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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In the key extended one-generation reproductive toxicity study, conducted according to OECD Test Guideline 443 and in compliance with GLP, the NOAEL for general systemic toxicity for P0, P1 (F1 Cohort 1B) and F1 (Cohort 1A) animals was concluded to be 40 mg/kg bw/day based on test substance related effects in the urinary system at 100 and 300 mg/kg bw/day; the reproductive toxicity NOAEL for P0 and F1 (Cohort 1A and 1B) animals was at least 300 mg/kg bw/day based on no adverse effects on male and female reproductive organs, on oestrous cycle and sperm parameters, and male and female fertility and performance; the developmental toxicity NOAEL for F1 (Cohort 1A and 1B) and F2 (Cohort 1B extension) was at least 300 mg/kg bw/day based on no adverse effects on pre- and post-natal development; the developmental immunotoxicity NOAEL for F1 (Cohort 3) was at least 300 mg/kg bw/day based on no adverse effects on immune system development and response (BSL Bioservice, 2021).

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity – with F2 generation and developmental immunotoxicity (Cohorts 1A, 1B with extension, and 3)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 November 2019 to 21 October 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals : 2 weeks as requested in ECHA Final Decision No CCH-D-2114405896-41-01/F.
- Basis for dose level selection : Based on the findings of a 28-day dose range finding study (BSL Bioservice, 2020) and a combined repeated dose and reproduction/developmental toxicity screening test (Hashima Laboratory, 2005) with trimethoxy(vinyl)silane. The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
- Inclusion/exclusion of extension of Cohort 1B: Included, because it was requested in ECHA Final Decision No CCH-D-2114405896-41-01/F.
- Termination time for F2 : F2 animals were terminated at weaning (PND 22) according to OECD Test Guideline 443.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : Not included, because they were not requested in ECHA Final Decision No CCH-D-2114405896-41-01/F and based on no evidence of neurotoxicity in the available data for trimethoxy(vinyl)silane.
- Inclusion/exclusion of developmental immunotoxicity Cohort 3 : Included, because Cohort 3 was requested in ECHA Final Decision No CCH-D-2114405896-41-01/F.
- Route of administration : The oral gavage route was selected based on test item characteristics and stability.
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han) (Full Barrier)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 12-13 weeks old; (F1) x wks
- Weight at study initiation: (P) Males: 268 - 317 g; Females:163 - 206 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: no
- Housing:
Pre-mating period of P and F1: male and female animals were housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages.
Mating period: During mating period males and females (Parental and F1) were housed together in ratio 1:1 (male to female)
Post-mating period: during post-mating period males (Parental and F1) were housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages.. After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages.
- Diet (e.g. ad libitum): Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least five days

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

IN-LIFE DATES: From: 6th November 2019 To: 3rd April 2020
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulations were vortexed and/or stirred until visual homogeneity was achieved.
After homogenization the formulation was overlaid with argon to prevent hydrolysis caused by contact of the test item formulation with water.
Based on the results of stability testing, the test item formulations were prepared at least every 12 days. The prepared formulations were stored at room temperature and overlaid with argon.


VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle for test item formulations had been selected in consultation with the sponsor based on the test item’s characteristics.
- Concentration in vehicle: 0, 10, 25, 75 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no. (if required): MKCH1635
Details on mating procedure:
- M/F ratio per cage:
Parental animals: 1:1 pairing
Cohort 1B animals: 1:1 pairing
- Length of cohabitation: until pregnancy occurred or up to 2 weeks
- Proof of pregnancy: vaginal sperm or a vaginal copulation plug referred to as day 0 of pregnancy
- In case mating had not occurred after 2 weeks, the animals were separated without further opportunity for mating.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability analysis: conducted before the study start on the samples from HD and LD groups and the investigation was made for 0 h, 6 h (RT), 12 days (RT), 12 days (2 to 8 °C) and 11 days (-15 to -35 °C). The test item formulations were stable for up to 12 days at room temperature.
- Homogeneity: conducted before the study start on the samples collected from various levels (top, middle and bottom) of HD and LD groups. Based on the results from the setup the test item formulation was a suspension. The test item was shown to be homogenous after 30 min without stirring, samples were not collected during the study for the investigation of homogeneity.
- Concentration analysis: duplicate samples were taken for substance concentration analysis. Concentration analysis of formulation samples was performed at three concentrations, 10.00 mg/mL, 25.00 mg/mL and 75.00 mg/mL in study weeks 1, 5, 9, 13 and in the last week of the study.
Duration of treatment / exposure:
- Parental males: dosed until the minimum total dosing of 10 weeks, i.e. during 14 days of pre-mating, maximum 14 days of mating and post-mating until terminal sacrifice.
- Parental females: dosed during 14 days of pre-mating, maximum 14 days of mating, during gestation and lactation until weaning (PND 21).
- F1 offspring: received treatment with the test item from weaning (PND 21) to terminal sacrifice of the respective cohort.
- Cohort 1A animals (F1 generation): treated from weaning to week 13 of age (10 weeks treatment), sacrifice at the age of 13 weeks
- Cohort 1B animals (F1 generation): treatment until weaning of the F2, or until termination of the study (week 20 - 25)
- Cohort 3 animals (F1 generation): treatment from weaning to week 8-10 of age (5-7 weeks treatment), sacrifice at the age of 8 -10 weeks.
Frequency of treatment:
daily
Details on study schedule:
- F1 parental animals not mated until PND 90 after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 90 days
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control group (corn oil); Group 1
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Remarks:
Low dose group (LD); Group 2
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Mid dose group (MD); Group 3
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
High dose group (HD); Group 4
No. of animals per sex per dose:
- Parental animals: 25 male and 25 female animals per group
- Cohort 1A: 20 M + 20 F / Group
- Cohort 1B: 20 M + 20 F / Group
- Cohort 3: 10 M + 10 F / Group

Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In consultation with the sponsor and based on the results of a 28-day dose range-finding study (BSL Bioservice, 2019). The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and NOAEL. In the dose range finding study male and female Wistar rats were treated with the test item at doses of 0 (vehicle), 50, 150, 600 and 1000 mg/kg bw/day. No mortality or marked clinical signs and no effects of the test item on functional observations, body weight development, food consumption, clinical biochemistry or urinary parameters were observed up to the highest tested. Test item-related increases in haematological parameters were observed in reticulocytes of males at =600 mg/kg bw/day and females at =150 mg/kg bw/day and in monocytes of males and females at =150 mg/kg bw/day. Test item-related lesions were observed in the kidneys (enlargement, renal pelvis dilatation), ureters (dilatation) and urinary bladder (thickened wall). Adverse histopathological treatment-related changes were observed in organs of the urinary system (i.e. urothelial hyperplasia, inflammatory reactions, liminal precipitate(s), granular casts, tubular dilatation, and tubular basophilia) in animals at =150 mg/kg bw/day, and in the small intestine with regional lymphoid tissues (lipid accumulation) in animals at =600 mg/kg bw/day.
In a combined repeated dose and reproduction/developmental toxicity screening test (Hashima Laboratory, 2005, OECD 422) male and female Sprague-Dawley rats were treated with the test item at doses of 0 (vehicle), 62.5, 250, and 1000 mg/kg bw/day. Two males and 1 female from the 1000 mg/kg group died on days 16 and 17 (males) and day 8 (female). Transient salivation, soiled hair, a decrease in locomotor activity, reddish urine, hypothermia, perioral smudges, perianal soiling, diarrhoea, bradypnea, and piloerection were noted in the dying animals. Transient salivation, soiled hair and reddish urine were noted in the surviving males and females at =250 mg/kg bw/day. Lower body weights were noted in males on days 4 to 42 during the administration period and days 1 and 4 during the recovery period. Females at 1000 mg/kg bw/day also showed a decrease in body weight.
In females, the number of oestrous cases before pairing at 1000 mg/kg bw/day was less than in the control group. In males, moderate atrophy of seminiferous tubule, slight degeneration of seminiferous tubule, slight vacuolization of Sertoli cell and slight retention of spermatid were observed at 1000 mg/kg bw/day. A slight decrease in sperm and slight or moderate cell debris were observed in lumen of the epididymis at 1000 mg/kg bw/day.
No changes were observed in reproductive parameters.
Body weight of males at 1000 mg/kg bw/day was increased after the administration period. No change of body weight was detected in males after the recovery period and no changes in body weight were detected in females after the administration period.
Decrease in absolute weight of thymus, increase in absolute weight of kidney, increase in relative weight of spleen, kidney and adrenals were observed in males at 1000 mg/kg bw/day after the administration period. Relative weight of thymus was decreased in females at 62.5 and 250 mg/kg bw/day. Decreased absolute weight of thymus, decreased relative weight of thymus and increased relative weight of liver were observed in females after the administration period.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: yes, fasted overnight
- Cohort 1A animals (F1 generation): used to assess effect on reproductive system and toxicity; treated from weaning to week 13 of age (10 weeks treatment), sacrifice at the age of 13 weeks
- Cohort 1B animals (F1 generation): used to assess reproductive performance, additional histopathology data or endocrine toxicity; treatment until weaning of the F2, or until termination of the study (week 20 - 25)
- Cohort 3 animals (F1 generation): used to assess developmental immunotoxicity; treatment from weaning to week 8-10 of age (5-7 weeks treatment), sacrifice at the age of 8 -10 weeks.

Positive control:
Cohort 3: T-Cell Dependent Antibody Response Assay positive control group was administered oral gavage dose of 10 mg/kg bw/day cyclophosphamide (administration volume of 10 mL/kg bw) 7 days before immunization (i.e. PND 49 ± 3 days) until the day before the last blood sampling. Cyclophosphamide was diluted with 0.9 % sodium chloride to a concentration of 1 mg/mL.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: For Parental and selected F1 cohorts, general clinical observations of the animals were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. Twice daily all animals (Parental and F1 generation) were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily. Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality were recorded.
- Cage side observations included: health condition (once daily); morbidity and mortality (twice daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed clinical observations were made in all P animals and all F1 cohorts, when animals were weighed outside the home cage. 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:
- Parental animals (P0): weighed once before the assignment to the experimental groups, on the first day of dosing and weekly during the study period as well as at the terminal sacrifice. In addition, during pregnancy, females were weighed on gestation days (GD) 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 and within 24 hours of parturition (day 0 post-partum) as well as on day 4, 7, 14 and 21 post-partum along with pups.
- F1 (all cohorts) animals: F1 animals were weighed every two days during the first 2 weeks following weaning, weekly thereafter and at termination. In addition, F1 females from Cohort 1B were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as on day 4, 7, 14 and 21 post-partum along with F2 pups. Body weight of all cohorts was also taken on the day of attainment of vaginal patency or balano-preputial separation. Body weight was also measured on PND 4, at weaning (surplus pups after selection of cohorts) and weekly until terminal sacrifice.

FOOD CONSUMPTION: Yes
Time schedule for examinations: Food consumption was measured at intervals corresponding to the body weight measurements after the beginning of the dose administration except during the mating period for Parental and Cohort 1B F1 animals. Food consumption was also not measured during the post-mating period in Parental and Cohort 1B F1 males until all females were mated and all males were back to their original housing cage of 5 males per cage.

HAEMATOLOGY
Time schedule for examinations: at the end of the treatment prior to sacrifice
How many animals: 10 randomly selected Parental males and females
Fasted overnight: yes
Parameters examined: see tables 3 & 4

CLINICAL BIOCHEMISTRY
Time schedule for examinations: at the end of the treatment prior to sacrifice
How many animals: 10 randomly selected Parental males and females
Fasted overnight: yes
Parameters examined: see table 5

THYROID HORMONE ANALYSIS
Time schedule for examinations: at the end of the treatment prior to sacrifice
How many animals: 10 randomly selected Parental males and females
Parameters examined: T4 & TSH

URINALYSIS
Time schedule for examinations: at the end of the treatment prior to sacrifice
How many animals: 10 randomly selected Parental males and females
Parameters examined: see table 6. Additionally, urine colour/appearance and volume were recorded. Additionally, microscopic analysis of urine for sediment and presence of blood and/or blood cells or cell debris was conducted.
Oestrous cyclicity (parental animals):
- Parental females: vaginal smears were examined 2 weeks before beginning of treatment period, during 2 weeks premating period and until the confirmation of mating and/or the end of the 2 weeks mating period to record the oestrous cyclicity and also to confirm the evidence of mating. The oestrous cycle stage was recorded in P at termination.
- F1 females in Cohort 1A: vaginal smears were examined daily for all animals after the onset of vaginal patency until the first cornified smear was recorded to determine the time interval between these two events. Vaginal smears in Cohort 1A were examined for 2 weeks starting from PND 75. The oestrous cycle stage was recorded in Cohort 1A at termination.
- F1 females in Cohort 1B: vaginal smears were examined during mating period to confirm the evidence of mating. The oestrous cycle stage was recorded in Cohort 1B at termination.
- Cohort 3: The oestrous cycle stage was recorded in Cohort 3 at termination.
Sperm parameters (parental animals):
Parameters examined in P and F1 (Cohort 1A and 1B) male generations: testis weight, epididymis weight
Parameters examined in P and F1 (Cohort 1A) male generations: sperm motility, sperm morphology, testicular sperm head count, cauda epididymis sperm count
Litter observations:
STANDARDISATION OF LITTERS
- On PND 21, F1 pups were randomly (with the exception of obvious runts) assigned between three Cohorts 1A (20 animals / sex / group), 1B (20 animals / sex / group) and 3 (10 males and 10 females per group; one per litter)
- The surplus pups at PND 4 were subjected to gross necropsy.
- The pups not selected for cohorts, including runts, were terminated after weaning, on PND 22 or when they were not needed for further in-life investigations.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
- Cage-side observations: Twice daily all animals (F1 and F2 generation) were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
- General litter observations: Each F1 and F2 litter was examined after parturition (PND 0) to establish the number and sex of pups, stillbirths, live births and the presence of gross external anomalies, including cleft palate, subcutaneous haemorrhages, abnormal skin colour or texture, presence of umbilical cord, lack of milk in stomach and presence of dried secretions. In addition, the first clinical examination of the neonates included a qualitative assessment of body temperature, state of activity and reaction to handling. Pups found dead on PND 0 or later time were examined for the possible cause of death. Live pups were counted and sexed.
- Litter weight: F1 and F2 litters were weighed within 24 hours of parturition (PND 0) or PND 1, on PND 4, 7, 14 and PND 21. In addition to the observations on Parental animals, any abnormal behaviour of the offspring was recorded.
- Clinical signs: The clinical signs of pups were recorded on the corresponding days when offspring were weighed.
- Anogenital distance (AGD) and pup body weight: AGD of each pup was measured once between PND 0 and PND 4. Pup body weight was measured on the day of the AGD measurement and was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD / Cube root of pup weight).
- Nipples/areolae retention: Male pups were checked for the presence of nipples/areolae on PND 12.
- Sexual Maturity: All selected F1 male and female pups from all cohorts (1A, 1B, and 3 with exception of surplus pups not selected for cohorts) were checked daily for balano-preputial separation or vaginal patency, respectively, starting from PND 30 in males and PND 25 in females. Any abnormalities of genital organs were recorded.

HAEMATOLOGY
Time schedule for examinations: at the end of the treatment prior to sacrifice
How many animals: 10 randomly selected F1 Cohort 1A males and females
Fasted overnight: yes
Parameters examined: see tables 3 & 4

CLINICAL BIOCHEMISTRY
Time schedule for examinations: at the end of the treatment prior to sacrifice
How many animals: 10 randomly selected F1 Cohort 1A males and females
Fasted overnight: yes
Parameters examined: see table 5

THYROID HORMONE ANALYSIS
Time schedule for examinations: at the end of the treatment prior to sacrifice
How many animals: 10 randomly selected F1 Cohort 1A males and females. Also performed on 10 pups/sex/group pups from Parental females on PND 4 and after weaning (pups not allocated to cohorts) on PND 22 or when they were not needed for further in-life investigations. Neonatal (PND 4) blood was pooled by litters for thyroid hormone analysis.
Parameters examined: T4 & TSH

URINALYSIS
Time schedule for examinations: at the end of the treatment prior to sacrifice
How many animals: 10 randomly selected F1 Cohort 1A males and females
Parameters examined: see table 6. Additionally, urine colour/appearance and volume were recorded. Additionally, microscopic analysis of urine for sediment and presence of blood and/or blood cells or cell debris was conducted.

GROSS EXAMINATION OF DEAD PUPS: Dead or moribund pups were recorded and examined for possible defects and/or cause of death and preserved.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Yes
- Details: On PND 56 ± 3 days, Cohort 3 animals were used in a T-cell dependent antibody response assay.
The positive control group (PC) was administered cyclophosphamide 7 days before immunization (i.e. PND 49 ± 3 days) until the day before the last blood sampling. Cyclophosphamide was diluted with 0.9 % sodium chloride to a concentration of 1 mg/mL. Cyclophosphamide was administered by oral gavage at a dose of 10 mg/kg bw/day and with an administration volume of 10 mL/kg bw.
On PND 56 ± 3 days, each animal of each group (C, LD, MD, HD and PC) was injected intravenously into the tail vein with 0.300 µg/kg of KLH as single dose (at a dose volume of 0.75 mL/kg bw). On this particular day, the oral gavage treatment with the vehicle, test item or cyclophosphamide, was performed after the i.v. treatment with KLH.
The immune response to KLH was evaluated by determining the titre of KLH-specific IgM antibody in the serum by ELISA, at the peak of the response before and after immunization (day 6).
Blood was sampled from all animals of this cohort by sublingual bleeding (under isoflurane anaesthesia) at least one week before immunization. On day 6 after immunization, blood was sampled for determination of KLH-specific IgM antibodies. The blood volume and tube, as well as parameters of serum preparation were documented in raw data.

- Splenic Lymphocyte Subpopulation and Organ Weights (Cohort 1A): 10 Male and 10 female Cohort 1A animals from each treatment group (1 male or 1 female per litter; randomly selected) were subjected for the investigation of pre- and post-natally-induced immunotoxic effects at termination. This included measuring the weight of lymph nodes (axillary lymph nodes), adrenal gland, thymus and spleen. Analysis of splenic lymphocyte subpopulation (lymphocytes, T-cells, CD4+ (helper T cells), CD8+ (cytotoxic T cells), B-lymphocytes, and natural killer (NK) cells) using one half of the spleen was performed.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving P males were subjected to necropsy after 10 weeks of dose administration.
- Maternal animals: All surviving P females were subjected to necropsy post-weaning (PND 21).

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The animals of F1-generation were subjected to necropsy depending on the scheduled ages for each cohort (Cohort 1A: week 13, 1B: week 20-25, Cohort 3: 8 weeks). Special attention was paid to the sexual organs of P and F1 animals for structural abnormalities.
- The F1 surplus pups after standardization and F1 pups not selected for cohorts (including runts) were sacrificed and subjected to gross necropsy on PND 4 and weaning (PND 22), respectively.
- F2 pups from Cohort 1B mating were sacrificed and subjected to necropsy at weaning (PND 22).
- Cohort 3 F1 animals were subjected only to gross necropsy and examined macroscopically for any structural abnormalities or pathological changes.

NECROPSY EXAMINATIONS
- Organ weights: performed for P and F1 (Cohort 1A) animals. Paired organs were weighed together. Organ weights of animals found dead or euthanised for animal welfare reasons were not taken. See table 1 for organs weighed for P and F1 animals at necropsy. The reproductive organs and organs identified as target organs in P and Cohort 1A animals were also weighed for Cohort 1B animals. See table 2 for organs weighed for Cohort 1B.
- Macroscopic examination of organs: Performed for P and F1 (Cohort 1A) animals. See table 1 for organs examined at gross necropsy.The reproductive organs and organs identified as target organs in P and Cohort 1A animals were also examined macroscopically for Cohort 1B animals. See table 2 for organs examined for Cohort 1B.
- Histopathology:
P animals: Performed for for all HD and control P animals. Histopathological examinations were extended to animals of the other dose groups, as treatment-related changes were observed in the HD group. Reproductive organs of all P animals that failed to mate, conceive, sire, or deliver healthy offspring, or for which oestrous cyclicity or sperm number, motility, or morphology were affected and all gross lesions were subjected to histopathological evaluation. See table 1 for tissues examined for P animals.
F1 (Cohort 1A) animals: Full histopathology of the organs listed in table 1 was performed on control and HD adult Cohort 1A animals. All litters were represented by at least 1 pup per sex. Histopathological examinations were extended to animals of the other dose groups, as treatment-related changes were observed in the HD group. All gross lesions from all dose groups were subjected to histopathological evaluation. Histopathology of lymph nodes (mesenteric and axillary) and bone marrow was performed on 10 males and 10 females Cohort 1A animals. Histopathology of thymus, spleen, and the adrenal glands was performed in all Cohort 1A animals. See table 1 for tissues examined for Cohort 1A animals.
F1 (Cohort 1B) animals: As the results from Cohort 1A examination there were no concearns for reproductive / endocrine toxicity and there were no triggers for further histopathology. Therefore, the examination was not extended to Cohort 1B animals.
Statistics:
A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed animals with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, thyroid hormones and foetal evaluation parameters including external, visceral, craniofacial and skeletal parameters were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. The statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 is considered as statistically significant).
Reproductive indices:
percent male mating index, female mating index, male fertility index, female fertility index, gestation index and live birth index
Offspring viability indices:
survival index
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In terminally sacrificed Parental male and female animals, predominant clinical signs transiently observed in the majority of HD group animals were increased salivation (slight/moderate) and/or moving the bedding.
Low incidences of clinical signs including hairless area, crust, scratch/cut, eyelid closure, piloerection, chromodacryorrhea, corneal opacity and diarrhoea (single incidence), exophthalmos (left), wound, overgrown teeth and spontaneous reduced activity (slight) were observed in few animals on few days in all groups including controls.
As these findings showed no dose-dependency and were noted transiently, they were not considered to be toxicologically relevant.
The clinical signs salivation and moving the bedding in males and females were observed immediately after the dose administration and, therefore, were considered to be signs of a local reaction to the test item rather than a systemic adverse effect.

The weekly detailed clinical observations revealed no toxicologically relevant differences between the Parental treated and control groups during the entire study period. There were statistical significances observed in a few parameters in treated groups on a few occasions. However, observed statistically significant differences in a few parameters were either before initiation of treatment, not dose dependent/consistent or biologically relevant and, therefore, these findings were considered to be incidental and not related to the treatment with the test item.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related mortality was observed in males during the study period. In females, one LD animal (no. 138) was found dead on treatment day 6. The cause of death was due to gavage error during the dosing procedure.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In both male and female Parental animals, there was no test item-related effect observed on group mean body weight and mean body weight gain in the dose groups when compared with the controls. There were no statistically significant differences observed for these parameters between the dose groups and the control group, except for the following:
In males, statistically significantly lower body weight gain was seen between days 14-21 in LD and HD groups (46.49% and 37.84%, respectively) and between days 70-74 in LD and HD groups (135.23% and 204.55%, respectively) when compared to control. Statistically significantly increased body weight gain was found between days 49-56 in HD group (53.16%) males when compared to control. In females, statistically significantly higher mean body weight gain was observed between lactation days 14-21 (1.52%) in the HD group when compared to control. Due to the lack of dose dependency and consistency, these differences on Parental mean body weight gain and/or loss were not considered as test item-related and the mean body weight were found to be within the historical control range of this strain.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In correlation to body weight and body weight gain, food consumption in both males and females of the Parental generation tended to increase with the progress of the study in all groups.
No test item-related or statistically significant effect on food consumption was observed in males and females of the Parental generation during the whole study period except slight but statistically significantly higher group mean food consumption in Parental females between lactation days 0-4 in the MD group (24.77%) when compared to control. Due to the lack of dose dependency or consistency, this effect on food consumption was not considered to be adverse.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In 10 selected Parental males and females per group sacrificed at the end of the treatment period, no test item-related adverse effects were observed in haematological parameters in the dose groups when compared to the control group. In HD group males, moderately but statistically significantly higher mean WBC (35.1%) and marginally but statistically significantly higher mean RBC (5.4%), HGB (5.1%) and HCT (3.7%) were observed when compared to the control. In MD group males, higher mean HGB (6.0%) and HCT (4.9%) and lower RET percent (15.7%) were observed when compared to the control. In females, no statistical significance was observed when compared with the control. All these values were without any dose dependency or consistency; hence they were not considered to be adverse.
No test item-related effect was observed on coagulation parameters in Parental males and females when compared with the control, except for slight but statistically significantly higher mean PT value in HD group males (5.1%) and MD and HD group females (10.8-12.3%) when compared to the control.
All other group mean and most of the individual values for haematological parameters in male and females were comparable to the controls and within the normal range of variation; they were also within the range of historical control data of reproductive and developmental toxicity studies in this strain.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In 10 selected Parental males and females sacrificed at the end of the treatment period, marginal but statistically significantly lower creatinine (0.4%) was observed in HD group males when compared to the control; in HD group females, moderate but statistically significantly lower creatinine was observed in MD (30.4%) and HD (33.6%) groups when compared to the control. As the differences were marginal-to-moderate and all values were within the range of historical control data or without dose-dependency, this was not assumed to be toxicologically relevant.
All other group mean and most of the individual values for clinical chemistry parameters in male and females were comparable to the controls and within the normal range of variation.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The urinalysis performed in 10 selected males and females per group from Parental sacrificed at the end of treatment period revealed no test item-related effect in the dose groups when compared to control. All urinary parameters were in the normal range of variation.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
From the decedents in P-generation histopathological examination was available in one female from P-generation. Female No. 138 (P-generation, Group 2; found dead on Study Day 6): A limited set of organs and tissue was available for histopathology, which included the heart, lungs, esophagus and thymus. On the heart section, marked inflammation with tissue destruction, hemorrhage and granulation was found at/around the right atrium. There was also focal necrosis with hemorrhage in the esophagus. Thus, the cause of animal’s death was considered to be due to gavage error.
The most obvious finding in P-generation animals was diffuse urothelial hyperplasia in the urinary bladder, and this was observed in 20/25 males and 12/25 females of Group 3 and all males and females of Group 4. Diffuse urothelial hyperplasia was also found in the kidney of both sexes of Groups 3 and 4 and in the ureter of both sexes of Group 4, although it occurred at lower frequency or severity compared to that of the urinary bladder.
In the urinary bladder, the following findings that were considered to be treatment-related were also observed in both sexes of animals:
- Mixed inflammatory cell infiltration in the mucosa (epithelial layer and lamina propria): Groups 3 and 4;
- Increased incidence and/or severity of mononuclear cell focus/foci in lamina propria: Groups 3 and 4;
- Submucosal edema: Groups 3 and 4;
- Congestion: Group 4;
- Hemorrhage: one male of Group 3 and both sexes of Group 4.
In addition, the following changes recorded in the kidneys and ureters were also considered to be treatment-related.
Kidneys:
- Mixed inflammatory cell infiltration in the suburothelium: one male of Group 3 and in both sexes of Group 4;
- Fibrosis at/around the fornix and focal to multifocal interstitial fibrosis: males of Group 4;
There were also pelvic luminal precipitates, as well as appearance of foreign body gaint cells at/around the fornix, in males of Group 4;
- Increased incidence and/or severity of pelvic dilation, focal to multifocal mononuclear cell infiltration, tubular basophilia and tubular dilatation: males of Group 4;
Thus, the overall incidence and severity of the treatment-related renal lesions was higher in males than in females.
Ureters:
The ureters can be found by chance within or near the accessory sex organs, and in the present study, it was available in some sections from the prostate. Therefore, the observation of the ureters on the prostate sections was also made as a reference for histological evaluation.
- Increased incidence and severity of luminal dilatation: males of Group 4;
- Mixed inflammatory cell infiltration and submucosal edema were identified in the ureters on the prostate sections.
Small Intestine (Duodenum, Jejunum and Ileum)
Lipid accumulation in the lamina propria mucosa of the small intestine was observed in the HD animals from P-generation. This change was recognized histomorphologically as vacuolation or empty space of various sizes, most of which, especially larger vacuolation, were considered to be dilated lymphatic vessels, and some were within the cytoplasm of macrophages or present in the interstitium of the lamina propria.
Within the small intestinal segments, this lesion appeared predominantly in the jejunal region, and was not detected in the duodenum of females of P-generation.
The remaining microscopic findings recorded in this study were within the range of normal background lesions which may be observed in this study type and animals of this strain and age.
There was no test item-related histomorphological changes in the male and female reproductive organs in P-generation. In addition, no microscopic indicators for endocrine disruption were noted in the organs and tissues (including pituitary glands, adrenal glands and thyroid glands) examined in P-generation.
There were no statistically significant differences in all parameters including primordial follicles, growing follicles, the sum of primordial and growing follicles, antral follicles and corpora lutea.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
In Parental males and females (10/sex/group), group mean T4 and TSH levels in dose groups were comparable to control and there were no statistically significant changes observed.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The test item had no biologically or statistically significant effect on the oestrus cycle analysed during the 2 weeks pre-treatment and 2 weeks pre-mating period after the first administration in treatment groups when compared to the control. There were no considerable differences in the length or sequence of cycle stages between the treatment groups and the control group.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
The test item had no effect on epididymal sperm motility analysed from all males. Group mean motility values from Parental males of the dose groups were comparable to control.
Evaluation of sperm morphology from control and HD Parental males did not reveal any test item-related findings, except for a marginal but statistically significantly higher percent of abnormal sperm observed in the HD groups from Parental (8.42%) when compared to the control (6.91% in Parental). However, this was not considered to be adverse and these values were within the normal biological variation in this strain.
The test item had no effect on mean testis sperm count in Parental. Group mean sperm count from Parental males of dose groups were comparable to the control. The test item had no effect on mean testis weight in Parental males of dose groups when compared to the control.
Reproductive performance:
no effects observed
Description (incidence and severity):
None of the Parental females showed signs of abortion or premature delivery.
In Parental females, there were no test item-related or statistically significant effects on litter parameters including group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, stillbirth, runt on PND 0 as well as number of alive pups, alive male and female pups and sex ratio on PNDs 4, 7, 14 and 21 when compared to the control. Statistical analysis of litter data revealed no significant effects in treatment groups when compared to the control.
There was no test item-related effect on pup mean weight, total litter weight, male and female litter weight on PNDs 0, 4, 7, 14 and 21 observed in Parental treatment groups when compared with the controls. There was no statistically significant change in dose groups compared to control.
There was no test item-related effect observed on the duration of pre-coital interval and the duration of gestation in the Parental female dose groups when compared to control and within the range of biological variation in treatment groups when compared to control.
There was no test item-related effect observed on the number of corpora lutea, implantation sites, alive pups on PND 0, percent pre-implantation loss and post- implantation loss in Parental treatment group females when compared with the corresponding control group.
There was no test item-related effect observed on the reproductive indices (percent male mating index, female mating index, male fertility index, female fertility index, gestation index and live birth index) in Parental dose group animals when compared to the respective control group.
The survival index of pups during PND 0 to 4, PND 4 (after interim sacrifice) to PND 14 and PND 14 (after interim sacrifice) to PND 21 in Parental females remained unaffected and within the range of biological variation in treatment groups when compared to the control.
Key result
Dose descriptor:
NOAEL
Remarks:
Parental animals / reproductive toxicity
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects on reproductive function observed.
Key result
Dose descriptor:
NOAEL
Remarks:
Parental animals / general systemic toxicity
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In terminally sacrificed males and females from various Cohort 1B clinical signs of increased salivation (slight/moderate) and/or moving the bedding were observed in the HD group.
The clinical signs salivation and moving the bedding in males and females were observed immediately after the dose administration and, therefore, were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect.
The weekly detailed clinical observations revealed no toxicologically relevant differences between the Cohort 1B treated and control groups during the entire study period. There were statistical significances observed in a few parameters in treated groups on a few occasions. However, observed statistically significant differences in a few parameters were either before initiation of treatment, not dose dependent/consistent or biologically relevant and, therefore, these findings were considered to be incidental and not related to the treatment with the test item.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Cohort 1B: One HD male (no. 430) died on day 25 by accidental incidence during the study. Two control females (nos. 442, 452, on days 28, 36, respectively), four LD females (nos. 469, 470, 476, 478 on days 22, 58, 21, 31, respectively) and one MD female (no. 497, on day 21) were found dead during the study period. At necropsy, no abnormalities were observed for animal nos. 469, 470, and 476. Animal no. 442 showed abnormal coloured (red) with fluid-filled lungs. Animal no. 452 showed lung adhesion with fluid-filled thoracic. Both animals did not show any clinical signs beforehand. Animal no. 478 showed advanced autolysis and lungs with fluid-filled (orange). Animal no. 497 showed abnormal coloured lungs, dark red.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1B: In both male and female Cohort 1B animals, there was no test item-related effect observed on group mean body weight and mean body weight gain in the dose groups when compared to control. Slight but statistically significantly higher group mean body weight was observed on days 11-29, 43, 64-71 and 84-98 in LD group (5.33-7.25%) males and on days 7-9 a slight but statistically significantly lower mean body weight (8.00-9.69%) was observed in HD group females when compared to control. Moderate but statistically significantly higher group mean body weight gain was observed between days 57-64 in LD group males (40.87%) and between days 119-126, moderate but statistically significantly lower mean body weight gain (84.96%) was observed in HD group males when compared to control. In females, moderate but statistically significantly higher mean body weight gain (85.03%) was observed on days 11-13 in MD group females when compared to control. Statistically significantly lower mean body weight (94.1% compared to control) was observed during lactation on day 21 in HD group females. Mean body weight gain was found to be lower between days 14-21 of lactation in all females, including controls.
Overall, mean body weight and mean body weight gain remained unaffected by the treatment with test item and values were in the normal range of variation throughout the treatment period when compared to the control group and they are within the range of historical control data of reproductive and developmental toxicity studies in this strain.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In correlation to body weight and body weight gain, food consumption in both males and females of the F1 Cohort 1B tended to increase with the progress of the study in all groups.
No test item-related or statistically significant effect on food consumption was observed in males and females during the whole study period except statistically significantly higher food consumption in LD group males (Cohort 1B) on days 1-7 (11.51%) and 63-70 (5.67%) when compared to control. Due to the lack of dose dependency or consistency, this effect on food consumption was not considered to be adverse.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Thickened wall of the urinary bladder was recorded in 13/20 males of Group 4 (HD) and in 1/20 females of Group 3 (MD) and 2/20 females of Group 4 (HD). In addition, dilatation of the ureter was recorded in 1/20 females of Group 4 (HD).
All other macroscopic findings recorded were lesions within the range of normal background changes which may be observed in rats of this strain and age, incidental appearances without corresponding histomorphological changes, or alteration representing normal physiology, and therefore, were deemed not to be related to treatment with the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
None of the Cohort 1B females showed signs of abortion or premature delivery.
Litter parameters of Cohort 1B females, including group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, stillbirth, runt on PND 0, as well as number of live pups, male pups, number of female pups and sex ratio on PNDs 4, 7, 14 and 21 remained unaffected when compared with the controls. Statistical analysis of litter data revealed no significant effects in treatment groups when compared to control.
There was no test item-related effect on pup mean weight, total litter weight, male and female litter weight on PNDs 0, 4, 7, 14 and 21 observed in Cohort 1B treatment groups when compared with the controls. There was no statistically significant change in dose groups compared to control.
There was no test item-related effect observed on the duration of pre-coital interval and the duration of gestation in the Cohort 1B female dose groups when compared to control and within the range of biological variation in treatment groups when compared to control.
There was no test item-related effect observed on the number of corpora lutea, implantation sites, alive pups on PND 0, percent pre-implantation loss and post- implantation loss in Cohort 1B treatment group females when compared with the corresponding control group.
There was no test item-related effect observed on the reproductive indices (percent male mating index, female mating index, male fertility index, female fertility index, gestation index and live birth index) in Cohort 1B dose group animals when compared to the respective control group. The survival index of pups during PND 0 to 4, PND 4 (after interim sacrifice) to PND 14 and PND 14 to PND 21 in Cohort 1B females remained unaffected and within the range of biological variation in treatment groups when compared to the control.
Key result
Dose descriptor:
NOAEL
Remarks:
P1 (Cohort 1B) / reproductive toxicity
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects on reproductive function observed
Key result
Dose descriptor:
NOAEL
Remarks:
P (Cohort 1B) / general systemic toxicity
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
ureter
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In terminally sacrificed males and females from various F1 Cohorts (1A and 1B), clinical signs of increased salivation (slight/moderate) and/or moving the bedding were observed in the HD group.
The clinical signs salivation and moving the bedding in males and females were observed immediately after the dose administration and, therefore, were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect.
The weekly detailed clinical observations revealed no toxicologically relevant differences between the F1 (Cohort 1A, 1B and 3) treated and control groups during the entire study period. There were statistical significances observed in a few parameters in treated groups on a few occasions. However, observed statistically significant differences in a few parameters were either before initiation of treatment, not dose dependent/consistent or biologically relevant and, therefore, these findings were considered to be incidental and not related to the treatment with the test item.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F1 pup survival data: No test item-related effect on mean mortality of pups from PND 0 to 4, PND 4 (after interim sacrifice) to PND 14 and PND 14 (after interim sacrifice) to PND 21 in pups from Parental females was observed when compared to the respective control group.
Mean mortality of pups of the dose groups was comparable to the respective control and slight differences are considered as incidental and not related to treatment with the test item and they are within the range of historical control data of reproductive and developmental toxicity studies in this strain.
Adult F1 mortality data:
Cohort 1A: One LD female (no. 316) was found dead on day 22 and was cannibalized. One LD male (no. 239) and two females (no. 338, and no. 347) were moribund sacrificed for the animal welfare reasons on day 46 and on day 8 or 10, respectiively. Animal no. 338 showed abnormal and dark coloured lungs and animal no. 347 did not show any abnormalities at necropsy. Animal no. 239 showed wound/scar/crust, abdominal region of skin.
Cohort 3: No deaths.
The cause of the unscheduled deaths in Cohorts 1A and 1B was deemed to be due to accidental (e.g. gavage error) and not considered to be test item-related.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A: In both male and female Cohort 1A animals, there was no test item-related effect observed on group mean body weight and mean body weight gain in the dose groups when compared with the control. Slight but statistically significantly higher group mean body weight was observed on days 1-3 (10.34-10.78%) and 7-15 (7.41-9.38%) in LD group males and on days 5-11 (6.26-6.89%) in LD group females when compared with the control. Slight but statistically significantly higher mean body weight gain was observed on days 3-5 in LD group females (15.5%) when compared to the control. However, in males, no statistical significance in mean body weight gain was observed between the groups at any time. As the observed differences on mean body weight and body weight gain in males and females were marginal and without dose dependence, they were not considered to be adverse.
Cohort 3: In both male and female Cohort 3 animals, there was no test item-related effect observed on group mean body weight and mean body weight gain in the dose groups when compared to control. Slight but statistically significantly higher mean body weight was observed in LD group males on days 9-11 and 22-36 (8.15-10.75%), in HD group males on day 29 (8.90), and in positive control group males on days 5, 9-11, and 22-29 (8.94-20.62%) when compared to control. Slight but statistically significantly lower mean body weight was observed in positive control group females on day 36 (10.04%) when compared to control. Slight but statistically significantly higher mean body weight gain was observed in LD group males on days 3-5 (79.1%) and days 1-36 (7.9%) when compared to control. In positive control group males, a statistically significantly lower mean body weight gain was observed on days 29-36 (60.7%) when compared to control. In females, a statistically significantly lower mean body weight gain was observed in the MD group on days 9-11 (37.7%) and in the positive control group on days 29-36 (48.6%) and days 1-36 (12.6%) when compared to control.
Overall in all F1 Cohorts, mean body weight and mean body weight gain remained unaffected by the treatment with test item and values were in the normal range of variation throughout the treatment period when compared to the control group and they are within the range of historical control data of reproductive and developmental toxicity studies in this strain
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In correlation to body weight and body weight gain, food consumption in both males and females of F1 cohorts (Cohort 1A and 3) tended to increase with the progress of the study in all groups.
No test item-related or statistically significant effect on food consumption was observed in males and females of the F1 cohorts during the whole study period except statistically significantly higher food consumption LD group females (Cohort 1A) on days 1-7 (10.77%) when compared to control. Due to the lack of dose dependency or consistency, this effect on food consumption was not considered to be adverse.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In 10 selected Cohort 1A males and females per group sacrificed at the end of the treatment period, marginally but statistically significantly higher mean RBC (8.4%) was observed in MD group males when compared with the control. Marginally but statistically significantly higher mean RBC (5.9%), HGB (7.0%) and HCT (6.1%) were observed in MD group females when compared with the control. As the differences were marginal and all values were within the range of historical control data, this was not assumed to be toxicologically relevant. All other group mean and most of the individual values for haematological parameters in male and females were comparable to the controls and within the normal range of variation.
In the absence of test item-related histopathological findings and effect on splenic lymphocyte subpopulation in the study, the above-mentioned increase or decrease in a few haematology parameters was not considered to be adverse.
No test item-related effect was observed on coagulation parameters in Cohort 1A males and females when compared with the respective control.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In 10 selected Cohort 1A males and females per group sacrificed at the end of treatment period, marginal but statistically significantly higher potassium (2.6%) in LD group males and lower total protein (6.0%) in HD group males were observed when compared to the control. In HD group females, moderate but statistically significantly lower ASAT (25.8%) and higher cholesterol (42.5%) were observed when compared to the control. In absence of test item-related histopathological findings and clinical signs, these effects in Cohort 1A animals were not considered to be toxicologically relevant.
All other group mean and most of the individual values for clinical biochemistry parameters in male and female Cohort 1A animals were comparable to the controls and within the normal range of variation.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The urinalysis performed in 10 selected males and females per group from Cohort 1A sacrificed at the end of treatment period revealed no test item-related effect in the dose groups when compared to control. All urinary parameters were in the normal range of variation.
Sexual maturation:
no effects observed
Description (incidence and severity):
All selected F1 male and female pups from all cohorts were checked daily for balano-preputial separation or vaginal patency, respectively, starting from PND 30 in males and PND 25 in females. No abnormalities of genital organs were observed in any of the pups.
Cohort 1A: No significant difference in group mean body weight and day of onset of vaginal opening in females and balano-preputial separation in males was observed in treatment groups when compared to control, except for a marginal but statistically significant reduction in vaginal opening observed in the LD group (PND 29.20) when compared to the control (PND 30.65) and this could be due to persistent diestrus of two female animals (nos. 309 and 311). It is not considered to be adverse and these values were within the normal biological variation in this strain.
Cohort 1B: No significant difference in group mean body weight and day of onset of the vaginal opening in females and balano-preputial separation in males was observed in treatment groups when compared to control.
Cohort 3: No statistically significant difference in group mean body weight and day of onset of the vaginal opening in females and balano-preputial separation in males was observed in treatment groups when compared to control, however marginal but statistically significant higher mean body weight on the day of balano-preputial separation was observed in the positive control (18.9%) and LD (15.7%) group when compared to the control. It is not considered to be adverse and these values were within the normal biological variation in this strain.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A: There were no statistically significant differences in the terminal body weights (TBW) in both sexes of F1-generation Cohort 1A.
In the organ weight determinations, the weight changes that could be related to treatment with the test item were found in the kidney of F1-generation Cohort 1A animals.
Kidneys: There was a statistically significant increase in relative (to TBW) weights in males of the HD group. In the HD group, there was also a tendency for an increase in the group mean values of absolute weights of males and of absolute and relative weights of females, although there were no statistically significant differences. These higher weight values were considered to be treatment-related.
The following statistically significant changes in the absolute and/or relative organ weights were recorded in females. However, there were no corresponding histological changes that could be related to the organ weight changes or no dose-response relationships were observed. Therefore, the weight differences recorded were considered to be incidental and not treatment-related.
- increase in absolute liver weights in the HD group;
- decrease in relative brain weights in the HD group;
- increases in absolute and relative spleen weights in the MD group.
Weights of spleen and thymus of Cohort 1A animals revealed no considerable changes that could indicate a test item-related immunotoxic effect.
F1 Pups not Selected for Cohorts: No test item-related effects on brain, spleen and thymus weights were observed.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related gross external abnormalities of toxicological relevance on PND 0-20 were observed in the F1 pups of any of the groups from Parental females.
Cohort 1A adults: Test item-related gross findings were not recorded in F1-generation Cohort 1A animals.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
From the decedents in F1-generation Cohort 1A, histopathological examination was available in one male and one female from F1-generation Cohort 1A.
Male No. 239 (F1-generation Cohort 1A, Group 2; sacrificed moribund on Study Day 46): Grossly identified wound/scar/crust correlated microscopically with ulceration that arose on the surface of basal cell carcinoma of the skin, and this lesion was deemed to be the cause of animal’s morbidity. In this animal, enhanced hemopoiesis was found in the bone marrow (increased cellularity), spleen (massive extramedullary hematopoiesis) and liver (hemopoietic foci). This was considered to be secondary to the inflammatory response and possible loss of blood in the ulcerative lesion of the surface of the mass. In addition, there were stress-related reactions in the thymus (slight atrophy) and adrenal glands (minimal diffuse cortical hypertrophy).
Female No. 338 (F1-generation Cohort 1A, Group 3; sacrificed moribund on Study Day 8): Histopathological examination was available only for the lung sample. Only a minimum congestion was found in the lung sample, and the direct cause of animal's morbidity could not be established histologically. However, in the HD group of F1-generation, there was no unscheduled deaths likely to be due to the effects of the test item, and therefore, the morbidity in this animal was deemed not to be treatment-related.
The treatment-related findings qualitatively identical with that of P-generation animals were also observed in F1-generation Cohort 1A animals.
The most obvious finding was diffuse urothelial hyperplasia in the urinary bladder which was observed in 12/20 males and 4/19 females of Group 3 and all males and females of Group 4. This finding was recorded in the kidney of both sexes from Group 4 at a high frequency (all males and 16/19 females of Group 4), and it was also found in both sexes of Group 3 (two males and one female). Diffuse urothelial hyperplasia was also found in the ureter of both sexes of Group 4, at a lower frequency in comparison with the urinary bladder and kidneys.
Kidneys: The incidence and severity of some findings in the kidney were lesser in F1-generation Cohort 1A compared to that in P-generation, and these included mixed inflammatory cell infiltration in suburothelium, tubular dilatation and pelvic luminal precipitates. Further, there were no apparent differences in the incidence/severity of tubular basophilia and mononuclear cell infiltration between groups. Interstitial fibrosis, as well as fibrosis and foreign body giant cells at/around fornix, were not observed in the kidney of F1-generation Cohort 1A, although granuloma at/around fornix was found in one male of Group 4 instead.
Small Intestine (Duodenum, Jejunum and Ileum)
Lipid accumulation in the lamina propria mucosa of the small intestine was observed in the HD animals from F1-generation Cohort 1A. This change was recognized histomorphologically as vacuolation or empty space of various sizes, most of which, especially larger vacuolation, were considered to be dilated lymphatic vessels, and some were within the cytoplasm of macrophages or present in the interstitium of the lamina propria.
Within the small intestinal segments, this lesion appeared predominantly in the jejunal region, and was not detected in the duodenum and ileum of both sexes of F1-generation Cohort 1A.
The remaining microscopic findings recorded in this study were within the range of normal background lesions which may be observed in this study type and animals of this strain and age.
There was no test item-related histomorphological changes in the male and female reproductive organs in F1-generation Cohort 1A. In addition, no microscopic indicators for endocrine disruption were noted in the organs and tissues (including pituitary glands, adrenal glands and thyroid glands) examined in F1-generation Cohort 1A.
There were no statistically significant differences in all parameters including primordial follicles, growing follicles, the sum of primordial and growing follicles, antral follicles and corpora lutea.
Other effects:
no effects observed
Description (incidence and severity):
In Cohort 1A females, no biologically significant effect was observed on the duration of vaginal opening until first oestrus cycle in treatment groups when compared to control.
In this cohort, no biologically significant effect was observed on the oestrus cycle length or sequence of cycle stages between the treatment groups and the control group analysed from PND 75 for 2 weeks.

The test item had no effect on epididymal sperm motility analysed from all Cohort 1A males. Group mean motility values from Cohort 1A males of the dose groups were comparable to control.
Evaluation of sperm morphology from control and HD Cohort 1A males did not reveal any test item-related findings, except for a marginal but statistically significantly higher percent of abnormal sperm observed in the HD groups from Cohort 1A (8.23%) when compared to the control (5.84% in Cohort 1A). However, this was not considered to be adverse and these values were within the normal biological variation in this strain.
The test item had no effect on mean testis sperm count in Cohort 1A males. Group mean sperm count from Cohort 1A males of dose groups were comparable to the control. The test item had no effect on mean testis weight in PCohort 1A males of dose groups when compared to the control.

F1 pups on PND 4 and PND 21: In pups sacrificed on PND 4 (10/sex/group - pooled samples), T4 and TSH levels in treatment groups were comparable to the controls. In pups sacrificed on PND 21 (10/sex/group), T4 and TSH levels in treatment groups were comparable to the controls.
Cohort 1A adults: In males and females of Cohort 1A (10/sex/group), group mean T4 and TSH levels in treatment groups were comparable to the controls. Moreover, no test item-related effect of toxicological relevance was observed on thyroid weight and thyroid histopathology.

Behaviour (functional findings):
not examined
Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
Cohort 3: Control Group
Male: All males (10/10) showed an immune response after injection with KLH. It showed elevated anti-KLH specific IgM titers on day 6 after KLH injection, when compared to pre-levels (in average 2.8-fold higher). Animal no. 521 showed a 9.2-fold higher titer on day 6. Animal no. 528 did not show an elevated level of anti-KLH specific IgM titers on day 6 when compared to other animals in this group.
Female: All females (10/10) showed an immune response after injection with KLH. It showed increased anti-KLH specific IgM titers on day 6 when compared to pre-levels (in average a 5.1-fold higher). Animal no. 576 showed a 8.3-fold higher titer on day 6. Animal no. 577 showed a 13.6-fold higher titer on day 6 when compared to other animals in this group.
Cohort 3: Positive Control Group
Male: After immunization with KLH, decreased anti-KLH IgM titers (1.6-fold) were observed on day 6 in 4/10 male animals treated with the immunosuppressant cyclophosphamide when compared to pre-levels. Anti-KLH IgM titers of 10/10 animals were below the level of quantification (BLQ) or negative when compared to pre-levels. This showed the immune response to KLH in male animals treated with cyclophosphamide was effectively decreased.
Female: After immunization with KLH, decreased anti-KLH IgM titers (1.8-fold) were observed on day 6 in 7/10 female animals treated with the immunosuppressant cyclophosphamide when compared to pre-levels. Anti-KLH IgM titers of 9/10 animals were below the level of quantification (BLQ) or negative when compared to pre-levels. This showed the immune response to KLH in female animals treated with cyclophosphamide was effectively decreased.
Cohort 3: Dose Groups
Male: All male animals treated with test item (10/10 for each dose group) showed elevated anti-KLH specific IgM levels on day 6 after KLH-injection, when compared to pre-levels (on average 4.1-, 4.4- and 6.0-fold higher in the LD, MD and HD groups, respectively). This shows a clear immunological response in these male animals. Animal nos. 557 and 559 in the HD group showed 9.6-20.7-fold higher titer on day 6, when compared to other animals in this group. The slightly higher fold difference in the HD group could be due to these two individual animals with higher titer values.
Female: All female animals treated with test item (10/10 for each dose group) showed elevated anti-KLH specific IgM levels on day 6 after KLH-injection, when compared to pre-levels (on average 4.9-, 3.3- and 9.7-fold higher in the LD, MD and HD groups, respectively). This shows a clear immunological response in these female animals. Animal nos. 603, 605, 606, 607 and 610 in the HD group showed 9.9-77.7-fold higher titer on day 6, when compared to other animals in this group. The higher fold difference in the HD group could be due to these five individual animals with higher titer values.

Cohort 1A: Splenic Lymphocyte Subpopulation analysis
Analysis of spleen samples for splenic lymphocyte subpopulation (lymphocytes, T cells, CD4+ (helper T cells), CD8+ (cytotoxic T cells), B lymphocytes, and natural killer (NK) cells) from 10 male and 10 female animals per group from Cohort 1A revealed that there was a slight increase in mean lymphocytes and T cell populations in both males and females when compared to the control. There was no test item-related or statistically significant change in the number of splenic B cells. They were found to be comparable between all dose groups and control group in both males and females, except for a slightly higher mean splenic B cell value due to a high level of B cells in HD group female no. 263. Thus, there was no indication of an immunosuppressive effect of the test item on lymphocyte subpopulations.
Key result
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Generation:
F1 (cohort 1A)
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Generation:
other: F1 (Cohort 1A) & F1 (Cohort 1B)
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects on reproductive organs observed
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
other: F1 (Cohort 1A) & F1 (Cohort 1B)
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse developmental effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
developmental immunotoxicity
Generation:
F1 (cohort 3)
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse immunotoxic effects observed.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
F2 pup data: No test item-related effect on mean mortality of pups from PND 0 to 4, PND 4 to 21 in F2 pups from Cohort 1B female treatment groups was observed when compared to the respective control group.
Mean mortality of pups of the dose groups was comparable to the respective control and slight differences are considered as incidental and not related to treatment with the test item and they are within the range of historical control data of reproductive and developmental toxicity studies in this strain.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no test item-related effect on pup mean weight, total litter weight, male and female litter weight on PNDs 0, 4, 7, 14 and 21 observed in F2 from Cohort 1B treatment groups when compared with the controls. There was no statistically significant change in dose groups compared to control.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item-related effects on brain, spleen and thymus weights were observed in F2 pups from Cohort 1B females.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related gross external abnormalities of toxicological relevance on PND 0-20 were observed in the F2 pups of any of the groups from Cohort 1B females.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F2 (cohort 1B)
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse developmental effects observed.
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Concentration analysis of formulation samples was performed at three concentrations, 10.00 mg/mL, 25.00 mg/mL and 75.00 mg/mL in study weeks 1, 5, 9, 13 and in the last week of the study. The mean recoveries observed for the LD group were between 91.3% and 99.0% of the nominal value, between 91.8% and 97.1% of the nominal value for the MD group and between 88.5% and 96.3% of the nominal value for HD group. The mean recoveries observed in the LD, MD and HD groups were 95.8%, 94.5%, and 93.0% of the nominal concentration, respectively.

Nominal concentrations were confirmed for all dose groups, as mean measured concentrations were within acceptance criterion of 10%.

However, HD group sample no. 16 in week 13 did not meet this criterion with a recovery of 88.5%. It was decided that no reanalysis was necessary, since the mean value was still within the acceptance criteria.

Conclusions:
In the extended one-generation reproductive toxicity study, conducted according to OECD Test Guideline 443 and in compliance with GLP, the NOAEL for general systemic toxicity for P and F1 (Cohort 1A) animals was concluded to be 40 mg/kg bw/day based on test substance related effects in the urinary system at 100 and 300 mg/kg bw/day; the reproductive toxicity NOAEL for P and F1 (Cohort 1A and 1B) animals was at least 300 mg/kg bw/day based on no adverse effects on male and female reproductive organs, on oestrous cycle and sperm parameters, and male and female fertility and performance; the developmental toxicity NOAEL for F1 (Cohort 1A and 1B) and F2 (Cohort 1B extension) was at least 300 mg/kg bw/day based on no adverse effects on pre- and post-natal development; the developmental immunotoxicity NOAEL for F1 (Cohort 3) was at least 300 mg/kg bw/day based on no adverse effects on immune system development and response.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Conducted according to OECD Test Guideline 443 and in compliance with GLP.
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 extended one-generation reproductive toxicity study (BSL Bioservice, 2021), conducted according to OECD Test Guideline 443 and in compliance with GLP, 25 male and 25 female Parental (P0) Wistar rats per group, were exposed to 0, 40, 100, 300 mg/kg bw/day trimethoxy(vinyl)silane in corn oil by oral gavage for 2 weeks during pre-mating (males and females), for up to 2 weeks during mating (males and females), for 6 weeks during post-mating and up to termination after weaning of F1 (males; 10 weeks total treatment) or during pregnancy and lactation up to termination after weaning of F1 (females; 8-10 weeks total treatment), respectively. At weaning, selected F1 offspring were assigned to specific cohorts for investigations consisting of sexual maturation, reproductive organ integrity and function, neurological and behavioural endpoints, and immune functions. In F1 males and females, the direct exposure to test item was started at weaning until the scheduled termination, i.e., until an age of 13 weeks (Cohort 1A, 20 animals per sex and group) or until study termination (weeks 20-25: Cohort 1B, 20 animals per sex and group). Furthermore, Cohort 3 animals underwent evaluation of developmental immunotoxicity and were sacrificed at an age of 8-10 weeks (10 animals per sex and group). The dose levels were selected based on the findings of the 28-day oral repeated dose toxicity study (BSL Bioservice, 2020a).

During the administration period the animals (P0, F1 and F2) were observed closely each day for signs of toxicity. Detailed clinical observations were made once before the first exposure, and once a week thereafter, in all P0 animals and all cohorts.

Body weight and food consumption were measured in all animals at specific intervals, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

After 2 weeks pre-mating period, P0 male and female rats from the same dose group were mated (1:1 pairing). F1 (P1) males and females from Cohort 1B were bred (1:1 pairing) after minimum treatment up to PND 90 to obtain a F2 generation.

Each F1 and F2 litter was examined as soon as possible after the delivery of the dam (PND 0) to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on days 4, 7, 14 and 21 post-partum.

The anogenital distance (AGD) of each F1 and F2 (Cohort 1B extension) pup was measured on PND 0 and all male pups were checked for the presence of nipples/areolae on PND 12.

All selected F1 male and female pups from all cohorts (except surplus not selected for cohorts) were checked daily for balano-preputial separation or vaginal patency, respectively, starting from PND 30 in males and PND 25 in females.

Vaginal smears of P0 females were examined 2 weeks before the beginning of the treatment period, during the 2-week premating period and until confirmation of mating. Vaginal smears were examined daily for all F1 females in Cohort 1A after the onset of vaginal patency until the first cornified smear was recorded. Vaginal smear in Cohort 1A was also examined for 2 weeks starting from PND 75. The vaginal smear in Cohort 1B was examined during the mating period to confirm evidence of mating.

Haematological, coagulation, thyroid hormone analysis (T4 and TSH) and clinical biochemistry parameters were determined with blood samples obtained from 10 randomly selected P0 males and females and 10 randomly selected F1 Cohort 1A male and female animals at terminal sacrifice. Urinalysis was also performed on samples collected from these animals prior to or as part of their terminal sacrifice. Thyroid hormone analysis (T4 and TSH) was also performed on 10 pups/sex/group at PND 4 and after weaning (pups not allocated to cohorts) on PND 22.

To identify possible toxic effects on male fertility, sperm motility and testicular sperm head count an evaluation was performed at the end of the treatment period from all Parental generation males and all Cohort 1A males of each group by using a Hamilton Thorn sperm analyser. Sperm morphology was evaluated at the end of the treatment period from all P0 generation males and all F1 Cohort 1A males from control and HD group.

Cohort 3 animals consisting of 10 males and 10 females (on PND 56±3 days) from each treatment group were used for a T-cell dependent antibody response assay to measure KLH-specific IgM antibodies. Pre- and postnatally induced immunotoxic effects at termination from 10 male and 10 female F1 Cohort 1A animals from each treatment group were evaluated by analysis of splenic lymphocyte subpopulations (lymphocytes, T cells, CD4+ (helper T cells) CD8+ (cytotoxic T cells), B lymphocytes and natural killer (NK) cells using one half of the spleen.

At the conclusion of the treatment period of P0 animals and various cohorts, all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved. Animals that died or were sacrificed in a moribund condition were examined macroscopically and histopathologically.

A full histopathological evaluation of the collected tissues was performed on HD and control P0 and F1 Cohort 1A animals. The histopathological examination of F1 Cohort 1A female ovaries included quantitative evaluation of primordial and small growing follicles and corpora lutea. From F1 Cohort 1A males, for the testes, a detailed qualitative examination was made taking into account the tubular stages of the spermatogenic cycle at evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides. These examinations were not extended to animals of the other dosage groups as no treatment-related changes were observed in the HD group.

Any gross lesion macroscopically identified was examined microscopically in all animals including found dead or moribund sacrificed animals. Additionally, based on the histopathology findings of the HD group, lower dose levels (LD and MD groups) were evaluated in the P0 generation including kidneys, ureters, urinary bladder, duodenum, jejunum and ileum and F1 Cohort 1A with kidneys, ureters, urinary bladder and jejunum.

In the P0 generation, 1/25 LD group female was found dead during the study period. In F1 Cohort 1A, 1/20 LD male, 1/20 MD female and 1/20 HD female were moribund sacrificed and 1/20 LD female was found dead during the study period. In F1 Cohort 1B, 2/20 control, 4/20 LD group, 1/20 MD group females and 1/20 HD male were found dead during the study period. No mortality was observed in F1 Cohort 3. The cause of the remaining unscheduled deaths was deemed to be accidental due to gavage error, and none were considered to be treatment-related.

In terminally sacrificed P0 and F1 male and female animals, predominant clinical signs transiently observed in the majority of the HD group were increased salivation (slight/moderate) and/or moving the bedding. Low incidences of clinical signs including hairless area, crust, scratch/cut, eyelid closure, piloerection, chromodacryorrhea, corneal opacity and diarrhoea (single incidence), exophthalmos (left), wound, overgrown teeth and spontaneous reduced activity (slight) were observed in few animals on few days in all groups including control. As these findings showed no dose-dependency and were transient, they were not considered to be toxicologically relevant.

The clinical signs salivation and moving the bedding in males and females were observed immediately after the dose administration and, therefore, were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect. None of the P0 or F1 Cohort 1B females showed signs of abortion or premature delivery. During the weekly detailed clinical observation, no toxicologically relevant differences between the treated groups and the control group were observed in P0 and F1 Cohorts (1A, 1B and 3) during the entire study period. In all P0 and F1 cohorts, mean body weight and mean body weight gain remained unaffected by the treatment with the test item. There were no test item-related effects on food consumption for P0 and F1 animals. The test item had no biologically or statistically significant effect on the oestrus cycle of P0 and F1 Cohort 1A females.

In P0 and Cohort 1B females, there were no test item-related or statistically significant effects on litter parameters including group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, stillbirth, or runt on PND 0, as well as number of alive pups, alive male and female pups and sex ratio on PNDs 4, 7, 14 and 21 when compared to the control. Statistical analysis of litter data revealed no significant effects in treatment groups when compared with the control. There was no test item-related effect on pup mean weight, total litter weight, male and female litter weight on PNDs 0, 4, 7, 14 and 21 observed in P0 and F1 Cohort 1B treatment groups when compared with the controls. There was no test item-related effect observed on the duration of pre-coital interval and the duration of gestation in the P0 and F1 Cohort 1B treatment group females when compared to the control.

There were no test item-related effects observed on the number of corpora lutea, implantation sites, alive pups on PND 0, percent preimplantation loss and post implantation loss in P0 and F1 Cohort 1B treatment group females when compared with the corresponding control group. There was no test item-related effect observed on the reproductive indices (percent male mating index, female mating index, male fertility index, female fertility index, gestation index and live birth index) in P0 and F1 Cohort 1B treatment group animals when compared to the respective control group. No test item-related effect on mean mortality of pups from PND 0 to 4, PND 4 (after interim sacrifice) to PND 14 and PND 14 (after interim sacrifice) to PND 21 in P females and from PND 0 to 4, PND 4 to 21 in F1 Cohort 1B female treatment groups was observed when compared to the control group. There was no test item-related effect on anogenital distance and nipple retention in F1 and F2. No test item-related gross external abnormalities of toxicological relevance on PND 0-20 were observed in the F1 and F2 pups. No test item-related adverse effects were observed in haematological parameters in the dose groups when compared to the control group.

In HD group males, statistically significantly higher mean WBC, RBC, HGB and HCT were observed when compared to the control. In MD group males, higher mean HGB and HCT and lower RET percent were observed when compared to the control. In females, no statistical significance was observed when compared with the control. All these values were without any dose dependency or consistency; hence they were not considered to be adverse. No test item-related effect was observed on coagulation parameters in P0 males and females when compared with the control, except for slight but statistically significantly higher mean PT value in HD group males and MD and HD group females when compared to the control. All other group mean and most of the individual values for haematological parameters in males and females were comparable to controls and within the normal range of variation, and they were also in line with historical control data of reproductive and developmental toxicity studies in this strain.

In F1 Cohort 1A males, marginally but statistically significantly higher mean RBC was observed in MD group males when compared with the control. Marginally but statistically significantly higher mean RBC, HGB and HCT were observed in MD group females when compared with the control. As the differences were marginal and all values were within the range of historical control data, these findings were not assumed to be toxicologically relevant. All other group means and most of the individual values for haematological parameters in male and females were comparable to the controls and within the normal range of variation. In the absence of test item-related histopathological findings and effect on splenic lymphocyte subpopulation in the study, the above-mentioned increase or decrease in a few haematology parameters was not considered to be adverse. No test item-related effect was observed on coagulation parameters in Cohort 1A males and females when compared with the respective control. There were a few marginal but statistically significant differences in clinical biochemistry parameters of male and female animals of P0 and F1 Cohort 1A. In Parental animals, lower creatinine was observed in HD group males and in MD and HD group females. In F1 Cohort 1A animals, differences in potassium (LD group males), protein (HD group males), and ASAT and Cholesterol (HD group females) were observed. As they were within the range of historical control data, not dose-dependent, and did not coincide with histopathological findings, these findings were not considered toxicologically relevant. No test item-related changes were observed in urinary parameters in P0 and F1 Cohort 1A groups. No effects were noted in thyroid hormone analysis for P0, F1 Cohort 1A and F1 pups on PND 4 and PND 21. No significant difference in group mean body weight and day of onset of vaginal opening in females and balano-preputial separation in males was observed in F1 (Cohort 1A, 1B and 3) treatment groups when compared to the control. There were no test item-related effects on sperm analysis in P0 and F1 Cohort 1A animals. There was a slight increase in mean lymphocytes and T cell populations in both male and female F1 Cohort 1A animals when compared to the control. There was no test item-related or statistically significant change in the number of splenic B cells. They were found to be comparable between all dose groups and control group in both males and females. There was no significant indication of immunosuppressive effect of the test item on lymphocyte subpopulations.

The results of the TDAR in F1 Cohort 3 indicate a functional immune system. KLH-specific IgM levels fold difference in the HD group was found to be variable but LD and MD groups were comparable to the negative control and did not show any sign of effect on the specific immune response. Macroscopic findings were not recorded in animals with unscheduled death. In the decedents, there were no gross lesions that could be attributed to treatment with the test item. In animals that survived their scheduled treatment period, gross lesions that were considered to be treatment-related were recorded in the urinary bladder of females from P0-generation and in the urinary bladder and/or ureters of both sexes of animals from F1-generation Cohort 1B. Test item-related gross findings were not recorded in F1-generation Cohort 1A animals. There were no statistically significant differences in the terminal body weights (TBW) in both sexes of both generations (P0-generation and F1-generation Cohort 1A). Test item-related changes were found in the kidney of P0-generation and F1-generation Cohort 1A animals. In the histopathological examination, for both the P0- and F1 (Cohort 1A)-generations, microscopic changes that could be attributed to treatment with the test item were observed in the kidneys and urinary bladder of both sexes of animals treated with 100 mg/kg bw/day and higher, and in the ureters and small intestine of both sexes of animals treated with 300 mg/kg bw/day.

In the kidneys of  P0-generation the following changes were observed: diffuse urothelial hyperplasia and mixed inflammatory cell infiltration in the suburothelium in both sexes; fibrosis at/around the fornix, focal to multifocal interstitial fibrosis, pelvic luminal precipitates, and foreign body giant cells at/around the fornix in males; and increased incidence and/or severity of pelvic dilation, focal to multifocal mononuclear cell infiltration, tubular basophilia and tubular dilatation in males. In the urinary bladder of P0-generation the following changes were observed: diffuse urothelial hyperplasia, mixed inflammatory cell infiltration in the mucosa, increased incidence and/or severity of mononuclear cell focus/foci in lamina propria, submucosal oedema, congestion, and haemorrhage in both sexes. In the ureters of P0-generation the following changes were observed: diffuse urothelial hyperplasia in both sexes; increased incidence and severity of luminal dilatation in males; and mixed inflammatory cell infiltration and submucosal oedema, as well as diffuse urothelial hyperplasia and luminal dilatation, in the ureters present by chance on the prostate sections. In the small intestine of P0-generation the following changes were observed: lipid accumulation in lamina propria was observed in the duodenum, jejunum and ileum of males and in the jejunum and ileum of females.

In the kidneys of F1-generation Cohort 1A the following changes were observed: diffuse urothelial hyperplasia and mixed inflammatory cell infiltration in the suburothelium in both sexes; tubular dilatation, granuloma at/around the fornix and increased incidence of pelvic dilation in males; and pelvic luminal precipitates in female. In the urinary bladder of F1-generation Cohort 1A the following changes were observed: diffuse urothelial hyperplasia, mixed inflammatory cell infiltration in the mucosa, mononuclear cell focus/foci in lamina propria, haemorrhage, and increased incidence of submucosal oedema in both sexes; and congestion in males. In the ureters of F1-generation Cohort 1A the following changes were observed: diffuse urothelial hyperplasia and increased incidence of luminal dilatation in both sexes; mixed inflammatory cell infiltration and submucosal oedema, as well as diffuse urothelial hyperplasia and luminal dilatation, were identified in the ureters present by chance on the prostate sections. In the small intestine of F1-generation Cohort 1A the following changes were observed: lipid accumulation in lamina propria was observed in the jejunum of both sexes. There was no test item-related histomorphological changes in the male and female reproductive organs in either generation. In addition, no microscopic indicators for endocrine disruption were noted in the organs and tissues (including pituitary glands, adrenal glands and thyroid glands) examined in either generation.

The NOAEL for general systemic toxicity for P0 and F1 (Cohort 1A and 1B [P1]) animals was concluded to be 40 mg/kg bw/day based on test substance related effects in the urinary system at 100 and 300 mg/kg bw/day; the reproductive toxicity NOAEL for P0 and F1 (Cohort 1A and 1B) animals was at least 300 mg/kg bw/day based on no adverse effects on reproductive organs and performance; the developmental toxicity NOAEL for F1 (Cohort 1A and 1B) and F2 (Cohort 1B extension) was at least 300 mg/kg bw/day based on no adverse effects on pre- and post-natal development; the developmental immunotoxicity NOAEL for F1 (Cohort 3) was at least 300 mg/kg bw/day based on no adverse effects on immune system development and response.

In the oral combined repeated dose toxicity study with the reproduction / developmental toxicity screening test, conducted according to OECD Test Guideline 422 and in compliance with GLP (Hashima Laboratory, 2005), trimethoxy(vinyl)silane (CAS 2768-02-7; EC No. 220-449-8) was administered by gavage at doses of 62.5, 250 or 1000 mg/kg bw/day. The NOAELs for reproductive performance of parental animals were estimated to be 1000 mg/kg bw/day for males and 250 mg/kg bw/day for females. The NOAEL for development was 1000 mg/kg bw/day. Regarding reproductive toxicity, a low number of estrous cases was noted in the 1000 mg/kg bw/day group. No changes attributable to the test article were noted for the copulation index, number of conceiving days, number of pregnant females, fertility index, gestation length, gestation index, delivery conditions, nursing conditions, number of corpora lutea, number of implantation sites, or the implantation rate. No effects were noted for the offspring.

 

In the 14-week repeated dose vapour inhalation toxicity study in rats which was conducted according to current guideline and in compliance with GLP (Bushy Run Research Center, 1990), rats were repeatedly exposed to nominal concentrations of  10, 100 or 400 ppm of the registered substance for 6 hours per day over 14 weeks. The reproductive organs of male and female animals were examined microscopically in response to treatment with the test article. After 14 weeks of exposure, the absolute testes weight was statistically significantly lower in the 400 ppm group when compared to control mean values. When expressed as a percentage of body weight, the testes weights were equivalent to control values. There was no effect on absolute testes weight in the 400 ppm recovery group animals. There were no histopathological lesions reported for any of the reproductive organs examined in any group. No effect was noted on male reproductive organs in the study.

Effects on developmental toxicity

Description of key information

In the prenatal developmental toxicity study, conducted according to EPA OTS 798.4350 (Inhalation Developmental Toxicity Screen) and in compliance with GLP, pregnant female rats were exposed to the test concentrations of 25, 100 or 300 ppm via whole body inhalation during gestation days 6 to 15. Exposure of pregnant rats during organogenesis to trimethoxy(vinyl)silane (CAS 2768-02-7; EC No. 220-449-8) by inhalation resulted in slight maternal toxicity at 100 and 300 ppm as evidenced by concentration-dependent reductions in gestational body weight gain (gestation days 6-9). There was evidence of slightly delayed development in fetuses from the 300 ppm group as indicated by delayed ossification in several skeletal districts. No exposure-related embryotoxicity or teratogenicity was observed in this study (Bushy Run Research Center, 1993). The observed variations are not considered toxicologically relevant, particularly in the presence of maternal toxicity, and therefore the NOAEC is greater than or equal to 300 ppm (approximately 1730 mg/m3).

In the prenatal developmental toxicity study in rabbits, conducted according to OECD Test Guideline 414 and in compliance with GLP, the LOAEL for maternal toxicity was concluded to be 7.5 mg/kg bw/day based on degenerative changes characterized by tubular dilatation and tubular basophilia in all dose groups; the NOAEL for developmental toxicity was concluded to be equal to or greater than 75 mg/kg bw/day (the highest dose tested) based on no observed adverse effects in any of the foetuses (BSL Bioservice, 2020b).

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25th of October 2019 to 9th of June 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted 25 June 2018
Deviations:
yes
Remarks:
General clinical observation were not recorded for several animals on one weekend
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Remarks:
Crl: KBL
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: Approximately 19-25 weeks
- Weight at study initiation: males: 4060 – 3377 g (mean: 3681 g ± 20 % = 2945 – 4418 g)
females: 4190 - 2519 g (mean: 3472 g ± 20 % = 2777 – 4166 g)
- Fasting period before study: no
- Housing: Housed in ABS-plastic or Noryl rabbit cages, floor 4200 cm2, semi barrier housing in an air-conditioned room.
- Diet: Altromin 2123 (manufactured by Altromin Spezialfutter GmbH & Co.KG, Im Seelenkamp 20, D-32791 Lage) maintenance diet for rabbits, rich in crude fibre, ad libitum
- Water: tap water (with municipal residue and microbiological controls at regular intervals), ad libitum
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 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 substance was weighted and the vehicle was added to the appropriate final concentrations. The formulation was vortexed and/or stirred until visual homogeneity was achieved. After homogenisation, the formulation was overlaid with argon to prevent instability caused by repeated contact of the test substance formulation with air. The test substance formulations were prepared once every 9 days (within the stability time frame). Formulates were kept under magnetic stirring during the daily administration. The prepared formulation was stored protected from light and at room temperature.

VEHICLE
- Justification for use and choice of vehicle: The vehicle has been selected in consultation with the sponsor based on the test item’s characteristics. The test item was dissolved in dried and de-acidified corn oil.
- Concentration in vehicle: 0, 7.5, 25, 75 mg/mL
- Amount of vehicle: 1 mL/kg bw
- Lot/batch no.: Sigma-MKCH1635, MKCK6411, Caelo-19112904
- Purity: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle. The test item was shown to be homogenous and samples were not collected during the main study for the investigation of homogeneity. Samples were taken in duplicate during the main study only for substance concentration analysis in the first, third, fifth and last weeks of the study for all doses including the vehicle control.
Details on mating procedure:
- Impregnation procedure: Cohoused
- If cohoused: A female was taken to a male’s cage
- M/F ratio per cage: 1:1 ratio (male to female)
- Length of cohabitation: Until mating was confirmed
- In case of unsuccessful mating, the female was taken back to its original cage and mating of the female with another male or at another time was considered.
- Further matings after two unsuccessful attempts: Not specified
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: Visual inspection and based on the falling of male on its back or side and emittance of a typical cry. The day on which a confirmed mating was observed is referred to as day 0 of pregnancy
- Any other deviations from standard protocol: No
Duration of treatment / exposure:
Between GD 6 to GD 27
Frequency of treatment:
Daily
Duration of test:
28 days
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
7.5 mg/kg bw/day
Remarks:
Low Dose, LD
Dose / conc.:
25 mg/kg bw/day
Remarks:
Middle Dose, MD
Dose / conc.:
75 mg/kg bw/day
Remarks:
High Dose, HD
No. of animals per sex per dose:
20 pregnant females per group.
16 males for mating only
100 females in total
In total 116 animals
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A previous dose range finding study examined the developmental effects at doses of 0, 100, 250 and 400 mg/kg bw/day. Test item-related maternal toxicological effects in terms of reduced body weight gain, food consumption and gravid uterine weight were observed at 100, 250 and 400 mg/kg bw/day; slightly lower carcass weight was observed at the 400 mg/kg bw/day dose group; reduced male and female foetal weight was seen in all the dose groups and inflammatory, degenerative and hyperplastic lesions in the urinary tract organs were reported in all dose groups. Based on these results and and in consultation with the sponsor, the following doses were selected for the 3 dose groups: 0 mg/kg bw/day for the control (C) group, treated with the vehicle corn oil, 7.5 mg/kg bw/day for the low dose (LD), 25 mg/kg bw/day for the middle dose (MD) and 75 mg/kg bw/day for the high dose (HD).
- Rationale for animal assignment: randomised
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed for morbidity and mortality twice daily except on weekends and public holidays when observations were made once daily. However, one deviation from the protocol was that clinical signs were not recorded for some animals for 2 days, on a weekend.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once per day. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling, as well as the presence of colonic or tonic movements, stereotypes or bizarre behaviour were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before initiation of pairing to ensure that the body weights were within ±20 % variation.
The mated females were weighed on GDs 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28.
Males were not weighed in this study except once before initiation of pairing

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Food consumption of pregnant females were measured for the following intervals: GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 28.
Food consumption was not measured for males during the entire study or for females before confirmation of mating.

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 28
- Organs examined: The urinary tract (kidneys, ureter and urinary bladder) and liver were examined histopathologically.
Ovaries and uterine content:
The ovaries and uterine content were examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]
- Other: Once the soft tissue evaluation of all foetuses was completed, half of the foetuses were decapitated. Foetuses with intact heads and without heads were then processed for skeletal and cartilaginous double staining by Alcian blue and Alizarin red and evaluated for skeletal anomalies.
Statistics:
Toxicology and pathology data were captured either on paper according to appropriate SOPs or using the validated computerised system Ascentos® System (version 1.3.4, Pathology Data Systems Ltd.).
A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed animals with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, thyroid hormones and foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. The statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 is considered as statistically significant).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related clinical signs observed in the females of any of the treatment group.

Clinical signs observed on a few days during the treatment period of the study. LD group: skin and fur, hairless area (right hind paw) in one animal on GDs 26-27; in one animal on the right or left hind paw on GDs 20-28; skin and fur, hairless area (left forelimb and hind limb) in one animal on GDs 25-28 and skin and fur, hairless area (left hind limb) in one animal on GDs 19-22 and GDs 25-28.
MD group: skin and fur, scratch/cut (neck) was observed in one animal on GD 0. Skin and fur, hairless area (left fore and hind paw, right forepaw) was observed in one animal on GDs 7-28 and 8-17.
HD group: skin and fur, hairless area (left and right hind paw) was observed in one animal on GD 25-28; in one animal on the left and right hind limb on GDs 24-28 and 26-28 and in one animal on the left and right hind paw on GDs 21-25 and GDs 21-28, respectively.
These clinical signs are considered to be incidental findings and not related to treatment with test item.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No test item-related mortality was observed during the treatment period and all animals survived until the end of the study.

One control group rabbit was found dead immediately after dosing on GD 7. It showed clinical signs of cough/sneezing at the post dose observation. Macroscopic observation revealed dark and abnormal red colour lungs. The cause of the death is assumed to be a gavage error during administration.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weight remained unaffected by the treatment with the test item and slightly increased with the progress of the study in all groups throughout the study period. No statistical significance was achieved in any treatment groups on any day or interval of body weight measurement and all values in the treatment groups were comparable to the controls. The inconsistent and statistically non-significant decreases or increases observed on different days of body weights measurement in the treatment groups between GD 3 and GD 27 are considered to be incidental and not test item-related.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption in the LD, MD and HD groups was comparable to the control. Slightly lower food consumption was observed on GDs 9-12 and 21-24 in the LD group (9.31% and 16.38 % respectively, below control). Other slight differences in food consumption did not follow any dose-dependency in the treated groups and thus were not considered toxicologically relevant.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item-related effects of statistical or toxicological relevance were noted for the uterine weight
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related gross pathological changes of toxicological significance were observed during the macroscopic examination of the terminally sacrificed females of the control, LD, MD and HD groups.
A few specific macroscopic changes recorded in the female animals during the macroscopic examination at terminal sacrifice were dark/dark red colour lungs (1/25 in each control, LD and MD groups; abnormal coloured liver (1/25 in control), enlarged liver (1/25 in each control and MD group), red/white gallbladder (1/25 in control), small gallbladder (2/25 in LD group), enlarged gallbladder (1/25 in control, 2/25 in LD female, 2/25 in MD and 1/25 in HD), abnormal shape kidneys in one control, oviduct cyst (1/25 in control female and 2/25 in LD), abnormal shape uterus right horn in LD group (1/25 in LD group) and cyst in the uterus horns (2/25 in control, 1/25 in LD, 2/25 in MD group and 2/25 in HD group) and right uterus horn absent in LD female.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic changes that could be attributed to treatment with the test item were observed in the kidneys, urinary bladder and ureters of all groups treated with the test item.
In kidneys, diffuse transitional cell hyperplasia (urothelial hyperplasia) was observed in 5/25 and 4/25 animals from the MD and HD groups, respectively. Further, degenerative changes characterized by tubular dilatation (2/25, 10/25 and 8/25 in LD, MD and HD groups, respectively) and tubular basophilia (5/25, 8/25 and 1/25 in LD, MD and HD groups, respectively) were noted in animals from all dose groups.
In the urinary bladder, diffuse transitional cell hyperplasia (urothelial hyperplasia) was observed in 9/25, 12/25 and 17/25 animals from the LD, MD and HD treatment groups, respectively. The transitional cell hyperplasia was accompanied by submucosal oedema (2/25, 5/25 and 13/25 in LD, MD and HD groups, respectively) and congestion (5/25, 6/25 and 13/25 in LD, MD and HD groups, respectively) and in one MD group animal and one HD group animal, by accumulation of mixed cell infiltrates in the submucosa.
In ureters, diffuse transitional cell hyperplasia (urothelial hyperplasia) was noted in 10/25, 12/25 and 13/25 animals from the LD, MD and HD treatment groups, respectively.
Overall, the incidence and/or severity of the transitional cell hyperplasia increased in a dose-dependent manner, whereas for all the other changes described above, no clear dose-dependency was observed.
All the above-mentioned findings observed in the urinary tract were deemed to be adverse changes.
In the liver, no test item-related changes were observed. All observed hepatic changes were within the range of normal background lesions which may be observed in animals of this strain and age or were considered incidental lesions.
The remainder of microscopic findings recorded were within the range of normal background lesions which may be observed in animals of this strain and age or were incidental lesions that were not related to treatment with the test item.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice, except one LD female on GD 28.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Mean preimplantation loss was 0.27, 0.09 and 0.26 in the LD, MD and HD groups, respectively, when compared to control (0.04). Mean post implantation loss was 0.91, 0.23 and 0.30 in the LD, MD and HD groups, respectively, when compared to control (1.50). These findings were observed without dose dependency and were considered to be a biological variation in this species.
Total litter losses by resorption:
not specified
Early or late resorptions:
no effects observed
Description (incidence and severity):
Mean early resorption was 0.27, 0.14 and 0.09 in the LD, MD and HD groups, respectively, when compared to control (0.79). Mean late resorption was 0.18, 0.09 and 0.22 in the LD, MD and HD groups, respectively, when compared to control (0.25). These findings were observed without dose dependency and were considered to be a biological variation in this species.
Dead fetuses:
no effects observed
Description (incidence and severity):
One pup in the control group and one pup in the LD group were dead at scheduled sacrifice. These findings were observed without dose dependency and were considered to be a biological variation in this species.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
None of the females showed signs of premature delivery prior to the scheduled sacrifice.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Three LD, three MD, and two HD females were confirmed to be non-pregnant at scheduled sacrifice.
Other effects:
no effects observed
Description (incidence and severity):
Number of corpora lutea was not affected by the treatment.
Key result
Dose descriptor:
LOAEL
Effect level:
ca. 7.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: Urinary tract
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no test item-related effects of toxicological relevance observed for the mean foetus weight, male and female foetus weight on a per litter basis (group mean of individual litter mean) in any of the treatment groups when compared to control.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no test item-related effects of toxicological relevance observed for the number of live offspring.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The number of the difference in the sex ratio was unaffected by the test substance.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
Foetal external examination on the day of terminal sacrifice revealed no test item-related external findings in foetuses of any of the treated groups.
Statistical analysis of data revealed no significant differences compared to the control group. One incidence of cleft palate was observed in one male pup no. 4 from animal no. 2 and considered to be incidental in nature.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal examination of the foetuses observed for skeletal and cartilaginous findings revealed a range of findings that were of a type or occurred at an incidence generally comparable to or slightly lower/higher in the treatment groups when compared to the control group.

A statistically significant lower foetal incidence of pelvic girdle, caudal shift was observed in the MD and HD groups (0%) when compared to control (13%). A statistically significant higher litter incidence of vertebra thoracic centrum/centra was observed in the MD group (81.8%) when compared to control (30.4%) and higher foetal incidences in the MD group (21.3) when compared to control (8.3%). A statistically significant higher litter incidence of dumbbell ossification in vertebra thoracic centrum was observed in the MD group (50%) when compared to control (13%). A statistically significant higher foetal incidence of bipartite ossification in vertebra thoracic centrum was observed in the MD group (14.4%) when compared to control (4.2%). A statistically significant higher foetal incidence of misaligned ossification of hind limb proximal phalanx was observed in the HD group (3.2%) when compared to control (0%). A statistically significant lower foetal incidence of unossified forelimb medial phalanx was observed in the LD group (27.1%) when compared to control (52.4%). A statistically significant higher litter incidence of unossified 6th sternebra was observed in the MD group (54.5%) when compared to control (13%). A statistically significant lower foetal incidence of long costal cartilage was observed in the HD group (32.7%) when compared to control (59%). A statistically significant lower foetal incidence of interrupted costal cartilage was observed in the LD, MD and HD groups (0% in each) when compared to control (4.6%). All these statistically significant findings were observed without any dose dependency, hence they are not considered to be test item-related, but rather spontaneous in nature.
Higher or lower litter incidences, but without achieving statistical significance were observed as mentioned below. Higher litter incidences of hole in the xiphoid (LD, MD and HD groups (19%, 22.7% and 34.8%, respectively) were observed compared to 26.1 % in control. Higher litter incidences of unossified forelimb proximal phalanx (LD, MD and HD groups (14.3%, 18.2% and 30.4%, respectively) were observed compared to 8.7% in control. Higher or lower litter incidence of skull hyoid body (incomplete ossification, unossified) was observed in treated groups (76.2 %, 81.8 % and 91.3 % in the LD, MD and HD groups, respectively) and control group (91.3 %). Higher or lower litter incidence of hind limb talus (incomplete ossification, unossified) was observed in treated groups (19 %, 18.2 % and 30.4 % in the LD, MD and HD groups, respectively) and control group (21.7 %).
Higher or comparable litter incidence of pelvic girdle, caudal shift was observed in the treated groups 52.4 %, 50 % and 52.2 % in the LD, MD and HD groups, respectively) and control group (52.2 %). Higher or lower litter incidence of unossified 5th sternebra was observed in treated groups (42.9 %, 54.5 % and 73.9 % in the LD, MD and HD groups, respectively) and control group (69.6 %). Higher or lower litter incidence of incomplete ossification of 5th sternebra was observed in treated groups (76.2 %, 77.3 % and 91.3 % in the LD, MD and HD groups, respectively) and control group (87 %). Lower litter incidence of interrupted vertebra cervical cartilaginous ventral plate was observed in treated groups (42.9 %, 40.9 % and 43.5 % in the LD, MD and HD groups, respectively) and control group (52.2 %). Higher litter incidence of rudimentary ribs was observed in treated groups (76.2 %, 95.5 % and 78.3 % in the LD, MD and HD groups, respectively) and control group (65.2 %). Higher or lower litter incidence of unossified hind limb medial phalanx was observed in treated groups (23.8 %, 27.3 % and 60.9 % in the LD, MD and HD groups, respectively) and control group (30.4 %).
Higher or lower litter incidence of unossified and incomplete ossification of pelvic girdle pubis was observed in treated groups (28.6 %, 22.7 % and 52.2 % in the LD, MD and HD groups, respectively) and control group (30.4 %). Higher litter incidence of incomplete ossification of 6th sternebra was observed in treated groups (42.9 %, 68.2 % and 52.2 % in the LD, MD and HD groups, respectively) and control group (39.1 %). Higher litter incidence of misaligned costal cartilage was observed in treated groups (28.6 %, 27.3 % and 39.1 % in the LD, MD and HD groups, respectively) and control group (8.7 %). Higher litter incidence of nodulated costal cartilage was observed in treated groups (0 %, 18.2 % and 30.4 % in the LD, MD and HD groups, respectively) and control group (4.3 %). Higher or lower litter incidence of short costal cartilage was observed in treated groups (14.3 %, 45.5 % and 56.5 % in the LD, MD and HD groups, respectively) and control group (21.7 %).
The observed incomplete ossification of a few bones and other skeletal findings in the treatment groups were either marginally lower or higher and were not considered to be adverse. Generally delayed ossification is not regarded to persist postnatally as it is not associated with long-term consequences on survival, general growth and development. There was no indication of a test item-related trend in the type and/or incidences of other skeletal findings, therefore they were considered to be spontaneous in nature.

All the above findings are considered to be incidental and without dose dependency, as frequencies were even less in number compared to controls. Therefore, these findings are not considered to be treatment-related, but rather spontaneous in nature.
There were further non-statistically significant findings that were considered to be spontaneous in nature and not related to the treatment with the test item.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Internal observation of the foetal viscera revealed a range of visceral findings in all groups including control.

Higher litter incidence of dilated aortic arch - was observed in the MD group (9.1%) when compared to control (4.3%). Higher litter incidence of enlarged liver was observed in the HD group (8.7%) when compared to control (0%). Higher litter incidence of supernumerary liver lobe was observed in the MD and HD groups (36.4% and 30.4%, respectively) when compared to control (26.1%). Higher litter incidence of cleft at liver lobe was observed in the LD, MD and HD groups (23.8%, 50% and 34.8%, respectively) when compared to control (60.9%). Higher litter incidence of heart with large ventricle, thin ventricular wall and dilated ductus arteriosus was observed in the MD group (9.1% for each finding) when compared to control (0%). Higher litter incidences of small lung were observed in the MD group (9.1%) when compared to control (0%). Higher litter incidences of fluid-filled abdomen was observed in MD group (9.1%) when compared to control (4.3%). Higher litter incidences of discoloured kidney were observed in the MD group (9.1%) and dilated pelvis (13%) in the HD group when compared to control (0% for each observation). Higher litter incidences of large gall bladder were observed in the LD, MD and MD groups (14.3%, 13.6%, and 17.4%, respectively) when compared to control (0%).

Higher or lower litter incidences of small gall bladder were observed in LD, MD and MD groups (33.3%, 18.2%, and 17.4%, respectively) when compared to control (34.8%). Higher or lower litter incidences of discoloured gallbladder contents were observed in all treated groups (81%, 95.5%, and 87% in LD, MD and HD groups, respectively) when compared to control (87%).

Higher litter incidences of discoloured spleen were observed in the MD and HD groups (18.2%, and 8.7%, respectively) and small spleen in HD group (13%) when compared to control (4.3% for both observations). Higher litter incidences of discoloured thymus were observed in the LD, MD and HD groups (28.6%, 27.3%, and 30.4%, respectively) when compared to control (26.1%) and long thymus in the MD and HD groups (40.9% and 43.5%, respectively) when compared to control (39.1%).

Visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls. As observed findings were either minor variation and/or showed a lack of dose dependency and consistency, no serious toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature.

