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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:
12 March - 11 April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
27 June 2018
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
clear liquid, colourless to yellowish
Stored at RT, protected from light and humidity
Expiry date: 6 February 2020
Specific details on test material used for the study:
already provided

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Species and strain: Hannover Wistar rats (CRL:WI(Han))
Age of animals: Young adult female rats, nulliparous and non-pregnant, at least 11 weeks old at mating
Starting body weight: 189-230 g (the variation did not exceed ± 20% of the mean weight)
Acclimation period: at least 12 days
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.7-24.6°C (target: 22 ± 3°C)
Relative humidity: 33-56% (target: 30-70%)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Successfully mated animals were housed individually.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
A constant volume of 5 mL/kg body weight was administered to all dose groups, including the controls. The individual volume of the treatment was based on the most recent individual body weight of the animals.
The control or test item dose formulations were administered to mated, sperm positive (assumed pregnant) female rats daily by oral gavage at approximately the same time each day, from gestation day 6 (GD 6) to gestation
day 19 (GD 19).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for test item concentration and homogeneity determination of the dosing formulations were collected four times during the treatment period. Formulation stability was established before this study. The test item concentration was analysed at the Test Site using a HPLC-UV method. The mean concentration of all measured formulations were found to be in the range of 91.8-95.6% of their nominal concentrations (20, 60 and 200 mg/mL) and were found to be homogenous.
Details on mating procedure:
The oestrus cycle of female animals was examined shortly before start of pairing. After acclimation, the females were paired according to their oestrus cycle with males in the morning for approximately 2-3 hours (1 male : 1 female) until at least 24 sperm positive females/group were attained. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (gestation day 0, GD 0). Sperm positive females were separated and caged individually.
Duration of treatment / exposure:
Days 6 - 19 of gestation
Frequency of treatment:
Once daily during exposure period
Duration of test:
Until Day 20 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
98 female animals, 25-26 mated female animals/group, 4 groups (one control and 3 test item-treated groups)
Control animals:
yes, concurrent vehicle
Details on study design:
The sperm-positive, assumed pregnant females were allocated to the experimental groups (on each mating day) in such a way that the group averages of the body weight were as similar as possible. A computer software (SPSS PC+4.0) was used to verify homogeneity/variation among/within groups.

Examinations

Maternal examinations:
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). Detailed clinical observations were made on all animals at the onset of treatment (GD 6) then weekly.
Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentas were examined macroscopically.
Ovaries and uterine content:
The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses.
The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes.
The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted and the number and percentage of pre- and postimplantation losses were calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.
Fetal examinations:
Each foetus was weighed individually (accuracy ±0.01 g) and subjected to external examination, plus an additional examination of the great arteries. The anogenital distance of each foetus was measured. The sex of the fetuses was assigned based on the external observations and was reconfirmed by examining the internal sex organs. All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the foetal examinations were recorded; photographic records were made additionally.
Statistics:
The statistical evaluation of data was performed with the program package SAS v9.2 in case of Provantis v.9, or SPSS PC+4.0 (SPSS Hungary Kft, Budapest) in the case of data tabulated in Excel, by an appropriate statistical method.
Indices:
No information available.
Historical control data:
HC data available

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Transient signs of piloerection in some Mid and High dose animals, slightly noisy respiration in some High dose animals, and hunched back, red coloured faeces and reduced faeces for a two-days period in one
High dose animals were considered to be treatment related but not adverse.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No test item related mortality was observed in the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item related effect on body weight was observed in the test item related groups when compared to the controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item related effect on food consumption was observed in the test item related groups when compared to the controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no treatment-related effects.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no findings note at necropsy that were considered to be related to the treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No test item related effect on the weight of the thyroid gland (with parathyroid glands) was observed in the test item related groups when compared to the controls.
No test item-related changes were observed in the thyroid hormone levels of the dams.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
The early and the late embryonic loss values of the test item treated groups, and the number of dead foetuses were comparable with the controls. There was no statistically significant difference in the postimplantation loss or
total intrauterine mortality between the treated and control groups.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no test item related effect on the intrauterine mortality parameters.

Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There was no test item related effect on the intrauterine mortality parameters.

Early or late resorptions:
no effects observed
Description (incidence and severity):
The early and the late embryonic loss values of the test item treated groups, were comparable with the controls.
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of dead foetuses were comparable with the controls.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The number of confirmed pregnant, evaluated dams was 24 in the Control and the Mid dose groups, and 25 in the Low and High dose groups.
Other effects:
no effects observed
Description (incidence and severity):
The mean foetal weight per litter in the test item treated groups did not differ significantly from the control mean value in any treated group.
No abnormalities were observed on the placentas of any treated or control animals.
Details on maternal toxic effects:
As there were no maternal findings, it was considered that the test item had no maternal toxicity effect at dose levels up to 1000 mg/kg bw/day.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No maternal toxicity observed at the highest dose level

Maternal abnormalities

Key result
Abnormalities:
no effects observed
Description (incidence and severity):
Up to 1000 mg/kg bw/day did not induce maternal toxicity

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean foetal weight per litter values and the number of retarded foetuses (runts) were comparable with the control in all test item treated groups.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The mean number of viable foetuses was comparable with the control mean in all test item treated groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups and no effect on anogenital distance of either sex.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean foetal weight per litter values and the number of retarded foetuses (runts) were comparable with the control in all test item treated groups.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
Foetal malformations observed in the study were considered to be incidental. The malformations showed no dose dependency, and were not regarded as a test item related effect.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There was no test item related effects on external, visceral, or skeletal development of foetuses in the study.
Visceral malformations:
no effects observed
Description (incidence and severity):
There was no test item related effects on external, visceral, or skeletal development of foetuses in the study.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
At dose levels up to 1000 mg/kg bw/day did not induce maternal toxicity, embryotoxicity, foetotoxicity, or teratogenicity.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: At dose levels up to 1000 mg/kg bw/day did not induce foetotoxicity

Fetal abnormalities

Key result
Abnormalities:
no effects observed
Description (incidence and severity):
At the highest dose levels up to 1000 mg/kg bw/day did not induce embryotoxicity or foetotoxicity.

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The test item, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at dose levels up to 1000 mg/kg bw/day did not induce maternal toxicity, embryotoxicity, foetotoxicity, or teratogenicity. There were no malformations or developmental effects attributed to the test item at any dose level.

NOAELmaternal toxicity: 1000 mg/kg bw/day
Based on the lack of any test-item related maternal toxicity effect in any treatment group.
NOAELembryotoxicity: 1000 mg/kg bw/day
Based on the lack of any test-item related intrauterine effect in any treatment group.
NOAELfoetotoxicity: 1000 mg/kg bw/day
Based on the lack of any test-item related foetotoxicity effect in any treatment group.
NOAELteratogenecity: 1000 mg/kg bw/day
Based on the lack of any developmental effects in any treatment group. 
Executive summary:

The teratogenic potential of the test item was evaluated in female Hannover Wistar rats in year 2019 according to OECD 414 and GLP. The test substance was administered daily by oral gavage to pregnant rats on gestation days 6 -19, at dose levels of 100, 300 and 1000 mg/kg bw/day. The dams were sacrificed at gestation day 20 and the foetuses were examined for visceral and skeletal variations and malformations.

Up to dose level of 1000 mg/kg bw/day did not induce maternal toxicity, embryotoxicity, foetotoxicity, or teratogenicity. There were no malformations or developmental effects attributed to the test item at any dose level

The following no-observed-adverse-effect (NOAEL) levels were derived:

 NOAELmaternal toxicity:                  1000 mg/kg bw/day

Based on the lack of any test-item related maternal toxicity effect in any treatment group.

NOAELembryotoxicity:                    1000 mg/kg bw/day

Based on the lack of any test-item related intrauterine effect in any treatment group.

NOAELfoetotoxicity:                               1000 mg/kg bw/day

Based on the lack of any test-item related foetotoxicity effect in any treatment group.

NOAELteratogenecity:                     1000 mg/kg bw/day

Based on the lack of any developmental effects in any treatment group.