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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Jul - 10 Sep 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Dichloro(diphenyl)silane
EC Number:
201-251-0
EC Name:
Dichloro(diphenyl)silane
Cas Number:
80-10-4
Molecular formula:
C12H10Cl2Si
IUPAC Name:
dichlorodiphenylsilane
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: females (at arrival): 11-12 weeks; males (at start of pairing): 12-13 weeks
- Weight at study initiation: males: 326 – 377 g (mean: 348.88 g, ± 20% = 279.10 – 418.65 g), females: 207 - 257 g (mean: 224.29 g, ± 20% = 179.43 – 269.15 g)
- Housing: individually in IVC cages (type III H, polysulphone cages) on 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: Altromin 1324 maintenance diet for rats and mice, provided ad libitum
- Water: Tap water, sulphur acidified to a pH of approximately 2.8, provided ad libitum
- Acclimation period: At least 5 days

DETAILS OF FOOD AND WATER QUALITY: Drinking water, municipal residue control, microbiological controls at regular intervals;
Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins BioPharma Product Testing Munich GmbH.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
dried and de-acidified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tared plastic vial and the vehicle was added to give the appropriate final concentration of the test item. The formulation was vortexed and/or stirred until visual homogeneity was achieved. After homogenization the formulation was overlaid with argon to prevent instability caused by repeated contact of the test item formulation with air.
Based on the results of stability testing, the test item formulations were prepared daily. The prepared formulations were stored protected from light and at appropriate storage conditions tested (max. 3 h at room temperature or up to 2 days at below -15 °C).

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected as suggested by the sponsor based on the test item’s characteristics.
Corn oil was filtered through a mixture of activated silica gel 60 and aluminum oxide (1:1, volume/volume), which had been filled into a glass chromatography column to three quarters of its height. For filtering, a vacuum of 75 mbar was applied. The dried and de-acidified vehicle was overlaid with argon or nitrogen and stored until usage.
- Concentration in vehicle: 12.5, 25 and 50 mg/mL
- Amount of vehicle: 4 mL/kg bw
- Lot/batch no.: MKCK6411
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle as part of a separate GLP study.
The formulations (2.50 to 31.25 mg/mL) were stable for 2 hours at room temperature. Therefore, the formulations were prepared freshly within the stability time frame on each administration day. An additional validation was conducted prior to the start of the study including up to a concentration of 50 mg/mL.
As the test item formulation was shown to be homogenous (after 30 minutes without stirring), samples were not collected during the study for the investigation of homogeneity and samples were only taken for substance concentration in the first and in the last week of the study for all doses (8 samples in total). The mean recoveries observed for the low-dose group was between 95.1% and 84.9% of the nominal value, between 92.4% and 88.2% for the mid-dose group and between 99.0% and 94.5% of the nominal value for high-dose group. The mean recoveries observed in the low-, mid- and high-dose groups were 90.0%, 90.3%, and 96.8% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 15%.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: Females were paired for cohabitation in batches in order to control the number of animals for terminal sacrifice on a particular day. After getting 92 sperm positive females, the remaining females and males will be discarded without any observations.
- 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:
Day 5-19 of gestation
Frequency of treatment:
daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
control, low- and high-dose group: each 23 pregnant females
mid-dose group: 24 pregnant females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: On the basis of a 14-day dose range finding study with dichloro(diphenyl)silane in male and female Wistar rats with dose levels of 25, 50, 100, 500 and 375 mg/kg bw/day the following conclusions can be made: Mortality occurred in 2/2 males and 2/2 females at the dose of 500 mg/kg bw/day and 1/2 females at 375 mg/kg bw/day, Inflammatory lesions in stomach, small and, large intestine were considered as the cause of morbidity. Adverse clinical signs were observed at 500 mg/kg bw/day in both males and females including increased salivation (slight), apathy, hypotonia (muscle), reduced spontaneous activity (slight/severe), slow movements, piloerection (moderate) and half eyelid close (both), hypothermia, lacrimation (left) on treatment days.
