Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 April 2003 to 07 June 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
but none that affected the study validity
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
PURITY: 99.92% Butyltrichlorostannane (or monobutyltin trichloride).

The alkyl group distribution of the test substance was determined using a Grignard ethylation (ethylmagnesium bromide) followed by GC-analysis with FID detection. The concentration of the parent compound in feed was determined by GC-MS analysis of the hexane extracts. The homogeneity and stability of the test substance was determined for each group by a one way ANOVA (sample location and time as grouping factor, respectively).

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Species: rats
- Strain: Wistar (Crl:(WI)WU BR)
- Age at study initiation: 13-14 weeks old females in the satellite repro study (approximately 17 weeks old males from the main 13-week study, after 10 weeks of treatment)
- Weight at study initiation: body weights for females in the satellite groups at the start of treatment ranged from 194.2 - 229.6 g (mean 208.5 g)
- Feed and drinking water were provided ad libitum.
- Number of animals: 
Dose-range finding study: 21 males and 21 females; 20 of each sex selected (4/sex/group)
Main study (13-week subchronic study) - 43 males and 43 females (4 dose groups of 10 rats/sex/dose group)
Satellite study (reproduction/developmental toxicity screening): 44 females (4 dose groups of 10 females/dose group)
- Acclimation: Upon arrival animals were put in a quarantine room and were given time to acclimate to the new surroundings. The quarantine and acclimatisation periods between arrival and experimental start date were 9, 13, and 13 days for the dose-range finding study, 13-week study, and satellite repro study, respectively.
- Housing: The animals were housed in one room, in macrolon cages, with sterilised wood shavings as bedding material. 2 (dose-range finding study) or 5 rats (13-week study) were housed per cage, separated by sex. During the premating period, females of the satellite groups were housed 3 or 4 per dose group per cage. During gestation and lactation, the females were housed individually.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30-70 %
- Air changes: 10 air changes per hour
- Photoperiod: 12 hr light cycle.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: plain diet
Details on exposure:
ADMINISTRATION / EXPOSURE:
- Duration of exposure: 14 days or 13 consecutive weeks, dose-range finding study and subchronic study (from which the males were used for mating, after 10 weeks of treatment), respectively; females in the satellite reproductive-developmental screening study were exposed for about 5 weeks.
- Type of exposure: via the diet
- Doses: 0, 300, 1500 and 7500 mg/kg diet for the main subchronic study and satellite (reproductive/developmental) study
Details on mating procedure:
- During the mating period (weeks 10 and 11), one male from the 13-week main study and one female of the satellite group of the same dose group were caged until copulation occurred or two weeks had elapsed.
- Every morning vaginal smears were made to ascertain copulation by detection of sperms.
- The day a smear was sperm-positive was considered gestation day 0.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The analytical method was validated in the matrix under examination, viz. RM3 diet, before the start of the study. The test material and other organotin chlorides used as internal standards were converted into the corresponding ethylated tetraorganotin derivatives, which were extracted into the hexane layer. GC-MS analysis was then performed.
- GC-MS was used to determine the achieved concentration, homogeneous distribution and stability of the test material in diet samples. The method of analysis involved derivatisation. This method only measures the amount of the alkyltin moiety, MBT, present and does not identify the other ligands attached to the tin. All measured MBT was attributed to the parent substance, the test material.
- The homogeneity, stability, and achieved concentration (content) of the test material in RM3 rat feed was analysed in the batch of diets prepared for the dose-range finding study and the 13-week study.
- From these analyses, it was concluded that the test material was homogenously distributed in all diets, the test material was stable in the diets at room temperature for 7 days and in the freezer at <-18 °C for 6 weeks, and the content of the test material was close to the nominal level for all diets.
Duration of treatment / exposure:
Dose-range finding study: 14 days
Males derived from the main subchronic 13-week study were treated for 10 weeks before mating with females from the satellite groups around 17 weeks of age. They then continued treatment to complete the 13-week study.
Females in the satellite study were treated via the diet starting 2 weeks before mating, then during mating (up to 2 weeks), gestation, and up to euthanasia at or shortly after postnatal day 4 (ca. 6 weeks).
Frequency of treatment:
Daily
Details on study schedule:
Duration of test: About 6-8 weeks for females in the satellite study (2-week pre-mating, up to 2 weeks for mating, gestation and up to euthanasia at around PND 4).
Doses / concentrationsopen allclose all
Dose / conc.:
300 mg/kg diet
Dose / conc.:
1 500 mg/kg diet
Dose / conc.:
7 500 mg/kg diet
No. of animals per sex per dose:
Dose-range finding study: 4 rats/sex/dose group; 5 dose groups including controls
13-week main study and satellite study: 10 rats/sex/dose group and 10 females/dose group, respectively; 4 dose groups including controls
Control animals:
yes, plain diet
Details on study design:
During the premating period females were housed 3 or 4 per group per cage. Male rats of the 13 week study were mated after a premating period of 10 weeks with the satellite female rats.  
During the gestation and lactation periods the females were housed individually. If a male died before or during the mating period before the female was found sperm positive, the female was mated with another, proven male of the same group (i.e. a male which already had a successful copulation, sperm positive smear with another female).

