tert-butylchlorodimethylsilane

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 29th July 2016

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

tert-butylchlorodimethylsilane

EC Number:

242-042-4

EC Name:

tert-butylchlorodimethylsilane

Cas Number:

18162-48-6

Molecular formula:

C6H15ClSi

IUPAC Name:

tert-butyl(chloro)dimethylsilane

Test material form:

solid

Test animals

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: 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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral 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 the 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.

Duration of treatment / exposure:

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.

Frequency of treatment:

Daily administration, 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Low dose, LD

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Middle dose, MD

Doses / concentrations 4

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 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

Examinations

Observations and examinations performed and frequency:

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 thereafter, at least once a week. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter 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 post-partum) as well as 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/females or during the post-mating period in males.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: ophthalmoscopy examination (anterior chamber of the eye and fundus of eye) was included in the functional observations that occurred in the week before the first treatment, during the last week of the treatment and during the last week of the lactation period.
- Dose groups that were examined: 5 randomly selected males and 5 randomly selected females (only lactating) of each group outside their home cage.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment, prior to or as part of the sacrifice of the animals.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 5 randomly selected males and females (only lactating) from each dose group.
- Parameters checked in table were examined: yes, see table 1.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment prior to or as part of the sacrifice of the animals.
- Animals fasted: not specified
- How many animals: 5 randomly selected males and females (only lactating females) of each group
- Parameters checked in table were examined: yes, see table 1

URINALYSIS: Yes
- Time schedule for collection of urine: prior to or as part of the sacrifice of the animals.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table were examined: yes, see table 1.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in the week before the first treatment and during the last week of the treatment
- Dose groups that were examined: All animals were examined during the week before the first treatment; 5 randomly selected males and females (only lactating females) of each group were examined during the last week of treatment.
- Battery of functions tested: Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, see Table 2

HISTOPATHOLOGY: Yes, see Table 2

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 homogeneity 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).

Results and discussion

Results of examinations

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 the clinical findings of apathy, reduced spontaneous activity, abnormal breathing dehydration, piloerection, 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.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the control group was euthanized in moribund condition. In the histopathological examination, necrotizing 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 comparable 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

Description (incidence and severity):

No toxicologically relevant effect was 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 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. In addition, 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 as 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 histopathological 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, 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.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects observed.

2

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

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Applicant's summary and conclusion

Conclusions:

In a 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, a NOAEL for systemic effects was concluded to be ≥90 mg/kg bw/day based on no adverse effects observed. A local LOAEL of 10 mg/kg bw/day was also concluded based on local effects in the stomach.