Ethoxytrimethylsilane

Endpoint summary

Description of key information

Oral

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422), rat: NOAEL (systemic) = 150 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 May - 06 Nov 2017

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:

29 July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, approximately 9 - 10 weeks old at start of the study (11 - 12 weeks old at start of treatment and 13 - 14 weeks old at mating)
- Weight at study initiation: Males: 373 - 484 g, females: 244 - 316 g at the start of the treatment
- Housing: Rodents were group-housed, up to 2 animals of the same sex and dose group/cage (Type II polycarbonate) with the exception of the mating and gestation/delivery period when they were paired or individually housed (with pups), respectively
- Diet: ssniff SM R/M "Autoclavable complete diet for rats and mice - breeding and maintenance" provided ad libitum
- Water: tap water from the municipal supply provided ad libitum
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY:
The food is considered not to contain any contaminants at levels that could reasonably be expected to affect the purpose or integrity of the study.
Water quality control analysis was performed at least once every three months and microbiological assessment is performed monthly.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 - 23.4
- Humidity (%): 30 - 71
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle (as a visibly stable homogenous suspension). Formulations were prepared at appropriate concentrations according to the dose level and treatment volume selected.
Formulations were prepared at given intervals (formulations were stored at room temperature in tightly closed glass containers for not more than 5 days before use) as follows. The calculated volume of the vehicle and the calculated amount of test item were added into a beaker; they were mixed vigorously by a vortex or repeated manual inversion to make a homogenous formulation. Glass containers were closed immediately after preparation. Based on the
analytical method validation study formulations in the 10 - 250 mg/mL concentration range were found to be stable after 7 days of storage at room temperature.

VEHICLE
- Concentration in vehicle: 12.5, 37.5, 125 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no.: A0381515 / A0384372

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test item formulations for concentration and/or homogeneity was performed at the Analytical Department of the Test Facility using a validated GC-FID (Gas Chromatography – Flame Ionization Detector) method. Duplicate samples were taken from the top, middle and bottom of the test item formulations three times during the study (during the first and approximately last weeks of treatment and approximately midway during the treatment period), one set to analyse (which could be collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement. Formulation samples were kept at room temperature in a closed glass or plastic container until analysis.
The measured test item concentrations of the individual test item containing dose formulations varied between 93% and 101% of their nominal concentrations. No test item was detected in the control samples. These results were within the acceptable ranges (85% - 115%) and were considered suitable for the study purposes.
All test item formulation samples were found to be homogeneous. Acceptance criteria of the homogeneity was that the coefficient of variation (CV%) of replicates (top, middle and bottom of test item formulations) must be less than 10%.
Formulations were considered to be adequately stable under the study conditions (stability period of 7 days).

Duration of treatment / exposure:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating) and then euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, during the mating period, throughout gestation and until the day before the necropsy (13-day post-partum dosing).

Frequency of treatment:

once daily, 7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on available data including the results of an OECD TG 414 study with trimethyl silanol (CAS 1066-40-6) in rats and a 14-day (repeated dose) Dose Range Finding (DRF) study in the rat performed at the Test Facility. For the DRF, the test material was administered by oral gavage to Wistar rats for 14 consecutive days at dose levels of 100, 250 and 600 mg/kg bw/day. At 600 mg/kg bw/day, test item-related clinical signs (ataxia and splayed gait) and test item-related body weight loss in the first days of treatment, with an overall body weight gain of less than 50% of the controls, were observed in male and female animals. There were no clearly adverse effects in clinical pathology, organ weights or at necropsy. No clear effects were observed at 250 mg/kg bw/day. Based on these results, it was considered that the high dose may be above the maximum tolerated dose (MTD).
- Rationale for animal assignment: All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges before the pre-exposure period.
- Fasting period before blood sampling for clinical biochemistry: All animals were fasted overnight.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were made once a day (except the days when detailed clinical observations were made).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: More detailed examinations were performed once before the start, before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment.
- The animals were monitored outside the home cage in a standard arena for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed with an accuracy of 1 g for randomisation purposes, then at least weekly during the pre-exposure period, on Day 0, afterwards at least weekly and at termination. Parental females were weighed on gestation days (GD) 0, 3, 7, 10, 14, 17 and 20 and on post-partum days (PPD) 0 (within 24 hours after parturition), 4, 7, 10, 13 and 14 (before termination). The body weight of the parental female animals measured on GD3, 10 and 17 were only additional measurements as aid for the calculation of accurate treatment volumes, but these data were not evaluated statistically.

