Amino functional alkoxysilanes

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

In the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted according to OECD Test Guideline 422 and in compliance with GLP

with trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine (EC 701 -408 -8), no reproductive or developmental toxicity was observed up to the highest dose of 250 mg/kg bw/day. The NOAEL for reproductive toxicity was concluded to be at least 250 mg/kg bw/day (Charles River Laboratories, 2018).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD 421, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from moisture/water as well as heat and ignition sources
- Stability under test conditions: Not stable above 180°C (potential formaldehyde release). Stable under test conditions.
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dosed undiluted. The required amount of test item for daily dosing was transferred into a container and stored at room temperature.
- Preliminary purification step: No correction factor required
- Final dilution of a dissolved solid, stock liquid or gel: The test item was used undiluted.
- Final preparation of a solid: not applicable

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River,
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks old males and 13 weeks old females
- Weight at study initiation: 260 and 295g for males and 194 and 249g for females
- Fasting period before study: none
- Housing: On arrival and following the pre-test (females only) and pre-mating period, main animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. Recovery males and females were group housed during the entire study period. During the mating phase, Main males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, Main males were housed in their home cage with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages. During the lactation phase, Main females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water.
- Diet: Pelleted rodent diet, ad libitum
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: The feed and water were analyzed by the supplier for nutritional components and environmental contaminants. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C (target) and 20 to 22°C (actual)
- Humidity (%): 40 to 70% (target) and 49 to 73% (actual)
- Air changes (per hr): 10x/hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was administered undiluted.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: for 14 days
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not specified
- Further matings after two unsuccessful attempts: not specified
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: none

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance administered neat. No analytical verification conducted as not needed.

Duration of treatment / exposure:

Main and Recovery Males were treated for 29 days. Females that delivered were treated for 50-63 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 41 or 53 days. Recovery females (not participating in the reproduction part of the study) were treated for 50 days.

Frequency of treatment:

7 days per week

Details on study schedule:

Not applicable

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

Test animals: 10 males and 10 females
Recovery groups: 5 males and 5 females for high dose and control groups

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 14-day oral dose range finder with oral administration of Reaction mass of trimethoxy(aminoalkyl)-silanes and modified alkylether oligomers in rats, and in an attempt to produce graded responses to the test item.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: Recovery groups were selected for low and high dose treatment groups.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): random

Positive control:

Not used

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.
- Cage side observations checked included: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: hree times daily, shortly before, immediately after and 1 to 2 hours after dosing. During the recovery period, animals were observed at least once daily up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION:
- Food consumption was quantitatively measured weekly, except for Main males and Main females which were housed together for mating and for Main females without evidence of mating. Food consumption of mated Main females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
Thyroid hormone:
Blood samples were processed for serum for possible analysis for the thyroid hormone parameters total thyroxine (T4) and/or thyroid-stimulating hormone (TSH). These serum samples were stored until (possible) analysis in a freezer (≤-75°C).
Samples for T4 of F0-males and PND 13-15 pups were analysed.
Samples for T4 of F0-females and PND 4 pups and samples for TSH of F0-males, F0-females and PND 13-15 pups were not analysed.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females (Main and Recovery) during 14 days prior to treatment (pre-test period) and the first 14 days of treatment. For Main females, daily vaginal lavage was continued during mating until evidence of copulation was observed.
On the day of necropsy, a vaginal lavage was also taken from Main females to determine the stage of estrous. This was done for all Main females, except for females with total litter loss.

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight, epididymis weight

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, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS: yes
Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation


ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were euthanised following completion of the mating period (a minimum of 28 days of administration)
- Maternal animals: All surviving animals that delivered were euthanised on PND 14-16.

GROSS NECROPSY
- Gross necropsy consisted of a full post mortem examination, with special attention being paid to the reproductive organs

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [# 1] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 13-15 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of sex determination both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

Mating index, precoital index, gestation index, fertility index, duration of gestations, post-implantation survival index, live birth index, percentage of live males at first litter check, percentage of live females at first litter check, viability index, lactation index.

