Hexadecyltrimethoxysilane

Endpoint summary

Description of key information

RDT oral (OECD TG 408, GLP), rat: NOAEL = 100 mg/kg bw/day

RDT inhalation: no data available

RDT dermal: no data available

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Mar - 04 Aug 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 7-8 weeks
- Weight at study initiation: males: 155 – 192 g; females: 113 – 163 g
- Fasting period before study: Not reported
- Housing: Housed in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet for rats and mice available ad libitum
- Water: Tap water sulphur acidified to a pH of approximately 2.8 available ad libitum
- Acclimation period: At least 5 days

DETAILS OF FOOD AND WATER QUALITY:
Drinking water: municipal residue control, microbiological controls at regular intervals;
Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins BioPharma Product Testing Munich GmbH. There were no known contaminants in the feed or water at levels that would interfere with the objectives of the study.

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

Route of administration:

oral: gavage

Details on route of administration:

The test item formulation and vehicle were administered as a single daily dose to the animals by oral gavage. The application volume for all groups was 4 mL/kg body weight.

Vehicle:

corn oil

Remarks:

dried and de-acidified

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tared plastic vial and the vehicle was added to give the appropriate final concentration of the test item (w/w). The formulation was vortexed and/or stirred until visual homogeneity was achieved. After homogenization the formulation was overlaid with argon to prevent instability caused by repeated contact of the test item formulation with air. Formulates were kept under magnetic stirring during the daily administration.
Based on the results of stability testing, the test item formulations were prepared at least every 15 days. The prepared formulation was stored protected from light and at room temperature.


VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected in consultation with the sponsor based on the test item’s characteristics.
The corn oil was dried and de-acidified as follows: corn oil was filtered through a mixture of activated silica gel 60 and aluminium oxide (1:1, volume/volume), which has been filled into a glass chromatography column to three quarters of its height.
For filtering, a vacuum of 75 mbar was applied. The dried and de-acidified vehicle was overlaid with argon and stored until usage.
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Amount of vehicle: 4 mg/mL
- Lot/batch no.: MKCK6411

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 analyzed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle as part of a separate GLP study.
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) at high- and low-dose concentrations.
As the test item was shown to be homogenous according to the separate GLP study (after 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and only samples were taken for substance concentration verification in study weeks 1, 5, 9 and in the last week of treatment (16 samples in total). The mean recoveries observed were between 97.4% and 104.7% of the nominal value for the low-dose dose group, between 97.7% and 102.8% of the nominal value for the mid-dose group and between 91.5% and 101.5% of the nominal value for the high-dose group. The mean overall recoveries observed in the low-, mid- and high dose groups were 100.4%, 100.4%, and 97.4% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within the acceptance criterion of 10%.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 (treatment and control groups)
5 (recovery group for control and high-dose)

20 additional animals (10 males and 10 females) will be used in satellite recovery groups for assessment of possible delayed toxic effects (5 male and 5 female animals per group).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: I n a previous dose range finding study in which 3 animals / sex / dose were dosed at 100, 300 and 1000 mg/kg bw/day, respectively, for 14 consecutive days test item-related effects on body weight development of mid- and high-dose male animals and food consumption of male and female mid and high-dose animals were observed.
- Rationale for animal assignment: Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with Microsoft Excel 2010 software).
- Fasting period before blood sampling for clinical biochemistry: yes; overnight
- Rationale for selecting satellite groups: In order to allow a detection of possible delayed occurrence or persistence of, or recovery from toxic effects, the animals in the recovery groups are observed for a period of 28 days following the last administration.
- Post-exposure recovery period in satellite groups: 28 days

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations are made once daily.
The health condition of the animals was recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure and at least once a week thereafter.
- Detailed cage side observations (outside the home cage in a standard arena) included: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment and recovery period.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly during the treatment and recovery periods