FOOD CONSUMPTION: Yes
- Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g weekly.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples from dedicated (randomly selected) animals were collected immediately prior to scheduled necropsy.
- Anaesthetic used for blood collection: Yes; pentobarbital anaesthesia
- Animals fasted: Yes; animals were fasted (overnight period of food deprivation)
- How many animals: 5 males and 5 females/group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: blood samples from dedicated (randomly selected) animals were collected immediately prior to scheduled necropsy.
- Animals fasted: Yes; animals were fasted (overnight period of food deprivation)
- How many animals: 5 males and 5 females/group
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine sampling was performed prior to necropsy
- Metabolism cages used for collection of urine: Yes; animals placed in metabolism cages for 16 hours
- Animals fasted: Yes; animals were fasted (overnight period of food deprivation)
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males on Day 26; females on PPD9 - PPD12
- Dose groups that were examined: all dose groups
- Battery of functions tested: Selected animals were subjected to the functional observation battery including quantitative assessment of grip strength and measurement of landing foot splay and fore/hind limb grip strength.

IMMUNOLOGY: No

OTHER: For thyroid hormone analysis, blood samples were taken by cardiac puncture or venepuncture into tubes containing K3-EDTA as anticoagulant as follows: from up to two pups per litter on PND4, from all dams at termination (PPD14), and from all adult males at termination.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; gross necropsy was performed on all animals. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system. Additionally, at the time of termination, the weight of the following organs from all adult animals were recorded: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus, adrenals, ovaries, and thyroids with parathyroids. Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights.

HISTOPATHOLOGY: Yes (see table 4). For the adult animals, a detailed histological examination was performed as follows: on the selected list of retained organs in the control and high-dose groups (selected 5 animals/sex/group); the trachea and lungs of one low-dose female because macroscopic findings (abnormalities) were seen at necropsy; all kidney samples of mid- and low-dose males, plus the remaining control and high-dose males; and retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the control and high-dose groups, and of all males and all females that failed to sire / conceive or unable to delivering healthy pups.
Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Other examinations:

Oestrus cycle monitoring
Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the treatments starts. Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating.

Statistics:

SPSS PC+4.0 program: The heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If a significant result was obtained, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of non-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. The Chi-squared test was used for non-continuous data.

SAS v9.2 software package: The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests. Where both tests showed no significant heterogeneity, an ANOVA / ANCOVA (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess inter-group differences; identifying differences of <0.05 or <0.01 as appropriate.

If either of the Shapiro-Wilk or Levene tests showed significance, a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate. For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control.

For pathology data, first a Chi-squared test was used; the system then checked the significance of the result against a user-defined value (0.05) and where suitable, also performed pairwise tests. The Fisher’s Exact Test was performed replacing the Chi-squared test as the final evaluation method if the group size was <5.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment related clinical signs were observed during the study in all high-dose (500 mg/kg bw/day) males and females. Treatment-related effects consisted of splayed gait, which was observed in all high dose males from Day 2 to Day 27. High-dose females displayed splayed gait from Day 2 until the end of the lactation period. Slightly or moderately decreased activity was recorded for 4 high-dose males on Day 14 and/or Day 15 and for 1 high-dose female on Day 14.
No clinical signs were observed for mid- (150 mg/kg bw/day) and low-dose (50 mg/kg bw/day) animals.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test item related effect (reduction) on body weight gain was detected during the study in the high-dose males (500 mg/kg bw/day), while no such effect was observed in high-dose females or in other groups (Table 5).
There were no statistically significant differences in the absolute body weight values of the control and test item treated groups.