Offspring viability indices:

viability index

Clinical signs:

no effects observed

Description (incidence and severity):

Rales were observed in several 75 and 250 mg/kg bw/day treated main and recovery animals predominantly in the first two weeks of treatment. In one female at 250 mg/kg bw/day the rales were accompanied by laboured respiration. Rales were also observed in a single 25 mg/kg bw/day treated male and female on one day in the first week of treatment. The rales were always observed temporarily, lasting between one observation to maximally three days.
Piloerection was observed in one 250 mg/kg bw/day treated female (no.94) for two days in the first week of mating.
No specific clinical signs were noted in the animals of all dose groups during the weekly arena observations.
During the treatment period, salivation was observed among animals of the 75 and 250 mg/kg bw/day dose group immediately after dosing on one or more occasions. Salivation was observed on a single occasion in a 25 mg/kg bw/day treated female. Dose response relationship was observed. Salivation observed in this study was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Other clinical signs noted during the treatment period, including alopecia, scales and/or scabs, occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the morning of Day 3 (prior to dosing) male no.42 (Group 4) was found moribund (gasping) and died shortly thereafter. Macroscopic examination of this animal revealed an enlarged mandibular lymph node (unilateral) foamy contents in the trachea, dark red foci in the thymus and reddish foci in the lungs. Based on the time of occurrence and the absence of similar signs in the other animals, it was considered an accidental event, rather than indicative of test item related toxicity.
No further mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Lower body weights and body weight gain were observed in main and recovery males at 250 mg/kg bw/day, achieving levels of statistical significance for body weights on days 15 and 22 of treatment and on all occasions during treatment for body weight gain when compared to controls. At the end of treatment, a body weight difference of only approximately 4% between controls and males at 250 mg/kg bw/day was noted.

During the recovery period, the difference in body weights persisted during the recovery period, achieving levels of statistical significance on all occasions. The body weight gain data indicated that growth of the recovery males ran parallel. The body weight gain (when compared to study day 1) in high dose recovery males at start of recovery, was statistically significantly lower than in control recovery males. The increase in difference in mean body weights between control and high dose males over one day (from end of treatment to start of recovery) was due to relatively high body weights of the control males that continued in the recovery period compared to those of the whole group. Since the body weight gain over the recovery period was comparable for the control and high dose males, no toxicological significance was attached to this finding.

Body weights and body weight gain in 25 and 75 mg/kg bw/day treated males were considered not to be affected by treatment.
Body weights and body weight gain in female rats were considered to have been unaffected by treatment.
Over the recovery period, body weights and body weight gain were also unaffected by cessation of treatment in female rats.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight in main and recovery male and recovery (not-mated) female rats was similar to the control level over the treatment period and recovery period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

At the end of the treatment period, haematological parameters of treated rats and recovery rats were considered not to have been affected by treatment.
The statistical significance for the mean number of lymphocytes in 75 mg/kg bw/day treated males and for the mean corpuscular haemoglobin concentration (MCHC) in 75 mg/kg bw/day treated females were fortuitous findings. In the absence of a treatment-related distribution or corroborative findings these changes were considered to be of no toxicological significance.
Coagulation parameters of treated rats were considered not to be different from controls at the end of treatment as well as after a subsequent fourteen-day recovery period.
The lower prothrombin time (PT) seen in 250 mg/kg be/day treated males, achieving a level of statistical significance was considered not to be of toxicological relevance as the opposite effect (i.e. an increase) would be expected in case of target organ toxicity.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

At the end of treatment, lower mean levels for bile acids were observed in 250 mg/kg bw/day treated males and recovery females, achieving a level of statistical significance in recovery females. The high dose animals did not recover from this difference and the levels of bile acids remained lower at the end of the subsequent fourteen-day treatment free period, now achieving a level of statistical significance in 250 mg/kg be/day treated males.
In main females, the mean levels for bile acids did not indicate an effect by treatment and were comparable between the treated and control animals at the end of treatment. However, it should be noted that a greater individual variation was observed and the mean level for bile acids was approximately two times higher than in the recovery females. This was likely to be related to the difference in physiological status between the primiparous and nulliparous females.
The statistical significances for the mean level for inorganic phosphate in 75 and 250 mg/kg bw/day treated males and for the mean level of calcium in 250 mg/kg bw/day treated main females occurred by chance. As the changes in these parameters were minimal (<10%) and the values for these parameters remained within the historical range for rats of this strain and age, these findings were considered to be of no toxicological significance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis parameters of treated rats were considered not to be different from controls at the end of treatment as well as after a subsequent fourteen-day recovery period.
The pH value of the urine in 75 mg/kg bw/day treated (main) females achieving a level of statistical significance when compared to controls, was considered to have arisen as a result of slightly low control value and in the absence of a treatment-related distribution considered to be of no toxicological significance.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

The functional observation parameters, hearing ability, pupillary reflex, static righting reflex and grip strength, were considered not to be affected by treatment.