Examinations

Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS AND FREQUENCY
- Clinical signs: at least once daily (in the morning) and on working days also once in the afternoon.
- Mortality: at least once daily (in the morning) and on working days also at least once in the afternoon.
- Body weight: once during the acclimatisation period, once at initiation of the study prior to introduction of feed and once weekly thereafter. The females of the satellite groups were weighed 4 days before start of dosing at randomisation, on gestation days 0, 7, 14 and 21 and on postnatal days 1 and 4. Furthermore, all surviving animals were weighed on the day of necropsy in order to determine their correct organ to body weight ratios.
- Food consumption: measured per cage over weekly periods during premating and gestation period and from PN 1-4 by weighing the feeders (in g/animal/day and g/kg bw).
- Water consumption: provided ad libitum, the amount consumed was not measured.
- Intake of test material: the intake of test material per kg bw/day was calculated from the nominal dietary concentration of the test material, the food consumption and the mean body weight in the period for which the intake of the test material is calculated.
- Mating: during the mating period every consecutive morning vaginal smears were made to ascertain copulation by detection of sperm cells in the smear.
Oestrous cyclicity (parental animals):
no
Sperm parameters (parental animals):
no
Litter observations:
- Parturition and litter evaluation: at the end of the gestation period, females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded. To keep nest disturbance to a minimum, the litters were examined only once daily for dead pups.
- Litter size, sexes and weights: the total litter size and numbers of each sex as well as the number of stillbirths, live and dead pups and grossly malformed pups were evaluated on days 1 and 4 of lactation. The pups were weighed individually and litter weight was calculated for days 1 and 4 of lactation. Mean pup weights were calculated as litter weight/number pups. The number of runts (pup weight < 2 sd from the control litter mean) were noted and reported as well.
Postmortem examinations (parental animals):
- Macroscopic: all adult animals were subjected to a complete gross necropsy. Organs weighed ifor satellite repro females included: ovaries, uterus (after counting of the implantation sites), thymus and all gross lesions. Samples of latter organs were preserved for microscopic examination.  
- Microscopic: microscopic examination of the ovaries, uterus and thymus of the control and 7500 mg/kg groups was performed. Examination was extended to the thymus of the females of the 300 and 1500 mg/kg groups because of the effects observed in the 7500 mg/kg group for this tissue/organ. Furthermore, the reproductive organs of the males of the 300 and 1500 mg/kg groups that failed to sire (did not mate or female was not pregnant) and the reproductive organs of females of the 300 and 1500 mg/kg groups that were non-mated or non-pregnant were microscopically examined.
Postmortem examinations (offspring):
Necropsy was performed on stillborn pups and pups dying during the study; macroscopic abnormalities were recorded. Pups were examined externally for gross abnormalities and killed by hypothermia at < -18 °C.
Statistics:
- Test material analysis:
Homogeneity: one way analysis of variance (Anova) using the sample location (1-5) as grouping factor. The test material was considered to be homogeneously distributed in the diets if p > 0.01 and/or if the relative standard deviation (RSD) between the samples means was less than or equal to 15 %.
Stability: one way analysis of variance (Anova) using time as grouping factor. The test material was considered to be stable in the diets if p > 0.01 and/or if the mean concentration on the last day was between 80 and 120 % of the mean concentration on the first day (t =0).
Achieved concentration: for each concentration level, the mean of the concentrations, as measured in the diet samples used for the assessment of the homogeneity, was considered to represent the achieved concentration. The content of the test material in the diet was considered to be 'close to intended' if the mean measured concentration was between 80 and 120 % of the intended concentration.
- Body weight: one way analysis of covariance (covariate: body weight on day 0) followed by Dunnett's multiple comparison tests.
- Food consumption and food efficiency: one way analysis of variance (Anova) followed by L.S.D. tests.
- Fisher's exact probability test was used to evaluate the number of mated and pregnant females and females with live pups. Number of implantation sites, live and dead pups were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U-test.
- Histopathological changes: Fisher's exact probability test.
The litter was used as the statistical unit for calculations of foetal values.
All tests were two-sided. Probability values of p<0.05 were considered significant.
Reproductive indices:
With regard to fertility and reproductive performance, the following parameters were calculated:
- pre-coital time = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- mating index = (number of females mated/number of females placed with males) x 100
- male fertility index = (number of males that became sires/number of males placed with females) x 100
- female fertility index = (number of pregnant females/number of females placed with males) x 100
- female fecundity index = (number of pregnant females/number of females mated) x 100
- gestation index = (number of females with live pups/number of females pregnant) x 100
- live birth index = (number of pups born alive/number of pups born) x 100
Offspring viability indices:
- pup mortality day n = (number of dead pups on day n/total number of  pups on day n) x 100
- viability index day 1-4 = (number of pups surviving 4 days/total number  of live pups on day 1) x 100
- sex ratio day n = (number of live male pups on day n/ number of live pups on day n) x 100
- number of lost implantations = number of implantations sites - number  of pups born alive
- post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100 

