% of body surface (25 cm 2 ) Occlusion Semiocclusive Vehicle Distilled water Concentration in vehicle Total volume applied Duration of exposure Removal of test substance Controls TI435 (dry powder) wetted with.2 ml distilled water See (dose level TI435: 2 mg/kg bw) 24 h Swabbed with moist cotton wool 4 RESULTS AND DISCUSSION 4.1 Clinical signs No animals died at the limit dose level (2 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 2week observation period. 4.2 Pathology No gross lesions were apparent during post mortem examination of the animals. 4.3 Other 4.4 LD 5 No lethal effect at maximal dose dermal LD 5 (males, females, males + females) >2 mg/kg bw 5.1 Materials and methods 5 APPLICANT'S SUMMARY AND CONCLUSION 5.2 Results and discussion Dermal LD 5 in rats (males, females, males + females) >2 mg/kg bw no mortality at limit dose 5.3 Conclusion No classification required for TI435 according to Directive 21/59/EC (adaptation to 67/548/EEC) Reliability Deficiencies No RCC project no page 2 of 3

15 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.2 Annex Point IIA VI.6.1.2/1 Acute Toxicity Dermal Acute dermal toxicity study in rats (Limit Test) Date 2417 Evaluation by Competent Authorities Use separate "evaluation boxes" to provide transparency as to the comments and views submitted EVALUATION BY RAPPORTEUR MEMBER STATE Materials and Methods Acceptable, no deviations from OECD guideline 42 Results and discussion Conclusion Reliability Acceptability Remarks Acute dermal LD 5 in rats is >2 mg/kg bw for both sexes. No animal died at the limit dose level of 2 mg/kg bw. Nor were there clinical signs in reaction to the treatment neither pathological findings during post mortem examination of the animals. No classification is needed for TI435 according to Directive 21/59/EC (adaptation to 67/548/EEC). Date Materials and Methods Results and discussion Conclusion Reliability Acceptability Remarks COMMENTS FROM... Give date of comments submitted Discuss additional relevant discrepancies referring to the (sub)heading numbers and to applicant's summary and conclusion. RCC project no page 3 of 3

16 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.3 Annex Point IIA VI.6.1.3/1 Acute Toxicity Inhalation Acute inhalation toxicity study in rats (Limit Test) 1 REFERENCE 1.1 Reference Shepherd, N. M. (1998); TI435: Single dose inhalation (headonly) toxicity study in the rat; Covance Laboratories, Harrogate, UK; unpublished report number 586/129D6154, Data protection Yes Data owner Sumitomo Chemical Takeda Agro Co. Ltd Companies with letter of access Criteria for data protection None Data submitted on existing a.s. for its first entry into Annex I. Official use only 2 GUIDELINES AND QUALITY ASSURANCE 2.1 Guideline study Yes 92/69/EEC (method B2), EPA FIFRA, Subdivision F, Section 813 (1982), OPPTS (1996), Japan MAFF (59 NohSan No. 42, 1985), OECD No. 43 (1981) 2.2 GLP Yes 2.3 Deviations No 3.1 Test material TI Lot/Batch number 33478b Specification As given in section Description Pale, yellow powder Purity 3 MATERIALS AND METHODS 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 g, supplied by Charles River UK Ltd) 3.3 Administration/ Exposure Postexposure period Control group included Inhalation 14 days RCC project no page 1 of 4

17 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.3 Annex Point IIA VI.6.1.3/1 Acute Toxicity Inhalation Acute inhalation toxicity study in rats (Limit Test) Inhalation Concentrations Nominal concentration 14 mg/m³ Gravimetrical concentration 6141 mg/m³ (concentration over the totally 4.5 h exposure period was 5538 mg/m 3 including the first half hour where difficulties with the aerosol generation occurred) Particle size MMAD (mass median aerodynamic diameter) 2.78 µm + GSD (geometric standard deviation) 2.38 µm Type or preparation of particles 13% particles <1 µm 68% particles <4 µm Type of exposure nose/head only Vehicle Concentration in vehicle Duration of exposure Controls sham exposition 4 h (due to technical difficulties with the aerosol generation in the first.5 h of exposure, the exposure period was prolonged to totally 4.5 hours to guarantee a 4 h exposure at the target concentration) 4 RESULTS AND DISCUSSION 4.1 Clinical signs No deaths occurred in either experimental group. Treatmentrelated clinical signs comprised ataxia and semiclosed 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 treatmentrelated transient weight loss occurred in all test group animals on day 2 (see table A6_13/1). Thereafter all treated animals gained weight at a comparable rate to the controls. 4.2 Pathology There were no treatmentrelated gross or microscopic lesions and the lung weights of test group animals were unaffected by exposure to TI Other 4.4 LC 5 LC 5 (male, females, males + females) >6141 mg/m 3 No lethal effect at the limit dose RCC project no page 2 of 4