The group mean data for motor activity, comprising total movements and ambulation, showed a large variation between the groups in both males and females.

Whereas the group mean values for total movements and ambulation in the 25 and 75 mg/kg bw/day treated main and recovery males were approximately 20% lower than controls, these group mean values in the 250 mg/kg bw/day treated males were similar to that of controls. In the absence of clear dose response relationship and since the motor activity data of individual animals of all groups were within the historical control range for male rats of this strain, age and used in this type of studies, it was concluded that the variation in motor activity between groups was not treatment related. Moreover, a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period was observed in all groups.
In lactating (main) females, the motor activity was higher in the 25 and 75 mg/kg bw/day treated animals, whereas a marked decrease was observed in the 250 mg/kg bw/day treated females in comparison with controls. The increases in group mean total movements were 19% and 16% and for ambulation were 36% and 27% for the 25 and 75 mg/kg bw/day treated females, respectively. In the 250 mg/kg bw/day treated females, the group mean values were 43% lower for total movements, achieving a level of statistical significance, and 40% lower for the ambulation, when compared to controls.

The motor activity in not-mated (recovery) females at 250 mg/kg bw/day was in general higher than in lactating females, but a similar difference between control and high dose females was observed with a 30% lower value for total movements and a 40% lower value for ambulation in the latter animals, but not achieving a level of statistical significance in comparison with controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone
At the end of treatment, the serum levels of T4 in F0-males were comparable between the treated and control animals and considered not to be affected by treatment.
At the end of the subsequent fourteen-day recovery period, a difference in levels of T4 between the high dose males and controls was observed, achieving a level of statistical significance. The difference was considered to have arisen as a result of slightly high control value and since the T4 values of the high dose males were well within the historical control range for rats of this strain and age no toxicological significance was attached to this finding.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment.
All females had regular cycles of 4 days. During the mating period, extended di-estrus occurred in two control females (nos.58 and 59) until mating. Both females delivered normal litters. Extended di-estrus was also observed in one 250 mg/kg bw/day treated female no.88 during the mating period.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Testis and epididymis weight were unchanged following treatment.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating index was considered not to be affected by treatment. One 250 mg/kg bw/day treated female showed no evidence of mating, which was considered an incidental finding and not related to treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on reduced motor activity in main and recovery females at 250 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect on reproductive parameters

Key result

Critical effects observed:

yes

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
A pale and cold appearance and a wound on the right leg was observed for the pup of control litter no.51 before it went missing on Day 5.
From Day 9-10 of lactation onwards all pups in litter no.93 (at 250 mg/kg bw/day) showed alopecia.
The nature and incidence of these and other clinical signs remained within the range considered normal, and were therefore considered not to be toxicologically relevant.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

For the treated females, the number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment.
One pup (of litter 72 at 25 mg/kg bw/day) was found dead on Day 2. For control females, 6 pups (out of three litters, nos.51, 52 and 59) were found dead or went missing between lactation Days 2 and 4. The missing pups were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

At birth, body weights of male and female pups in litters at 250 mg/kg bw/day were higher (slightly over 10%) in comparison with that in the other groups, achieving levels of statistical significance when compared to controls. During lactation this difference remained, but had reduced to less than 10% on lactation Day 13.

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 examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 250 mg/kg bw/day had no effect on areola/nipple retention.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.
No milk in the stomach was noted for all stillborn pups in litter 84.
The nature and incidence of the other macroscopic findings remained within the range considered normal for pups of this age, were related to the death or confirmed the in-life observation of the pup.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 13-15 pups were considered not to be affected by treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect observed at any dose level.

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

In the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted according to OECD Test Guideline 422 and in compliance with GLP, no reproductive or developmental toxicity was observed up to the maximum dose of 250 mg/kg bw/day. The NOAEL for reproductive and developmental effects was concluded to be greater than 250 mg/kg bw/day for trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch score of 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In the key combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test

conducted according to OECD Test Guideline 422 and in compliance with GLP

for trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine, undiluted test material was administered by oral gavage to 10 male and female Wistar Han rats respectively per dose groups (Charles River Laboratories, 2018). The administered concentrations were 25, 75 or 250 mg/kg bw/day with a treatment period for a minimum of 28 days. A control group was also included that received water. A 14-day recovery period was included for 5 additional male and female rats respectively, from the high and low dose groups. The recovery animals (used to study the potential reversibility of possible toxic effects) were not mated and consequently were not used for the assessment of reproduction/ developmental toxicity.

Mortality/moribundity, clinical signs, functional observations, body weight and food consumption, oestrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations were performed. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4.

No toxicological significant clinical signs were observed in male and female rats treated up to 250 mg/kg bw/day.  

The motor activity in male and female rats showed a large variation between the groups. Despite the fact that the motor activity recorded in the animals of all dose groups was within the historical control range, there was no indication of a treatment related change in males, but it could not be ruled out that the marked decrease in group mean values for total movements and ambulation in both main and recovery females at 250 mg/kg bw/day was a treatment related effect. Any corroborating evidence from other possibly related parameters was not observed in the current study, e.g. no other behavioural changes, no effects on grip strength and absence of histopathological alterations in nerve and muscle tissue in these females. Nevertheless, in case the changes in motor activity in females at 250 mg/kg bw/day were treatment related the magnitude of the decrease of approximately 40% lower group mean values for total movements and ambulation should be an indication of an adverse effect.

Slightly reduced body weight gain was observed in males at 250 mg/kg bw/day during treatment, resulting in approximately 4% lower group mean body weight compared to that of controls. During the recovery period, the body weight gain was similar between these two groups and the difference in body weight continued until termination of the recovery males. Based on the magnitude of the changes in body weight the effect of treatment was considered to be non-adverse.

At the end of treatment, lower mean levels for bile acids were observed in 250 mg/kg bw/day treated males and recovery females. These high dose animals had not recovered from this difference and the levels of bile acids remained lower at the end of the subsequent fourteen-day treatment free period in the recovery animals.

At the end of treatment, a dose related increase in splenic weight (in males) and decrease in adrenal weight (in females) was observed. The changes in weight of these organs in high dose animals were still within the historical control range and there were no microscopic correlates for these organ weight changes. In addition, the splenic weights in control and high dose recovery males and the adrenal weights in control and high dose recovery females were comparable at the end of the fourteen-day treatment free period. Therefore, these changes in organ weights at the end of treatment were considered to be non-adverse after treatment up to 250 mg/kg bw/day. 

No treatment-related changes were noted in the other parameters investigated in this study (i.e. food consumption, haematology, coagulation and (male) T4 thyroid hormone levels, macroscopic and microscopic examination) at the end of treatment.

Furthermore, the fourteen-day recovery period after cessation of treatment did not induce changes between the 250 mg/kg bw/day treated males and females compared to their concurrent controls in any of the parameters investigated. 

No reproduction toxicity was observed up to the highest dose level tested (250 mg/kg bw/day) and the NOAEL was concluded to be at least 250 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

In the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test

conducted according to OECD Test Guideline 422 and in compliance with GLP

with trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine (EC 701 -408 -8) no reproductive or developmental toxicity was observed up to the highest dose of 250 mg/kg bw/day. The NOAEL for developmental toxicity was concluded to be at least 250 mg/kg bw/day (Charles River Laboratories, 2018).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD 421, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from moisture/water as well as heat and ignition sources
- Stability under test conditions: Not stable above 180°C (potential formaldehyde release). Stable under test conditions.
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dosed undiluted. The required amount of test item for daily dosing was transferred into a container and stored at room temperature.
- Preliminary purification step: No correction factor required
- Final dilution of a dissolved solid, stock liquid or gel: The test item was used undiluted.
- Final preparation of a solid: not applicable

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River,
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks old males and 13 weeks old females
- Weight at study initiation: 260 and 295g for males and 194 and 249g for females
- Fasting period before study: none
- Housing: On arrival and following the pre-test (females only) and pre-mating period, Main animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. Recovery males and females were group housed during the entire study period. During the mating phase, Main males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, Main males were housed in their home cage with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages. During the lactation phase, Main females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water.
- Diet: Pelleted rodent diet, ad libitum
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: The feed and water were analyzed by the supplier for nutritional components and environmental contaminants. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C (target) and 20 to 22°C (actual)
- Humidity (%): 40 to 70% (target) and 49 to 73% (actual)
- Air changes (per hr): 10x/hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was administered undiluted.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance administered neat. No analytical verification conducted as not needed.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: for 14 days
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not specified
- Further matings after two unsuccessful attempts: not specified
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: none

Duration of treatment / exposure:

Main and recovery males were treated for 29 days. Females that delivered were treated for 50-63 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 41 or 53 days. Recovery females (not participating in the reproduction part of the study) were treated for 50 days.

Frequency of treatment:

7 days per week

Duration of test:

up to 50 days

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

Test animals: 10 males and 10 females
Recovery groups: 5 males and 5 females for high dose and control groups

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 14-day oral dose range finder with oral administration of Reaction mass of trimethoxy(aminoalkyl)-silanes and modified alkylether oligomers in rats, and in an attempt to produce graded responses to the test item.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: Recovery groups were selected for low and high dose treatment groups.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.
- Cage side observations checked included: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Three times daily, shortly before, immediately after and 1 to 2 hours after dosing. During the recovery period, animals were observed at least once daily up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION:
- Food consumption was quantitatively measured weekly, except for Main males and Main females which were housed together for mating and for Main females without evidence of mating. Food consumption of mated Main females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

Thyroid hormone:
Blood samples were processed for serum for possible analysis for the thyroid hormone parameters total thyroxine (T4) and/or thyroid-stimulating hormone (TSH). These serum samples were stored until (possible) analysis in a freezer (≤-75°C).
Samples for T4 of F0-males and PND 13-15 pups were analysed.
Samples for T4 of F0-females and PND 4 pups and samples for TSH of F0-males, F0-females and PND 13-15 pups were not analysed.

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: No
- Skeletal examinations: No
- Head examinations: Yes [all per litter]

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Indices:

Mating index, precoital index, gestation index, fertility index, duration of gestations, post-implantation survival index, live birth index, percentage of live males at first litter check, percentage of live females at first litter check, viability index, lactation index.

Clinical signs:

no effects observed

Description (incidence and severity):

Rales were observed in several 75 and 250 mg/kg bw/day treated main and recovery animals predominantly in the first two weeks of treatment. In one female at 250 mg/kg bw/day the rales were accompanied by laboured respiration. Rales were also observed in a single 25 mg/kg bw/day treated male and female on one day in the first week of treatment. The rales were always observed temporarily, lasting between one observation to maximally three days.
Piloerection was observed in one 250 mg/kg bw/day treated female (no.94) for two days in the first week of mating.
No specific clinical signs were noted in the animals of all dose groups during the weekly arena observations.
During the treatment period, salivation was observed among animals of the 75 and 250 mg/kg be/day dose group immediately after dosing on one or more occasions. Salivation was observed on a single occasion in a 25 mg/kg bw/day treated female. Dose response relationship was observed. Salivation observed in this study was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Other clinical signs noted during the treatment period, including alopecia, scales and/or scabs, occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the morning of Day 3 (prior to dosing) male no.42 (Group 4) was found moribund (gasping) and died shortly thereafter. Macroscopic examination of this animal revealed an enlarged mandibular lymph node (unilateral) foamy contents in the trachea, dark red foci in the thymus and reddish foci in the lungs. Based on the time of occurrence and the absence of similar signs in the other animals, it was considered an accidental event, rather than indicative of test item related toxicity.
No further mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Lower body weights and body weight gain were observed in main and recovery males at 250 mg/kg bw/day, achieving levels of statistical significance for body weights on days 15 and 22 of treatment and on all occasions during treatment for body weight gain when compared to controls. At the end of treatment, a body weight difference of only approximately 4% between controls and males at 250 mg/kg bw/day was noted.

During the recovery period, the difference in body weights persisted during the recovery period, achieving levels of statistical significance on all occasions. The body weight gain data indicated that growth of the recovery males ran parallel. The body weight gain (when compared to study day 1) in high dose recovery males at start of recovery, was statistically significantly lower than in control recovery males. The increase in difference in mean body weights between control and high dose males over one day (from end of treatment to start of recovery) was due to relatively high body weights of the control males that continued in the recovery period compared to those of the whole group. Since the body weight gain over the recovery period was comparable for the control and high dose males, no toxicological significance was attached to this finding.

