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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / 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:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29th of July 2016
Deviations:
yes
Remarks:
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:
trimethoxyoctylsilane
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl: WI(Han)
Sex:
male/female
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:
- 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
IN-LIFE DATES: From: To:

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: 4 mL/kg bw/day
- Lot/batch no.: MKCC9871 / MCKD1021
- Purity: not specified
Details on mating procedure:
- 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.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged in individually cages during the gestation/lactation period in type III H, polysulphone cages.
- Any other deviations from standard protocol: no
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).
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
Details on study schedule:
Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to assess the occurrence of mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
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
- Fasting period before blood sampling for clinical biochemistry: not specified
- Rationale for selecting satellite groups: additional 12 males and females from the control and high dose groups.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale: n/a

Examinations

Parental animals: Observations and 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
Oestrous cyclicity (parental animals):
Estrous cycles were monitored before treatment starts to select females with regular cyclicity (using vaginal smears) for the study. Subsequently, vaginal smears were also examined daily from the beginning of the treatment period until evidence for mating of main study females.
Sperm parameters (parental animals):
Parameters examined in male parental generations: testis weight, epididymis weight, sperm count in testes.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other: runts.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; possible cause of death was not determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: The surviving males of the main groups were sacrificed after the completion of the mating period (after a minimum dosing period of 28 days).
- Maternal animals: Sacrificed on their respective PND 13.

GROSS NECROPSY
- Gross necropsy consisted of external examinations including the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities plus their contents. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved (See table 1).

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.

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

HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid/parathyroid glands from 1 pup/sex/litter/group sacrificed on PND 13 and non-selected adult animals were preserved for potential histopathological examination.
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).
Reproductive indices:
copulation, fertility, viability and delivery indices

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 treatmentrelated. 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 were 29 animals (8 females from MD, 3 male and 10 female from HD, 2 male and 6 females from HD recovery) 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 statistical significance 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 statistic 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 was 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
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In LD females, statistically significant lower amount of platelets compared to controls were observed. However, the group mean value was within the range of historical control data and was therefore considered to be incidental and not related to the treatment of the test item. No haematology data were available for MD, HD and HD recovery females.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Compared to recovery controls, males in the HD recovery group had a statistical significantly lower TBA, although, this was within the historical control data range. Generally, all mean and most of the individual values were comparable with controls and within the historical control data range. In females sacrificed at the end of the treatment period, no treatment-related or statistically significant effects were observed on clinical biochemistry parameters in LD and MD groups when compared to the control. No clinical biochemistry data for the HD and HD recovery females was available as those females were sacrificed before the scheduled terminal sacrifice for animal welfare reasons.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
In a few males and females, high protein and erythrocytes levels were found in the urine. However, this was found in all groups, including the control group. Therefore, this effect was considered to be non-treatment related.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
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 HDR female, indicators for gavage accidents (perica
rdial 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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
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.
Reproductive function: sperm measures:
not specified
Reproductive performance:
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.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed in all dose groups for which data could be obtained

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality 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 in LD and MD groups. However, a slightly higher mean mortality of pups between PND 0 and PND 4 was observed in the control group (11.43 %) and LD group (4.17 %). All pups from 2 dams were sacrificed on PND 3, due to animal welfare reasons. No female from the HD group littered with live pups or these females were euthanised for animal welfare reasons before littering. Therefore no pup survival data were available for the HD group.
Body weight and 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.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not examined
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
In male pups, higher absolute and relative anogenital distance (AGD) (statistical significantly) were observed in MD group when compared to control. Similarly, slightly but statistical significantly increase of the absolute and relative anogenital distance was observed in female pups compared to the controls. However, all anogenital values in male and female pups were within historical control data range and is therefore considered to not be adverse.
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

No test item-related effect of toxicological relevance or statistical significance was observed on pup thyroid weight, male and PND 13 pup thyroxine hormone (T4) in the treatment groups when compared to the controls. There was also no test item-related effect observed on male and female thyroxine hormone (T4) in the male recovery groups, and data from the female HD recovery group could not be obtained.
No female from the HD group littered with live pups or these females were euthanised for animal welfare reasons before littering. Therefore, no male and female pup thyroid weight and pup T4 data on PND 13 were available from the HD group.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects on litter data, litter weight data, pup survival data, anogenital distance and nipple retention, pup thyroid weight and thyroid hormone analysis, and external findings in all dose groups for which data could be obtained

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive 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 reproduction toxicity based on 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 and thyroid hormone analysis in parental males and pups sacrificed on PND 13 and male sacrificed at the end of recovery period and pup external findings in all dose groups for which data could be obtained.