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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:
2019-06-17 to 2019-12-13
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
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest Cserkesz u. 90. Hungary
- Age at study initiation: females: 8 weeks; males: 33-34 weeks
- Weight at study initiation: 169-230 g
- Fasting period before study: no
- Housing: Before mating: 1-3 females per cage 1-2 males per cage; During mating: 1 male and 1-3 females / cage; During gestation: 2 sperm positive females per cage, if not possible 1 sperm positive female per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days (females); 21 weeks (males)

ENVIRONMENTAL CONDITIONS
- Temperature: 19.3 - 23.7 °C
- Humidity: 42-68 %
- Air changes: > 10 per hr
- Photoperiod: 12/12 hrs dark / hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared in the formulation laboratory of the Test Facility not longer than for three days before the administration and stored in the refrigerator.

VEHICLE
- Justification for use and choice of vehicle: The test item was not soluble in water therefore PEG 400 was used for preparing formulations appropriate for oral administration. PEG 400 was a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: The test item was formulated in the vehicle in concentrations of 200 mg/mL, 60 mg/mL and 20 mg/mL.
- Amount of vehicle: final volume applied: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing solutions (control of concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility twice during the study. Five samples from different places were taken from each concentration. Similarly, five samples were taken from the vehicle (Control group) and analyzed. The suitability of the chosen vehicle for the test item at the intended concentrations was verified up front. Test item recovery was 105 and 99 % of nominal concentrations at 1 and 200 mg/mL in PEG 400, respectively. The test item proved to be stable at room temperature for 24 hours (recovery was 110 % of starting concentration at 1 mg/mL and 107 % at 200 mg/mL) and at 5 ± 3°C for 3 days (recovery was 110 % of starting concentration at 1 mg/mL and 107 % at 200 mg/mL).
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male and 1-3 females/cage
- Length of cohabitation: The females were paired to males in the mornings for two to four hours (one male: one to three females) until the number of sperm positive females per group achieved at least twenty-five.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and/or sperm in the vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
from gestational day 5 to 19.
Frequency of treatment:
daily, 7 days per week
Duration of test:
14 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 sperm positive females per dose
Control animals:
yes, concurrent vehicle
yes, historical
Details on study design:
- Dose selection rationale: The dose setting is based on findings obtained in Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with the test item in the Rat. where the NOAEL for the adult animals (including reproductive performance) and offspring was determined as 1000 mg/kg bw/day which is the limit dose according to the OECD 414 test guideline.
- Rationale for animal assignment: random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day, after treatment at approximately the same time, considering the peak period of anticipated effects after dosing
- Cage side observations checked: behavior and general condition, duration and severity of the clinical signs, for signs of morbidity and mortality were made twice daily, at the beginning and end of the working period.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 3, 5, 8, 11, 14, 17 and 20

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Food consumption was measured between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined:
- gross pathology of dams´viscera
- organ weight: Thyroid glands together with the parathyroid glands of all sperm positive females
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- 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: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
Data were individually recorded on data sheets, transferred, and compiled by computer or compiled manually.
The statistical evaluation of data was performed with the program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result is significant Duncan’s Multiple Range test was used to assess the significance of inter-group differences. If the result of the Bartlett’s test was significant, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Dams or litters were excluded from the data evaluation in cases of:
- Non pregnant females (no implantation, no corpora lutea), total exclusion
Although these animals were excluded from the data evaluation the Study Report contains all data of these animals, too.
A male/female fetus was considered as retarded in body weight, when its weight was below the average minus twofold standard deviation of the control male/female fetuses.
Indices:
Pre-implantation loss:
Number of corpora lutea – Number of implantations / Number of corpora lutea * 100

Post-Implanation loss:
Number of Implantations – Number of live fetuses / Number of implantations * 100

Sex distribution:
Number of Male (Female) fetuses / Number of fetuses * 100

External abnormalities/litter:
Number of fetuses with abnormality / Number of fetuses * 100

Visceral abnormalities/litter:
Number of fetuses with abnormality / Number of fetuses examined * 100

Skeletal abnormalities/litter:
Number of fetuses with abnormality / Number of fetuses examined * 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
None of the females died in the course of the study and there were no test item related clinical signs observed in the females of all dose groups. Soft faeces was observed in all females (including control animals) from gestation day 13 during the whole in-life phase. This was attributed as an effect of the vehicle and did not influence the general condition of the animals.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight, body weight gain in the different periods as well as corrected body weight of the females was similar in all groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Minimal (7.5%) but statistically significant decrease of the food consumption was observed in the 1000 mg/kg bw/day group if compared to the control between G.D. 11 and 14. This difference was considered as biologically not relevant considering the minimal manner and single incidence. Moreover the slightly reduced food consumption did not result in a reduced body weight development.
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
Description (incidence and severity):
Thyroid weight: There were no significant differences in the thyroid weights among the experimental groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Dilated renal pelvis were found in 3 females in the control group, two both in the 100 and 300 mg/kg bw/day groups and none in the high dose. This finding was not attributed to the treatment considering the lack of dose response. Diaphragmatic hernia as a developmental disorder (hence not related to the treatment) was observed in one female in the 300 mg/kg bw/day group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were not lesions observed upon histological examinations in the thyroid tissue.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The free thyroid hormone levels (FT3 and FT4) were not affected by the treatment. TSH levels were all below the level of detection, including the values of the control animals, and could thus not be further evaluated.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no increase indicated in the mean percent of pre- and post-implantation loss (early embryonic, late- and fetal death).
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Remarks:
maternal
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed at highest dose tested (limit dose)

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no treatment related differences in the body weight of the fetuses and placental weight.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The mean number of viable fetuses was similar in all dose groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses was similar in all dose groups.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
Malformations
There were no malformations found in the test item treated groups.
Exencephaly was observed in one fetus in the control group.

