Trimethoxyoctylsilane

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 August 2020 to 28 October 2020

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:

adopted 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trimethoxyoctylsilane

EC Number:

221-338-7

EC Name:

Trimethoxyoctylsilane

Cas Number:

3069-40-7

Molecular formula:

C11H26O3Si

IUPAC Name:

trimethoxyoctylsilane

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han) (Full Barrier)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 11-12 weeks old (females at arrival); 17-25 weeks old (males at start of pairing)
- Weight at study initiation: females: 193 - 251 g; males: 366 - 487 g
- Fasting period before study: no
- Housing: kept individually in IVC cages except during the pre-mating period when females were kept in groups of two animals and during mating period when two females were paired with one male.
- Diet (e.g. ad libitum): Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water (e.g. ad libitum): tap water, sulphur acidified to a pH of approximately 2.8, ad libitum
- Acclimation period: at least 5 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

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 glass vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item (w/w). The formulation was alternately vortexed and/or stirred until visual homogeneity is achieved. After homogenization the formulation was overlaid with a protective gas 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 at least every 11 days as given by Eurofins Munich Study No. STUG7.59AA2169-3. The prepared formulation was stored protected from light and at -15 to -35°C.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected in consultation with the sponsor based on the test item’s characteristics.
- Concentration in vehicle: 0, 7.5, 25, 75 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no. (if required): 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. Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups. As 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. Samples were taken for substance concentration in the first and last week of the study for all doses.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:2 ratio
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm positive vaginal smears referred to as day 0 of pregnancy

Duration of treatment / exposure:

from gestation day (GD) 5 to gestation day (GD) 19

Frequency of treatment:

daily

Duration of test:

20 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

control group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

low dose (LD)

Doses / concentrations 3

Dose / conc.:

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

Remarks:

middle dose (MD)

Doses / concentrations 4

Dose / conc.:

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

Remarks:

high dose (HD)

No. of animals per sex per dose:

22-23 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In consultation with the sponsor, based on a dose range finding study as well as an OECD 422 study.
In the 14-day dose range finding study, male and female Wistar rats were treated for 14 consecutive days at dose levels of 100, 300, and 1000 mg/kg bw/day or 600 mg/kg bw/day, respectively. Test item-related mortality occurred in 2/3 males and 3/3 females of the HD group (1000 mg/kg bw/day). Subsequently, the HD was reduced to 600 mg/kg bw/day on study day 12. Clinical findings such as paresis, ataxia, apathy, reduced spontaneous activity, wasp waist, abnormal breathing, dehydration, hypothermia, and chromodacryorrhea were observed at 1000/600 mg/kg bw/day. The animals of the MD (300 mg/kg bw/day) and LD (100 mg/kg bw/day) did not show any mortality or clinical signs. No toxic effects of the test item were observed in the haematology and clinical biochemistry as well as on organ weights. There were no macroscopic abnormalities at necropsy.
In the OECD 422 study, male and female Wistar rats were treated at dose levels of 100 (LD), 300/200 (MD), and 600/400 (HD) mg/kg bw/day. Test item-related mortality occurred in 1 male and 5 females at a dose of 600 mg/kg bw/day (HD and HD recovery). After a dose reduction to 400 mg/kg bw/day, additional 4 males and 10 females were sacrificed in a moribund condition. At the MD dose of 300 mg/kg bw/day, test item-related mortality occurred in 3 females and even after a dose reduction to 200 mg/kg bw/day additional 4 females were euthanized in a moribund condition. Already within the first 15 days of treatment, which is a duration of daily dosing similar to the one applied in the current OECD 414 study, test-item related mortality occurred in 5 males and 10 females of the HD/HD recovery (600 or 400 mg/kg) and in 1 female of the MD (300 mg/kg) groups.
Animals sacrificed in a moribund condition or animals at their terminal sacrifice from the MD, HD and HD recovery groups, mainly displayed test item related clinical signs like piloerection, reduced spontaneous activity, abdominal breathing, half eyelid closure, hypotonia, altered locomotion associated to general weakness, reduced grip strength, hunched posture and hypothermia. However, there were no effects on estrous cyclicity, litter data, litter weight data, precoital interval and duration of gestation, pre- and post-natal data, reproductive indices, pup survival data, anogenital distance and nipple retention, pup thyroid weight as well as thyroid hormone levels in parental males, pups sacrificed on PND 13 and males sacrificed at the end of their recovery period, and pup external findings in all groups with available data. For the current study, the highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Therefore, a dose of 300 mg/kg bw/day was chosen as high dose. A descending sequence of dose levels was selected with an aim to demonstrate any dose-related responses and to identify a NOAEL.

- Rationale for animal assignment (if not random): random

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: general clinical observations were made once per day; twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily;
- Cage side observations included: 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 or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The sperm positive females were weighed on GDs 0, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption of sperm positive females was measured on GDs 5, 8, 11, 14, 17 and 20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: macroscopic examination of structural abnormalities or pathological changes which may have influenced the pregnancy; uteri; gravid uterus with the cervix was weighed; thyroid/parathyroid glands were weighed; a full histopathology was carried out on the preserved thyroid/parathyroid glands.

OTHER: Thyroid hormones
- Blood samples were collected prior to sacrifice for thyroid hormone (T3, T4, TSH) analysis.

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

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]
- Other: anogenital distance; foetal weight; examination of reproductive tract; external foetal sex was compared to gonadal sex; testicular descent/cryptorchidism was examined.

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 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).

Historical control data:

See attached historical control data tables.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs such as animals displaying hairless area or slight diarrhoea were considered incidental by nature and not test-item related.
Slightly increased salivation, moving the bedding were occasionally observed in single females of the high dose group, and several high dose dose group animals occasionally showed slight piloerection and slightly to moderately reduced spontaneous activity towards the end of the treatment period. These mild clinical signs were considered as signs of general discomfort of the animals due to oral administration or local reactions of the test item and were therefore assessed as test item-related but not indicative of systemic toxicity.

Mortality:

no mortality observed

Description (incidence):

No test item-related mortality was observed during the study period and all females survived until the end of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The high dose group females showed moderately lower body weight gain when compared to the values of the control group. This difference was statistically significant on GDs 17-20 and for the total weight gained over the whole treatment period (GDs 5-20). This result was also reflected by the statistically significantly reduced mean carcass weight and the statistically significantly lower mean net weight change (= carcass weight – body weight from GD0) in high dose females compared to control females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The observed test item-related effect on the body weight and body weight gain of the HD females was reflected by the statistically significantly lower food consumption of high dose females on GDs 17-20 and over the whole treatment period of GDs. 5-20. This effect was considered to be test item-related.

Food efficiency:

not examined

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

not specified

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 statistically significant differences in uterine weight data were observed between the control group and the treatment groups.
There was no statistically significant or toxicologically relevant effect on the absolute and relative (to body weight) weights of post-fixed thyroid/parathyroids of the dose groups when compared to controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related macroscopic findings were observed at necropsy. Animal no. 34 of the LD group showed a cyst at the ovaries and animal no. 50 of the MD group showed an abnormal content (gelatinous, brown) in the vagina, which were considered to be incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no gross lesions or histology changes in the thyroid glands that could be related to treatment with the test item.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No statistically significant or toxicologically relevant effect was observed on group mean T3, T4 and TSH hormone levels of terminally sacrificed females. The values were comparable to those of controls.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

No female showed signs of abortion or premature delivery prior to the scheduled sacrifice.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Treatment with the test item had no toxicologically relevant effect on prenatal data including the number of corpora lutea, implantation sites, living foetuses, early resorptions and late resorption and pre- and post-implantation loss when comparing test item-treated groups to the control group.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

Treatment with the test item had no toxicologically relevant effect on prenatal data including the number of corpora lutea, implantation sites, living foetuses, early resorptions and late resorption and pre- and post-implantation loss when comparing test item-treated groups to the control group.

Dead fetuses:

no effects observed

Description (incidence and severity):

Dead or malformed foetuses were not observed in any of the groups.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Successful mating resulted in 20/23 pregnancies in the control group, 23/23 pregnancies in the LD group, 20/22 pregnancies in the MD group and 21/22 pregnancies in the HD group. No female showed signs of abortion or premature delivery prior to the scheduled sacrifice.

Other effects:

not specified

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

100 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

Maternal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: reduced mean body weight gain and mean food consumption

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Despite the lower body weight and body weight gain of the HD females, the mean foetal weight was comparable between the treatment and control groups.

Mean male and mean female foetal weight was slightly, but statistically significantly higher in litters of the LD females compared to control. As the mean male and mean female foetal weight of MD and HD groups were comparable to values of the control group, the effect was not considered to be test item-related.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No statistically significant effect was observed on number of male and female foetuses per pregnant female and on the sex ratio of foetuses.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No statistically significant effect was observed on number of male and female foetuses per pregnant female and on the sex ratio of foetuses.

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There were no test item-related external abnormalities observed in any of the foetuses of treated groups.

One MD foetus of dam no. 64 was observed with a hyperflexion of the right hindlimb. As all other foetuses were without findings, this was considered to be incidental.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

As usual for foetuses at the stage of GD 20, skeletal examination of the Alizarin red-stained foetuses revealed a range of findings in several bones of several litters in all groups including control. Mainly bones of the skull, sternum, limbs, and vertebra and ribs were affected by variations in the status of expected ossification in terms of incomplete ossification, unossification, irregular ossification or increased ossification throughout all groups. However, there were only statistically significantly more litters affected in the MD group for the finding “incomplete ossification of the frontal skull”. As there was no incomplete ossification of the frontal skull observed for foetuses of the HD group, there was no dose-dependency and the findings were therefore considered to be incidence in nature.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Internal observation of the foetal viscera by free hand microdissection technique revealed a range of visceral findings in all groups including the control group.

Statistically significantly higher litter incidences for a supernumerary liver lobe were found in the HD group compared to control. However, these results were within the historical control data and, therefore, not considered as adverse. All other 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 the observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Craniofacial examination by razor blade serial sectioning technique revealed a range of findings in all dose groups at similar frequencies or in some cases in slightly lower or higher frequency than in litters of the control group. A statistically significantly higher litter incidence was not observed. Thus, none of the craniofacial findings were considered to be test item-related, but incidental in nature.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

In the prenatal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP, the NOAEL for maternal toxicity was concluded to be 100 mg/kg bw/day based on statistically significant test item-related effects in maternal animals at the highest dose tested of 300 mg/kg bw/day including decreased mean body weight, body weight gain and food consumption. The NOAEL for foetal toxicity was concluded to be at least 300 mg/kg bw/day based on no significant effect observed in any of the foetal developmental parameters.