Body weights and body weight gain in 25 and 75 mg/kg bw/day treated males were considered not to be affected by treatment.
Body weights and body weight gain in female rats were considered to have been unaffected by treatment.
Over the recovery period, body weights and body weight gain were also unaffected by cessation of treatment in female rats.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight in main and recovery male and recovery (not-mated) female rats was similar to the control level over the treatment period and recovery period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

At the end of the treatment period, haematological parameters of treated rats and recovery rats were considered not to have been affected by treatment.
The statistical significance for the mean number of lymphocytes in 75 mg/kg bw/day treated males and for the mean corpuscular haemoglobin concentration (MCHC) in 75 mg/kg bw/day treated females were fortuitous findings. In the absence of a treatment-related distribution or corroborative findings these changes were considered to be of no toxicological significance.
Coagulation parameters of treated rats were considered not to be different from controls at the end of treatment as well as after a subsequent fourteen-day recovery period.
The lower prothrombin time (PT) seen in 250 mg/kg bw/day treated males, achieving a level of statistical significance was considered not to be of toxicological relevance as the opposite effect (i.e. an increase) would be expected in case of target organ toxicity.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

At the end of treatment, lower mean levels for bile acids were observed in 250 mg/kg bw/day treated males and recovery females, achieving a level of statistical significance in recovery females. The high dose animals did not recover from this difference and the levels of bile acids remained lower at the end of the subsequent fourteen-day treatment free period, now achieving a level of statistical significance in 250 mg/kg bw/day treated males.
In main females, the mean levels for bile acids did not indicate an effect by treatment and were comparable between the treated and control animals at the end of treatment. However, it should be noted that a greater individual variation was observed and the mean level for bile acids was approximately two times higher than in the recovery females. This was likely to be related to the difference in physiological status between the primiparous and nulliparous females.
The statistical significances for the mean level for inorganic phosphate in 75 and 250 mg/kg bw/day treated males and for the mean level of calcium in 250 mg/kg bw/day treated main females occurred by chance. As the changes in these parameters were minimal (<10%) and the values for these parameters remained within the historical range for rats of this strain and age, these findings were considered to be of no toxicological significance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis parameters of treated rats were considered not to be different from controls at the end of treatment as well as after a subsequent fourteen-day recovery period.
The pH value of the urine in 75 mg/kg bw/day treated (main) females achieving a level of statistical significance when compared to controls, was considered to have arisen as a result of slightly low control value and in the absence of a treatment-related distribution considered to be of no toxicological significance.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

The functional observation parameters, hearing ability, pupillary reflex, static righting reflex and grip strength, were considered not to be affected by treatment.

The group mean data for motor activity, comprising total movements and ambulation, showed a large variation between the groups in both males and females.

Whereas the group mean values for total movements and ambulation in the 25 and 75 mg/kg bw/day treated main and recovery males were approximately 20% lower than controls, these group mean values in the 250 mg/kg bw/day treated males were similar to that of controls. In the absence of clear dose response relationship and since the motor activity data of individual animals of all groups were within the historical control range for male rats of this strain, age and used in this type of studies, it was concluded that the variation in motor activity between groups was not treatment related. Moreover, a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period was observed in all groups.
In lactating (main) females, the motor activity was higher in the 25 and 75 mg/kg bw/day treated animals, whereas a marked decrease was observed in the 250 mg/kg bw/day treated females in comparison with controls. The increases in group mean total movements were 19% and 16% and for ambulations were 36% and 27% for the 25 and 75 mg/kg bw/day treated females, respectively. In the 250 mg/kg bw/day treated females, the group mean values were 43% lower for total movements, achieving a level of statistical significance, and 40% lower for the ambulation, when compared to controls.

