Dichlorocyclohexylmethylsilane

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 March 2019 to 09 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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

Test material information

Constituent 1

Reference substance name:

Dichlorocyclohexylmethylsilane

EC Number:

226-956-0

EC Name:

Dichlorocyclohexylmethylsilane

Cas Number:

5578-42-7

Molecular formula:

C7H14Cl2Si

IUPAC Name:

dichloro(cyclohexyl)methylsilane

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han) (Full Barrier)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany

- Age at study initiation:
Age of the females at the arrival at BSL Munich: approx. 11-12 weeks old
Age of the males at the start of pairing: between 13-14 weeks old

- Weight at study initiation:
males: 378 – 469 g (mean: 419.7 g, ± 20 % = 335.7 – 503.6 g)
females: 225 - 272 g (mean: 241.7 g, ± 20 % = 193.4 – 290.0 g)

- Fasting period before study: no
- Housing: The animals were kept individually in IVC 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): Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water (e.g. ad libitum): tap water, sulphur acidified to a pH of approximately 2.8, ad libitum
- Acclimation period: at least 5 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Based on the results of stability testing, the test item formulations were prepared freshly on each administration day before the administration procedure. The prepared formulation was stored protected from light and at room temperature or at appropriate storage conditions tested.
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. The formulation was vortexed and/or stirred until visual homogeneity was achieved.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected in consultation with the sponsor based on the test item’s characteristics. The test item was dissolved in dried and de-acidified corn oil.
- Concentration in vehicle: 0, 2.5, 6.25, 18.75 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no. (if required): MKCH1635

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 at Eurofins Munich as part of a separate GLP study (Eurofins Munich Study No. 187148).
Study pre-start stability analysis was included on the samples from high dose and low dose group and the investigation was made for 0 h, 6 h (RT), 10 day (RT), 10 day (2 8 °C) and 10 day -15 to -35 °C.
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
As the test item was shown to be homogenous according to Eurofins Study No. 187148 (after 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and only samples were taken for substance concentration in the first and last week of the study for all doses (8 samples in total).
Concentration analysis of formulation samples was determined at three concentrations, 2.5 mg/mL, 6.25 mg/mL and 18.75 mg/mL in study weeks 1 and in the last week of the study.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2 (male to female)
- Length of cohabitation: At the subsequent mornings, the vaginal smear of the female was checked to confirm mating.
- Further matings after two unsuccessful attempts: cohabitation was continued
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: The day on which sperms were observed in the vaginal smear was considered as GD 0

Duration of treatment / exposure:

From gestation day 5 until 19

Frequency of treatment:

daily

Duration of test:

20 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control group

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Remarks:

Low dose group (LD)

Doses / concentrations 3

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Remarks:

Middle dose group (MD)

Doses / concentrations 4

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Remarks:

High dose group (HD)

No. of animals per sex per dose:

23 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were based on the findings of the 14-day dose range-finding study (Bioservice, 2019; BSL Munich Study No. 187146). In the study male and female Wistar rats were treated with the test item at doses of 50, 100, 175, 250 and 375 mg/kg bw/day for 14 days. Mortality occurred at the dose of 375 mg/kg bw/day with inflammatory lesions in stomach, large intestine and lungs considered as the cause of morbidity.No treatment-related mortality or marked clinical signs were observed at 50, 100, 175 and 250 mg/kg bw/day. Test item-related gross lesions were noted in the stomach and large intestine at 175 and 375 mg/kg bw/day and these were evident during additional histopathological examination. At the microscopic examination, specific findings were found, beginning at the lowest dose of 50 mg/kg bw/day and continuing in all doses above. These findings consisted at 50 mg/kg bw/day of acute inflammation in the glandular stomach mucosa and single cell necrosis in the glandular mucosa. At 100 mg/kg bw/day, there were forestomach ulceration, glandular stomach erosion, inflammations in the forestomach submucosa and/or epithelial hyperplasia of the forestomach in one male and two females. From 175 to 375 mg/kg bw/day, the findings consisted of forestomach and glandular stomach ulceration, inflammation of the forestomach and/or glandular stomach submucosa, and squamous cell hyperplasia of the forestomach. The highest dose level was therefore 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.

- Rationale for animal assignment (if not random): random

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily all animals were observed for morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time each day. 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.