18 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.3 Annex Point IIA VI.6.1.3/1 Acute Toxicity Inhalation Acute inhalation toxicity study in rats (Limit Test) 5.1 Materials and methods 5 APPLICANT'S SUMMARY AND CONCLUSION 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 5.2 Results and discussion OECD 43) LC 5 of TI435 in rats (male, females, males + females) >6141 mg/m 3 No lethal effect at limit dose 5.3 Conclusion No classification required for TI435 according to Directive 21/59/EC (adaptation to 67/548/EEC) Reliability Deficiencies No Date 2417 Materials and Methods Results and discussion Conclusion Reliability 1 Acceptability Remarks Evaluation by Competent Authorities Use separate "evaluation boxes" to provide transparency as to the comments and views submitted EVALUATION BY RAPPORTEUR MEMBER STATE Apart from minor deviations as regards contents the study complies with the requirements of OECD guideline 43. 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. Acute inhalation LC 5 in rats is >6141 mg/m 3 air for both sexes. No animal died at the limit concentration of 6141 mg/m 3 air. Treatmentrelated clinical signs comprised ataxia and semiclosed 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 treatmentrelated transient weight loss occurred in all test group animals on day 2 (see table A6_13/1). Thereafter all treated animals gained weight at a comparable rate to the controls. There were no treatmentrelated gross or microscopic lesions apparent during post mortem examination of the animals. No classification required for TI435 according to Directive 21/59/EC (adaptation to 67/548/EEC) Acceptable None RCC project no page 3 of 4

19 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.3 Annex Point IIA VI.6.1.3/1 Acute Toxicity Inhalation Acute inhalation toxicity study in rats (Limit Test) Date Materials and Methods Results and discussion Conclusion Reliability Acceptability Remarks COMMENTS FROM... Give date of comments submitted Discuss additional relevant discrepancies referring to the (sub)heading numbers and to applicant's summary and conclusion. Table A6_13/1. Table for Acute Inhalation Toxicity: body weights [g] Dose [mg/ m 3 air] Day 1 (predose) Males 1 (postdose) (predose) Females 1 (postdose) RCC project no page 4 of 4

20 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.4 Annex Point IIA VI.6.1.4/1 Acute Toxicity Skin irritation Acute skin irritation study in rabbits 1 REFERENCE 1.1 Reference Gardner, J. R. (1997d); TI435: Skin irritation study in the rabbit; Covance Laboratories, Harrogate, UK; unpublished report number 586/124132, Data protection Yes Data owner Sumitomo Chemical Takeda Agro Co. Ltd Companies with letter of access Criteria for data protection None Data submitted on existing a.s. for its first entry into Annex I. Official use only 2 GUIDELINES AND QUALITY ASSURANCE 2.1 Guideline study Yes 92/69/EEC (method B4), EPA FIFRA, Subdivision F, Section 815 (1982), Japan MAFF (59 NohSan No. 42, 1985), OECD No. 44 (1992) 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 EPAFIFRA and JMAFF 3.1 Test material TI Lot/Batch number Specification As given in section Description Pale, yellow powder Purity 3 MATERIALS AND METHODS Stability Considered stable under conditions of this study 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 3.3 Administration/ Exposure Application Preparation of test substance Test site and Preparation of Test Site Dermal Occlusion Semiocclusive Test substance was used as delivered; the application site was moistened with.1 ml distilled water immediately before application of 5 mg TI435 (powder). Dorsal area of the trunk (6 cm 2 ) Shaved skin (clipped) RCC project no page 1 of 4

