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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:
23/06/2015 to 05/10/2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany.
- Age at study initiation: Females: 11-12 weeks old; Males: 12-13 weeks old.
- Weight at study initiation: Females: 200 - 244 g; Males: 304 - 351 g.
- Fasting period before study:
- Housing: The animals were kept individually in type III H, polysulphone cages on Altromin saw fibre bedding (except during the pre-mating period when females were kept in groups of two animals and during mating period when two females were paired with one male)
- Diet (e.g. ad libitum): Ad libitum.
- Water (e.g. ad libitum): Ad libitum.
- Acclimation period: At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 29-25
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18/06/2015 To: No data.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item plus further vortexing for 2-3 minutes. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared formulation thoroughly before every dose administration. The test item formulation was prepared freshly on each administration day before the administration procedure.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on the test item characteristics and testing guideline.
- Lot/batch no. (if required): BCBM3643V.
- Purity: No data.
- Concentration in vehicle: 0, 25, 75 and 250 mg/l.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for analysis of concentration of test item in the dosing formulations were taken on the first (treatment day 1), third and last week of the study for all doses (12 samples in total). Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation. Samples were taken in the first (treatment day 1), third and last week of the study (18 samples in total). These samples were either measured within 6 hours after arrival at the analytical department on the day of sampling (week 3 and last week samples) or were stored under appropriate conditions (-15 to -35°C) for 16 days until analysis (week 1 samples).
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: No data.
- Matings were continued until 100 'sperm positive' females were obtained.
- Further matings after two unsuccessful attempts: No data.
- 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:
15 days (gestation days 5-19)
Frequency of treatment:
Daily
Duration of test:
15 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
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:
25 'sperm positive' females.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were based on the results of a dose-range finding study. The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once per day. Twice daily all animals checked for mortality and morbidity. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before initiation of pairing to ensure that the body weights were within + 20% variation. The sperm positive females were weighed on gestations days 0, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION: Gestations days 5, 8, 11, 14, 17 and 20.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20. The animals were examined macroscopically for structural abnormalities or pathological changes.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus (with cervix) 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 fetuses in each litter.
- Soft tissue examinations: Yes: half fetuses per litter.
- Skeletal examinations: Yes: half fetuses per litter.
- Head examinations: Yes: half fetuses per litter (from soft tissue examination).
Statistics:
A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Fetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using Fisher’s exact test. The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
One animal (of the control group) was euthanised on study day 19 due to littering before the scheduled sacrifice. This was considered as incidental in nature. There were no deaths in the test groups. Low incidences of the slight clinical sign alopecia were noted in isolated females of the dose groups and the control group. This finding was considered incidental. Moving the bedding was noted in five females of the high dose group on a few treatment days. Salivation was observed in one single female of the high dose group on one occasion. These transient findings were noted shortly after administration and were considered to be signs of discomfort without toxicological relevance.

Body weight gain was not affected by treatment. Some animals in the mid and high dose groups had a statistically significantly reduced food consumption, but as this did not impact on body weights it was not considered to be an adverse finding.

Successful mating resulted in 20/25 pregnancies in the low dose group, 22/25 in the mid dose group and 22/25 in the high dose group compared to 19/25 pregnancies in the control group. Low pregnancy rate (no. of pregnancies / no. of females mated or sperm positive x 100) of 76 % in the control group was considered to be a biological variation.

Prenatal parameters such as group mean terminal body weight, gravid uterus weight, adjusted maternal weight, number of corpora lutea, implantation sites, early and late resorptions, and percent pre- and post-implantation loss were not affected by treatment.

No gross pathological changes related to treatment were observed during the macroscopic examination of the females of any group.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The number of live fetuses, male and female fetuses, sex ratio, number of fetuses in each uterine horn were unaffected in the dose groups when compared to the control group. No dead fetuses were noted in any of the groups. Therefore overall there were no statistically significant differences for prenatal data.

There were no test substance-related effects of toxicological relevance for the total number of fetuses, number of male and female fetuses, mean fetus weight, total litter weight and male and female litter weight. Therefore overall there were no statistically significant differences for litter data.

Fetal examinations did not reveal any dose-related adverse findings. All findings were similar to controls and concluded to be incidental.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse findings for developmental toxicity

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In a Prenatal Developmental Toxicity study (BSL Bioservice, 2016) conducted to OECD Test Guideline 414 and in compliance with GLP 4,4,13,13-tetraethoxy-3,14-dioxa-8,9-dithia-4,13-disilahexadecane did not induce maternal toxicity or developmental toxicity at doses up to 1000 mg/kg bw/day. Therefore the NOAEL for this study was at least 1000 mg/kg bw/day.