No treatment-related mortality or marked clinical signs were observed up to and including 100 mg/kg bw/day. Treatment did not affect the body weight gain up to and including 100 mg/kg bw/day in males and females and body weight was comparable to the respective controls. Test item-related gross lesions were noted in the stomach, small and, large intestine at ≥375 mg/kg bw/day and these were evident during the histopathological examination. The test item caused inflammatory and degenerative findings in the stomach of animals at 375 and 500 mg/kg bw/day. Based on the histopathological data, no adverse effects were noted up to and including 100 mg/kg bw/day.
The highest dose level of 200 mg/kg bw/day was chosen for this study 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.
- Rationale for animal assignment (if not random): Females were assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups (randomisation will be performed with IDBS Workbook 10.1.2 software).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time each day. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
- 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, or bizarre behaviour were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of the mating a detailed clinical observation was made outside the home cage.

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 GD 0, 5, 8, 11, 14, 17 and 20. Males were not be weighed in this study except once before initiation of pairing.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption of sperm-positive females was determined for the following intervals: GD 0-5, 5-8, 8-11, 11-14, 14-17 and 17-20. Food consumption was not measured for males during the entire study or for both males and females during the mating period.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Each dam was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Any macroscopic findings were preserved in 4% neutral-buffered formaldehyde.
Thyroid/parathyroid glands from all dams were preserved in 4% neutral-buffered formaldehyde. The weight of thyroid/parathyroid glands was measured after 24 hours fixation.
Special attention was paid to gastrointestinal tract in order to examine corrosivity of the test item.
All organs listed in Table 1 (see below) from all dams underwent a detailed macroscopic observation. The organs were then preserved in 4% neutral-buffered formaldehyde for possible histopathological examination. For animals of control and high-dose group, the organs listed in Table 1 were histopathologically analysed.

OTHER: Thyroid hormones levels from samples from all dams were assessed at the end of the treatment prior to or as part of the sacrifice of the animals. At termination, blood samples were collected from the defined site will be collected in serum separator tubes and obtained serum were stored under appropriate conditions. Serum samples were assessed for thyroid hormone levels (T3, T4, TSH) using ELISA.
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
Blood sampling:
- Plasma: No
- Serum: Yes (for evaluation of thyroid hormones)
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:
A statistical assessment of the results of the body weight and food consumption will be performed by comparing values of dosed animals with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, thyroid hormones and foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters will be statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. The statistics will be performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 is considered as statistically significant).
Historical control data:
Fetal historical control data were presented in the report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test item-related clinical signs observed in any of the treated groups. No female showed signs of abortion or premature delivery prior to the scheduled sacrifice.
One animal of the mid-dose group was moribund and sacrificed due to animal welfare reasons on GD 5. When the animal was dosed on first day of treatment (gestation day 5), accidently the animal had bitten the gavage tube and swallowed it. The animal showed abnormal breathing as well as severe salivation immediately after dosing and was then moribund sacrificed. In consultation with the study monitor, an additional animal was added to the mid-dose group.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No test item-related mortality was observed during the treatment period and all females survived until scheduled sacrifice.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weight remained unaffected by the test item and increased with the progress of the study in all the groups.
Moderate, but statistically significant lower mean body weight gain was observed in the high-dose group (20.5%) on GDs 0-5 when compared to control. Lower mean body weight gain of the mid- (28.8%) and high-dose (52.6%) groups were observed on GDs 5-8 when compared to control without achieving statistical significance. Lower mean body weight gain of the high-dose group was observed on GDs 8-11 (20.4%) and GDs 11-14 (9.8%) when compared to control, without statistical significance.
Overall, there were no test item-related effects on mean body weight gain on GDs 5-20 in any of the groups when compared to the control.

Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant lower food consumption was observed in the high-dose group on GDs 5-8, 8-11, 11-14 and 14-17 (14-32% below control) and the mean food consumption on GDs 5-20 was found to be statistically significantly lower in the high-dose group (18% below control). The group mean food consumption of the low- and mid-dose groups was comparable to the control. The difference in statistical significance could be due to slightly higher food consumptions in control group and in view of the increased body weight gain, the statistically significant changes observed in mean food consumption are not considered to be test item related effects.