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examinations, using an ophthalmoscope were made on all animals before the first administration and in the last week of the treatment period. At the end of the recovery period the ophthalmological examinations were not recorded for the recovery animals.
- Dose groups that were examined: All groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment and recovery period as part of the sacrifice of the animals
- Anaesthetic used for blood collection: Yes; ketamine/xylazin
- Animals fasted: Yes; overnight
- How many animals: All surviving animals of the treatment period and 4 weeks after the last administration all surviving animals of the recovery period (study day 119)
- Parameters checked in tables 1 and 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment and recovery period as part of the sacrifice of the animals
- Animals fasted: Yes; overnight
- How many animals: All surviving animals of the treatment period and 4 weeks after the last administration all surviving animals of the recovery period (study day 119)
- Parameters checked in table 3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis performed on all animals as part of the scrifice of the animals
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table 5 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before the first exposure and towards the end of the exposure period but not earlier than in week 11. In the last week of the recovery period the behavioural observations were not performed for the recovery animals.
- Dose groups that were examined: All dose groups
- Battery of functions tested:

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. One day after the last administration (study day 91) all animals of the treatment period and 4 weeks after the last administration all animals of the recovery period (study day 119) were sacrificed using anesthesia (ketamine, xylazin) followed by exsanguination and were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Vaginal smears were examined on the day of necropsy to determine the stage of oestrous cycle.
The wet weight of the organs presented in Table 6 were taken from sacrificed animals as soon as possible. Paired organs were weighed together. Weight of thyroid/parathyroid glands was measured after fixation.
The tissues presented in Table 7 from all animals were preserved in 4% neutral-buffered formaldehyde except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol.

HISTOPATHOLOGY: Yes; organs presented in Table 7 were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for the animals of the control and high-dose groups sacrificed at the end of the treatment period.
For testis, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
The examinations mentioned above were not extended to animals of all other dosage groups as there were no treatment-related changes observed in the high-dose group.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.

Other examinations:

T3, T4, TSH: For an evaluation of test item-related effects on the pituitary-thyroid axis and thyroid hormones, serum samples of all animals was retained at the end of treatment (80 animals) and recovery (20 animals) and stored at < -15 °C. T3, T4 and TSH serum levels were determined of main study animals (80 animals; Table 4).

Examination of fertility parameters:
Daily over a period of 8 days, the oestrous cycle of all female animals was monitored in the last week of treatment. In the recovery animals the oestrous cycle was also monitored during the last week of the recovery period.
At necropsy (one day after the last administration) and at the end of the recovery period, left epididymis, left testis and left vas deferens were separated and used for evaluation of sperm parameters.
Epididymal sperm motility and testicular sperm count were evaluated in all male animals using Hamilton Thorne Sperm Analyser. Sperm morphology slides were prepared and evaluated from all male animals (main and recovery).

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights was performed for each gender 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. Furthermore, statistical comparisons of data acquired during the recovery period may be performed with a Student’s t-Test or Mann-Whitney U-Test when appropriate. These statistics were performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Slight to severe salivation was noted in 4/10 males and 3/10 females of the mid-dose group, 5/15 males and 6/15 females of the high-dose group. Furthermore, moving the bedding was noted in 2/10 males and in 5/10 females in the mid-dose group and 8/15 males and 13/15 females in the high-dose group. Slight salivation was recorded for 1/15 females in the control group on one single day. The clinical signs of moving the bedding and salivation in the mid- and high-dose groups were observed immediately after the dose administration during the treatment period and were therefore considered to be signs of discomfort, rather than a systemic adverse effect of the test item. These signs were still observed on the first day of the recovery period in the male and female high-dose group.
Further observations in the high-dose group were hunched posture in 3/15 males and 3/15 females, piloerection in 7/15 males and 4/15 females. 1/15 Male high-dose animals showed abnormal breathing. These findings were observed on several or single days in most of the animals. As no such findings were observed in other dose groups a treatment-related effect of the test item is considered.
Clinical symptoms like hairless area and scratch/cut were observed mostly transiently or on single days in animals of the control, low-, mid- or high-dose group in males or females and are were within the normal background frequency without toxicological relevance.
On the first day of the recovery phase moving bedding was noted in 3/5 high-dose males and 4/5 high-dose females, piloerection in 1/5 high-dose males and 2/5 high-dose females, slight salivation on two days in 1/5 high-dose females and hunched posture in 1/5 high-dose females. Hairless area was seen on one day in 1/5 female control animals. These findings were seen at the beginning of the recovery phase and therefore considered to be incidental and without toxicological relevance.

No statistically significant effects were noted on detailed clinical examination parameters during the treatment and recovery period when compared to the control group.

For a detailed desription of (detailed) clinical observations in tabular form please refer to the attachment.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in the control or any of the dose groups during the treatment period of this study and the recovery period. All male and female animals survived to the scheduled necropsy.
For a detailed desription of the findings in tabular form please refer to the attachment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment with the test item had a toxicological relevant effect on the body weight development in high-dose males and females and mid-dose males during the treatment period.
A statistically significant decrease of body weight was found in high-dose males on day 8 (4.64% below control) and continued during the entire treatment period in the high- and mid-dose group (high-dose group: up to 20.94% below control, mid-dose group: up to 15.63% below control). In high-dose females, statistically significant decreases were found on study days 29 and 36 and additionally, from day 57 until the end of the treatment period (up to 8.88% below control). Furthermore, the weekly body weight change, was seen regularly during the treatment period with statistical significant decrease in the male mid- and high-dose group and in single weeks in the female mid- and high-dose groups. One single statistical significance was found in the first week of the treatment period in the female low-dose group (7.8 g in the low-dose group, 13.20 g in control). Overall, the body weight change between day 1 to day 90 was statistically significantly decreased amounting 132.5 g in the male mid-dose and 104.20 g in the male high-dose group when compared to the control with 200.40 g. The statistically significant decrease in body weight change in the female high-dose group was 55.07 g compared to 73.93 g in the control group. In the control and all dose groups mean body weight was increased on treatment day 90 when compared to day 1.
During the entire recovery period the mean body weight was statistically significantly decreased in the male and female high-dose group when compared to the respective control groups, but the mean body weight change increased with statistical significance in the first two weeks in the male high-dose group and in the first week in the female high-dose group. A statistical significant decrease in body weight change was noted in week 3 of the recovery period in the female high-dose group when compared to control. Overall, the body weight change showed an increase on day 118 compared to day 90 in males and females with statistical significance in the male high-dose group (38.80 g compared to 18.60g in the control).
For a detailed desription of the body weight and body weight change in tabular form please refer to the attachment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The measurement of food consumption showed effects in the male and female high-dose group, which were considered to be related to the treatment with test item.
Decreased food consumption in the high-dose male group was found with statistically significant differences compared to the control group in several weeks during the treatment period amounting up to 23.15% below control. Additionally, reduced food consumption with statistical significance was found in the female high-dose group in most weeks of the treatment period (up to 20.05% below control).
The food consumption during the recovery phase was comparable to the respective control group in the male and female high-dose group.
For a detailed desription of the food consumption in tabular form please refer to the attachment.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The test item had no toxicologically relevant effects on haematological and coagulation parameters of male and female animals analysed at the end of the treatment and recovery period of this study.
At the end of the treatment period, the measurement of WBC showed an increase of the group mean value in the low-dose and the high-dose group in males compared to the control, but only for mid- and high-dose in females. The WBC mean value was statistically significantly increased in the male high-dose group (75.75% above control) and in the female mid-dose (80.99% above control) and high-dose group (140.52% above control). At the end of the recovery period, no such increase was found in high-dose males and females. The group mean values for WBC were 11.84% below control in males and statistically significantly 56.45% below control in females. In the absence of specific findings in differential blood cell count and no test item-related abnormalities during histopathological evaluation, the statistical significances in WBC count are assumed to be of no toxicological relevance.
The red blood cell parameters RBC, HGB and HCT were slightly and statistically significantly increased in the high-dose male group (7.95%, 6.50% and 7.95% above control) whereas in females, a slight statistically significant increase was seen in the mid-dose group for RBC (6.62% above control) and for HGB and HCT in the mid- and high-dose group (mid-dose group: 6.84% and 6.28% above control, high-dose group: 5.24% and 5.67% above control). The changes were slight in both genders and seen without dose dependency in the female groups. Furthermore, histopathological evaluation showed no test item-related findings. Therefore, no toxicological relevant effect is concluded for red blood cell parameters. The single statistically significant finding in differential blood cell count in mid-dose males for neutrophils (36.76% above control) is considered to be incidental.
At the end of the recovery period, statistical significances from the respective control group were noted in high-dose males for RBC, MCH, MCHC, RET, NEUT and LUC and in females for HCT, LYM, NEUT and BASO. As the statistically significant differences were only slight or were not observed at the end of the treatment period, no toxicological relevance is concluded.
The coagulation parameter PT, was found with a slight statistically significant increase in the mid-dose (5.30% above control) and high-dose female group (6.71% above control) at the end of the treatment period and is assumed to be of no toxicological relevance as no statistical significant increase for PT was seen in the male dose groups. No statistical significant changes were observed for any parameter of the coagulation parameters for the male and female high-dose group at the end of the recovery period.
For a detailed desription of the findings in tabular form please refer to the attachment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The test item is considered to have no toxicologically relevant effect on clinical biochemical parameters and hormone analysis of male and female animals analysed at the end of the treatment and recovery period of this study.
At the end of the treatment period, a statistically significant increase of the parameters TBIL (36.07% above control), CHOL (24.67% above control) and LDL (99.53% above control) was found in the male high-dose group and for LDL additionally in the male mid-dose group (57.85% above control).
A statistically significant increase of TBIL was also seen in high-dose females (31.55% above control), but not for CHOL. The slight increase for CHOL between the female dose groups when compared to the control group was not statistically significant. An increase of 140.07% above control was found at the end of treatment in high-dose females for TBA, whereas the increase in the high-dose male group was found without statistical significance. In the absence of toxicological relevant findings of liver specific parameters at clinical pathological evaluation and, additionally, the histopathological evaluation, which showed no test item-related findings, no toxicological relevance is assumed for the statistically significant changes in males or females at the end of the treatment period.
In females, the parameters TP and ALB showed a statistically significant decrease in the high-dose group when compared to control (6.34% and 5.52% below control) without dose dependency and were, therefore, considered to be without toxicological relevance.
The statistically significant decrease of 32.61% below control for ALAT in the male low-dose group and Crea (17.83% below control) in the female mid-dose group are considered to be incidental.
At the end of the recovery period, a statistical significance was only found for ALAT in the high-dose female group (36.11% below control).
There were no statistically significant changes in hormone analysis (T3, T4, TSH) at the end of the treatment and recovery period. Mean values of all male and female dose groups were comparable to the control group and no toxicological effect of the test item is concluded.
For a detailed desription of the findings in tabular form please refer to the attachment.