21 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.4 Annex Point IIA VI.6.1.4/1 Acute Toxicity Skin irritation Acute skin irritation study in rabbits Vehicle Distilled water Concentration in vehicle Total volume applied Removal of test substance Duration of exposure Postexposure period Controls 3.4 Examinations See See Swabbed with moist cotton wool 4 h 3 days Clinical signs Yes Dermal examination Yes scoring system Erythema and Eschar: no erythema very slight erythema (barely perceptible) 1 welldefined erythema 2 moderate to severe erythema 3 severe erythema (beet redness) or eschar preventing reading of erythema 4 Oedema: no oedema very slight oedema (barely perceptible) 1 slight oedema (edges of area welldefined by definite raising) 2 moderate oedema (edges raised approximately 1 mm) 3 severe oedema (raised >1 mm and expanding beyond area of exposure) Examination time points Other examinations 3.5 Further remarks 4.1 Average score 6 min, 24 h, 48 h, 72 h 4 RESULTS AND DISCUSSION Erythema There were no skin reactions in any rabbit at any of the observation points 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 TI435 did not induce any skin reaction at all after dermal application for 4 hours. RCC project no page 2 of 4

22 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.4 Annex Point IIA VI.6.1.4/1 Table A6_14S/1. Acute Toxicity Skin irritation Acute skin irritation study in rabbits Table for skin irritation study score (average animals investigated) time Erythema Edema average score Draize scores ( to maximum 4) 6 min 24 h 48 h 72 h other times State time average score 24h, 48h, 72h reversibility: * average time for reversibility * c : completely reversible n c : not completely reversible n : not reversible 5.1 Materials and methods 5.2 Results and discussion 5 APPLICANT'S SUMMARY AND CONCLUSION Evaluation of skin reactions in rabbits after 4 hour dermal exposure; no relevant deviation from guidelines (92/69/EEC B4, EPA FIFRA 815, Japan MAFF, OECD 44) TI435 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 TI435 according to Directive 21/59/EC (adaptation of 67/548/EEC) Reliability Deficiencies No Date 2417 Evaluation by Competent Authorities Use separate "evaluation boxes" to provide transparency as to the comments and views submitted EVALUATION BY RAPPORTEUR MEMBER STATE Materials and Methods Acceptable, no deviations from OECD guideline 44 Results and discussion Conclusion Reliability 1 Acceptability Remarks There were neither erythema or edema nor any skin reaction in any rabbit during the study. TI435 is not irritating to the skin. No classification for skin irritation is required for TI435 according to Directive 21/59/EC (adaptation of 67/548/EEC). RCC project no page 3 of 4

23 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.4 Annex Point IIA VI.6.1.4/1 Acute Toxicity Skin irritation Acute skin irritation study in rabbits Date Materials and Methods Results and discussion Conclusion Reliability Acceptability Remarks COMMENTS FROM... Give date of comments submitted Discuss additional relevant discrepancies referring to the (sub)heading numbers and to applicant's summary and conclusion. RCC project no page 4 of 4

24 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section Annex Point IIA VI.6.1.4/2 Acute Toxicity Eye Irritation Acute eye irritation study in rabbits 1 REFERENCE 1.1 Reference Gardner, J. R. (1997e); TI435: Eye irritation study in the rabbit; Covance Laboratories, Harrogate, UK; unpublished report number 586/123132, Data protection Yes Data owner Sumitomo Chemical Takeda Agro Co. Ltd Companies with letter of access Criteria for data protection None Data submitted on existing a.s. for its first entry into Annex I. Official use only 2 GUIDELINES AND QUALITY ASSURANCE 2.1 Guideline study Yes 92/69/EEC (method B5), EPA FIFRA, Subdivision F, Section 814 (1982), Japan MAFF (59 NohSan No. 42, 1985), OECD No. 45 (1987) 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 EPAFIFRA and JMAFF 3.1 Test material TI Lot/Batch number Specification As given in section Description Pale, yellow powder Purity 3 MATERIALS AND METHODS Stability Considered stable under conditions of this study 3.2 Test Animals Six male New Zealand White rabbits (Crl:NZW/Kbl.BR strain, 11 to 14 weeks old, body weight 2.1 to 2.47 kg, supplied by Charles River UK Ltd) No control group 3.3 Administration/ Exposure Preparation of test substance Amount of active substance instilled Test substance was used as delivered 66 mg corresponding to.1ml Exposure period The eyes remained unwashed Postexposure period 3 days RCC project no page 1 of 5

