tert-butylchlorodimethylsilane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In the key combined repeated dose oral toxicity study with the reproduction / developmental toxicity screening test, with the registered substance tert-butyl(chloro)dimethylsilane, conducted according to OECD Test Guideline 422 and in compliance with GLP, the NOAEL for reproductive toxicity was concluded to be ≥90 mg/kg bw/day (the highest dose tested) based on no adverse effects on reproduction parameters (BSL Bioservice / Eurofins, 2020). The high dose was limited to 90 mg/kg bw/day based on severe local effects observed in a 14-day range finding study at doses of 100 mg/kg bw/day and above (BSL Bioservice, 2019).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18th of April 2019 to 28th of January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Adopted 29th of July, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: health Effects guidelines, OPPTS 870/3650

Version / remarks:

Combined repeated dose toxicity study with the reproduction / developmental toxicity screening test, EPA 712-C-00-368, July 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl: WI(Han) (Full barrier), derived from a controlled full-barrier maintained breeding system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 14-15 weeks old
- Weight at study initiation:
males: 318 – 376 g (mean: 348.5 g, ± 20% = 278.8 – 418.2 g)
females: 221 – 258 g (mean: 238.4 g, ± 20% = 190.7 – 286.0 g)
- Fasting period before study: not specified
- Housing: Type IV polysulphone cages or in double decker IVC cages during the premating period for both males and females as well as during the post-mating period for males depending on the m ating status. During the mating period, males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Diet: Altromin 1324 maintenance diet for rats, ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8, ad libitum.
- Acclimation period: at least 5 days.

DETAILS OF FOOD AND WATER QUALITY: Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich

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

IN-LIFE DATES: not specified

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was dissolved in dried and de-acidified corn oil and thereafter, administered daily.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected in consultation with the sponsor based on the test item's characteristics.
- Concentration in vehicle: 0; 2.5; 7.5; 22.5 mg/mL.
- Amount of vehicle: 4 mL/kg bw/day
- Lot/batch no.: MKCG3257 and MKCH1635
- Purity: not specified

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 days.
- Proof of pregnancy: sperm in vaginal smear, referred to as day 0 of gestation
- Mating period was up to 14 days long.
- Further matings after two unsuccessful attempts: up to 14 days were allowed for matings
- After successful mating each pregnant female was caged: females were kept individually during gestation/lactation period in type III H, polysulphone cages.
- Any other deviations from standard protocol: no
- other: If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the oestrous cycle on that day was documented

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration analysis of formulation samples was performed on the three concentrations, 2.5 mg/mL, 7.5 mg/mL and 22.5 mg/mL in study weeks 1, 4, 7 and in the last week of the study. The mean recoveries observed in the low, medium and high dose groups were 98.0%, 98.6%, and 97.7% of the nominal concentration, respectively. The low recoveries found in those samples were considered due to technical error in the preparation of formulation samples. Thus, both mentioned samples were excluded from the evaluation of concentration verification.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily administration, 7 days per week

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Low dose, LD

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Middle dose, MD

Dose / conc.:

90 mg/kg bw/day (nominal)

Remarks:

High dose, HD

No. of animals per sex per dose:

10 females/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were based on a previous dose range finding study and in consultation with the sponsor. In the dose range-finding study inflammatory and degenerative lesions (e.g. erosion, ulceration) of the stomach were noted in male and female animals treated with 100 mg/kg bw/day and higher. Although the highest dose of 90 mg/kg bw/day may not induce toxic effects, higher doses were not considered due to animal welfare reasons. A descending sequence of dose levels were selected to demonstrate any dose-related response and to determine a NOAEL.
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: not reported
- Rationale for selecting satellite groups: no satellite groups were included
- Post-exposure recovery period in satellite groups: no
- Section schedule rationale: random
- Other: n/a

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once per day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily, all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure and at least once a week thereafter. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, as phyxia, 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: once before the assignment to the experimental groups, on the first day of dosing and thereafter, weekly as well as at the end of the study. During pregnancy, females were weighed on gestation days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 pos t-partum), on PND 4, PND 9 and PND 13 along with pups. In total, 2 animals from the control group; 4 animals from the low dose group and 2 animals from the middle dose group were weighed on GD 21 instead of GD 20. All animals were weighed directly before termination. Any animals prematurely sacrificed were weighed prior to the sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating per iod in males and females and the post-mating period in males.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

Oestrous cyclicity (parental animals):

60 females were screened for regular oestrous cycles for 14 days before the treatment initiation and 40 females only (10 females/ group) showing regular oestrous cycles were continued in the study.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight and a detailed qualitative examination taking into account the tubular stages of the spermatogenic cycle at evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, runts born, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance AGD, pup weight on the day of AGD and presence of nipples/areolae in male pups. For 2 dams, litter observations scheduled for PND 0 were performed on PND 1 and PND 3, respectively.

