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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
Study period:
From confirmation of pregnancy to Day 20 post coitum
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wi-AF/Han SPF
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Institute of Toxicology, E. Merck, Darmstadt, Germany
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 170-191 g
- Fasting period before study: NA
- Housing: Individually housed in Makrolon cages (type III)
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 1-2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 40-80
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light):12/12 (6 a.m. to 6 p.m.)

IN-LIFE DATES: From: 12th April 1988 To: 10th May 1988

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared every 3 to 4 days and stored refrigerated and protected from light.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Stability had been proven for at least 14 days in the chosen vehicle
- Concentration in vehicle: 2, 6 and 20 g/L
- Amount of vehicle (if gavage): 5 mL/Kg
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1 male to 4 females
- Length of cohabitation: Overnight
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Treatment was carried out daily on 10 consecutive days from the 6th to the 15th day post coitum
Frequency of treatment:
Daily on 10 consecutive days from the 6th to the 15th day post coitum
Duration of test:
From day of successful mating (designated Day 0) to sacrifice of all dams on Day 20.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control - oleum arachidis
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In subchronic toxicity studies in rats dose levels of 50 and 125 mg/kg/day had shown clear, dose-dependent, toxici effects (increased TSH and T3 concentrations in serum and morphological alterations as a consequence of thyroid stimulation)
- Rationale for animal assignment: Animals were allocated to groups according to random lists, and the animals within each group were allocated to positions in the rows by random numbers. Than animal sequence at sacrifice and allocation of litters to transverse section were also randomised.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations included behaviour and appearance

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Dams were weighed on Days 0, 3 and 6 post coitum and then daily until the end of the study

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined once per week on Days 6, 10, 15 and 20

WATER CONSUMPTION: Water consumption determined on days 3, 6, 9, 12, 15, 18, 20 by weighing unconsumed water.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Macroscopic examination of organs and detailed examination of ovaries and uterus (containing foetuses) from each animal.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: Litters from one third of females
- Skeletal examinations: Yes: Litters from two thirds of females
- Head examinations: No data
Statistics:
Body weight, food and water consumption:
For each measuring point the values of the treated animals were compared with the controls using Dunnett's multiple t-test (1955). Various nonhomogeneity between the control and dose groups were taken into account by Dunnett's procedure (1964) and, in case of variance nonhomogeneity by Welch's correction of degrees of freedom.
Reproduction and embryotoxic effects data:
Statistical evaluation was carried out by the Pitman randomisation test using the procedure described by Stucky and Vollmar (1976). Any instance of data where normal distribution could be assumed, the procedure for body weight data was followed.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly less weight was gained by those dams treated at 100 mg/kg/day when compared to the controls (not significant).
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was slightly increased during the treatment period at the highest dose level, and further until day 20 (significant from day 12 until day 18).
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
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
The dose levels of 10 and 30 mg/kg/day proved to be non-toxic to pregnant female rats dosed daily over days 6 to 15 of gestation. The dose level of 100 mg/kg/day was shown to be minimally toxic to the dams as demonstrated by a slightly lower body weight gain by these females.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The number of pregnant rats per goup were as follows: Group 1 - 22/25; Group 2 - 24/25; Grop 3 - 24/25; Group 4 - 23/25
Other effects:
no effects observed
Description (incidence and severity):
The distribution of the numbers of corpora lutea in treated groups was similar to that in controls.
Details on maternal toxic effects:
There was no evidence of an effect of treatment on maternal reproductive parameters at any of the dose levels examined.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Remarks on result:
other:
Remarks:
The effect on body weight gain of the dams at 100 mg/kg/day was minimal and not considered adverse.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight of male and female foetuses from Group 4 (100 mg/kg/day) when compared with the controls.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One foetus with hernia umbilicalis from Group 2 (10 mg/kg/day) and one foetus with anasarca, micrognathia, squatty trunk and short extremities from Group 4 (100 mg/kg/day). The malformations are the same as those that occurred spontaneously in the historical control.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One foetus from Group 4 (100 mg/kg/day) had retarded skeletal development, and pelvic bones, humerus, ulna, femur, tibia, fibula and cranial bones were rudimentary. All ribs were kinked with incomplete ossification. The malformations are the same as those that occurred spontaneously in the historical control.
Visceral malformations:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Skeletal variations: The degree of ossification of the extremities was slightly lower in some foetuses of Group 4 (100 mg/kg/day). This finding was regarded as a consequence of maternal toxicity. Also the number of rudimentary lumbar ribs was dose-dependently increased in Groups 3 (30 mg/kg/day) and Group 4 (100 mg/kg/day) in the male and female foetuses. A finding which was similarly attributed to adverse effects on the dams.
Details on embryotoxic / teratogenic effects:
A small but statistically significant reduction in body weight was recorded for foetuses from Group 4 (100 mg/kg/day). Corresponding to these lighter foetal weights in Group 4 was the lower degree of ossification of the sternum and the extremities seen in foetuses from this treatment group. Since a level of maternal toxicity was seen in this treatment group (slightly reduced weight gain), this incidence of reduced ossification was considered to be secondary to the effect on the dams. The dose-dependent increase of rudimentary lumbar ribs in both sexes in Groups 3 and 4 (30 and 100 mg/kg/day) was also attributed to stress in the dams being sufficient to express the developmental instability inherent in the species.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Skeletal variations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: sternum
skeletal: rib
Description (incidence and severity):
In creased incidence of reduced ossification of sternum and extremities in foetuses of Group 4 (100 mg/kg/day) and increased incidence of rudimentary lumbar ribs in males and females of Groups 4 and 3 (100 and 30 mg/kg/day respectively).

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL for maternal toxicity, based on a slight adverse effect on body weight, was determined to be 30 mg/kg/day although the NOAEL for reproductive effects was considered to be >100 mg/kg/day. The NOAEL for foetal toxicity was considered to be 30 mg/kg/day based on the increased incidence of skeletal anomalies in Group 4 foetuses (100 mg/kg/day).
Executive summary:

Groups of female rats were dosed with the test substance at dose levels of 0, 10, 30 and 100 mg/kg/day by oral gavage between days 6 to 15 of gestation. These females were killed on day 20 of gestation and the uterine contents examined in detail to evaluate any potential effects on reproduction and the embryo. Over this treatment regime, the dose levels of 10 and 30 mg/kg/day proved to be non-toxic to the pregnant female rat. However, the dose level of 100 mg/kg/day was shown to be minimally toxic to the dams as demonstrated by a slightly lower body weight gain by these females. There was no evidence of an effect of treatment on maternal reproductive parameters at any of the dose levels examined.

A small but statistically significant reduction in body weight was recorded for foetuses from Group 4 (100 mg/kg/day). Corresponding to these lighter foetal weights in Group 4 was a lower degree of ossification of the sternum and the extremities seen in foetuses from this treatment group. Since a level of maternal toxicity was seen in this treatment group (slightly reduced weight gain), this incidence of reduced ossification was considered to be secondary to this effect on the dams. The dose-dependent increase of rudimentary lumbar ribs in both sexes of foetuses from Groups 3 and 4 (30 and 100 mg/kg/day) was also attributed to stress in the dams being sufficient to express the developmental instability inherent in this species.

Consequently, the NOAEL for both maternal and foetal toxicity in this study was determined to be 30 mg/kg/day.This study was assigned a reliability rating of 1: reliable without restrictions.