25 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section Annex Point IIA VI.6.1.4/2 Acute Toxicity Eye Irritation Acute eye irritation study in rabbits 3.4 Examinations Ophthalmoscopic examination yes Scoring system Cornea (degree of opacity: area most dense assessed): no ulceration or opacity 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 <5% of corneal area 2 area of opacity amounts to 5 <75% of corneal area 3 area of opacity amounts to 75 or more of corneal area 4 Iris: no reaction 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 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 any swelling above normal 1 obvious swelling with partial eversion of lids 2 swelling with lids about halfclosed 3 swelling with lids more than halfclosed Examination time points, 3 min, 6 min, 4 h, 24 h, 48 h, 72 h Other investigations Initial 'sting response' (immediately after instillation): no response 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 Further remarks As requested by the USEPAguideline, conjunctival discharge was also assessed. RCC project no page 2 of 5

26 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section Annex Point IIA VI.6.1.4/2 Acute Toxicity Eye Irritation Acute eye irritation study in rabbits 4.1 Clinical signs No effects 4.2 Average score (2472 hours) Cornea Iris Conjunctiva Redness Chemosis 4 RESULTS AND DISCUSSION 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 3 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.5 hours after treatment. TI435 is considered to be practically nonirritant to the eye. 5.1 Materials and methods 5.2 Results and discussion 5 APPLICANT'S SUMMARY AND CONCLUSION Evaluation of eye reactions in rabbits after instillation into the eye; no relevant deviation from guidelines (92/69/EEC B5, EPA FIFRA 814, Japan MAFF, OECD 45) Cornea and iris were unaffected by treatment. Slight conjunctival redness was recorded up to 4 hours and slight chemosis up to.5 hours after treatment. 5.3 Conclusion No classification for eye irritation is considered required for TI435 according to Directive 21/59/EC (adaptation of 67/548/EEC) Reliability Deficiencies No RCC project no page 3 of 5

27 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section Annex Point IIA VI.6.1.4/2 Acute Toxicity Eye Irritation Acute eye irritation study in rabbits Date 2417 Evaluation by Competent Authorities Use separate "evaluation boxes" to provide transparency as to the comments and views submitted EVALUATION BY RAPPORTEUR MEMBER STATE Materials and Methods Acceptable, no relevant deviations from OECD guideline 45 Results and discussion Conclusion Reliability 1 Acceptability Remarks Cornea and iris were unaffected by treatment. Slight conjunctival redness was recorded up to 4 hours and slight chemosis up to.5 hours after treatment. Under the terms of Directive 21/59/EC (adaptation of 67/548/EEC) TI435 is not irritating to the eye. No classification for eye irritation is required for TI435 according to Directive 21/59/EC (adaptation of 67/548/EEC). Acceptable None Date Materials and Methods Results and discussion Conclusion Reliability Acceptability Remarks COMMENTS FROM... Give date of comments submitted Discuss additional relevant discrepancies referring to the (sub)heading numbers and to applicant's summary and conclusion. RCC project no page 4 of 5

28 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section Annex Point IIA VI.6.1.4/2 Table A6_1_4E/2. Acute Toxicity Eye Irritation Acute eye irritation study in rabbits Appendix Results of eye irritation study Cornea Iris Conjunctiva redness chemosis score (average of animals investigated) to 4 to 2 to 3 to4 6 min h 48 h 72 h Average 24h, 48h, 72h Area effected Maximum average score (including area affected, max 11) 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 RCC project no page 5 of 5