GROSS EXAMINATION OF DEAD PUPS:
yes, external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents were examined.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Males were dosed for a minimum of four weeks until up to one day before the scheduled sacrifice (this includes a minimum of two weeks prior to mating, during the mating period and up to two weeks post-mating). The males were sacrificed after completion of the mating period on treatment days 29 and 30.
- Maternal animals: a maximum exposure of 63 days in total in females (at least 14 days pre-mating, up to 14 days mating, approximately 22 days of gestation and up to post-natal day 12). The females along with their pups were sacrificed on PND 13. Non-pregnant females were sacrificed on day 26.

GROSS NECROPSY
- Gross necropsy consisted of: external surface body examination as well as examination of all orifices, the cranial, thoracic and abdominal cavities and their contents.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

GROSS NECROPSY - Gross necropsy consisted of external examination as well as examination of all orifices, the cranial, thoracic and abdominal cavities and their contents.

ORGAN WEIGTHS
Thyroid/parathyroid glands from 1 pup/sex/litter/group (sacrificed on PND 13). Weight of thyroid/ parathyroid glands was measured after fixation.

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using an one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry 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 homogenei ty and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 was considered as statistically significant).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

A female animal from the control group which was euthanized in moribund condition had previously shown clinical findings of apathy, reduced spontaneous activity, abnormal breathing dehydration, piloer ection, half eyelid closure, lacrimation, exophthalmos and chromodacryorrhea on the day of sacrifice. Other observed clinical signs in treatment animals included moving the bedding and increased salivation, however, this appeared directly after the administration and thus, considered to be a sign of discomfort or a local reaction of the test item and not adverse systemic effects.
Furthermore, piloerection, crust, hairless area and diarrhoea occurred in single animals without any dose dependency and therefore, not considered as test substance related

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the control group was euthanized in moribund condition. In the histopathological examination, necrotising inflammation along with foreign materials containing feed composition in the lesions was observed in the tissues around the intrathoracic organs and tissues. Such inflammatory lesions were noted in the thymus and lungs (recorded as pleuritis), and observed in the subcutis at the neck region, as well. From these findings, the cause of animal’s morbidity was considered to be technical error that happened during the oral gavaging procedure. All remaining animals survived the scheduled study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Treatment with the test item showed no statistically significant or toxicologically relevant effects on body weights or body weight development of male and female animals. Body weight development was co mparable between test item-treated groups and their respective control group throughout the treatment period. Slight changes were not considered to be of toxicological relevance. There were no statistically significant differences in final body weights in both sexes.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No considerable and statistically significant difference in food consumption of male and female animals between the dose groups and the control group were observed. Slight differences between the test item-treated animals and corresponding controls were only temporary and without a clear dose-dependent trend and thus were not assumed toxicologically relevant.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The mean percentage of reticulocytes was slightly lower in males compared to control groups, however, due to a lack of dose dependency and without statistical significance, the finding was not considered test substance related. Moreover, the percentage of eosinophils was higher in males in all dose groups with a statistical significance in the low dose group when compared to the control group. Furthermore, higher mean percentage of basophils and higher mean percentage of large unstained cells were observed to be higher in males in the middle dose group compared to the control group. In both cases, the elevated values were mainly based on the results of one single animal and therefore, not considered to be toxicologically relevant.
The mean percentage of monocytes in females was higher in all dose groups compared to female controls. Middle dose group females had a statistically significantly lower percentage of lymphocytes and a statistically significant higher percentage of neutrophils compared to the females in the control group. In the low dose group females, the mean percentage of basophils was higher in females compared to the control group. In the middle and high dose group females, the mean percentage of large unstained cells was lower compared to the females control group. Due to the lack of dose-dependency or gender consistency, these finding were not considered as toxicologically relevant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Lower serum TBA levels were observed in the males in all dose groups whereas slightly higher serum ALAT levels were found in the high group when compared to the control group. Nevertheless, no statistical significance or dose dependency were observed and therefore, these differences were assumed to be not test substance-related.
Statistically higher urea levels in females were observed in the low dose group compared to the control females. Additionally, lower serum AP levels were observed in females in all the test substance treated groups. None of these findings were considered to be toxicologically relevant due to gender inconsistency or dose-dependency.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Findings of nitrite in the urine and glucose were considered incidental and not test substance related.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant lower body temperature in high dose group males was observed compared to that of the control group males before the treatment. However, lower temperature in the HD group was observed prior to treatment start and thus was not considered test-item related. Statistically significant lower body temperature in middle dose group males was observed compared that of the control group males during the last week of treatment. Lower temperature in the MD group in the last week of treatment was not considered toxicologically relevant as LD and HD group showed no statistically significant differences and no dose-dependent trend was observed.