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related clinical signs were observed during the premating, mating, gestation and lactation periods.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- Mean body weight of the females of the 7500 mg/kg group was statistically significantly increased on GD 21 and body weight change was also statistically significantly increased from GD 14-21. During the premating, gestation and lactation periods, no other significant differences in mean body weights and body weight changes of the females were observed.
- Mean food consumption of the females (in g/animal/day and g/kg bw/day) of the 7500 mg/kg group was statistically significantly increased from GD 14-21. During the premating, gestation and lactation periods, no other significant differences in mean food consumption of the females were observed.

Please refer to "Any other information on results incl. tables" for further information
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The test material intake of the female animals of the 300, 1500 and 7500 mg/kg groups was respectively:
Premating period
days 0-7: 19.1, 92.2 and 433.4 mg/kg bw/day
days 7-14: 18.7, 91.4 and 476.4 mg/kg bw/day
Gestation period
GD 0-7: 21.7, 101.8 and 526.2 mg/kg bw/day
GD 7-14: 21.0, 96.5 and 544.0 mg/kg bw/day
GD 14-21: 15.2, 77.3 and 494.3 mg/kg bw/day
Lactation period PN 1-4: 25.3, 140.8 and 684.7 mg/kg bw/day
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- Reproductive organs and the thymuses of the control and 7500 mg/kg dose groups were examined microscopically. In addition, reproductive organs of the 300 and 1500 mg/kg groups of non-pregnant females and males that failed to sire were examined.
- All histopathological changes in the testes, epididymides, prostate and in the ovaries, uterus and thymuses represent common findings in rats of this strain and age. Moreover, they occurred only incidentally or at similar incidences amongst the groups, including controls. Therefore, they were not considered to be related to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Analysis of the test material in diet samples revealed that the test material dose was close to the nominal level for all diets.
Homogeneity: The test material was considered to be homogeneously distributed in all diets.
Stability: The test material was considered to be stable in the diets upon storage at room temperature for 7 days and in the freezer at < -18 °C for 6 weeks.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
- Pre-coital time: comparable among the control and treated groups.
- Mating index: 90 - 100 %
- Number pregnant per dose level: 6, 6, 7 and 7 for the control, 300, 1500 and 7500 mg/kg groups, respectively.
- Female fecundity index: comparable among the control and treated groups.
- Female fertility index: comparable among the control and treated groups.
- Male fertility index: comparable among the control and treated groups.
- Number of implantations: 11-13 (control group), 10-14 (300 mg/kg), 11-14 (1500 mg/kg), 11-13 (7500 mg/kg).
- Gestation index: 100 % in the control, 300, 1500 and 7500 mg/kg groups.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
7 500 mg/kg diet
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No significant health effects were observed at the highest dose tested. This dietary dose level is equivalent to 433-685 mg/kg bw/day for females.
Key result
Dose descriptor:
NOAEL
Remarks:
fertility and reproduction
Effect level:
7 500 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean pup weight on PN 1 and 4 of the 7500 mg/kg group was statistically significantly increased. Pup weight change of the pups from the 7500 mg/kg group between PN 1-4 was not statistically significantly increased. No effect on pup weight and pup weight change was observed in the other treated groups when compared to the control group.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
The incidence of runts in the 1500 mg/kg group was statistically significantly increased on PN 1; this observation was only significant on pup basis. No statistically significant difference was observed between the other treated groups and the control group. Macroscopic observation of the stillborn pups revealed no abnormalities of the pups of the 300 and 1500 mg/kg dose groups. The stillborn pup of the 7500 mg/kg group was partly cannibalised; as far as possible no abnormalities were observed in this pup.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Litter size: The mean number of pups delivered per litter amounted to 8.7, 10.8, 11.0 and 11.3 for the control, 300, 1500 and 7500 mg/kg groups, respectively.
- Live birth index: 100, 100, 100 and 96 % in the control, 300, 1500 and 7500 mg/kg groups, respectively.
- Number of females with live born pups: 6, 6, 7 and 7 in the control, 300, 1500 and 7500 mg/kg groups, respectively.
- Number of females with stillborn pups: 0, 0, 0 and 3 for the control, 300, 1500 and 7500 mg/kg groups, respectively.
- Number of females with all stillborn pups: 0 for the control and all treated groups.
- Post implantation loss: 29.1, 11.4, 17.2 and 10.7 % for the control, 300, 1500 and 7500 mg/kg
- Pup mortality: 0, 0, 0 and 3.8 % for the control, 300, 1500 and 7500 mg/kg groups, respectively (PN 1)
- Number viable: The viability index (PN 1-4) was 100 % in the control, 300, 1500 and 7500 mg/kg groups.
- Number of live pups per litter: 8.7, 10.8, 11.0 and 10.9 for the control, 300, 1500 and 7500 mg/kg groups, respectively (PN 1); 8.7, 10.8, 10.1 and 10.9 for the control, 300, 1500 and 7500 mg/kg groups, respectively (PN 4).
- Sex ratio: No difference was observed in the sex ratio between the groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
7 500 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects on pups up to and including lactation day 4