A greater than 40% reduction in the body weight gain of high-dose males was detected on the first week of treatment compared to the control data (the difference was statistically significant at p<0.01). Although the animals showed recovery, the reduction (by 17%) in the total body gain during the entire treatment period (Days 0-27) was still statistically significant (p<0.05) compared to control. No similar effect was seen in high-dose females or in mid- or low-dose groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test item related reduced food consumption was observed only in the first week of treatment in high-dose males and females (500 mg/kg bw/day). There were no test item related differences in the mean daily food consumption of mid- or low-dose groups (Table 6).
A 16% and 8% reduction in food consumption was noted in the high-dose males and females, respectively in the first week of treatment. For males, the difference compared to the control value was statistically significant at p<0.01; for females the difference compared to the control value was statistically significant at p<0.05 level). However, it was only a transient effect, the animals recovered quickly and food consumption returned to the control level, and no statistically significant difference was recorded in the high-dose males at the end of the treatment period, or in the high-dose females in the gestation or lactation period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no differences in males and females of the test item treated groups compared to control that were statistically significant or could be considered toxicologically significant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Significantly increased urea concentration was observed in high-dose males (500 mg/kg bw/day), this finding might be test item related and connected to the kidney findings recorded during histopathology evaluation (Table 7). There were no other changes in the evaluated serum chemistry parameters that could be ascribed to the test item administration.
Statistically significant increase (p<0.05) in urea concentration was seen in high-dose males (500 mg/kg bw/day) when compared to the control. However, the observed values were within the historical control range. No significant change was seen in mid- or low- dose males, or in any female groups (although it should be noted that values for all groups including control showed higher mean value than the upper limit of the historical control range). There were no statistically significant changes in other parameters (creatinine, calcium, potassium, phosphate and sodium; although creatinine in high-dose males decreased by 12% compared to control), but the relationship to test item treatment is plausible based on the observed histopathology findings in the kidney samples of high-dose males. No statistically significant changes were recorded for any other parameters in the test item treated males and females.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Significantly (p<0.01) lower urinary pH was detected in high-dose males when compared to control, the mean pH was close to the lower end of the historical control rage (Table 8). The specific gravity of the urine of high-dose males was significantly higher than control (p<0.05). Although the absolute difference was small in this case, but slight or moderate presence of protein in the urine was noted in all high-dose male samples while 3/5 of the control samples were negative and two control samples showed slight protein content. These findings may be related to the test item treatment based on the observed histopathology findings in the kidney samples of high-dose males. The pH of the mid-dose male urine samples was also significantly (p<0.05) lower than control in case of mid dose males. No similar changes in pH or specific gravity were seen in the low dose males, or high-, mid- or low-dose females.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Although splayed gait was observed for all high-dose animals after dosing, the signs recovered each day by the afternoon. There were no test item related effects in the neurological assessment as there were no observed differences in animal behaviour, general physical condition or in the reactions to different types of stimuli in the control or test item treated groups.
No test item related effect was noted in the Irwin test.
No statistically significant or biologically relevant differences were noted during the quantitative assessment of landing foot splay test or grip strength of any dose groups when compared to control during the last week of treatment.
In case of motor activity measurements (SMART), all dose groups of males and females had a normal locomotor activity; in all cases the initial activity was high, with generally reduced activity in the 5-minute periods to an approximate plateau by about 20-30 minutes. The high-dose females showed slightly lower data than control females (due to one control female showing higher than usual activity), but there was no statistical significance between dose group when evaluating the total travelled distance (period of 0-60 minutes). Despite the observation of splayed gait (shortly after treatment each day) the quantitative assessment of activity did not show any clear effect of treatment on movement, although the high-dose males and females had slightly low activity in the first 5-minute period. Taking into account the single female control animal with a high activity, and the lack of a clear dose effect, the single statistical difference was considered as not being a test item related effect.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item related increased relative kidney weights were observed in high-dose males by about 25% relative to body weight (Table 9 and 10). These findings were considered to be related to the observed microscopic changes of the affected animals. No differences compared to the control were seen in high-dose females or mid- or low-dose males and females.

