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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(limited documentation of: males and mating procedure; treatment not through the entire period of gestation, but on days 6-15)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: white powder
- Storage condition of test material: in the dark at ambient temperature

Test animals

Species:
rat
Strain:
other: (CrL: COBS CD (SD) BR strain) SPF
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Manston Road, Margate, Kent
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 170-204 g
- Housing: groups of five in suspended, galvanised metal cages (Bowman R) equipped with solid sides and back, wire mesh front, floor and top
- Diet (ad libitum): Spratt's Laboratory Diet No. 1 (LAD 1)
- Tap water (ad libitum)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 4
- Humidity (%): 59 +/- 7
- Air changes (per hr): 13
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
During preparation of suspensions on the first day of dosing, a check of the specific gravity of the highest, intended concentration (30% w/v) indicated that an excessive amount of air was being trapped in the mixture. This problem had not been encountered in the feasibility study where a smaller volume was prepared. Attempts to disperse the air ultrasonically were unsatisfactory and absence of stability data precluded overnight preparation. Therefore, in order to ensure greater accuracy of formulation, the concentration was halved and the dosage volume doubled for each group, thereby maintaining the same dosage levels.
Each concentration was prepared separately by suspending a weighed amount of the test material in an appropriate amount of 1% aqueous methylcellulose.
The test suspensions were formulated each day, and to ensure homogeneity, suspensions were stirred with a magnetic stirrer during the dosing procedure.
Dosage volumes were calculated for individual animals on Day 6 of pregnancy and adjusted according to bodyweight on Days 10 and 14.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 1.5, 5 and 15 % w/v at 300, 1000 and 3000 mg/kg bw
- Amount of vehicle (if gavage): 2 ml/ 100 g bw
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Proof of pregnancy: The day of mating, as judged by the appearance of sperm in the vaginal smear or by the presence of a vaginal plug was considered as Day 0 of pregnancy.
No further details on mating procedure were given.
Duration of treatment / exposure:
Treatment commenced on Day 6 of pregnancy and continued daily up to and including Day 15 of pregnancy.
Frequency of treatment:
daily
Duration of test:
Animals were killed on day 20 of gestation.
Doses / concentrationsopen allclose all
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
3 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Prior to the main study, the feasibility of administering 3000 mg/kg/day - the highest intended dosage - was investigated in non-pregnant females which were treated for ten consecutive days. No clinical signs of reaction to treatment were observed in this feasibility study and there were no deaths; other parameters, such as overall food intake and mean bodyweight gain, also appeared to be unaffected and no macroscopic changes attributable to treatment were recorded at terminal autopsy.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed initially (=Day 1 of gestation) and on Days 3, 6, 10, 14, 17 and 20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: 20
- Organs examined: On Day 20 of pregnancy the animals were killed by CO2 asphyxiation, dissected and examined for congenital abnormalities and macroscopic pathological changes in maternal organs.

OTHER: All animals that died were weighed and subjected to post mortem examination.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [half per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No data
Statistics:
Statistical analyses were performed routinely on litter data, using the litter as the basic sample unit; non-parametric tests (Jonckheere and Kruskal-Wallisl) were employed for mean values of litter size, pre- and post implantation loss, litter weight, mean pup weight and the incidence of anomalous offspring as these values rarely follow a 'normal' distribution.
Historical control data:
No data.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Pale coloured faeces were observed amongst rats treated at 3000 mg/kg bw/d, the incidence - occurring between Days 8 and 16 of gestation - basically coinciding with the dosing period. No signs of reaction were recorded at either 300 or 1000 mg/kg bw/d.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two deaths occurred, one at 1000 mg/kg bw/d and another at 3000 mg/kg bw/d, but both were consistent with intubation error.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 3000 mg/kg bw/d, a gradual retardation of mean bodyweight gain was apparent during the dosing period. No further retardation occurred after cessation of dosing, but parity with the controls had not been regained by termination. Mean weight gains at 300 and 1000 mg/kg bw/d were comparable to that of the control group throughout the study.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
When surviving animals were killed on Day 20 of gestation, autopsy findings did not reveal any macroscopic changes considered to be attributable to treatment with the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Pregnancy rate, as assessed by the number of pregnant animals, was essentially comparable for all groups. Slight differences between test groups and the control, in respect of mean values for corpora lutea count and preimplantation loss, were neither statistically significant (P>0.05) nor adversely dosage-related. Mean post implantation loss of all treated groups was lower than that of the control group.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
not examined
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL SIGNS and MORTALITY
Pale coloured faeces were observed amongst rats treated at 3000 mg/kg bw/d, the incidence - occurring between Days 8 and 16 of gestation - basically coinciding with the dosing period. No signs of reaction were recorded at either 300 or 1000 mg/kg bw/d. Two deaths occurred, one at 1000 mg/kg bw/d and another at 3000 mg/kg bw/d, but both were consistent with intubation error.