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Among the available studies for the substance investigating pre-natal developmental toxicity in rats, decreased body weight and/or body weight gain in mothers were observed in groups for which toxic effects on mothers were noted. A statistically significant increased mean body weight of females of the high dose group at gestation day (GD) 21, together with statistically significant increases in body weight gain and food consumption for the same group on GD 14-21, was reported in this study and is the only exception.

The above ‘exception’ is apparent due to the greater mean litter size of the high dose females compared to controls.

Of the 10 satellite females per group, 2 control females did not mate and 2 were confirmed as not pregnant. The final number of pregnant females was 6, 6, 7 and 7 in order of ascending dose level. From the table below, it seems that all the 6 females were included in the mean value of the control group whereas 5, 7, and 5 females were included for the treated groups. One female of the low dose and 2 females of the high dose appear missing.

Mean body weights, females (g: satellite groups; gestation period)

 

Group A

control

Group B

300 mg/kg

Group C

1500 mg/kg

Group D

7500 mg/kg

Day 0

213.60 d

220.66

220.80

208.77

Day 7

230.00 d

244.52

237.84

226.83

Day 14

250.55 d

266.98

259.31

249.84

Day 21

283.43 d

300.28

287.74

310.46 *

Statistical key:
d = ANOVA & Dunnett test
* p < 0.05

Individual data reveal that body weight for the missing female of the low dose (No. 131) is not included because it was misjudged to be not mated, while body weights for the 2 missing females of the high dose (No.s 165 and 169) were not recorded at GD 21 because they started delivering in that day. These same females were also not included for the calculation of mean food consumption over GD 14-21.

Individual body weights (g) of rats of the satellite groups (gestation period), Group B: 300 mg/kg

Day of gestation

Female

0

7

14

21

121

213.4

233.7

252.5

285.0

123

217.2

236.6

261.0

282.4

125x  NP

185.6

211.8

212.5

216.1

127

216.7

234.8

262.2

293.0

129x  NP

217.3

241.7

236.5

240.7

131

nm-12

nm-12

nm-12

nm-12

133x  NP

209.9

233.5

230.6

242.8

135

223.0

259.5

274.2

307.0

137x  NP

194.0

217.3

213.8

218.0

139

233.0

258.0

285.0

334.0

Mean

220.66

244.52

266.98

300.28

 

NP: not pregnant
x: excluded from mean
nm-12 : not measured because female was misjudged to be not mated

Individual body weights (g) of rats of the satellite groups (gestation period), Group D: 7500 mg/kg

Day of gestation

Female

0

7

14

21

161

199.2

215.6

235.3

293.4

163x  NP

216.7

219.2

222.7

221.2

165

nm-2

224.4

247.8

nm-12

167x  NP

208.5

233.5

232.8

237.2

169

214.7

241.5

266.4

nm-12

171

204.9

220.4

245.7

314.8

173

207.3

223.1

249.7

304.7

175

208.5

225.7

244.1

311.2

177x  NP

205.7

228.8

232.9

231.3

179

218.0

237.1

259.9

328.2

Mean

208.77

226.83

249.84

310.46

NP: not pregnant
x: excluded from mean
nm-2 : erroneously not measured/ recorded
nm-12 : not measured because female was misjudged to be not mated

In addition to these, although statistical analysis did not show any significant difference between the control and the treated groups, the mean number of pups per litter in the high dose was approximately 30% greater than that of the control group (i.e., 11.29 pups for the high dose vs. 8.67 pups for the control).