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study: T3, T4 and TSH; histopathology of epididymides, mammay gland, ovary, oviduct, prostate (with seminal vesicles and coagulation gland), testes, thyroid, uterus with cervix, vagina, adrenals and pituitary; organ weights of epididymides, liver, ovary, prostate, seminal vesicles, testes, thyroid, uterus, adrenals, brain and pituitary; estrous cyclicity; sperm motility, testicular sperm count and sperm morphology. For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The urinalysis of animals sacrificed at the end of the treatment and recovery period revealed no toxicological relevant effects and all urinary parameters were in the normal range of variation. No marked differences with toxicological relevance were noted between the dose groups and compared to the control groups.
For a detailed desription of the findings in tabular form please refer to the attachment.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

In both males and females, no toxicological effects were observed in any of the parameters of the functional observational battery before and at the end of the treatment when compared to controls.
Before the start of the treatment period (week -1) statistically significant differences were observed for animal sleeping (50% below control), moving in the cage and grooming in the male low-dose group and in females for animal sleeping (100% below control), moving in the cage (170% above control) in the mid-dose group.
In the last week of the treatment period (week 13), statistical significance was found for animals sleeping (50% above control) and moving in the cage (100% below control) in all male dose groups, for twitches in the low-dose male group and a decrease in defaecation in all male dose groups (low-dose: 86.36%, mid-dose: 72.73%, high-dose: 90.91% below control). No statistically significant differences to the control group were observed for all female dose groups.
The single statistically significant differences were noted before start of the first treatment or were observed in one gender and without dose dependency in week 13. Therefore, no toxicological effect of the test item is considered.
Functional observation was not recorded for all dose groups at the end of the recovery period. As the functional observation showed no toxicological effects observed during the treatment period and clinical observation was without toxicological effects during the recovery phase, no toxicological relevant effects are assumed during the recovery of the animals.
For a detailed desription of the findings in tabular form please refer to the attachment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant changes to the control group were found in male animals of the mid- and high-dose groups at the end of the treatment period, but there were no statistically significant differences in organ weight between all female dose groups and the control group with the exception of spleen in the mid-dose group (relative to body weight 0.02% above control).
There were statistically significant differences for brain relative to body weight (mid-dose: 19.83% and high-dose: 24.25% above control), for spleen relative to body weight (high-dose: 0.03% above control), for thymus (absolute high-dose: 21.51% below control, relative to brain weight (mid-dose: 20.80% below control), for testes relative to body weight (mid-dose: 19.10% and high-dose: 24.19% above control) and for kidney (absolute high-dose: 11.13% below control, relative to body weight mid-dose: 8.89% and high-dose: 13.61% above control). Further organ weights with statistical significance in males were seen for pituitary gland (absolute high-dose: 22.35% below control, relative to brain weight 20.17% below control), for adrenal glands relative to body weight (high-dose: 32.72% above control) and heart relative to body weight (high-dose: 10.17% above control). Statistical significance in the male mid-dose and high-dose group was found for the reproductive organs epididymides (absolute 24.65% and 17.37% below control, relative to brain weight 24.97% and 15.12% below control) and prostate (absolute 16.40% and 30.92% below control, relative to brain weight 17.15% and 29.20% below control). Furthermore, the absolute liver weight was statistically significantly decreased (mid-dose: 12.63%, high-dose: 11.44%) and additionally decreased in relation to brain weight (13.07%) in the mid-dose and relatively to body weight increased in the high-dose group (12.98%).
Considering the statistically significant decrease of final body weight in mid-dose and high-dose males, the statistically significant absolute and relative weight changes of several organs in the male groups are considered to be related to the decrease in body weight when compared to control. Furthermore, the histopathological evaluation showed no correlating test item-related findings. Therefore, the statistical significances are considered not to be adverse effects of the test item.
The statistically significant differences relative to body weight at the end of the recovery period in the male and female high-dose groups for brain (male: 15.91% above control), testes (37.72% above control), kidneys (male 17.77%, female: 13.81% above control), adrenal glands (male 11.14%, female 33.65% above control), heart (male 18.22%, female 10.09% above control) and liver (male 11.21% above control) are considered to be an effect of the statistically significant decrease in final body weight. Additionally, the absolute weight of pituitary gland showed a statistically significant decrease in high-dose males (13.78% below control).
For a detailed desription of the findings in tabular form please refer to the attachment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic findings at the end of the treatment period were red coloured mesenteric lymph nodes in one male and three females of the high-dose group. For one high-dose female, the uterus was dilated and for one low-dose male the thymus was abnormally coloured.
Furthermore, findings were noted for one high-dose female. For this animal the uterus with cervix, oviducts and ovaries were not found at necropsy and the vagina was irregularly shaped, but a testis with normal appearance was found. This finding is considered to be incidental and not related to an effect of the test item.
The necropsy at the end of the recovery period showed no macroscopic findings in all animals of the control and high-dose groups in males and females.
Under consideration of the histopathological evaluation, all observed gross lesions were incidental findings and were considered not related to the treatment with the test item.
For a detailed desription of the findings in tabular form please refer to the attachment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were observed during the histopathological evaluation.
There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix and vagina in any of the investigated animals. The testes were checked on completeness of cell populations, completeness of stages and degenerative changes. No treatment-related effects on the testicular histomorphology were observed and the histological appearance reflected the animal physiology. Further, no treatment-related effect on interstitial cell structure was noticed.
Other findings recorded at histopathological evaluation are described in the following.
Mesenteric lymph nodes - minimal to moderate hemorrhages, minimal to slight lymphoid depletion and minimal hemosiderin deposition within macrophages was observed in some control and high-dose animals. The observed hemorrhages and the hemosiderin deposition occurred most likely perimortem and were deemed incidental. The minimal lymphoid depletion was considered to be most likely related to a stressful condition and also deemed to be incidental.
Urinary bladder - minimal hemorrhage was observed in the urinary bladder wall in two high-dose males. This change occurred most likely around the time of death and was deemed to be incidental.
All other findings recorded at histopathological evaluation were deemed to be incidental or were within the range of background alterations that may be observed in animals of this strain and age and in this study type.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

In the daily evaluation of the estrus cycle in the last week of the treatment and recovery period, showed no toxicological relevant differences for all female animals in all dose groups. There were no statistical significances on the testis weight, mean sperm count and mean sperm motility for all dose groups after the treatment period or after the recovery period. Mean total number of abnormal and normal sperms/findings and therefore the relative number of normal and abnormal sperms showed statistical significances after the end of the treatment period in the mid- and high-dose male groups and after the recovery period in high-dose males. For a detailed desription of the findings in tabular form please refer to the attachment.