Immunological findings:

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the forestomach, inflammation with focal submucosal haemorrhage was observed in one low dose group male as well as in one middle dose group male. Focal/single ulceration in the lesion was also seen for the latter animal. No forestomach lesions were observed in any high dose group animals. In the preliminary study doses higher than 90 mg/kg bw/day were administered to male and female rats and stomach injuries indicating local irritative effects of the test item were observed at all dose levels for both sexes. The forestomach lesions observed in the study were identical to the lesions in the dose range-finding study and therefore they were considered to be treatment-related and adverse changes. Mucous neck cell hyperplasia was observed in the glandular stomach in three high dose males and one high dose female, but it was considered to be an adaptive response to local stimuli and not to be adverse as no necrotic/degenerative or inflammatory changes were noted.
In the kidneys of high dose group males, hyaline droplet accumulation with sporadic single cell death in the proximal tubules as well as increased incidence of tubular basophilia were observed. No such effects were observed in any of the female animals. Hyaline droplets were due to an excessive accumulation of secondary lysosomes within the cytoplasm containing alpha-2u-globulin. This is considered to be a male rat specific effect.
Moreover, the hyaline droplet accumulation in high dose males was accompanied by tubular single cell necrosis and increased incidence of tubular basophilia. These effects were considered to be male rat-specific and non-human-relevant.
No histomorphological changes could be detected in the reproductive organs and tissues. Squamous hyperplasia and hyperkeratosis were observed in one low dose group male as well as one middle dose group male.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Treatment with the test item had no effect on serum T4 levels of parental males. No considerable differences were found between dose groups and the corresponding control group of this study.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no test item-related statistically significant or biologically relevant effect on the oestrous cycle of any of the female animals when analysed during the 2 weeks premating period. There were no considerable differences in the mean length or sequence of cycle stages. An abnormal cycle was noted for one high dose group female which was considered incidental.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

There were slight differences in the reproductive indices of test item-treated females when compared to control females. The changes followed no dose-dependency and were within the normal range of variation and therefore, were considered to not be toxicologically relevant.

Dose descriptor:

NOAEL

Remarks:

systemic and reproductive effects

Effect level:

>= 90 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects were observed

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

kidney

stomach

Treatment related:

yes

Dose response relationship:

yes

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

One control group pup was found dead on postnatal day 1. This was within the normal range of variation and therefore, not considered as toxicologically relevant. No other pup mortality was observed during the study duration.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Male litter weight was statistically significantly lower in the low dose group compared to the control group on postnatal day 0 and 4. However, males of higher dose groups showed no relevant differences in litter weight compared to the control group. Thus, lower male litter weight was not considered toxicologically relevant.

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, treatment-related

Description (incidence and severity):

A statistically significantly shorter absolute and relative anogenital distance was observed in middle and high dose group males when compared to the control group. Similarly, shorter absolute and relative anogenital distance was observed in high dose group female animals compared to the control group. Since all values were within the historical control data range and no changes were seen in several other parameters, e.g. histopathology of parental reproductive organs and their weights, litter seize, sex ration, pup thyroid /parathyroid weight and thyroxine hormone (T4), these effects were not considered adverse.

