Amino functional alkoxysilanes

Endpoint summary

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 for trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine (EC 701 -408 -8) the NOAEL was 75 mg/kg bw/day based on a treatment-related lower motor activity observed in main and recovery females at 250 mg/kg bw/day in comparison with that in females in the other groups. 

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28th April 2017 to May 2018

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/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:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study 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

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River,
- Females 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 cohabited 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 oral 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.

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

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 the registration substance 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

Positive control:

not used

Observations and examinations performed and frequency:

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.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected at the end of the treatment period on the day of scheduled necropsy. Blood of Recovery animals was collected at the end of the treatment period and on the day of scheduled necropsy at the end of the recovery period.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected at the end of the treatment period on the day of scheduled necropsy. Blood of Recovery animals was collected at the end of the treatment period and on the day of scheduled necropsy at the end of the recovery period.
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of treatment and from all recovery F0-animals at the end of treatment and at the end of recovery.
- Metabolism cages used for collection of urine: not specified
- Animals fasted: Yes
- Parameters checked in table [No.1] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 of treatment
- Dose groups that were examined: Selected 5 main males and 5 main females, and all recovery males and females
- Battery of functions tested: sensory activity / grip strength / motor activity / hearing ability / pupillary reflex / static righting reflex

IMMUNOLOGY: NO

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table No 2)

HISTOPATHOLOGY: Yes (see table No 2)

Other examinations:

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.

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.

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.

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, 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 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

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.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

System:

central nervous system

Organ:

not specified

Treatment related:

yes

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, with trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine the NOAEL for systemic effects was concluded to be 75 mg/kg bw/day based on a treatment-related lower motor activity observed in main and recovery females at 250 mg/kg bw/day in comparison with that in females in the other groups. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch score 1

System:

central nervous system

Organ:

not specified

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

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 orally by gavage for a minimum of 28 days to 10 male and 10 female Wistar Han rats per dose at three dose levels of 25, 75 and 250 mg/kg bw/day (Charles River Laboratories, 2018). A control group was also included that received water. A 14-day recovery period was included for 5 additional male and 5 female rats from the high and low dose groups. The recovery animals (used to study the potential reversibility of possible adverse 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, estrous 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 toxicologically 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 ambulations 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 ambulations 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 14-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.

Based on a treatment-related lower motor activity observed in main and recovery females after treatment at 250 mg/kg bw/day, a NOAEL of 75 mg/kg bw/day was concluded for systemic toxicity.  

Justification for classification or non-classification

Based on the available data for trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine, no classification is required for specific target organ toxicity following repeated exposure according to Regulation (EC) No 1272/2008.