BODY WEIGHT: Yes
- Time schedule for examinations: The females were weighed prior to mating. The sperm positive females were weighed during GD 0, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION: Yes
- Food consumption of sperm positive females was measured on GD 5, 8, 11, 14, 17 and 20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: See table 1
- Other: The weight of thyroid/parathyroid glands was measured after 24 hours fixation. At termination, blood samples were collected and serum samples were assessed for serum levels for thyroid hormones (T3, T4, TSH). A full histopathology was carried out on the preserved thyroid/parathyroid glands from all dams of all dose groups.

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: [one half of each litter]
- Skeletal examinations: Yes: [the second half of each litter]
- Head examinations: Yes: [the foetuses used for the soft tissue examination of 20 litters per group]

Statistics:

A statistical assessment of the results of the body weight and food consumption was 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 were 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 were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 is considered as statistically significant).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment related clinical signs of toxicological relevance observed in any of the treated groups.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weight remained unaffected by treatment with the test item and increased with the progress of the study in the control and all the treated groups throughout the study period. No statistical significance was achieved in any of the treated groups on any day or interval of body weight measurement and all values in the treated groups were comparable to the control.
No test item-related effects of toxicological relevance were noted for terminal body weight, adjusted maternal weight (carcass weight), uterus weight or net weight change from GD 0.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption in the treated groups was comparable to the control.

Food efficiency:

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

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 dose groups when compared to the control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No gross pathological changes were observed during the macroscopic examination of the females of the control, LD, MD and HD groups, except single incidence of abnormal colour and surface in liver in one animal of HD group, focus-dark red at caudate liver lobe in one HD animal, and uterus dilatation with fluid filled in one LD group animal. This are considered to be an incidental finding.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Forestomach: Test item induced inflammatory and degenerative changes at MD (25 mg/kg bw/day) and HD (75 mg/kg bw/day) group were observed that consisted of ulceration, erosions, mixed inflammatory cell infiltrates, edema, hemorrhage, necrosis, hyperkeratosis and squamous hyperplasia.
In the animals from the LD (10 mg/kg bw/day) group minimal inflammatory cell infiltrates, minimal hyperkeratosis and minimal to slight squamous hyperplasia were observed. The gastric changes in the LD group animals were located mainly at the border between the forestomach and the glandular stomach (limiting ridge) and were considered to be most likely incidental.
Nasal cavity: Acute necrotizing inflammation characterized by epithelial necrosis, mixed cell infiltrates, hemorrhage and the intraluminal presence of inflammatory exudate containing desquamated epithelial cells and inflammatory cells was observed at different sectioning levels at HD group. Further mixed inflammatory infiltrates were also observed in the nasal cavity of some control animals at a lower incidence and severity than those in the HD group animals. The changes observed in the nasal cavity of a few HD animals were generated due to reflux after oral gavage and the subsequent irritative action of the HCl released by the test item hydrolysis. Further, the histopathology changes observed in trachea of HD group animal no. 86 were deemed to be a consequence of aspiration.
In the lung of a few control and HD animals minimal to mild mixed inflammatory cell infiltrates were observed. Further, in one HD animal slight interstitial fibrosis was also observed. All the above-mentioned changes were considered to be related to an inflammatory process most likely unrelated to the test item administration and were deemed to be incidental.
In all other investigated organs including the thyroid and parathyroid glands, no test item related changes were observed and all recorded findings were deemed to be incidental or were within the range of background alterations that may be recorded in Wistar rats.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

In all terminally sacrificed females, no statistically significant or toxicologically relevant effect was observed on group mean T3, T4 and TSH hormone levels and values were comparable with the control.

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 sacrifice. No test item-related effects of toxicological relevance were noted for implantation sites.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No test item-related effects of toxicological relevance were noted for pre- and post-implantation loss in treatment groups when compared to the controls.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No test item-related effects of toxicological relevance were noted for early and late resorptions.

Early or late resorptions:

no effects observed

Description (incidence and severity):

No test item-related effects of toxicological relevance were noted for early and late resorptions.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead foetuses were noted in any of the groups.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No test item-related effects of toxicological relevance were noted for number of corpora lutea.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

>= 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No adverse systemic effects were observed.

2

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Maternal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

other: forestomach and nasal cavity

Description (incidence and severity):

Forestomach changes in MD and HD group animals: ulceration, erosions, mixed inflammatory cell infiltrates, edema, hemorrhage, necrosis, hyperkeratosis and squamous hyperplasia. No test item-related changes were observed in the thyroid and parathyroid glands.
Nasal cavity changes in HD group animals: Acute necrotizing inflammation characterized by epithelial necrosis, mixed cell infiltrates, hemorrhage and the intraluminal presence of inflammatory exudate containing desquamated epithelial cells and inflammatory cells.

