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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
27th of March 2018 to 30th of May 2018
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: The study is considered invalid, because it did not adequately meet the requirements or the objectives of the guideline. See attachment for further justification for reliability.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developme ntal Toxicity Screening Test)
Version / remarks:
29th of July 2016
Deviations:
yes
Remarks:
yes Inadvertently, animals of the HD group were treated for 8 days with a concentration of 75 mg/ mL instead of 100 mg/mL. Additionally, blood samples from the adult main study males were not assessed for serum levels for thyroid hormones (T4).
Qualifier:
according to guideline
Guideline:
other: Health Effects guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-368
Version / remarks:
July, 2000
Deviations:
yes
Remarks:
yes Inadvertently, animals of the HD group were treated for 8 days with a concentration of 75 mg/ mL instead of 100 mg/mL. Additionally, blood samples from the adult main study males were not assessed for serum levels for thyroid hormones (T4).
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Trimethoxyoctylsilane
EC Number:
221-338-7
EC Name:
Trimethoxyoctylsilane
Cas Number:
3069-40-7
Molecular formula:
C11H26O3Si
IUPAC Name:
trimethoxy(octyl)silane
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on test animals or test system and environmental conditions:
- Source: Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 14-15 weeks
- Weight at study initiation: males: 344 - 389 g (mean: 365.31 g, ± 20 % = 292.25 – 438.37 g)
females: 204 - 243 g (mean: 225.58 g, ± 20 % = 180.46 – 270.69 g)
- Fasting period before study: not specified
- Housing: Animals were housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages during the premating period for both main males and main group females and during the post-mating period for males depending on the mating status. During the mating period, males and females were housed together in 1:1 (male to female) ratio. After the confirmation of mating, females were kept individually during the gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage. In each cage, Altromin saw fibre were used as bedding. Animals of the recovery group were housed in groups of 3 animals / sex / cage in the type III H, polysulphone cages.
- Diet: ad libitum (altromin 1324)
- Water: Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals).
- 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): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulation was prepared at least every 10 days. The test item was suspended in corn oil. Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 4 mL/kg body weight.


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.
- Concentration in vehicle: 0, 25, 75, 50, 150, 100 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw/day
- Lot/batch no.: MKCC9871 / MCKD1021
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation analysis for nominal concentrations revealed that nominal concentrations of all formulations were confirmed throughout the study period during each occasions (study week 1, 3, 5 and in the last week of the study) as measured concentrations were within acceptance criterion of 15%. However, one sample (no. 8, HD week 3) did not meet this criterion with a recovery of 72.8% (measured concentration equates to MD).
Details on mating procedure:
- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
Duration of treatment / exposure:
The treatment period was 63 days, i.e. 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed. Animals in the recovery groups were not mated and dosed for 28 days (males) or until the first scheduled kill of dams (females).
Frequency of treatment:
The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose, LD
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Middle dose, MD
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Middle dose, MD
The dosing for the MD animals was 300 mg/kg bw/ day up to study day 16 (for animals numbers. 21-25, 61-65) and up to study day 15 (for animals number 26-30, 66-90), then paused for one day (study day 17 or 16, respectively), and then continued with 200 mg/kg bw/day from study day 18 or 17 onwards.
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
High dose, HD
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
High Dose, HD
The dosing of the HD group was 600 mg/kg bw/ day up to the study day 10 (for animals with the number 31-35, 71-75, and all recovery animals: number 87-92, 99-104) and up to study day 9 for animal number 36-40, 76-80), then paused for one day (study day 11 or 10, respectively), and then continued with 400 mg/kg bw/ day from study day 12 or 11 onwards.
No. of animals per sex per dose:
10 and 12 additional for the recovery group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected according to the results of a previous dose range finding study (BSL Munich Study No. 178264, non GLP) and in consultation with the sponsor.
- Rationale for animal assignment: random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: Once a day for signs of toxicity and twice a day for mortality and morbidity.
- Cage side observations checked included: signs of toxicity; mortality; morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure and at least once a week thereafter, detailed clinical observations were made with all animals of the main and the recovery groups outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes of 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:
- Time schedule for examinations: The animals were weighed once before the assignment to experimental groups, on the first day of dosing and weekly thereafter, as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours after parturition (day 0 post-partum), on PND 4, PND 9 and PND 13 along with their pups. All animals were weighed directly before termination. Any animal prematurely sacrificed was weighed prior to the sacrifice.

FOOD CONSUMPTION: Food consumption was measured on the days corresponding with the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: No data
- Skeletal examinations: No
- Head examinations: No data

Other: Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. In addition to the observations of the parent animals, any abnormal behaviour of the offspring was recorded.
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD/Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.
Statistics:
One-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a posthoc Dunn’s Test, based on the results of homogeneity and normality tests. Statistical comparisons of data acquired during the recovery period were performed with a Student’s t-Test. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos software version 1.1.3 or version 1.3.4 (p<0.05 was considered as statistically significant).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In male and female animals (MD and HD, HD Recovery) from treatment and recovery groups sacrificed in moribund condition, predominant clinical signs observed during the treatment period and before sacrifice due to animal welfare reasons were moderate to severe piloerection, reduced spontaneous activity, wasp waist, ptosis, moving the bedding, abdominal breathing, half eyelid closure, hypotonia, altered locomotion associated with general weakness, reduced grip strength, hunches posture, hypothermia, nasal discharge, dehydration, salivation and sunken flanks. These clinical signs observed in moribund animals were considered as test item related.

In terminally sacrificed male and female animals (MD, HD and HD recovery) from treatment and recovery groups during their treatment period, major clinical signs observed were moving the bedding, salivation, hypotonia, piloerection, hypothermia and reduced spontaneous activity. In LD group males and females, no major clinical signs were observed.

In HD males during recovery period, few clinical signs observed were piloerection, moving the bedding, reduced spontaneous activity and salivation. No female animals were available for clinical observations during the recovery period.

Isolated incidences of crust/alopecia on various body parts, regurgitation of test item, vocalisation and pale skin were observed on a few occasions in certain female animals. However, this was also observed in the control group and is thereby considered to be incidental in nature rather than treatment-related.

Immediately after the administration of doses, salivation and moving of the bedding were observed. Therefore, it was considered to be a sign of discomfort due to a local reaction to the test item rather than a systemic adverse effect and thus of no toxicological relevance.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
In total 29 animals (8 females from MD, 3 male and 10 female from HD, 2 male and 6 females from HD recovery) were sacrificed due to animal welfare reasons. In three of these animals, indicators for gavage accidents (pericardial inflammation) were noted. Additionally, fluid and/or pleural changes in the thoracic cavity occurred in two animals. In the remaining animals, no morphological cause of death could be established.


Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):

Lower group mean body weight was observed in the HD group for males when compared with the control, although a statistically significant difference was achieved on premating day 14 only. In recovery males, lower group mean body weight was observed on various days from day 8 to 42 during treatment and recovery period, although statistical significance was achieved only on day 15, 22 and day 28 in HD recovery group when compared with recovery control group.

The group mean body weight gain in the HD group was statistically significantly lower during premating day 1-7 and higher during premating day 14 - mating and post mating day 7 when compared to the control group. Additionally, incidental but statistically significant higher group mean body weight gain were observed in the LD group during premating day 14 - mating and post mating day 7 when compared to the control group.

In recovery males, there was a statistically significant lower group mean body weight gain observed in HD recovery group males during day 1-7 and 7-14 when compared with the controls. However, during the recovery period (Day 28-42), body weight gain observed in HD recovery group males was comparable to the control group.

Lower group mean body weight and body weight gain in the HD group for females was observed between premating day 7 to gestation day 7. Between gestation day 7-14 to terminal sacrifice, no body weight and body weight gain data were available for the HD group, since these animals were euthanised for animal welfare reasons before the scheduled terminal sacrifice.

In the female recovery group, lower group mean body weight in HD recovery group was observed on various days from day 7 to 28 during the treatment period although statistical significance was achieved only on day 14 when compared to the recovery control group. Between the treatment days 35-49 and during the recovery period, no body weight and body weight gain data were available for the HD recovery group, since these animals were euthanised for animal welfare reasons before the scheduled terminal sacrifice.

Mean body weight gain in the HD recovery female group was lower from day 1-7 to 21-28 during the treatment period although statistical significance was not achieved on any occasion when compared to recovery control group. Between treatment day 28 to 49 and during the recovery period, no body weight gain data were available for the HD recovery group since these animals were euthanised for animal welfare reasons before the scheduled terminal sacrifice.

These statistically and/or biologically significant effects on group mean body weight and body weight gain in HD and HD recovery group males or HD females were considered as test item-related and toxicologically relevant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males and females, the food consumption in main study males during the premating period and recovery males during treatment and recovery period was comparable to control in LD, MD and HD group males. However, In HD group main study females during premating and day 0-7 gestation period and HD recovery group females during treatment period day 1-14, food consumption was lower compared to the respective control group. From gestation day 7 to lactation day 13 in main study HD females and from treatment day 28-35 and during recovery period in HD recovery females, no food consumption data were available because these females were euthanised for animal welfare reasons before the scheduled terminal sacrifice.
This effect on food consumption in HD and HD recovery females was considered to be test item related and toxicologically relevant.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
See summary in Section 7.5.1
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
See summary in Section 7.5.1
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
See summary in Section 7.5.1
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
See summary in Section 7.5.1
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
See summary in Section 7.5.1
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
In males and females sacrificed at the end of treatment and recovery period, there were no statistically significant differences in the absolute and relative organ weights for the all dose groups in males and the LD and MD group in females at the end of treatment period when compared to the control groups. However, statistically significant higher absolute and relative brain weights and relative liver weights as well as statistically significantly lower absolute weights of prostate with seminal vesicles and coagulating gland in the male HD recovery group were observed compared to the control recovery group. This effect on organ weight was not considered to be adverse.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Some macroscopic alterations could be observed which were not considered to be of toxicological relevance since the changes were within the range of normal background alterations. These includes: lung – yellow hard mass 1.5 cm (male of LD group), lung- abnormal color, red, left caudal lobe (male of HD recovery group), urinary bladder- abnormal color, red, coagulating glands- abnormal color, clear, seminal vesicles- fluid filled (male of HD recovery group), thoracic cavity- fluid filled, yellow (female of MD group), thoracic cavity- fluid filled, white viscous, oily, pleura- focus, numerous (>12), thoracic, white , movable (female of MD group), heart- pericardium, fluid filled, yellow creamy (female of LD group), lung- focus, white, several (7-12), all lobes (female of control recovery group), lung- focus, numerous (>12), white (female of MD group), liver- diaphragmal herniation, thymus- abnormal color, red, spotted (female of HD group), thymus- abnormal color, spotted (female of control group), thymus- focus, several (7-12), 0.1 cm, red (female of HD group), uterus- complete dilatation (female of control group), uterus- fluid filled (three females of control recovery group) and skin/subcutis- wound/scar/crust, 0.2 cm on back (female of HD group).
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
A high proportion of the MD females were sacrificed during the course of the study due to poor health (eight females). From the HD group including HD recovery, five males and all females were sacrificed during the study. In two MD females and one HD recovery female, indicators for gavage accidents (pericardial inflammation) were noted. In two of these animals, there were also fluid and/or pleural changes in the thoracic cavity. However, in the remaining animals, no morphological cause of death could be established. The clinical symptoms were associated with the indicator altered locomotion associated with general weakness. A general intoxication by unknown reasons is supposed.
No special macroscopic lesions were observed that could be attributed to treatment with the test item. Microscopic evaluation of tissues revealed no specific morphological change in the treated animals that could be attributed to treatment with the test item.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
See summary in Section 7.5.1
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no test item related effects observed on implantation sites, percent pre-implantation loss and post implantation loss in the LD and MD groups when compared with the control group.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The number of dead foetuses were within the control data.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There were no test item related or statistically significant effects observed on the duration of precoital interval and the duration of gestation in the LD and MD dose groups and precoital interval in all treatment groups when compared to the control group.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
A reduced delivery index (number of females with live new borns/ No. of pregnant females X 100) of 80 % in the MD group was observed compared to 100 % in control group. This effect on delivery index was attributed to one out of five females which was pregnant but no live pups were born and considered as biological variation. The lower fertility index (Number of females pregnant / No. of females copulated) X 100) in MD group (55.56 %) was attributed to euthanasia of few females between evidence mating and implantation of zygote (gestation day 6) and therefore pregnancy status of few females could not be established at the time of necropsy. The lower copulation index (Number of rats copulated / No. of pairs) X 100) in HD group (50%) was attributed to euthanasia of one out of 2 paired females during mating period. No female from the HD group littered with live pups or these females were euthanised for animal welfare reasons before littering. Therefore no delivery index and viability data were available for the HD group.
Other effects:
no effects observed
Description (incidence and severity):
The test item had no biologically significant effect on the estrous cycle analysed over 2 weeks of the premating period following the first administration in treatment groups when compared to the controls.

There were no test item related effects on the reproductive indices (copulation, fertility, viability and delivery indices) in all dose groups when compared to the control group.
Details on maternal toxic effects:
Clinical signs and some systemic toxicity were observed in maternal animals.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
>= 300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed in all dose groups for which data could be obtained

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In female pups, slightly but statistically significant increased cube root of pup weight were observed when compared to the control group. This was considered as not an adverse finding since it was within the historical control data.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 was noted in all treatment groups when compared to the control group.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There was no test item-related or statistically significant effect on pup mean weight, total litter weight, female litter weight on PND 0, PND 4 and PND 13 observed in LD and MD groups when compared with the controls.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
No test item related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 was noted in all treatment groups when compared to the control group.
External malformations:
no effects observed
Description (incidence and severity):
No test item related gross external abnormalities of toxicological relevance were observed in the pups of any of the groups on PND 0-12
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
not specified
Description (incidence and severity):
No test item-related effect of toxicological relevance or statistical significance was observed on pup thyroid weights and PND 13 pup thyroxine hormone (T4) levels in treatment groups when compared to the controls.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on litter data, litter weight data, pup survival data, anogenital distance and nipple retention, pup thyroid weight and thyroid hormone analysis and pup external findings in all dose groups for which data could be obtained.

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test with trimethoxy(octyl)silane, conducted according to OECD Test Guideline 422 and in compliance with GLP, a NOAEL of 300 mg/kg bw/day was concluded for developmental toxicity based on no effects on litter data, litter weight data, pup survival data, anogenital distance and nipple retention, pup thyroid weight and thyroid hormone analysis and pup external findings in all dose groups for which data could be obtained.