The absolute and relative (to body and brain) kidney weights were statistically significant higher than control in the high-dose males (these included increases by 19.1% (p<0.01) absolute, by 24.6% (p<0.01) adjusted to body and by 20.9% (p<0.01) brain related). No similar effect was seen in mid- and low-dose males. Histopathology of the kidney supported an effect of the treatment with the test item, while no test item-related organ weight changes and microscopic findings were observed in females’ kidney.

Additionally, there were statistically significant higher relative (to body and brain) seminal vesicle (with coagulating gland) weights in the high-dose group, but the observed values were within the historical control range, these differences were not confirmed by histopathological findings and were therefore considered to not reflect an effect of the test item.

There was a statistically significant (p<0.05 or 0.01) decrease in the absolute and/or body or brain adjusted spleen weights in low-, mid-, and high-females, but this inconsistent decrease (without dose response) did not suggest treatment-related effects and was not confirmed by microscopic findings in examined high-dose females. Furthermore, the male animals showed opposite trend (approximately 12% increase in the high-dose group without statistical significance). Therefore, these changes were considered as not being test item related.

There were no other treatment-related statistically significant differences among groups in the weights of organs measured when compared to controls.

There was no effect on absolute and relative (to body) thyroid gland weights.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related macroscopic findings were noted at necropsy.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related findings were observed in the kidney of the high-dose males (500 mg/kg bw/day). These findings correlated with increase of organ weights. No test item-related changes were seen in high-dose females.

Minimal to moderate eosinophilic droplets within the cortical tubules (cytoplasm and lumen) were present in 5/5 examined high-dose males. Droplets were mainly multifocal and bilaterally distributed in the cortex. Droplets tended to be rounded form or polyangular of varying size. In 3/5 of these males, minimal to slight focal/multifocal unilateral/bilateral tubule degeneration (with some regenerated
tubules) was developed. The presence of degeneration suggests the effect in high-dose males was adverse.

All other changes are regarded as incidental or a common background and included: minimal to slight extramedullary haematopoiesis in the spleen of five control and seven high-dose animals; minimal inflammatory cell infiltrate in the prostate of two control, one low-dose and one high-dose male; minimal inflammatory cell infiltrate in the heart of one high-dose male; minimal hepatocellular macrovesicular vacuolation in the liver of a control male; severe tubular atrophy and degeneration in the left testis and moderate ductal atrophy in the left epididymis of a high-dose male; and moderate oedema in the lungs of a low-dose female.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis:
No endocrine disruptor effect of test item was observed in the study based on the results of thyroid hormone analysis, thyroid gland weights and available histopathology data.
Compared to the relevant control values, statistically significant decrease (by 20%, p<0.01) in the thyroxine hormone (T4) concentration was noted for parental males. However, the mean value of parental males was within the historical control range. The difference was most probably caused by one low value (28.7 ng/mL, outside the historical control range of 34.3-60.7 ng/mL) in the high-dose group.

Oestrus cycle:
No effect of test item on oestrus cycles was noted.
Each female selected for the treatment showed acceptable cycles (mean cycle length was in the range of 3.97-4.00 days for each group) before starting the treatment period. There was no effect on test item on the oestrus cycle of females (mean cycle length was in the range of 3.92-4.15 days for each group after the treatment started).

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 5: Selected body weight parameters of parental animals

	Dose groups
Parameters	Control	Low dose	Mid dose	High dose	Statistics
Male, Body weight on Day 27 (g)	510.3	514.3	497.1	490.6	NS
	difference (%)	0.8	-2.6	-3.9
Male, Body weight gain Day 0-7 (g)	27.0	29.2	25.5	15.0**	DN
	difference (%)	8.0	-5.6	-44.4
Male, Body weight gain Day 0-27 (g)	87.0	88.8	77.9	72.1*	DN
	difference (%)	2.1	-10.4	-17.1
Female, Body weight on GD20 (g)	421.4	425.6	451.6	410.5	NS
	difference (%)	1.0	7.2	-2.6
Female, Body weight on PPD13 (g)	378.5	385.4	402.6	374.4	NS
	difference (%)	1.8	6.4	-1.1
Female, Body weight gain Day 0- PPD13 (g)	98.7	111.8	120.9*	97.9	DC
	difference (%)	13.3	22.5	-0.8

Table 6: Selected food consumption parameters of parental animals

	Dose groups
Parameter	Control	Low dose	Mid dose	High dose	Statistics
Male, Daily food consumption Day 0-7 (g/day)	29.17	29.44	27.68	24.56**	D
	difference (%)	0.9	-5.1	-15.8
Male, Daily food consumption Day 0-27 (g/day)	27.38	27.81	27.13	26.73	NS
	difference (%)	1.6	-0.9	-2.3
Female, Daily food consumption Day 0-7 (g/day)	18.87	19.04	19.19	17.33*	DN
	difference (%)	0.9	1.7	-8.1
Female, Daily food consumption, gestation period GD0-20 (g/day)	23.29	23.87	25.64	24.27	NS
	difference (%)	2.5	10.1	4.2
Female, Daily food consumption, lactation period PPD0-13 (g/day)	50.22	56.61	60.41	48.92	NS
	difference (%)	12.7	20.3	-2.6

Table 7: Summary of selected clinical chemistry parameters

Parameters	Dose groups
	Control	Low dose	Mid dose	High dose	Statistics
Males
Urea (mmol/L)	5.72	5.76	6.16	7.29*	DN
HC range: 4.01-9.89	difference (%)	0.7	7.5	27.4
Females
Urea (mmol/L)	14.89	13.26	13.71	13.57	NS
HC range: 4.44-11.18	difference (%)	-11.0	-7.9	-8.9

Table 8: Summary of selected urinary analysis parameters

	Dose groups
Parameter	Control	Low dose	Mid dose	High dose	Statistics
Males
pH	7.6	7.0	6.4*	6.2**	DN
HC range: 6.0-8.0	difference (%)	-7.9	-15.8	-18.4
Specific gravity	1.006	1.009	1.012	1.017*	DN
HC range: 1.01-1.04	difference (%)	0.3	0.6	1.1
Females
pH	6.4	6.4	6.2	6.2	NS
HC range: 6.0-8.0	difference (%)	0.0	-3.1	-3.1
Specific gravity	1.020	1.022	1.021	1.019	NS
HC range: 1.01-1.05	difference (%)	0.2	0.1	-0.1

 

Table 9: Selected organ weight data (males)

	Dose groups
Organ weight	Control	Low dose	Mid dose	High dose	Statistics
Terminal body weight, g	490.4	493.1	478.2	467.9	NS
	(difference %)	0.5	-2.5	-4.6
Kidney (absolute), g	3.163	3.261	3.368	3.766**	DN
	(difference %)	3.1	6.5	19.1
Kidney (relative to body), %	0.645	0.662	0.706	0.804**	DN
	(difference %)	2.6	9.5	24.6
Kidney (relative to brain), %	143.3	146.9	147.6	173.2**	DN
	(difference %)	2.5	3.0	20.9
Seminal vesicle (absolute), g	2.603	2.739	2.903	3.040	NS
	(difference %)	5.3	11.5	16.8
Seminal vesicle (relative to body), %	0.530	0.559	0.608	0.655**	DN
	(difference %)	5.4	14.6	23.4
Seminal vesicle (relative to brain), %	118.2	123.4	127.2	140.1*	DN
	(difference %)	4.5	7.7	18.5
Thyroid (absolute), g	0.0289	0.0278	0.0278	0.0253	NS
	(difference %)	-3.7	-3.7	-12.7
Thyroid (relative to body), %	0.0059	0.0056	0.0058	0.0054	NS
	(difference %)	-4.2	-1.0	-8.5
Thyroid (relative to brain), %	1.313	1.257	1.220	1.159	NS
	(difference %)	-4.3	-7.1	-11.7

 

 

Table 10: Selected organ weight data (females)

