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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
The author had applied the criteria of 40 CFR 158.34 for flagging studies for potential adverse effects, to the results of the present study.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity >= 99.5%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Age: 8 to 10 weeks
Mean weight: 201 to 225 g

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
The treatment period was followed by a post-exposure period of 5 days
Details on mating procedure:
Follow an acclimatisation period of 10 days, the females were mated within 3 consecutive days. The females were paired with the sexually mature males at a ratio of 2 females:1male. The presence of sperm in the vaginal smear indicated day 0 of pregnancy.
Duration of treatment / exposure:
Day 6 to 15 of pregnancy
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 28, 80 mg/kg bw/day
Basis:

No. of animals per sex per dose:
24 females per test group
Control animals:
yes, concurrent no treatment
Details on study design:
Sex: female
Duration of test: 15 days (day 6 to day 20 of pregnancy)

Examinations

Maternal examinations:
Body weight: Body weights were recorded on day 0, daily from day 6 to day 15 of pregnancy, and finally on day 20.
Food consumption: Food consumption was measured over following intervals: day 0 to day 6, day 6 to day 9, day 9 to day 12, day 12 to day 15 and day 15 to day 20 of pregnancy.
Clinical signs: The animals were checked daily from day 0 to day 20 of pregnancy for clinical symptoms and mortality.
Necropsy: On day 20 of pregnancy, the females were sacrificed for the purpose of necropsy; they were examined for gross abnormalities. Females, which were sacrificed prematurely in extremis, also were subjected to necropsy. Organs or tissues showing abnormalities were fixed in neutral buffered formaldehyde for further examination.
Ovaries and uterine content:
The dams were examined for pregnancy status, gravid uterus weight, number of corpora lutea, and number and distribution of implantation sites. The implantations were classified in early resorptions, late resorptions, dead fetuses and live fetuses; they were further separated in numbers for each horn.
Following indices were calculated:
Pre-implantation loss (%):
(Number of corpora lutea - Total number of implantation sites) x 100/Number of corpora lutea

Post-implantation loss (%):
(Total number of implantation sites - Number of live fetuses) x 100/Total number of implantation sites

Fetal examinations:
General: The live fetuses were weighed and were examined for sex. All fetuses were examined for external abnormalities. The mean fetal body weights were calculated for each litter and sex; group mean body weights were calculated from the litter means.
Soft tissue: About 2/3 of the live fetuses from each litter were placed in 70% alcohol for the purpose of subsequent dissection and examination for visceral abnormalities. The remaining fetuses from each litter were fixed in Bouin´s fluid and were then transferred in 70% alcohol for slight fixation for subsequent sectioning and/or dissection.
Skelet: The carcasses of the 2/3 fetuses from each litter, which were fixed in 70% alcohol and were then dissected and examined for visceral abnormalities, were further processed for skelet examination. For this purpose, the carcasses were cleared in a potassium hydroxide solution, stained with Alizarin red S and preserved in aqueous glycerol with thymol crystals. The bones were identified and examined for shape, size and extent of ossification.
Statistics:
Group means and standard deviations (SD) were calculated where appropriate, and the data were subjected to analysis of variance or to the Kruskal-Wallis test. When significance was achieved (p>95%) and depending on the test method (analysis of variance or Krustal-Wallis test), each treated group was then compared to control using either Dunnett´s test or Dunn´s multiple comparison test.
Maternal body weights, gravid uterus weights and food consumption: These data were subjected to analysis of variance.
Number of corpora lutea, live fetuses and implantation sites: These data were subjected to analysis of variance; the results for each group were compared by means of the Kruskal-Wallis test.
Fetal body weights: Group mean fetal body weights were calculated from the litter means and were compared by analysis of variance.
Sex ratio: The sex ratio was calculated for each litter and the results for each group were compared by means of the Kruskal-Wallis test.
Fetuses with abnormalities: The percentages of fetuses with abnormalities in each litter were calculated, and the group mean percentages, which were calculated from the litter percentages, were compared by means of the Kruskal-Wallis test.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
Mortality and clinical symptoms of toxicity: No mortality was observed and no treatment-related clinical symptoms were reported.
Body weight: At the highest tested dose of 80 mg/kg bw/day, the maternal body weight gain was significantly below that of control animals over days 6 to 7 of pregnancy (1+/- 5 g versus 5 +/- 3 g for control). Thereafter, body weight gain in this group turned back to control level.
Food consumption: Food consumption was similar in all groups and therefore inconspicuous.
Necropsy: Necropsy of the dams revealed no treatment-related abnormalities.
Pregnancy and implantation data:
The number of pregnant females per test group was 24, 22, 24 and 24 for the control, the 10 mg/kg bw, the 28 mg/kg bw and the 80 mg/kg bw group respectively. None of the considered parameters was affected by the treatment.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOAEL
Effect level:
80 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Fetal parameters: None of the considered fetal parameters (sex ratio, fetal weight, gravid uterus weight) was affected by the treatment.
Fetal major abnormalities: A total of 1, 0, 1 and 3 fetuses showing major abnormalities (skeletal and external/visceral combined) was reported for the control, the 10 mg/kg bw, the 28 mg/kg bw and the 80 mg/kg bw group respectively In fact, one case of pulmonary valvular atresia was reported for the control group and one case of microphthalmia was reported for the 28 mg/kg bw group; in the 80 mg/kg bw, two cases of exencephaly (with associated skull abnormalities, open eye, protruding tongue and partial cleft palate) and one case of microphthalmia were reported. No abnormalities were found in the 10 mg/kg bw group. The reported abnormalities were of the type occurring spontaneously in the used rat strain, and the incidences were within background range. The increased incidence seen in the 80 mg/kg bw group was mainly due to one litter containing two fetuses with similar abnormalities (exencephaly with associated skull abnormalities, open eye, protruding tongue and partial cleft palate),and therefore, the finding was considered to rather be genetic than due to treatment.
Fetal minor abnormalities: When compared to control, the incidence of fetuses with incomplete ossification of one or more sacral neural arches was found to be significantly lowed in the 80 mg/kg bw group than in control, indicating a slightly advanced ossification when compared to control. Comparison to background mean revealed that the advance was not unusual. Furthermore, in both the 28 and the 80 mg/kg bw groups, advanced ossification of the forelimb phalanges was seen when compared to control; however, when compared to background mean, the advance was not unusual. The authors concluded that there might have been an association between treatment and advanced ossification of the sacral neural arches and forelimb phalanges; however, the findings still were within background mean range and therefore not unusal.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion