Hexadecyltrimethoxysilane

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Apr - 21 Aug 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

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

Test material

Test material information

Constituent 1

Reference substance name:

Hexadecyltrimethoxysilane

EC Number:

240-464-3

EC Name:

Hexadecyltrimethoxysilane

Cas Number:

16415-12-6

Molecular formula:

C19H42O3Si

IUPAC Name:

hexadecyltrimethoxysilane

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: approx. 11-12 weeks old for females; between 12 weeks and not older than 24 weeks for males
- Weight at study initiation: males: 351 – 428 g (mean: 392.3 g, ± 20% = 313.8 – 470.8 g), females: 201 - 252 g (mean: 225.1g, ± 20% = 180.1 – 270.1 g)
- Housing: individually in IVC cages (type III H, polysulphone cages) on saw fibre bedding (except during the pre-mating period when females will be kept in groups of two animals and during mating period when two females will be paired with one male)
- Diet: Altromin 1324 maintenance diet for rats and mice provided ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 provided ad libitum
- Acclimation period: at least 5 days


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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

dried and de-acidified

Details on 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. 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.
Based on the results of stability testing, the test item formulations were prepared within the stability period. The prepared formulation was stored protected from light and at room temperature.
The vehicle was also used as control item.


VEHICLE
- Justification for use and choice of vehicle:
The vehicle was selected in consultation with the sponsor based on the test item’s characteristics and testing guideline.
Corn oil was filtered through a mixture of activated silica gel 60 and aluminium oxide (1:1, volume/volume), which had 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 deacidified vehicle was overlaid with argon and stored until usage.
- Concentration in vehicle: 18.75, 75 and 250 mg/mL
- Amount of vehicle: 4 mL/kg bw
- 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 analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle as part of a separate GLP study. The formulations (18.75 and 250 mg/mL) were stable for at least 15 days at room temperature, at 2 to 8°C and at -15 to -35°C. Therefore, it was decided to store prepared formulations at room temperature and use them within 15 days after preparation.
Since 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 and samples were only taken for substance concentration (18.75 mg/mL, 75 mg/mL and 250 mg/mL) in the first and last week of the study for all doses (8 samples in total). The mean recoveries observed for the low-dose group was between 92.6% and 100.8% of the nominal value, between 91.8% and 104.9% of the nominal value for the mid-dose group and
between 94.4% and 103.3% of the nominal value for the high-dose group. The mean recoveries observed in the low-, mid- and high-dose groups were 96.7%, 98.4%, and 98.8% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within the acceptance criterion of 10%.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: Females were paired for cohabitation in batches in order to control the number of animals for terminal sacrifice on a particular day. After getting 92 sperm positive females, the remaining females and males will be discarded without any observations.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 5-19 of gestation

Frequency of treatment:

daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

23 pregnant females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on a dose range finding study, in which sperm positive female Wistar rats were treated with the test item at doses of 100, 300 and 1000 mg/kg bw/day. Test item-related maternal toxicological effects in terms of body weight gain and food consumption were observed at 100, 300 and 1000 mg/kg bw/day when compared to the control group. Test item-related reductions in uterine weight were observed at 1000 mg/kg bw/day when compared to the control group. A statistically significant reduction in mean foetal weight was observed in male foetuses at 1000 mg/kg bw/day when compared to control. This change was only due to the slightly reduced foetal body weight from one high-dose dam. No test item-related external foetal abnormalities were noted.

- Rationale for animal assignment: Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that group mean body weights were comparable with each other.

Examinations

Maternal examinations:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of the mating a detailed clinical observation was made outside the home cage.

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 sperm positive females were weighed on GD 0, 5, 8, 11, 14, 17 and 20. Males were not be weighed in this study except once before initiation of pairing.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption of sperm positive females was measured on GDs 5, 8, 11, 14, 17 and 20.
Food consumption was not measured for males during the entire study or for both male and females during the mating period.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: The dam was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Any macroscopic findings were preserved in 4% neutral-buffered formaldehyde.
Thyroid/parathyroid glands from all dams were preserved in 4% neutral-buffered formaldehyde. The weight of thyroid/parathyroid glands was measured after 24 hours fixation.

OTHER: Thyroid hormones levels from samples from all dams were assessed at the end of the treatment prior to or as part of the sacrifice of the animals. At termination, blood samples were collected from the defined site will be collected in serum separator tubes and obtained serum were stored at ≤-20°C. Serum samples were assessed for serum levels for thyroid hormones (T3, T4, TSH) using ELISA.

Ovaries and uterine content:

The ovaries and uterine content was 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

Blood sampling:

- Plasma: No
- Serum: Yes (for evaluation of thyroid hormones)

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

A statistical assessment of the results of the body weight and food consumption will be 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 will be 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 will be performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 is considered as statistically significant).