Nipple retention in male pups:

effects observed, treatment-related

Description (incidence and severity):

A statistically significantly higher nipple retention was noted in high dose male pups on postnatal day 12 when compared to the control group. Since the values are within the historical control data range, this change was considered non-adverse.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related gross external abnormalities of toxicological relevance were observed in the pups of any of the groups on PND 0-12 or the day of sacrifice/death.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Treatment with the test item had no effect on serum T4 levels in 13 day old pups. No considerable differences were found between dose groups and the corresponding control group of this study.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effect

Generation:

F1

Effect level:

>= 90 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

In the combined repeated dose oral toxicity study with the reproduction / developmental toxicity screening test, with the test substance tert-butyl(chloro)dimethylsilane, conducted according to OECD Test Guideline 422 and in compliance with GLP, a NOAEL for reproductive toxicity was concluded to be ≥90 mg/kg bw/day based on no adverse effects on reproduction and developmental parameters. Effects on the anogenital distance (AGD) and the nipple retention were within the historical control data, although a statistically significant difference was noted. In addition, AGD is not the only parameter to confirm potential endocrine disruption. No effects were reported for several other parameters, such as histopathology of parental reproductive organs and their weights, litter size, sex ratio, pup thyroid /parathyroid weight and thyroxine hormone (T4) to support a possible endocrine disruption modality of the test item..

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

90 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch score 1.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In the key combined repeated dose oral toxicity study with the reproduction / developmental toxicity screening test, conducted according to OECD Test Guideline 422 and in compliance with GLP, doses of 0, 10 (LD), 30 (MD) or 90 (HD) mg/kg bw/day tert-butyl(chloro)dimethylsilane in corn oil were administered daily via oral (gavage) route to male and female rats for a treatment period of up to 63 days. Male and female rats were exposed to the test item during the 14-day pre-mating period and during the mating period (maximum 14 days). Females were also exposed during the gestation period and up to post-natal day (PND) 12 (BSL Bioservice / Eurofins, 2020). During the study the animals were observed for mortality, clinical signs, functional observations, food consumption and body weights on a regular basis; haematological, clinical biochemistry and urinalysis evaluations were performed at terminal sacrifice; reproductive function and effects on reproductive organs were evaluated for males and females; litter observations were performed including the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities; anogenital distance of pups was also measured; all animals were examined for macroscopic lesions at gross necropsy; organ weights were recorded and a full histopathological evaluation was performed on preserved tissues.

No treatment-related mortality was observed for any of the test groups. No clinical signs indicating systemic toxicity were observed in any of the animals. No test item-related effects were observed on the parameters of the functional observation battery or body temperature of male and female animals and no relevant effects were noted on body weight development or food consumption in any of the dose groups. The test item had no toxicologically relevant effect on coagulation parameters, haematological parameters or parameters of clinical biochemistry of male and female animals. The test item had no toxicologically relevant effects on urinary parameters analysed in selected animals at the end of the treatment period. No effects on organ weights were reported for females. Histopathological treatment-related changes were observed in the stomachs of males, which were considered treatment-related and adverse for both sexes.

No treatment-related effects were observed on the oestrous cycle, pre-coital interval and duration of gestation, reproductive indices or pre- and post-natal parameters. Slightly dose-dependent trend towards higher pre-implantation loss in test item-treated groups was not considered toxicologically relevant as the data were within the historical control data range. No toxicologically relevant effects in litter observations and pup survival were noted. No toxicologically relevant external gross abnormalities were observed in the pups of test item-treated groups. Statistically significantly shorter anogenital distance was observed in male pups of the MD and HD group and mean nipple retention of male pups was found to be statistically significantly higher in the HD group. Female pups of the HD group showed a statistically significantly shorter anogenital distance.Even though, these changes were within the historical control data range, they could indicate possible endocrine disruption. However, no effects were observed in other parameters such as histopathology of parental reproductive organs and their weights, litter size, sex ratio, pup thyroid /parathyroid weight and thyroxine hormone (T4) to support a possible endocrine disruption modality of the test item. The study concluded the NOAELs for reproductive and developmental toxicity to be ≥90 mg/kg bw/day (the highest dose tested) based on no adverse effects on reproduction and developmental parameters. The high dose was limited to 90 mg/kg bw/day based on severe local effects observed in a 14-day range finding study at doses of 100 mg/kg bw/day and above (BSL Bioservice, 2019).