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):
Statistical analysis of post-fixed thyroid/parathyroid weights from all dams revealed no statistically significant differences in the absolute and relative (to body weight) thyroid/parathyroid weights of the treated dose groups when compared to the control group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related gross pathological changes were observed during the macroscopic examination in any of the groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histopathological examination revealed test item related forestomach findings which consisted of ulceration, erosion, hyperkeratosis, squamous hyperplasia, submucosal edema, and mixed cell infiltrates in the high-dose groups. In the mid-dose group, test item related forestomach findings was observed which consisted of erosion, hyperkeratosis, squamous hyperplasia, submucosal edema, and mixed cell infiltrates. In these animals, forestomach only mucosal erosions were present. The forestomach lesions were of lower severity and incidence as those observed in animals dosed with 200 mg/kg bw/day. Further, in the low-dose and control group, no ulcerations or erosions were noticed. Thus a dose dependency of the test item induced forestomach changes was shown in mid- and high-dose groups.
There were no test item-related findings at histopathological evaluation of the thyroid gland in any of the treated groups.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
In all terminally sacrificed females, statistically significant increase in thyroid stimulating hormone was observed in low- and mid-dose groups; however there were no statistically significant or toxicologically relevant effect observed on group mean T3 and T4 hormone levels and values were comparable with the control. As there were no test item-related findings at histopathological evaluation of the thyroid gland in any of the treated groups, the statistical significance is not considered to be toxicologically relevant.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
None of the females showed signs of abortion or premature delivery prior to the scheduled necropsy.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no biologically relevant or statistically significant effects on prenatal data. Prenatal parameters such as pre- and post-implantation loss remained unaffected in the dose groups when compared to the control.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
Early or late resorptions remained unaffected in the dose groups when compared to the control.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead foetuses were observed in any of the test item-treated groups or the control group.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Successful mating resulted in 21/23 pregnancies in the low-dose group, 21/24 in the mid-dose group and 21/23 in the high-dose group compared to 22/23 pregnancies in the control group. No female showed signs of abortion or premature delivery prior to the scheduled sacrifice.
Other effects:
not examined
Details on maternal toxic effects:
Net weight change (g) from GD 0 showed a slight trend towards lower mean weight in the high-dose group compared to the control group (23.9% below control). As there was no dose-dependency, it was not considered toxicologically relevant. Group mean carcass and uterine weight of treated groups were found to be comparable to control.
There were no biologically relevant or statistically significant effects on prenatal data. Prenatal parameters such as number of corpora lutea, implantation sites, live foetuses, early/late resorptions, pre- and post-implantation loss and sex ratio remained unaffected in the dose groups when compared to the control.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse systemic effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
In male and female fetuses, slight but statistically significant decrease in mean fetal body weight was observed in low-, mid- and high-dose groups (males- 3.2%, 3.9% and 7.4% and females- 2.4%, 2.1% and 7.1% below control, respectively) when compared to control. In both male and female fetuses, cube root of foetal weight was comparable to control and no test item-related findings were observed.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for the number of live fetuses.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio remained unaffected in the dose groups when compared to the control.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no test item-related effects of toxicological relevance observed for the mean fetal weight, male and female fetal weight on litter basis (group mean of individual litter mean) in any of the treatment groups when compared to control. However, statistically significant reductions in mean fetal weight and mean male and female fetal weight was observed in the high-dose group. All these values were within the historical control value for this strain of rat.
Anogenital distance of all rodent fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
In females, the absolute and relative anogenital distance (AGD) in the low-, mid-, and high-dose groups, respectively, was statistically significantly higher when compared to the control group. In males, absolute anogenital distance (AGD) in the high-dose groups was statistically significantly lower when compared to control. However, all these values were within the historical control values of this strain. Hence, this was not considered to be a test item-related effect.