The motor activity in not-mated (recovery) females at 250 mg/kg bw/day was in general higher than in lactating females, but a similar difference between control and high dose females was observed with a 30% lower value for total movements and a 40% lower value for ambulation in the latter animals, but not achieving a level of statistical significance in comparison with controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

At the end of treatment, a dose related increase for the splenic weight was observed in Main male rats, achieving a level of statistical significance for the relative spleen weight in high dose males when compared to controls. In Main females, a dose related decrease was observed for the adrenal glands, achieving levels of statistical significance for the absolute and relative adrenal gland weight in high dose females when compared to controls.
No test item-related alterations were observed in the other organ weights at the end of treatment.
At the end of recovery, in the males a difference in absolute liver weights and relative weights of the brain and seminal vesicles was observed between control and high dose males, achieving levels of statistical significance. These differences were considered to be related to the differences in terminal body weight and considered not related to treatment and of no toxicological significance.
In high dose females at the end of recovery, minimal higher weights of the heart, liver and kidneys were observed, achieving levels of statistical significance for absolute and relative heart weight and relative liver and kidney weights when compared to controls. Since all these organ weights remained within the historical control range for female rats of this strain and age and no treatment related effects were observed in main females at the end of treatment no toxicological significance was attached to these findings

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.
All of the recorded macroscopic findings at the end of treatment in main males and females and at the end of recovery in recovery males and females were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone
At the end of treatment, the serum levels of T4 in F0-males were comparable between the treated and control animals and considered not to be affected by treatment.
At the end of the subsequent fourteen-day recovery period, a difference in levels of T4 between the high dose males and controls was observed, achieving a level of statistical significance. The difference was considered to have arisen as a result of slightly high control value and since the T4 values of the high dose males were well within the historical control range for rats of this strain and age no toxicological significance was attached to this finding.

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Number of implantation sites was considered not to be affected by treatment.
One 250 mg/kg bw/day treated female (no. 90) had a relatively low number of implantation sites, i.e. 7 compared to the group mean value of 12.7. Since it was observed in a single female only this finding was considered to have occurred by chance and considered to be unrelated to treatment.

Total litter losses by resorption:

not examined

Early or late resorptions:

no effects observed

Description (incidence and severity):

The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment.
For female no.82 (at 75 mg/kg bw/day), the number of pups was higher than the number of implantations, i.e. 13 living pups compared to 12 implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during (the 14 days of) lactation. No toxicological relevance was attached to this finding in the current study.

Dead fetuses:

no effects observed

Description (incidence and severity):

The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment.
Female no.84 delivered eleven dead pups, which was regarded as total litter loss. One pup in a control group litter (no.51) was found dead at first litter check. No toxicological relevance was attributed to these dead pups/total litter loss since the mortality incidence did not show a dose-related trend and remained within the range considered normal.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Gestation index and duration of gestation were considered not to be affected by treatment.
One control female (no.54) had implantations only and a 75 mg/kg bw/day treated female (no.84) delivered (eleven) dead pups only, regarded as total litter loss. Moreover, the duration of gestation in the latter female was also slightly longer than normal, i.e. 23 days compared to 21-22 days in the other females. In the absence of a dose effect relationship, these events were considered not related to treatment and of no toxicological significance.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Fertility index was considered not to be affected by treatment. All impregnated females, except one 75 mg/kg bw/day treated female (no.81) developed a pregnancy.
Since the case of non-pregnancy occurred in a single female no.81 only and showed no dose-related incidence across the dose groups, this event was considered not to be related to treatment.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: Based on reduced motor activity in main and recovery females at 250 mg/kg bw/day

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

At birth, body weights of male and female pups in litters at 250 mg/kg bw/day were higher (slightly over 10%) in comparison with that in the other groups, achieving levels of statistical significance when compared to controls. During lactation this difference remained, but had reduced to less than 10% on lactation Day 13.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

For the treated females, the number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment.
One pup (of litter 72 at 25 mg/kg) was found dead on Day 2. For control females, 6 pups (out of three litters, nos. 51, 52 and 59) were found dead or went missing between lactation Days 2 and 4. The missing pups were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter size was considered not affected by treatment.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

For the treated females, the number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment.
One pup (of litter 72 at 25 mg/kg bw/day) was found dead on Day 2. For control females, 6 pups (out of three litters, nos.51, 52 and 59) were found dead or went missing between lactation Days 2 and 4. The missing pups were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

External malformations:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.
No milk in the stomach was noted for all stillborn pups in litter 84.
The nature and incidence of the other macroscopic findings remained within the range considered normal for pups of this age, were related to the death or confirmed the in-life observation of the pup.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
Treatment up to 250 mg/kg bw/day had no effect on areola/nipple retention. Serum T4 levels in male and female PND 13-15 pups were considered not to be affected by treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at any dose level.