Effects on developmental toxicity

Description of key information

In the key combined repeated dose toxicity study with the reproduction / developmental toxicity screening test, conducted according to OECD Test Guideline 422 and in compliance with GLP, a NOAEL for developmental toxicology was concluded to be ≥90 mg/kg bw/day based on no test substance related findings (BSL Bioservice / Eurofins, 2020). The high dose was limited to 90 mg/kg bw/day based on severe local effects observed in a 14-day range finding study at doses of 100 mg/kg bw/day and above (BSL Bioservice, 2019).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18th of April 2019 to 28th of January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Adopted 29th July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Health Effects guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test

Version / remarks:

EPA 712-C-00-368, July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 14-15 weeks old - Weight at study initiation:
males: 318 – 376 g (mean: 348.5 g, ± 20% = 278.8 – 418.2 g) females: 221 – 258 g (mean: 238.4 g, ± 20% = 190.7 – 286.0 g)
- Fasting period before study: not specified
- Housing: Type IV polysulphone cages or in double decker IVC cages during the premating period for both males and females as well as during the post-mating period for males depending on the mating status. During the mating period, males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage. In each cage, Altromin saw fibre was used as bedding.
- Diet: Altromin 1324 maintenance diet for rats, ad libitum.
- Water: tap water, sulphur acidified to a pH of approximately 2.8, ad libitum.
- Acclimation period: at least 5 days.


DETAILS OF FOOD AND WATER QUALITY: Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich.

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

IN-LIFE DATES: not specified

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was dissolved in dried and de-acidified corn oil and thereafter, administered daily.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected in consultation with the sponsor based on the test item's characteristics.
- Concentration in vehicle: 0; 2.5; 7.5; 22.5 mg/mL.
- Amount of vehicle: 4 mL/kg bw/day
- Lot/batch no.: MKCG3257 and MKCH1635
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration analysis of formulation samples was performed on the three concentrations, 2.5 mg/mL, 7.5 mg/mL and 22.5 mg/mL in study weeks 1, 4, 7 and in the last week of the study. The mean recoveries observed in the low, medium and high dose groups were 98.0%, 98.6%, and 97.7% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptable criterion of 10%.
However, two samples (one sample from the low dose week 7 and one sample from the middle dose week 7) did not meet this criterion with a recovery of 76.1% and 83.3%, respectively. The low recoveries found in those samples were considered due to technical error in the preparation of formulation samples. Thus, both mentioned samples were excluded from the evaluation of concentration verification.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 days.
- Proof of pregnancy: sperm in vaginal smear, referred to as day 0 of gestation
- Mating period was up to 14 days long.
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged: females were kept individually during gestation/lactation period in type III H, polysulphone cages
- Any other deviations from standard protocol: no
- other: If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the oestrous cycle on that day was documented

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily administration, 7 days per week

Duration of test:

In total, maximum of 63 days: The females were exposed during 14 days pre-mating period, up to 14 days mating period and approximately 22 days of gestation and up to post-natal day 12. The males were treated during pre-mating and mating period, up to 29-30 days of treatment. Non-pregnant females were treated for 26 days.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Low dose, LD

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Middle dose, MD

Dose / conc.:

90 mg/kg bw/day (nominal)

Remarks:

High dose, HD

No. of animals per sex per dose:

10 males and 10 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were based on a previous dose range finding study and in consultation with the sponsor. In the dose range-finding study, inflammatory and degenerative lesions (e.g. erosion, ulceration) of the stomach were noted in male and female animals treated with the dose of 100 mg/kg bw/day and higher. Although the highest dose of 90 mg/kg bw/day may not induce toxic effects, higher doses were not considered due to animal welfare reasons. A descending sequence of dose levels were selected to demonstrate any dose-related response and to determine a NOAEL.
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: not specified
- Rationale for selecting satellite groups: not specified
- Post-exposure recovery period in satellite groups: not specified
- Section schedule rationale: random
- Other: n/a

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once per day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily, all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure and at least once a week thereafter. 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 behav iour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: once before the assignment to the experimental groups, on the first day of dosing and thereafter, weekly as well as at the end of the study. During pregnancy, fe males were weighed on gestation days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 postpartum), on PND 4, PND 9 and PND 13 along with pups. In total, 2 animals from the control group; 4 animals from the low dose group and 2 animals from the middle dose group were weighed on GD 21 instead of GD 20.
All animals were weighed directly before termination. Any animals prematurely sacrificed were weighed prior to the sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 13 for females and their pups.
- Organs examined: see table 1.