All male fetuses were checked for indication of incomplete testicular descent/ cryptorchidism and evaluation revealed completion of testicular descent (abdominal) in all male fetuses from all groups.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item-related external abnormalities observed in any of the fetuses of treated groups. A single incidence of malrotated, hind limb was observed in the low- and high-dose groups and short tail in the high-dose group. These are considered to be incidental findings.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal examination of the Alizarin red stained fetuses revealed a range of findings which occurred at an incidence generally comparable to or slightly lower or higher in the dose groups when compared to the control group.
Statistically significantly higher litter incidences of pelvic girdle, caudal shift were observed in the high-dose group (35%) and higher fetal incidences in the high-dose group (9.84%) when compared to 0% in control. Statistically significantly lower litter incidences of skull parietal, incomplete ossification was observed in the high-dose group (5%) when compared to 45% in control and statistically significant lower fetal incidences in the mid- (2.43%) and high-dose (1.00%) groups were observed when compared to control (16.68%). Statistically significant lower fetal incidences of skull frontal, incomplete ossification in the low-, mid-, and high-dose (each 0%) groups were observed when compared to control (3.38%). Statistically significant higher fetal incidences of 14th rib (B), full was observed in the high-dose group (15.8%) when compared to control (0.83%). Statistically significantly higher litter incidences of 14th rib (B), full were observed in the mid- and high-dose group (45 and 50% respectively) when compared to 5% in control. Statistically significantly higher litter incidences of 14th rib (R) was observed in the high-dose group (85%) when compared to 40% in control and higher fetal incidences at high-dose group (24.4%) when compared to control (10.4%). Statistically significant lower litter incidences of ribs, wavy was observed in the high-dose group (5%) when compared to 55% in control and statistically significant lower fetal incidences in high-dose groups (0.71%) were observed when compared to control (22.2%). All these findings were observed without dose dependency or consistency; hence they are not considered to be test item-related.
Without achieving statistical significance, the observed incomplete ossification of some bones and other incidental skeletal findings in the treated group were either marginally lower or higher than concurrent control but within the historical control data range. Generally, delayed ossification is not regarded to persist post-natally and is not associated with long-term consequences on survival, general growth and development and, therefore, is not considered to be adverse.
There was no statistical significance and no indication of a test item-related trend in the type and/or incidences of other skeletal findings and they were, therefore, considered to be spontaneous in nature.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Internal observation of the fetal viscera by free hand microdissection technique revealed a range of visceral findings in all groups including the control group. Visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to concurrent controls. As the observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature.
Higher litter incidences of testes, malpositioned in the high-dose group were observed (20%) when compared to control (0%). There were higher litter incidences of umbilical artery malpositioned (10%, 15% and 45% in the low-, mid-, and high-dose groups, respectively, compared to 5% in control) observed, in which high-dose group showed statistically significant changes. There were higher litter incidences of azygos vein (bilateral) (5%, 15% and 20% in the low-, mid-, and high-dose groups, respectively, compared to 0% in control) observed. Higher litter incidence of small spleen (5%) and long thymus (10%) were observed in the low- and high-dose groups respectively (5% each). All these findings were considered to be incidental in nature.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Craniofacial examination by razor blade serial sectioning technique revealed incidences of head, subcutaneous hematoma in the treated groups (25% and 20% in the low- and high-dose groups, respectively) when compared to control (15%). Higher litter incidences of mid brain subdural oedema were observed in the treated groups (15% and 5% in mid- and high-dose groups, respectively) when compared to control (0%). Higher litter incidences of increased subcutaneous space in head was observed in the low- and mid-dose groups (5% and 10%, respectively) when compared to control (0%). All these findings were considered to be spontaneous in nature and not related to the treatment with the test item. Statistical analysis of the data revealed no statistical significance for any of these findings.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In an OECD 414 compliant study conducted according to GLP, the NOAEL was established at 200 mg/kg bw/day for both maternal systemic and fetal toxicity in rats administered dichloro(diphenyl)silane (CAS 80-10-4). Additionally, a NOAEL for local maternal effects based on histopathological findings in the forestomach was established at 50 mg/kg bw/day.