There was no statistical significance observed in litter incidences of findings in treatment groups when compared with the control and no indication of a test item-related trend in the type and incidences of visceral findings.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Craniofacial examination by razor blade serial sectioning technique revealed no treatment-related findings in the dose groups when compared to the control group. However, a few findings were observed as described below;
A statistically non-significant lower or higher litter incidence of haemorrhages in the nasal cavity was observed in the LD, MD and HD groups (19%, 22.7% and 52.2%, respectively) when compared to control (30.4%). Though the findings for the HD group were slightly higher when compared to control, these findings are not considered to be treatment-related, but rather spontaneous in nature.
Single incidences of hematoma in the subdural region and subcutaneous hematoma in the head in the LD group (4.8 % above control for each observation) and subcutaneous hematoma in the head in the HD group (4.3%) were observed, when compared to control. These findings are not considered to be test item-related, but rather spontaneous in nature.

Statistical analysis of the data revealed no statistical significance for any of these findings.
Key result
Dose descriptor:
NOAEL
Remarks:
foetal
Effect level:
>= 75 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
In a prenatal developmental toxicity study in rabbits, conducted according to OECD Test Guideline 414 and in compliance with GLP, the LOAEL for maternal toxicity was concluded to be 7.5 mg/kg bw/day based on degenerative changes characterized by tubular dilatation and tubular basophilia in all dose groups; the NOAEL for developmental toxicity was concluded to be equal to or greater than 75 mg/kg bw/day based on no observed adverse effects in any of the foetuses.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OTS 798.4350 (Inhalation Developmental Toxicity Screen)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: CD(R)
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytically determined concentrations of test item vapour were 24.6, 96.7 and 312.0 ppm for target concentrations of 25, 100 and 300 ppm, respectively.
Details on mating procedure:
Mating not performed; timed-pregnant CD® rats were exposed during gestation
Duration of treatment / exposure:
gestation days 6-15
Frequency of treatment:
6 hours/day
Duration of test:
Up to gestation day 21
Dose / conc.:
25 ppm
Dose / conc.:
100 ppm
Dose / conc.:
300 ppm
No. of animals per sex per dose:
25 timed-pregnant CD® rats/dose level
Control animals:
yes
Details on study design:
Four groups, each consisting of 25 timed-pregnant CD® rats, were exposed to vinyltrimethoxysilane; CAS No. 2768-02-7 vapour or filtered air for 6 hours/day on gestational days (gd) 6 through 15. Target concentrations of test item were 0 (control), 25, 100, and 300 ppm. The dams were sacrificed on gestation day 21.
Maternal examinations:
Clinical observations were made daily. In addition, each group of dams was observed from outside their respective exposure chambers for overt clinical signs during the actual exposures. Body weights were measured on gd 0, 6, 9, 12, 15, 18 and 21. Maternal food consumption was measured at
3-day intervals throughout gestation. At scheduled sacrifice on gd 21, the dams were evaluated for body weight, liver and kidney weights, and gravid uterine weight, . Maternal liver, kidneys, and the upper and lower respiratory tract were retained in 10% neutral buffered formalin.
Ovaries and uterine content:
The number of corpora lutea, and number and status of implantation sites (including early and late resorptions, dead fetuses, and live fetuses) were avaluated.
Fetal examinations:
All live and dead foetuses were dissected from the uterus, weighed and examined externally for malformations, variations, and gender determinations.
Approximately one-half of the live foetuses in each litter were examined for visceral and craniofacial malformations and variations. The remaining one-half of the foetuses were stained with alizarin red S and were examined for skeletal malformations and variations.
Clinical signs:
no effects observed
Description (incidence and severity):
One dam from the 300 ppm group was removed from the study on GD 14 due to trauma unrelated to exposure. There were no treatment-related effects clinical signs.
Mortality:
no mortality observed
Description (incidence):
No treatment-related mortality occurred during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment-related effects on gestational body weight. However, on GD 6 to 9 body weight gain was decreased by 28 and 34% in the 100 and 300 ppm groups, respectively.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no treatment-related effects on food consumption.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Macroscopic assessment of the dams and organ weights measured at necropsy did not reveal any treatment-related effects.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic assessment of the dams and organ weights measured at necropsy did not reveal any treatment-related effects.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
not specified
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
effects observed, treatment-related
Description (incidence and severity):
A 300 ppm female delivered early on GD 21, and therefore, was removed from the study and all data summaries with the exception of pregnancy calculations.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The pregnancy rate was equivalent for all groups and ranged from 96 to 100%. Two females, one each from the 25 and 300 ppm groups, had only non-viable implants and one female each from the 25 and 100 ppm groups were not pregnant.
Other effects:
no effects observed
Description (incidence and severity):
Assessment of the various reproductive endpoints did not reveal any differences among the groups.
Dose descriptor:
NOAEL
Effect level:
25 ppm
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Abnormalities:
effects observed, treatment-related
Localisation:
other: body weight gain
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean fetal body weight/litter were not different among control and treated groups.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
Fetal examinations indicated no evidence of treatment-related embryolethality or teratogenicity.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Developmental delay in the 300 ppm group was indicated by an increase in the incidence of delayed skeletal ossification of the anterior arch of the atlas, thoracic centra, interparietal, matatarsals, and phalanges. A statistical increase in the number of litters at 100 ppm with unossified anterior arch of the atlas was not considered to be biologically significant because no other endpoints were similarly affected and the incidence (79.2%) was close to that observed in historical controls (29.2-73.9%).
Visceral malformations:
no effects observed
Dose descriptor:
NOAEC
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Abnormalities:
effects observed, treatment-related
Localisation:
other: skeletal
Description (incidence and severity):
delayed ossification
Developmental effects observed:
yes
Lowest effective dose / conc.:
300 ppm
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
Exposure of pregnant CD® rats during organogenesis to the test substance by inhalation resulted in slight maternal toxicity at 100 and 300 ppm as evidenced by concentration-dependent reductions in gestational body weight gain (GD 6-9). There was evidence of slightly delayed development in foetuses from the 300 ppm group as indicated by delayed ossification in several skeletal districts in the presence of maternal toxicity only. No exposure-related embryotoxicity or teratogenicity was observed in this study.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
Conducted according to OECD Test Guideline 414 and in compliance with GLP.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
1 730 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Conducted according to EPA OTS 798.4350 (Inhalation Developmental Toxicity Screen) and in compliance with GLP.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In the key prenatal developmental toxicity study, conducted according to EPA OTS 798.4350 (Inhalation Developmental Toxicity Screen) and in compliance with GLP, pregnant female rats were exposed to the test concentrations of 25, 100 or 300 ppm via whole body inhalation during gestation days 6 to 15 (Bushy Run Research Center, 1993). The pregnancy rate was equivalent for all groups and ranged from 96 to 100%. No treatment-related mortality occurred during the study. One dam from the 300 ppm group was removed from the study on gestation day 14 due to trauma unrelated to exposure. Another 300 ppm female delivered early on gestation day 21, and was therefore removed from the study and all data summaries with the exception of pregnancy calculations. Two females, one each from the 25 and 300 ppm groups, had only non-viable implants and one female each from the 25 and 100 ppm groups were not pregnant. Assessment of the various reproductive endpoints did not reveal any differences among the groups. Foetal examinations indicated no evidence of treatment-related embryolethality or teratogenicity. Developmental delay in the 300 ppm group was indicated by an increase in the incidence of delayed skeletal ossification of the anterior arch of the atlas, thoracic centra, interparietal, metatarsals, and phalanges. A statistical increase in the number of litters at 100 ppm with unossified anterior arch of the atlas was not considered to be biologically significant because no other endpoints were similarly affected and the incidence (79.2%) was close to that observed in historical controls (29.2-73.9%). Mean foetal body weight/litter were not different among control and treated groups. The NOAEC for maternal toxicity was concluded to be 25 ppm based on concentration-dependent reductions in gestational body weight gain and the NOAEC delayed development is greater than or equal to 300 ppm based on evidence of slightly delayed development in foetuses from the 300 ppm group as indicated by delayed ossification in several skeletal districts, but only in the presence of maternal toxicity.

 

In the key prenatal developmental toxicity study in a second species, conducted according to OECD Test Guideline 414 and in compliance with GLP, pregnant female rabbits were exposed to the test item, trimethoxy(vinyl)silane, at doses of 0, 7.5, 25 or 75 mg/kg bw/day in corn oil via oral gavage during gestation days (GDs) 6 to 27. During the period of administration, the animals were observed each day for signs of toxicity and mortality. Animals that died during the study were examined macroscopically. All surviving animals were sacrificed on their respective GDs 28. Following the gross necropsy, the uteri and ovaries were removed, weighed and examined for number of implantations, resorptions (early and late), and live and dead foetuses. Foetuses were identified by colour strings, sexed and weighed. The foetuses were observed for external abnormalities, visceral, craniofacial abnormalities and skeletal abnormalities. Body weight and food consumption were measured on GDs 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28. The uteri of the non-pregnant females were processed and checked for the early embryonic deaths.

No test item-related mortality was observed in any of the treated groups during the treatment period of this study. Moribund sacrifice of one control female on GD 7 was attributed to a gavaging error. No treatment-related clinical signs or changes in body weight and food consumption were observed in any the treated groups when compared to control.

No treatment-related effect on prenatal and litter data parameters and gross pathology of terminally sacrificed females was observed at any dose tested.

Furthermore, no treatment-related and toxicologically relevant external, visceral, craniofacial and skeletal findings were observed in the treatment groups when compared with the controls.

In the urinary tract, diffuse transitional cell hyperplasia was observed in kidneys, ureters and urinary bladder of several animals from all groups treated with the test item. In the urinary bladder, the hyperplastic change was accompanied by submucosal congestion, oedema and, in two animals, by mixed cell infiltration. There were degenerative changes in the kidneys of the affected animals, which included tubular dilatation and slightly increased tubular basophilia. The LOAEL for maternal toxicity was concluded to be 7.5 mg/kg bw/day based on the degenerative changes characterized by tubular dilatation and tubular basophilia in all dose groups; the NOAEL for developmental toxicity was concluded to be equal to or greater than 75 mg/kg bw/day based on no observed adverse effects in any of the foetuses (BSL Bioservice, 2020b).

In a dose range finding study in rabbits, conducted according to a guideline similar to OECD Test Guideline 414 but not in compliance with GLP, pregnant female rabbits were exposed to the test item, trimethoxy(vinyl)silane, at doses of 0, 100, 250 or 400 mg/kg bw/day in corn oil via oral gavage during GDs 6 to 27 (BSL Bioservice, 2019). During the period of administration, the animals were observed each day for signs of toxicity and mortality. Animals that died during the study or were euthanised in moribund condition for animal welfare reasons were examined macroscopically. All surviving female animals were sacrificed on their respective GD 28. Following the gross necropsy, the uteri and cervix were removed, weighed and examined for number of corpora lutea, implantations, resorptions (early and late), and for live and dead foetuses. Foetuses were identified using numbered plates with strings tied around the neck or the abdomen, sexed and weighed. All foetuses were observed for external abnormalities. Body weight was measured on GD 0, 6, 9, 12, 15, 18, 21, 24, 27 and 28. Food consumption was measured on GD 6, 9, 12, 15, 18, 21, 24 and 27. The uteri of the non-pregnant females were processed and checked for early embryonic deaths. Tissues collected (urinary tract and liver) were processed and evaluated for histopathological changes.

Test item-related maternal toxicological effects in terms of reduced body weight gain, food consumption and gravid uterine weight were observed at 100, 250 and 400 mg/kg bw/day when compared to the control group. The carcass weight was slightly lower in the HD group when compared to the control.

No pre-implantation loss was found in any of the treated groups and control group. The post-implantation loss was slightly higher in the LD, MD and HD groups when compared to the controls. This was likely related to a slightly higher rate of early resorptions in the HD group, with no findings of early resorptions at MD and LD groups. These findings were not dose-dependent and are considered to be incidental findings and not toxicologically relevant.

The male and female foetal weight was found to be reduced in the LD, MD and HD groups when compared to the control. This reduction in mean body weight is considered to be toxicologically significant. No external foetal abnormalities were noted in this study.

Under the conditions of this study, test item at doses of 100, 250 or 400 mg/kg bw/day, produced treatment-related lesions (inflammatory, degenerative and hyperplastic lesions) in the urinary tract organs even in the LD animals, so a NOAEL could not be established.

 

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test via the oral route, conducted according to OECD Test Guideline 422 and in compliance with GLP (Hashima Laboratory, 2005), a low number of oestrous cases was noted in the 1000 mg/kg bw/day group. No changes attributable to the chemical were noted in the copulation index, number of conceiving days, number of pregnant females, fertility index, gestation length, gestation index, delivery conditions, nursing conditions, number of corpora lutea, number of implantation sites, or implantation rate. The NOAELs for reproductive performance of parental animals were estimated to be 1000 mg/kg bw/day for males and 250 mg/kg bw/day for females. No changes attributable to treatment were noted in the number of pups, number of stillbirths, number of pups born, sex ratio, delivery index, birth index, live birth index, general signs, and number of live pups on Day 4 of lactation, viability index of pups on Day 4 of lactation, appearance, body weight, or necropsy findings. The NOAEL for pups was estimated to be 1000 mg/ kg bw/day.

Justification for classification or non-classification

Based on the available data, trimethoxy(vinyl)silane does not require classification for reproductive or developmental toxicity according to Regulation (EC) No 1272/2008.

Additional information