Ovaries and uterine content:

The ovaries and uterine content were 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: No
- Number of late resorptions: No

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No data

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry 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 homogenei ty and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or A scentos 1.3.4 software (p<0.05 was considered as statistically significant).

Historical control data:

Yes, however, no further details.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

A female animal from the control group which was euthanized in moribund condition had previously shown clinical findings of apathy, reduced spontaneous activity, abnormal breathing dehydration, piloer ection, half eyelid closure, lacrimation, exophthalmos and chromodacryorrhea on the day of sacrifice. Other observed clinical signs in treatment animals included moving the bedding and increased salivation, however, this appeared directly after the administration and thus, considered to be a sign of discomfort or a local reaction of the test item and not adverse systemic effects.
Furthermore, piloerection, crust, hairless area and diarrhoea occurred in single animals without any dose dependency and therefore, not considered as test substance related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the control group was euthanized in moribund condition. In the histopathological exami nation, necrotising inflammation along with foreign materials containing feed composition in the lesions was observed in the tissues around the intrathoracic organs and tissues. Such inflammatory lesions were noted in the thymus and lungs (recorded as pleuritis), and observed in the subcutis at the neck region as well. From these findings, the cause of animal’s morbidity was considered to be a technical error that happened during the oral gavaging procedure. All remaining animals survived the scheduled study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Treatment with the test item showed no statistically significant or toxicologically relevant effects on body weights or body weight development of male and female animals. Body weight development was co mparable between test item-treated groups and their respective control group throughout the treatm ent period. Slight changes were not considered to be of toxicological relevance. There were no stat istically significant differences in final body weights in both sexes.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No considerable and statistically significant difference in food consumption of male and female anim als between the dose groups and the control group were observed. Slight differences between the tes t item-treated animals and corresponding controls were only temporary and without a clear dose-depe ndent trend and thus were not assumed toxicologically relevant.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant effects were observed.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The mean percentage of reticulocytes was slightly lower in males compared to control groups, however, due to a lack of dose dependency and without statistical significance, the finding was not considered test substance related. Moreover, the percentage of eosinophils was higher in males in all dose groups with a statistical significance in the low dose group compared to the control group. Furthermore, higher mean percentage of basophils and higher mean percentage of large unstained cells were observed to be higher in males in the middle dose group compared to the control group. In both cases, the elevated values were mainly based on the results of one single animal and therefore, not considered to be toxicologically relevant.
The mean percentage of monocytes in females was higher in all dose groups compared to female controls. Middle dose group females had a statistically significantly lower percentage of lymphocytes and a statistically significant higher percentage of neutrophils compared to the females in the control group. In the low dose group females, the mean percentage of basophils was higher in females compared to the control group. In the middle and high dose group females, the mean percentage of large unstained cells was lower compared to the females control group. Due to the lack of dose-dependency or gender consistency, these finding were not considered as toxicologically relevant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Lower serum TBA levels were observed in the males in all dose groups whereas slightly higher serum ALAT levels were found in the high group when compared to the control group. Nevertheless, no statistical significance or dose dependency were observed and therefore, these differences were assumed to be not test substance related.