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

In the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test for trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine, no reproduction and developmental toxicity was observed up to treatment at 250 mg/kg bw/day. The NOAEL for reproductive and developmental effects was concluded to be greater than 250 mg/kg bw/day (the highest dose tested).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch score of 1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In the key combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test

conducted according to OECD Test Guideline 422 and in compliance with GLP

for trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine, undiluted test material was administered by oral gavage to 10 male and female Wistar Han rats respectively per dose groups (Charles River Laboratories, 2018). The administered concentrations were 25, 75 or 250 mg/kg bw/day with a treatment period for a minimum of 28 days. A control group was also included that received water. A 14-day recovery period was performed to 5 additional male and female rats respectively, from the high and low dose groups. The recovery animals (used to study the potential reversibility of possible toxic effects) were not mated and consequently were not used for the assessment of reproduction/ developmental toxicity.

Mortality/moribundity, clinical signs, functional observations, body weight and food consumption, oestrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations were performed. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4.

No toxicological significant clinical signs were observed in rats treated up to 250 mg/kg bw/day.  

The motor activity in male and female rats showed a large variation between the groups. Despite the fact that the motor activity recorded in the animals of all dose groups was within the historical control range, there was no indication of a treatment related change in males, but it could not be ruled out that the marked decrease in group mean values for total movements and ambulation in both main and recovery females at 250 mg/kg bw/day was a treatment related effect. Any corroborating evidence from other possibly related parameters was not observed in the current study, e.g. no other behavioural changes, no effects on grip strength and absence of histopathological alterations in nerve and muscle tissue in these females. Nevertheless, in case the changes in motor activity in females at 250 mg/kg bw/day were treatment related the magnitude of the decrease of approximately 40% lower group mean values for total movements and ambulation should be an indication of an adverse effect.

Slightly reduced body weight gain was observed in males at 250 mg/kg bw/day during treatment, resulting in approximately 4% lower group mean body weight compared to that of controls. During the recovery period, the body weight gain was similar between these two groups and the difference in body weight continued until termination of the recovery males. Based on the magnitude of the changes in body weight the effect of treatment was considered to be non-adverse.

At the end of treatment, lower mean levels for bile acids were observed in 250 mg/kg bw/day treated males and recovery females. These high dose animals had not recovered from this difference and the levels of bile acids remained lower at the end of the subsequent fourteen-day treatment free period in the recovery animals.

At the end of treatment, a dose related increase in splenic weight (in males) and decrease in adrenal weight (in females) was observed. The changes in weight of these organs in high dose animals were still within the historical control range and there were no microscopic correlates for these organ weight changes. In addition, the splenic weights in control and high dose recovery males and the adrenal weights in control and high dose recovery females were comparable at the end of the fourteen-day treatment free period. Therefore, these changes in organ weights at the end of treatment were considered to be non-adverse after treatment up to 250 mg/kg bw/day. 

No treatment-related changes were noted in the other parameters investigated in this study (i.e. food consumption, haematology, coagulation and (male) T4 thyroid hormone levels, macroscopic and microscopic examination) at the end of treatment.

Furthermore, the fourteen-day recovery period after cessation of treatment did not induce changes between the 250 mg/kg bw/day treated males and females compared to their concurrent controls in any of the parameters investigated. 

No developmental toxicity was observed up to the highest dose level tested (250 mg/kg bw/day).

Higher pup weights in the litters at 250 mg/kg bw/day were observed at birth (+12%), for which the difference had slightly decreased on PND 13-15 (+8%) when compared to controls. Although a relation to treatment cannot be ruled out, this change in body weight was considered to be non-adverse based on the magnitude and direction of difference (a slight increase in pup weight compared to controls) and the fact that the overall development of the pups was normal. The NOAEL for developmental toxicity was concluded to be at least 250 mg/kg bw/day.

Justification for classification or non-classification

Based on the available data for trimethoxy(methyl)silane and its reaction products with 3-aminopropyl(trimethoxy)silane and [3-(2,3-epoxypropoxy)propyl]trimethoxysilane, no classification is required for reproductive or developmental toxicity according to Regulation (EC) No. 1272/2008.