Historical control data:

Comparisons with historical control data were performed.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related clinical signs observed in the low-dose and mid-dose groups. High-dose group animals showed clinical signs including piloerection (slight, moderate or severe- 10/23), spontaneous reduced activity (moderate- 5/23), hunched posture (4/23), nasal discharge (3/23), dehydration (2/23), wasp waist (slight/moderate- 3/23), moving the bedding (5/23) and increased salivation (slight to moderate- 2/23) at post dose observations occurring mainly between GD12 to GD20. These are considered to be treatment-related effects in the high-dose group.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All animals survived until the end of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight increased during the progress of the study in the control and all the treated groups, However, a slight but statistically significant decrease in mean body weight was observed in the high-dose group on GD 14, GD 17 and GD 20 (5%, 8% and 16% below control respectively) and a statistically significant increase in body weight was observed in the mid-dose group on GD 5 (3.9% above control). Mean body weight was statistically significantly reduced in the high-dose group from GDs 5-20 (68.1% below control) and also at all the separate intervals of body weight measurements (46-108% below control). A slight to moderate, decrease in mean body weight gain, which did not achieve statistical significance was observed on GDs 5-8 and GDs 8-11 in the low-dose group (16.8% and 11.7% below control respectively) and in the mid-dose group (58% and 13% below control respectively). A moderate but statistically significant reduction in mean body weight gain was observed in the mid-dose group on GDs 11-14.
Overall, there was a moderate decrease in mean body weight gain observed on GDs 5-20 in the high-dose group (68.10% below control) and a slight decrease in the mid-dose group (12.61% below control). The reduction in mean body weight and body weight gain in the high-dose and mid-dose groups are considered to be test item-related effects, as they are dose-dependent.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In association with the decrease in mean body weight and mean body weight gain, a slight to moderate, but statistically significantly decrease in food consumption was observed in the high-dose group at all treatment intervals of the gestation period (16.8-52.3% below control), and on GDs 5-20 (31.70% below control). A slight, decrease in mean food consumption,which did not achieve statistical significance was observed in the mid-dose group during all treatment intervals of the gestation period (4-12.56% below control) and between GDs 5-20 (10.85% below control).
Overall, there was a moderate decrease in mean food consumption observed between GDs 5-20 in the high-dose group (31.70% below control) and the mid-dose group (10.85% below control) and these are considered to be test item-related effects, as they are dose-dependent.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study: T3, T4 and TSH in dams, anogenital distance, thyroid weight and histopathology in dams, gravid uterus weight, litter size, litter/foetus weight, number of implantations, corpora lutea, numbers of embryonic or fetal deaths and viable foetuses, post- and preimplantation loss, presence of anomalies (external, visceral, skeletal) and sex ratio. For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Statistical analysis of post-fixed thyroid/parathyroid weights from all dams revealed no statistically significant differences in the absolute and relative (to body weight) thyroid/parathyroid weights of the dose groups when compared to the control. A slight reduction in mean thyroid/parathyroid weights was observed in the high-dose group (10.59% below control) when compared to control.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related gross pathological changes were observed during the macroscopic examination of any treatment group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item-related histopathological findings were observed in the thyroid glands and parathyroid of all females evaluated.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

In all terminally sacrificed females, no statistically significant or test item-related effects were observed for group mean T3 and TSH hormone levels and values were comparable between all animal groups. T4 was found to be statistically significantly reduced in the mid- and high-dose groups with no effects seen in the low-dose group. Due to the absence of weight effects and histopathological effects in the thyroid/parathyroid in any of the treated groups, the statistically significant reduction in group mean T4 levels is not considered to represent test item-related effects. In addition, there were no corresponding changes in either in T3 or TSH hormone levels.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

None of the females showed signs of abortion or premature delivery prior to their scheduled sacrifice.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Successful mating resulted in 21/23 pregnancies in the low-dose group, 23/23 pregnancies in the mid-dose group and 23/23 pregnancies in the high-dose group compared to 22/23 pregnancies in the control group.

Other effects:

not examined

Details on maternal toxic effects:

No test item-related effects of toxicological relevance were noted for any prenatal parameter including number of corpora lutea, implantation sites, late resorptions, number of live foetuses, anogenital distance (AGD) as well as number of male and female foetuses in treatment groups when compared to the control group. No dead foetuses were noted in any of the groups.
Terminal body weight, adjusted maternal weight (carcass weight) and net weight change from GD 0 did not show a treatment related effect in the low-dose group. In the mid-dose group a slight but non-significant reduction net weight change from GD 0 was seen. A statistically significant reduction in adjusted maternal weight (carcass weight) was observed in the high-dose group (17.1% below control). A statistically significant reduction in net weight change from GD 0 was observed in the high-dose group (122.6% below control). These are considered to be test item-related effects.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

A slightly but statistically significantly lower mean male and female foetal weight was observed on an individual basis (sum of weight of all foetuses in group divided by total number of foetuses in respective group) in the high-dose group (Male: 12.15%, Female: 12.51% below control). A slightly but statistically significantly lower mean male foetal weight was observed in the low-dose group (2.99% below control) when compared to control. This lower mean foetal weight is considered to be secondary to maternal toxicity i.e. lowered body weight and food consumption.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No test item-related effects of toxicological relevance were noted for sex ratios in treatment groups when compared to the control group.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related effects were observed for the mean foetal weight or male and female foetal weight on a per litter basis (group mean of individual litter mean) in the high-dose group when compared to the control. These effects are considered to be secondary effects to maternal toxicity i.e. lowered body weight and food consumption effects.

Anogenital distance of all rodent fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

In male and female foetuses, the absolute and relative anogenital distance (AGD) in treatment groups remained unaffected. Marginal but statistically significant decrease in relative AGD in mid-dose female foetuses was observed when compared to control. As these values were within the historical control range, this finding is considered to be an incidental finding.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There was no test item-related external abnormalities observed in any of the dose groups.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

At skeletal examination, incomplete ossification was seen in several bones of litters from all groups including control. Mostly bones of the skull, sternum, paws and vertebra were affected by variations in the status of expected ossifications in terms of incomplete ossification, irregular ossification, unossified or increased ossification throughout all groups. However, there were no dose dependent and no statistically significant increase in incomplete ossification of bones observed in the treated groups. Most of the findings in the treatment groups were comparable to the control. The slight change observed in the high-dose group is considered to be a secondary effect to lower fetal weight and maternal toxicity.

Ossification-related findings were observed at low or higher incidences and without dose-dependency and thus were not considered as toxicologically relevant.
There were also slightly higher or lower litter incidences of skeletal findings in low- and mid-dose groups, but these did not show any statistically significant changes and/or many of these findings were within historical control range for this strain. Hence, these are not considered as test item-related findings in the lower dose groups

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A slight but statistically significantly higher litter incidence of testis, malpositioned was observed in the low-dose group (30% compared to 5%) when compared to the control.
All other litter incidences were statistically insignificant when compared to the control group. There were higher litter incidences observed in which the umbilical artery was malpositioned (50% in the low-dose group compared to 25% in the control). Higher or lower litter incidences of abdomen internal haemorrhage were observed (55%, 35% and 25% in the low-, mid-, and high-dose groups, respectively, compared to 40% in the control). Higher or lower litter incidences of liver, supernumerary lobe were observed in the low-, mid-, and high-dose groups (30%, 10% and 0%, respectively, compared to 15% in the control group).
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 the control. As the observed findings were either minor variations and/or due to a lack of dose dependencyand consistency, no toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature. In addition, all these values were within the historical control range for this strain.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Craniofacial examination by razor blade serial sectioning technique revealed a finding of subcutaneous hematoma (general) in the head in all treated (10%, 10% and 15% in the low-, mid-, and high-dose groups, respectively) and control groups (10%) and a few incidences of subdural midbrain haematoma in control (15%) and low-dose (12%) groups, increased perimeningal space in the control (5%) and treated groups (15%, 10% and 5% in the low-, mid-, and high-dose groups, respectively). Single incidences of head, subcutaneous space increase were observed in the low-dose group (5%). These findings were considered to be spontaneous in nature and not related to the treatment with the test item. Statistical analysis of the data revealed no statistical significance for any of these findings. In addition, all these values were within the historical control range for this strain.

Effect levels (fetuses)

1

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

changes in litter size and weights

other: skeletal variations (increased incidences of altered ossification)

2

Key result

Dose descriptor:

NOAEL

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 observed

Fetal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

skeletal: scapule

skeletal: forelimb

skeletal: sternum

skeletal: rib

skeletal: vertebra

skeletal: pelvic girdle

other:

Description (incidence and severity):

incidence did not achieve statistical significance; the altered ossifications (classified as variation) were considered as secondary effect of lower fetal weight in the fetus as a result of maternal toxicity.

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

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:

not specified

Applicant's summary and conclusion

Conclusions:

In a developmental study according to OECD 414 and in compliance with GLP, a NOAEL for maternal and fetal toxicity was established at 300 mg/kg bw/day based on decreased body weight and food consumption in maternal animals. Decreased mean fetal weight and a decreased male and female fetal weight on a per litter basis as well as changes to skeletal ossification parameters (variations) observed in high-dose fetuses were considered to be secondary to maternal toxicity.