Fertility and reproduction

 

Pups delivered (total)

Group A

control

Group B

300 mg/kg

Group C

1500 mg/kg

Group D

7500 mg/kg

Liveborn (N)

52

65

66

79

Liveborn (mean)

8.67

10.83

11.00

11.29

Live Birth Index (%)

100

100

100

96

Stillborn (N)

0

0

0

3

Pup mortality day 1

0.0

0.0

0.0

3.8

 

There were 2 control litters which consisted of only 3 or 4 pups, whereas the lowest number of pups in a litter at the high dose was 9.

Fertility and reproduction (Group A: control)

Litter delivered

Female

Live (N)

Dead (N)

Total (N)

101

11

0

11

103

12

0

12

105  NM

 

 

 

107  NP

 

 

 

109

4

0

4

111

3

0

3

113  NM

 

 

 

115  NP

 

 

 

117

11

0

11

119

11

0

11

NP: not pregnant
NM: not mated

Fertility and reproduction (Group D: 7500 mg/kg)

Litter delivered

Female

Live (N)

Dead (N)

Total (N)

161

11

0

11

163  NP

 

 

 

165 

12

1

13

167   NP 

 

 

 

169

11

1

12

171

11

1

12

173  

10

0

10

175  

9

0

9

177  NP

 

 

 

179

12

0

12

NP: not pregnant

Additionally, group mean body weights for the control and high dose groups on Day 1 of lactation were not statistically different.

Mean body weights, females (g; satellite groups; lactation period)

 

 

Group A

control

Group B

300 mg/kg

Group C

1500 mg/kg

Group D

7500 mg/kg

Day 1

214.27 d

218.28

222.55

229.80

Day 4

230.32 d

236.03

239.74

238.68

d: ANOVA & Dunnett test

It can be concluded that the statistically significant difference in body weight at the end of gestation between the high dose and the control group is real, but has no biological significance other than to reflect litter size and therefore it is not an adverse effect.

Applicant's summary and conclusion

Conclusions:
As no maternal toxicity or reproductive effects in the females of the satellite groups after mating with the male animals of the main 13-wk study were observed at the high-dose level of this study, this dose level 7500 mg/kg diet (equivalent to 521 mg/ kg body weight/day in males and 433-685 mg/kg body weight/day for females) can be considered as a NOAEL for maternal toxicity and fertility and developmental effects.
Executive summary:

The toxicity of the test material in Wistar rats was examined using continuous administration via the diet for 13 consecutive weeks in accordance with OECD 408. In satellite groups of female rats a reproduction/developmental screening test was performed in accordance with OECD 421 to provide initial data on the possible reproductive and developmental effects of the test material. The study was performed under GLP conditions.

The main study used four groups of 10 rats/sex (13-week study) and the satellite study used four groups of 10 female rats (reproduction/developmental screening study). For both studies the control group was kept on untreated diet and three test groups received experimental diets containing 300, 1500 and 7500 mg/kg (ppm) of the test material. The dose levels used in both studies were selected based on the results of a preceding dose range finding study.

In the satellite study female rats were fed their respective diets beginning two weeks prior to the mating period, and continued through mating, gestation and up to PN 4 or shortly thereafter. Male rats from the main study were mated after a premating period of 10 weeks with female rats of the satellite groups which were fed the same dose of test diet.

Clinical observations, growth, food consumption, food conversion efficiency, neurobehavioural testing, ophthalmoscopy, haematology, clinical chemistry, renal concentration test, urinalysis, organ weights and gross examination at necropsy, microscopic examination of various organs and tissues and assessment of various reproductive and developmental parameters were used as

criteria for detecting the effects of treatment.

The calculated doses for the females receiving 300, 1500, or 7500 mg/kg test material in the diet ranged between 15.2 -25.3, 77.3-140.8, and 433.4-684.7 mg/kg body weight/day, depending on study period. None of the few changes observed in clinical signs, body weight, food consumption, fertility and reproductive performance, litter data, organ weights, gross macroscopy and microscopic examination were considered treatment-related.

As no maternal toxicity or reproductive effects in the females of the satellite groups after mating with the male animals of the main 13-wk study were observed at the high-dose level of this study, this dose level 7500 mg/kg diet (equivalent to 521 mg/ kg body weight/day in males and 433-685 mg/kg body weight/day for females) can be considered as a NOAEL for maternal toxicity and fertility and developmental effects.