Statistically higher urea levels in females were observed in the low dose group compared to the control females. Additionally, lower serum AP levels were observed in females in all the test substance treated groups. None of these findings were considered to be toxicologically relevant due to gender inconsistency or dose-dependency.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Findings of nitrite in the urine and glucose were considered incidental and not test substance related.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant lower body temperature in high dose group males was observed compared to that of the control group males before the treatment. However, lower temperature in the HD group was observed prior to treatment start and thus was not considered test-item related. Statistically significant lower body temperature in middle dose group males was observed compared that of the control group males during the last week of treatment. Lower temperature in the MD group in the last week of treatment was not considered toxicologically relevant as LD and HD group showed no statistically significant differences and no dose-dependent trend was observed.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly higher absolute and relative (to body weight) kidney weights were recorded in high dose group males. The increased renal weight correlated with the histopathologica l lesions present in the kidneys, particularly with enhanced accumulation of hyaline droplets in the proximal tubules. However, the process for hyaline droplet accumulations is limited to male rats and it is considered that the same process do not occur in female rats, mice of either sex, and higher species of either sex including dogs, primates and humans, because of lack of or little alpha-2u-globulin synthesis.
Other slight to moderate deviations in organ weights of test item treated animals compared to their respective controls followed no consistency or dose-dependency and thus were not considered toxicologically relevant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At necropsy, mass of the larynx, abnormal (hard) consistency of the salivary glands, yellow fluid-filled stomach and enlarged pancreas were observed in the female animal, which was euthanized for animal welfare reasons. The histopathological evaluation showed that lesions in the larynx and salivary glands had necrotising inflammations which occurred in the subcutis at the region around the neck and were not the lesions produced in the parenchyma of each organ.
Yellowish fluid-filled thoracic cavity was recorded in one low dose group female. No histological examination was performed to the organs and tissues of this animal. No gross or histomorphological lesions indicating abnormalities in respiratory or circulatory functions were observed in any high dose animals. Therefore, the fluid retention observed in this one low dose group female was considered to be incidental and not test substance-related.
Other findings included red coloured ileum in one middle dose group male as well as yellow fluid-filled thoracic cavity and red-spotted thymus in one control group female. These findings were within the range of normal background changes, observed in rats of this strain and age, and without any corresponding histopathological changes. They were therefore considered to be incidental and not test substance-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the forestomach, inflammation with focal submucosal haemorrhage was observed in one low dose group male as well as in one middle dose group male. Focal/single ulceration in the lesion was also seen for the latter animal. No forestomach lesions were observed in any high dose group animals. In the preliminary study doses higher than 90 mg/kg bw/day were administered to male and female rats and stomach injuries indicating local irritative effects of the test item were observed at all dose levels for both sexes. The forestomach lesions observed in the study were identical to the lesions in the dose range-finding study and therefore they were considered to be treatment-related and adverse changes. Mucous neck cell hyperplasia was observed in the glandular stomach in three high dose males and one high dose female, but it was considered to be an adaptive response to local stimuli and not to be adverse as no necrotic/degenerative or inflammatory changes were noted.
In the kidneys of high dose group males, hyaline droplet accumulation with sporadic single cell death in the proximal tubules as well as increased incidence of tubular basophilia were observed. No such effects were observed in any of the female animals. Hyaline droplets were due to an excessive accumulation of secondary lysosomes within the cytoplasm containing alpha-2u-globulin. This is considered to be a male rat specific effect.
Moreover, the hyaline droplet accumulation in high dose males was accompanied by tubular single cell necrosis and increased incidence of tubular basophilia. These effects were considered to be male rat-specific and non-human-relevant.
No histomorphological changes could be detected in the reproductive organs and tissues. Squamous hyperplasia and hyperkeratosis were observed in one low dose group male as well as one middle dose group male.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Treatment with the test item had no effect on serum T4 levels of parental males. No considerable differences were found between dose groups and the corresponding control group of this study.