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no test item-related effects observed for the mean foetus weight, male and female foetus weight on litter basis (group mean of individual litter mean) in any of the treatment groups when compared with the controls.
However, slight but statistically significantly higher (p < 0.01) mean female foetus weight was observed on an individual basis (sum of weight of all foetuses in group divided by total number of foetuses in respective group) in the LD (5.82% above control) and MD (5.84% above control) groups when compared with the control. As this difference was not dose dependent and no significant effect on male and female foetus weight was observed based on the litter means, it was not considered to be toxicologically relevant.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No dead foetuses were noted in any of the groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No test item-related effects of toxicological relevance were noted for sex ratios. No test item-related effects of toxicological relevance were noted for number of male and female foetuses.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no test item-related effects observed for the mean foetus weight, male and female foetus weight on litter basis (group mean of individual litter mean) in any of the treatment groups when compared with the controls.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There were no external abnormalities considered to be of toxicological relevance in any of the dose groups observed.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Craniofacial examination by razor blade serial sectioning technique revealed a predominant finding at all treated (56.2%, 47.6% and 10% in LD, MD and HD group respectively) and control (55%) groups {head, subcutaneous hematoma (general)} and few incidences of brain(small) in control group (5%), large perimeningal space in LD (12.5%) and HD (5%) groups and hematoma, subdural in control (5%) and HD (10%) groups. 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, except head, subcutaneous hematoma in HD group, which was found to be statistically lower. In addition, all these values were within the historical control range for this strain.
Skeletal examination of the Alizarin red stained foetuses 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.
The observed incomplete ossification without achieving statistical significance of a few bones and a few other skeletal findings in the HD group were either marginally lower or higher or within historical control data range. Generally delayed ossification is not regarded to persist postnatally and 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):

Visceral findings in all groups including control were observed. 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 controls. As the observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no toxicological significance was attributed to these findings and they were considered to be spontaneous in nature. All litter incidences from dose groups were well within historical control data range and statistically insignificant when compared with the control.

Other effects:

no effects observed

Description (incidence and severity):

In males and female, the absolute and relative anogenital distance (AGD) in treatment groups remained unaffected and no statistical significance was observed in any of the treatment group when compared to control. All male foetuses were checked for indication of incomplete testicular descent/cryptorchidism and evaluation revealed completion of testicular descent (abdominal) in all male foetuses from all group.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental effects

Effect level:

>= 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no adverse developmental effects seen.

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: Incidence and mean severity of gastric finding

Finding / Groups	C	LD	MD	HD
Dose (mg/kg bw/day)	0	10	25	75
Animals per Sex/ Affected / Mean Grade	23 (F)			23 (F)
Forestomach Ulceration	-	-	1/3.0	2/2.5
Forestomach Erosion	-	-	-	1/3.0
Mixed cell infiltrates	1/1.0	1/1.0	5/1.4	11/1.7
Forestomach Edema	-	-	1/3.0	3/2.3
Hemorrhage	-	-	-	1/2.0
Necrosis	-	-	-	3/2.3
Hyperkeratosis	-	6/1.0	6/1.3	20/1.7
Squamous hyperplasia	8/1.0	9/1.7	8/1.8	21/2.0

C - Control; LD - low dose; MD - medium dose; HD – High Dose

Table 2: Incidence and mean severity of findings in the nasal cavity

Finding / Groups	C	HD
Dose (mg/kg bw/day)	0	75
Animals per Sex/ Affected / Mean Grade	23 (F)	23 (F)
Nasal Cavity L1 + L2
Necrosis	-	1/2.0
Mixed Cell Infiltrates
2.      Nasal Cavity L3
Necrosis	-	1/3.0
Mixed cell infiltrates	3/1.7	5/2.2
Hemorrhage	-	1/1.0
3.      Nasal Cavity L4
Necrosis	-	3/1.7
Mixed Cell Infiltrates	-	2/2.0

C - Control; HD – High Dose

Applicant's summary and conclusion

Conclusions:

In a prenatal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP, a NOAEL for maternal local effects was concluded to be 10 mg/kg bw/day based on test item-induced necrosis and microscopic inflammatory and degenerative changes in the forestomach of middle dose and high dose groups, and in the nasal cavity of high dose group. No systemic effects were observed and no adverse effects could be detected in foetuses therefore the developmental and systemic NOAELs were concluded to be greater than or equal to 75 mg/kg bw/day, the highest dose tested.