	Dose groups
Organ weight	Control	Low dose	Mid dose	High dose	Statistics
Terminal body weight, g	349.6	355.9	369.1	351.5	NS
	(difference %)	1.8	5.6	0.5
Kidney (absolute), g	2.175	2.137	2.268	2.248	NS
	(difference %)	-1.7	4.3	3.4
Kidney (relative to body), %	0.623	0.602	0.615	0.639	NS
	(difference %)	-3.3	-1.3	2.6
Kidney (relative to brain), %	104.2	104.4	108.3	109.8	NS
	(difference %)	0.1	3.9	5.3
Spleen (absolute), g	0.982	0.807**	0.861	0.847*	DN
	(difference %)	-17.8	-12.3	-13.7
Spleen (relative to body), %	0.282	0.227**	0.233*	0.240*	DN
	(difference %)	-19.4	-17.3	-15.0
Spleen (relative to brain), %	47.07	39.46*	41.02	41.39	DN
	(difference %)	-16.2	-12.9	-12.1
Thyroid (absolute), g	0.0224	0.0198	0.0199	0.0231	NS
	(difference %)	-11.6	-11.1	3.1
Thyroid (relative to body), %	0.0064	0.0055	0.0054	0.0067	NS
	(difference %)	-13.9	-16.0	4.3
Thyroid (relative to brain), %	1.075	0.968	0.953	1.131	NS
	(difference %)	-10.0	-11.4	5.3

 

Statistical significance compared to control: * = p<0.05, ** = p<0.01
DN: Dunnett’s Multiple Range Test; DC: Duncan’s Multiple Range Test; NS: Statistically not significant when compared to control

Conclusions:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD TG 422 and in compliance with GLP, rats were exposed to the test substance at 50, 150, or 500 mg/kg bw/day by gavage. The NOAEL was 150 mg/kg bw/day based on clinical signs, urinary analysis, kidney organ weight and histopathology.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A reliable combined repeated dose oral toxicity study with the reproduction/developmental screening study according to OECD 422 and in compliance with GLP is available for ethoxy(trimethyl)silane in Wistar rats (Citoxlab, 2019). The test substance was administered in corn oil as vehicle at dose levels of 0, 50, 150, or 500 mg/kg bw/day. The control animals received the vehicle only. Male and female rats were treated for 2 weeks pre-mating and then during the mating / post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation day (PPD) 13.

 

No mortality was observed in the study. Treatment-related transient daily clinical signs (splayed gait and/or slightly decreased activity) were observed during the study at 500 mg/kg bw/day in both males and females; these findings were not observed at 150 mg/kg bw/day or 50 mg/kg bw/day.

Test item-related reduced food consumption was observed in the first week of treatment at 500 mg/kg bw/day in males and females. The effect was transient, but it caused significantly reduced body weight gain in high-dose males but not females (500 mg/kg bw/day), and was considered as an adverse effect for male animals.

No test item-related effects were observed in haematology parameters and during the quantitative neurotoxicity assessment in the last week of treatment.

The test material appeared to target the urinary system in high-dose males, specifically the kidneys. At 500 mg/kg bw/day, significantly increased blood urea concentrations, as well as significantly lower pH and slightly increased specific gravity of the collected urine samples was observed in males. The urinary pH was also significantly lower in mid-dose males (150 mg/kg bw/day). High-dose males also exhibited increased relative kidney weights. Histopathology of the kidney revealed minimal to moderate eosinophilic droplets within the cortical tubules (cytoplasm and lumen) and minimal to slight focal/multifocal unilateral/bilateral tubule degeneration in males at 500 mg/kg bw/day. These microscopic changes corresponded with an increase in kidney weights; they may be associated with differences in the urine parameters and urea in affected males. Degeneration may represent a reversible change but was considered to be an adverse finding.

 

In conclusion, under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for general toxicity of ethoxy(trimethyl)silane was considered to be 150 mg/kg bw/day (based on clinical signs, urinary analysis and kidney organ weight and histopathology).

Justification for classification or non-classification

The available data on repeated dose toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.