Number of abortions:

not specified

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

There was a trend of a dose-dependent higher pre-implantation loss, however, since all the values were within the range of historical control data, the observations were not considered to be toxicologically relevant.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Description (incidence and severity):

No toxicologically relevant effects were observed.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No toxicologically relevant effects were observed.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No toxicologically relevant effects were observed.

Other effects:

no effects observed

Description (incidence and severity):

No effects were seen in the number of corpora lutea.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic and developmental effects

Effect level:

>= 90 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed

Key result

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Abnormalities:

no effects observed

Description (incidence and severity):

No reproductive effects were observed.

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly higher weight of low dose male and female pups was noted. This change was not seen for any other of the treatment groups. In the absence of a dose-dependency, this effect is not considered toxicologically relevant.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No toxicologically relevant effects were observed.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No toxicologically relevant effects were observed.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly lower male litter weight in the low dose group was noted on postnatal day 0 and postnatal day 4. However, a similar effect was not seen in any of the higher dose groups. Thus, lower male litter weight was not considered toxicologically relevant.

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

Still births were reported for the low and middle dose group. This was not observed in the high dose group and thus, not considered as test item related.

External malformations:

no effects observed

Description (incidence and severity):

No gross external abnormalities of toxicological relevance were observed in the pups of test item-treated groups.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No statistically significant differences between test item-treated groups and the controls occurred.

Dose descriptor:

NOAEL

Effect level:

>= 90 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

In The combined repeated dose oral toxicity study with the reproduction / developmental toxicity screening test, with the test substance tert-butyl(chloro)dimethylsilane, conducted according to OECD Test Guideline 422 and in compliance with GLP, a NOAEL for reproductive toxicity was concluded to be ≥90 mg/kg bw/day based on no adverse effects on reproduction and developmental parameters. Effects on the anogenital distance (AGD) and the nipple retention were within the historical control data, although a statistically significant difference was noted. In addition, AGD is not the only parameter to confirm potential endocrine disruption. No effects were reported for several other parameters, such as histopathology of parental reproductive organs and their weights, litter size, sex ratio, pup thyroid /parathyroid weight and thyroxine hormone (T4) to support a possible endocrine disruption modality of the test item.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

90 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch score 1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In the key combined repeated dose oral toxicity study with the reproduction / developmental toxicity screening test, conducted according to OECD Test Guideline 422 and in compliance with GLP, 0, 10 (LD), 30 (MD) or 90 (HD) mg/kg bw/day of tert-butyl(chloro)dimethylsilane in corn oil was administered daily via oral (gavage) route to male and female rats for a treatment period of 63 days. Male and female rats were exposed to the test item during the 14-day pre-mating period and during the mating period (maximum 14 days). Females were also exposed during the gestation period and up to post-natal day (PND) 12 (BSL Bioservice / Eurofins, 2020). During the study the animals were observed for mortality, clinical signs, functional observations, food consumption and body weights on a regular basis; haematological, clinical biochemistry and urinalysis evaluations were performed at terminal sacrifice; reproductive function and effects on reproductive organs were evaluated for males and females; litter observations were performed including the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities; anogenital distance of pups was also measured; all animals were examined for macroscopic lesions at gross necropsy; organ weights were recorded and a full histopathological evaluation was performed on preserved tissues.

No treatment-related mortality was observed for any of the test groups. No clinical signs indicating systemic toxicity were observed in any of the animals. No test item-related effects were observed on the parameters of the functional observation battery or body temperature of male and female animals and no relevant effects were noted on body weight development or food consumption in any of the dose groups. The test item had no toxicologically relevant effect on coagulation parameters, haematological parameters or parameters of clinical biochemistry of male and female animals. The test item had no toxicologically relevant effects on urinary parameters analysed in selected animals at the end of the treatment period. No effects on organ weights were reported for females. Histopathological treatment-related changes were observed in the stomachs of males, which were considered treatment-related and adverse for both sexes.

No treatment-related effects were observed on the oestrous cycle, pre-coital interval and duration of gestation, reproductive indices or pre- and post-natal parameters. Slightly dose-dependent trend towards higher pre-implantation loss in test item-treated groups was not considered toxicologically relevant as the data were within the historical control data range.

No toxicologically relevant effects in litter observations and pup survival were noted. No toxicologically relevant external gross abnormalities were observed in the pups of test item-treated groups. Statistically significantly shorter anogenital distance was observed in male pups of the MD and HD group and mean nipple retention of male pups was found to be statistically significantly higher in the HD group. Female pups of the HD group showed a statistically significantly shorter anogenital distance. Even though, these changes were within the historical control data range, they could indicate possible endocrine disruption. However, no effects were observed in other parameters such as histopathology of parental reproductive organs and their weights, litter size, sex ratio, pup thyroid /parathyroid weight and thyroxine hormone (T4) to support a possible endocrine disruption modality of the test item. The study concluded the NOAELs for reproductive and developmental toxicity to be ≥90 mg/kg bw/day (the highest dose tested) based on no adverse effects on reproduction and developmental parameters. The high dose was limited to 90 mg/kg bw/day based on severe local effects observed in a 14-day range finding study at doses of 100 mg/kg bw/day and above (BSL Bioservice, 2019).

Justification for classification or non-classification

Based on the available data, no classification is required for reproductive and developmental toxicity for tert-butyl(chloro)dimethylsilane according to Regulation (EC) No 1272/2008.