The relative mean value for normal sperm was slightly decreased in the mid- and high-dose group (mid-dose: 90.95%, high-dose: 91.50%) when compared to control (93.05%) and the relative mean value for abnormal sperm slightly increased (mid-dose: 9.05%, high-dose 8.50%) when compared to control (6.95%). At the end of the recovery period, the relative mean value for normal sperm was 90.50% in the high-dose group and 93.80% in the control group, the relative mean value for abnormal sperm was 9.50% in the high-dose and 6.20% in the control. For a detailed desription of the findings in tabular form please refer to the attachment.
As the histopathological evaluation showed no toxicological findings for male reproductive organs and additionally, the sperm motility showed no abnormal findings, the statistical significances in sperm morphology are considered to be not related to the treatment with test item.

There were no statistically significant changes in hormone analysis (T3, T4, TSH) at the end of the treatment and recovery period. Mean values of all male and female dose groups were comparable to the control group and no toxicological effect of the test item is concluded. For a detailed desription of the findings in tabular form please refer to the attachment.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Key result

Critical effects observed:

no

Conclusions:

In a GLP compliant 90-Day Repeated Dose Oral Toxicity study acccording to OECD 408 with hexadecyl(trimethoxy)silane, the no observed adverse effect level (NOAEL) is considered to be 100 mg/kg bw/day based on the reduced male and female body development at 300 mg/kg bw/day (males) and 1000 mg/kg bw/day (males and females) and additional clinical findings in high-dose males and females. At histopathological evaluation, no test item-related changes were observed.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

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

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A reliable subchronic study in rats according to OECD TG 408 and in compliance with GLP is available by the oral route for hexadecyl(trimethoxy)silane (BSL, 2022). Male and female Wistar rats were administered the test substance at dose levels of 100, 300 and 1000 mg/kg bw/day for 90 days. The control group received the vehicle dried and deacidified corn oil. A recovery group was included in the control and high dose group and observed for a period of 28 days following the last administration to detect possible delayed occurrence or persistence of, or recovery from toxic effects.

No mortality occurred during the treatment or recovery phase of the study. Clinical observations such as hunched posture, piloerection and abnormal breathing were seen in the male and/or female high-dose group on several or single days during the treatment period and not during recovery of the animals. No such findings were observed in other dose groups. Therefore, a relation to the treatment with the test item is considered. No statistically significant effects were noted on detailed clinical examination parameters during the treatment and recovery period and no toxicological effects were observed in any of the parameters of the functional observational battery.

There were test item-related effects on body weight development in the mid-dose (male) and high-dose groups (males and females) correlating with decreased food consumption. The statistically significant decrease of body weight was up to 15.6% and 20.9% below control in the mid- and high-dose males, respectively and up to 8.9% below control in the high-dose females. The measurement of food consumption showed effects in the male and female high-dose groups, which were considered to be related to the treatment with test item. The food intake was statistically significantly reduced in the male and female high-dose group on a regular basis during the treatment period and was comparable to the respective control group in high-dose males and females during the recovery phase.

No toxicologically relevant effects were found on haematological, coagulation as well as clinical biochemical parameters, hormone analysis (T3, T4 and TSH) and urinalysis in this study. Fertility parameters, including the monitoring of the estrous cycle and the evaluation of sperm parameters, were not affected by the treatment with the test item. All observed gross lesions recorded at the end of the treatment period were incidental findings and were considered not related to the treatment with the test item. Furthermore, no adverse effects of the test item were considered for organ weights. At the histopathological evaluation, no test item-related changes were observed.

Under the conditions of this oral 90-day repeated dose toxicity study in rats, the NOAEL is considered to be 100 mg/kg bw/day based on the reduced body weight development at 300 mg/kg bw/day (males) and 1000 mg/kg bw/day (males and females) and additional clinical observations in the high-dose males and females.

 

Justification for classification or non-classification

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