Variations
Body weight retardation of the fetuses (below 2.39 g for males and 2.32 g for females) as well as paleness were evaluated as external variations. There was a statistically significant increased incidence of body weigh retardation indicated in the 100 mg/kg bw/day dose group. Considering that there was no dose response observed, this was not attributed to an effect of the test item. Further, there were two litters which were extraordinarily affected (dam#1238134 (6/7 pups retarded) and dam#1137192 (6/12 pups retarded)) which considerably affected the mean value evaluation, but should rather be regarded as biological variation.
Paleness was recorded for one single fetus in the 300 mg/kg bw/day group which was judged as incidental and not to be in relationship with the test item.

Placentas
There were no placental alterations recorded.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Malformations:
The number of the affected fetuses was 1 each in the control and high dose group.
The parietal and frontal bones missed in the control fetus (the same as with exencephaly according to the external examination No.: 1559111/11). Also this fetus had a hemicentric ossification of a thoracic vertebra. The malformed fetus in the high dose group had the same vertebral abnormality. Considering that only a single malformation was found in the high dose and the same occurred in the control group, this finding was not attributed to the test item.

Variations:
Incompletely or not ossified skull bones, bipartite supra-occipital, not ossified hyoid, incompletely ossified sternum or bipartite and/or asymmetrically ossified sternebra, wavy ribs, bipartite or/and asymmetrically or incompletely ossified vertebral centra, incompletely or not ossified lumbar centra or arches, or dumb-bell shaped ossification of lumbar centra, asymmetric ossification or lack of ossification of sacral arches, unossified pubic bone or ischii, and asymmetric or incomplete ossification of metacarpal and metatarsal were evaluated as variations during the skeletal examination.
There were no significant differences in the incidence of the different type of skeletal variations or if summarized.
Visceral malformations:
no effects observed
Description (incidence and severity):
Malformations:
There were no malformations found at visceral examination of the fetuses.

Variations:
Slightly dilated lateral brain ventricle (unilateral) was recorded for one fetus in the control group. Hydroureter were found in all groups except the high dose. Hydroureter accompanied with dilated renal pelvis was observed in the groups with similar incidences. Considering the lack of dose response these variations were not attributed to the treatment.
Other effects:
no effects observed
Description (incidence and severity):
There were no treatment related differences in the absolute or normalized ano-genital distance.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at highest dose tested (limit dose)

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Please refer to result tables attached.

Applicant's summary and conclusion

Conclusions:
Oral treatment of pregnant Han: Wistar rats from gestation day 5 up to day 19 (the day before Caesarean section) with the test item at the dose levels of 100, 300 and 1000 mg/kg bw/day did not cause death, clinical signs and macroscopic changes could have been revealed at necropsy. The test item did not influence the food intake and body weight development of the maternal animals. Treatment with the test item had no impact on thyroid hormones (FT3 and FT4) levels or weight of thyroid glands. Treatment of the dams with the test item did not result in histological changes of the thyroids.
Treatment with the test item did not reveal any adverse effect on the pre- and post-implantation loss, number of implantations, sex distribution, body and placental weight, ano-genital distance, external, visceral and skeletal development of the fetuses.
Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:
NOAEL (maternal toxicity): 1000 mg/kg bw/day
NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day
Executive summary:

The test item was examined for its possible prenatal developmental toxicity in accordance with OECD TG 414 and under GLP.

Groups of 25 sperm-positive female Han: of Wistar origin rats were treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 25 sperm positive females was included and the animals were given the vehicle PEG 400. The treatment volume was 5 mL/kg bw.

A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test item in PEG 400 was stable at room temperature for at least one day and for three days in the refrigerator (5 ± 3 °C) at the concentrations of 1 and 200 mg/mL. Analytical control of dosing solutions was performed during the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 93 and 98 % of nominal concentrations at both analytical occasions confirming proper dosing.

During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Blood sampling for determination of thyroid hormones FT3 and FT4 as well as TSH, Caesarean section and gross pathology were performed on gestational day 20. Thyroids were weighed and evaluated histologically. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

In total, on gestation day 20 there were 19, 20, 21 and 21 evaluated litters in the control, 100, 300 and 1000 mg/kg bw/day group respectively. None of the females died before scheduled necropsy and there were no test item related clinical signs recorded in the dose groups. No treatment related necropsy findings were observed. There were no adverse effects indicated in regards to food consumption or body weight development. Regarding FT3, FT4 and TSH level there was no test item effect indicated.

There was no significant differences in thyroid weights or histopathology among the groups. Number of implantations, intrauterine mortality and sex distribution of the fetuses were not influenced by the treatment. There were no test item related adverse effects on the fetal- and placental weight, ano-genital distance, external and visceral development of fetuses. There were no test item related malformations found. The number of litters with malformations was one in the control and one in the high dose group. There was no increase of variations during fetal examinations indicated.

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL (maternal toxicity): 1000 mg/kg bw/day

NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day