BODY WEIGHT
At 3000 mg/kg bw/d, a gradual retardation of mean bodyweight gain was apparent during the dosing period. No further retardation occurred after cessation of dosing, but parity with the controls had not been regained by termination. Mean weight gains at 300 and 1000 mg/kg bw/d were comparable to that of the control group throughout the study.

PREGNANCY RATE AND PRE-IMPLANTIATION LOSS
Pregnancy rate, as assessed by the number of pregnant animals, was essentially comparable for all groups. Slight differences between test groups and the control, in respect of mean values for corpora lutea count and preimplantation loss, were neither statistically significant (P>0.05) nor adversely dosage-related.

GROSS PATHOLOGY
When surviving animals were killed on Day 20 of gestation, autopsy findings did not reveal any macroscopic changes considered to be attributable to treatment with the test substance.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
3 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean foetal weight of all test groups was similar or superior to the control mean. Intergroup variation in mean values for litter weight reflected differences in mean litter size; there was no indication of any adverse effect of treatment.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no appreciable intergroup difference in mean sex ratio.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
The slightly lower mean values recorded for litter size in all test groups were not significantly different from the control value (P> 0.05) and appeared to be largely a perpetuation of differences in ovulation rate which would have pre-dated commencement of treatment.
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Of the nine foetuses which were recorded as showing malformations, none occurred at 3000 mg/kg bw/d: three were recorded in the control group, five at 300 mg/kg bw/d and one at 1000 mg/kg bw/d. Neither the types of defect seen nor their distribution indicated any association with treatment. The overall incidence of visceral and skeletal anomalies did not indicate any adverse treatment-related pattern. There were no foetuses with extra (14) ribs amongst treated groups and mean values for variant sternebrae did not indicate any adverse treatment-related effects.
Visceral malformations:
effects observed, non-treatment-related
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no instances of total litter loss (total resorption) in any group.

LITTER SIZE AND POST-IMPLANTATION LOSS
Mean post implantation loss of all treated groups was lower than that of the control group. The slightly lower mean values recorded for litter size in all test groups were not significantly different from the control value (P> 0.05) and appeared to be largely a perpetuation of differences in ovulation rate which would have pre-dated commencement of treatment. There was no appreciable intergroup difference in mean sex ratio.

LITTER AND MEAN FOETAL WEIGHT
Mean foetal weight of all test groups was similar or superior to the control mean. Intergroup variation in mean values for litter weight reflected differences in mean litter size; there was no indication of any adverse effect of treatment.

MALFORMATIONS AND ANOMALIES
Of the nine foetuses which were recorded as showing malformations, none occurred at 3000 mg/kg bw/d: three were recorded in the control group, five at 300 mg/kg bw/d and one at 1000 mg/kg bw/d. Neither the types of defect seen nor their distribution indicated any association with treatment. The overall incidence of visceral and skeletal anomalies did not indicate any adverse treatment-related pattern.

SKELETAL VARIANTS
There were no foetuses with extra (14) ribs amongst treated groups and mean values for variant sternebrae did not indicate any adverse treatment-related effects.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
3 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Embryonic and foetal development did not appear to have been adversely affected by treatment of the dam at any of the dosages used in this study.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

The test article was administered at dosages of 0 (Control), 300, 1000 and 3000 mg/kg/day by intra gastric intubation during Days 6 to 15 inclusive of gestation. The animals were killed on Day 20 of gestation, litter values determined and the foetuses examined for

structural malformations and anomalies. Prior to the main study, the feasibility of administering 3000 mg/kg/day - the highest intended dosage - was investigated in non-pregnant females which were treated for ten consecutive days. No clinical signs of reaction to treatment were observed in the feasibility study and there were no deaths; other parameters, such as overall food intake and mean bodyweight gain, also appeared to be unaffected and no macroscopic changes attributable to treatment were recorded at terminal autopsy. In the main study, pale coloured faeces were observed amongst rats treated at 3000 mg/kg/day, the incidence - occurring between Days 8 and 16 of gestation - basically coinciding with the dosing period. No signs of reaction were recorded at either 300 or

1000 mg/kg/day. Two deaths occurred, one at 1000 mg/kg/day and another at 3000 mg/kg/day, but both were consistent with intubation error. Mean bodyweight gain of dams treated at 3000 mg/kg/day was slightly retarded during the dosing period and parity with that of the control group had not been regained by Day 20. Mean weight gain at 300 and 1000 mg/kg/day remained essentially similar to that of the control group throughout. No macroscopic changes were observed at terminal autopsy of treated animals which were considered to be attributable to the test article. There were no instances of total litter loss (total resorption) at any dosage, nor any significant effects on litter parameters, as assessed by mean values for pre- and post implantation loss, litter size, sex ratio, litter weight and foetal weight. Embryonic and foetal development, as assessed by the overall incidence of malformations, anomalies and skeletal variants did not appear to have been adversely affected by treatment of the dam at any of the dosages used in this study. It was concluded that under the conditions of this study, the test item showed no evidence of teratogenicity or of significant effects on embryo-foetal development.