29 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.5 Acute Toxicity Skin sensitisation Annex Point IIA VI.6.1.5/1 Guinea pig maximisation test (GPMT) 1 REFERENCE Official use only 1.1 Reference Denton, S. M. (1997); TI435: Skin sensitisation study in the guinea pig; Covance Laboratories, Harrogate, UK; unpublished report number 586/125132, Data protection Yes Data owner Sumitomo Chemical Takeda Agro Co. Ltd Companies with letter of access Criteria for data protection None 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 B6), EPA FIFRA, Subdivision F, Section 816 (1982), Japan MAFF (59 NohSan No. 42, 1985), OECD No. 46 (1992) 2.2 GLP Yes 2.3 Deviations No 3.1 Test material TI Lot/Batch number Specification As given in section Description Pale, yellow powder Purity 3 MATERIALS AND METHODS Stability Considered stable under conditions of this study (testarticlevehicle formulations prepared at the day of application) Preparation of test substance for application Pretest performed on irritant effects for induction: intradermal: 1% w/v TI435 in Alembicol D topical: 55% w/w TI435 in Alembicol D for challenge: 1% and 2% w/w TI435 in Alembicol D Yes Intradermal application: one injection with,.1, 1, 2.5, 5, 7.5 and 1% w/v TI435 in Alembicol D Topical application for induction (exposure 48 h): 1, 25, 4 and 55% w/w TI435 in Alembicol D Topical application for challenge (exposure 24 h): 5, 1, 2 and 3% w/w TI435 in Alembicol D RCC project no page 1 of 5

30 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.5 Annex Point IIA VI.6.1.5/1 Acute Toxicity Skin sensitisation Guinea pig maximisation test (GPMT) 3.2 Test Animals 2 female Guinea pigs per treated and negative control group (Dunkin Hartley strain, 5 7 weeks old, body weight g, supplied by D. Hall Ltd., BurtononTrent, UK) concurrent positive control study 1 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 TI435 1 male Guinea pigs were treated with 2mercaptobenzothiazole (MBT) with further 5 males as negative control group (supplied by D. Hall, Newchurch, UK) 3.3 Administration/ Exposure Adjuvant Induction schedule day 1 (intradermal induction) day 8 (topical induction) day 21 (topical challenge) see table in appendix Way of Induction Intradermal and topical Concentrations used for induction Concentration Freunds Complete Adjuvant (FCA) Challenge schedule Concentrations used for challenge Rechallenge No Occlusive (topical) Intradermal: 1% w/v TI435 (causing mild to moderate irritation) 5% FCA in water (1:1 v/v) 1% and 2% w/w TI435 (usually maximum nonirritant concentration) Scoring schedule 24h, 48h after challenge Removal of the test substance Positive control substance 3.4 Examinations Pilot study Yes 3.5 Further remarks 24 hours after challenge application treated skin areas were washed with arachis oil Concurrent positive control: 2,4dinitrochlorobenzene (DNCB): Intradermal induction:.1% w/v in corn oil (and/or adjuvant) Topical induction:.5% w/v in corn oil Topical challenge:.2% and.5% w/v in corn oil Biannual positive control studies: 2mercaptobenzothiazole Intradermal induction: 2.5% w/w in Alembicol D (and/or adjuvant) Topical induction: 6% w/w in Alembicol D Topical challenge: 15% and 3% w/w in Alembicol D RCC project no page 2 of 5

31 Sumitomo Chemical Takeda Agro Co., Ltd. TI435 February 24 Section A6.1.5 Annex Point IIA VI.6.1.5/1 4.1 Results of pilot studies 4.2 Results of test h after challenge Acute Toxicity Skin sensitisation Guinea pig maximisation test (GPMT) 4 RESULTS AND DISCUSSION Intradermal application: no skin reactions at.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 1% w/v); 1% w/v selected for main part of study Topical induction: slight erythema in 1/2 Guinea pigs at 2555% w/w; no erythema at 1% w/w 55% w/w selected for main part of study Topical challenge: slight erythema in 1/3 Guinea pigs at 3% w/w; no erythema at 52% w/w 1% and 2% selected for main part of study h after challenge 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 TI435 concentration (55% w/w) is an irritant concentration. 4.3 Overall result The study director considered the results being negative for skin sensitisation in 19/2 treated animals and inconclusive for 1/2 treated Guinea pig. Therefore the positive sensitisation incidence was %. 5.1 Materials and methods 5.2 Results and discussion 5 APPLICANT'S SUMMARY AND CONCLUSION 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 816, Japan MAFF, OECD 46) The study director considered the results being negative for skin sensitisation in 19/2 treated animals and inconclusive for 1/2 treated Guinea pig. Therefore the positive sensitisation incidence was %. 5.3 Conclusion No classification for skin sensitisation is required for TI435 according to Directive 21/59/EC (adaptation of 67/548/EEC), as the positive skin reactions were below the threshold for classification (3%) defined in the Directive Reliability Deficiencies No RCC project no page 3 of 5