Trimethoxy(methyl)silane

Endpoint summary

Description of key information

In the key skin sensitisation local lymph node assay, conducted according to OECD Test Guideline 429 and in compliance with GLP, the SI values calculated for trimethoxy(methyl)silane were lower than 3, indicating that the test material is not a skin sensitiser (Charles River, 2021).

In addition, two trimethoxy(methyl)silane manufacturers have provided statements which confirm that there have been no cases of skin sensitisation amongst workers during more than 20 years of manufacture (see IUCLID Section 7.10.4) (Wacker, 2017; Momentive, 2017).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Jan 2021 - 10 Feb 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Remarks:

CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 11 weeks old
- Weight at study initiation: 20.6 to 25.0 g.
- Housing: up to 5 animals of the same sex and same dosing group together
- Diet (e.g. ad libitum): Pelleted rodent diet, ad libitum
- Water (e.g. ad libitum): Municipal tap-water, ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: Before the initiation of dosing, a health inspection was performed, and any assigned animal considered unsuitable for use in the study were replaced by alternate animals.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): 39 to 42%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

- IN-LIFE DATES: From: 20 Jan 2021 To: 08 Feb 2021

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0, 10, 25 and 50 % w/w

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: two test concentrations were tested - 25% and 50%. The highest concentration was the maximum concentration that could technically be applied.
- Irritation: very slight irritation at 25% and 50% concentration
- Systemic toxicity: no effects indicative of systemic toxicity
- Ear thickness measurements: yes
- Erythema scores: very slight irritation observed

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Skin Sensitization: Local Lymph Node Assay
- Criteria used to consider a positive response: Disintegrations Per Minute (DPM) values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitiser. Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3).


TREATMENT PREPARATION AND ADMINISTRATION:
- Preparation of test item - Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item. The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.
- Induction - The dorsal surface of both ears was topically treated (25 μL/ear) with the test item on days 1, 2 and 3, at approximately the same time on each day. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
- Excision of the Nodes - On day 6, each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine. After five hours, all animals were euthanised and the draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.
- Tissue Processing for Radioactivity - On day 6, following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) and then stored in the refrigerator until the next day.
- Radioactivity measurements - On day 7, precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter. Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Positive control results:

The six-month reliability check with Alpha-Hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity. The SI values calculated for the item concentrations 5, 10 and 25% were 2.1, 3.6 and 9.0 respectively. An EC3 value of 8.0% was calculated using linear interpolation.

Key result

Parameter:

SI

Value:

0.6

Test group / Remarks:

10 % w/w

Key result

Parameter:

SI

Value:

0.6

Test group / Remarks:

25 % w/w

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

50 % w/w

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA : All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

DETAILS ON STIMULATION INDEX CALCULATION : Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 472, 460 and 531 DPM, respectively. The mean DPM/animal value for the vehicle control group was 734 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 0.6, 0.6 and 0.7, respectively.

EC3 CALCULATION : The EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 50%.

CLINICAL OBSERVATIONS: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period, except for one animal dosed at 10%, which showed slight body weight loss (-10%), as there were no corroborative findings for these animals, it was considered that the results for these animals were not affected.

See attachment for data tables.

Interpretation of results:

GHS criteria not met

Conclusions:

In the skin sensitisation local lymph node assay, conducted according to OECD Test Guideline 429 and in compliance with GLP, the SI values calculated for trimethoxy(methyl)silane were lower than 3, indicating that the test material is not a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In the key skin sensitisation local lymph node assay with trimethoxy(methyl)silane, conducted according to OECD Test Guideline 429 and in compliance with GLP, the test concentrations selected for the main study were based on the results of a pre-screen test (Charles River, 2021). At a 25% and 50% test item concentration, no signs of systemic toxicity were noted, and only very slight irritation was observed. Therefore, a 50% concentration was selected as highest concentration for the main study.

In the main study, three experimental groups of five female CBA/J mice were treated with test concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 472, 460 and 531 DPM, respectively. The mean DPM/animal value for the vehicle control group was 734 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 0.6, 0.6 and 0.7, respectively. Since all SI values were below 3, it was concluded that trimethoxy(methyl)silane is not a skin sensitiser.

 

The result from the key study is supported in an in vivo skin sensitisation study, conducted according to OECD Test Guideline 406 and in compliance with GLP, with acceptable restrictions. The restriction was that the starting concentration selected for induction was not the highest to cause mild-to-moderate skin irritation as required by OECD test guideline 406. The highest, non-irritating concentration used in the preliminary test (test article and PEG at 50% dilution) was selected for use in the induction and challenge phases. No positive skin reactions were seen in any of the test animals, and appropriate responses were evident in all the control animals. The study concluded that the test material was not sensitising to guinea pig skin (Toxicon, 2013).

 

A third skin sensitisation study with trimethoxy(methyl)silane, conducted according to OECD Test Guideline 406 and in compliance with GLP, found that the test material is sensitising to guinea pigs skin (Harlan Laboratories, 2009).

In the study, trimethoxy(methyl)silane at 25% in PEG 300 was found non-irritating in two pre-screen experiments as well as in the second control group. Skin reactions were observed in the test and control group in the first challenge when treated at 25% in PEG 300. At second challenge, local skin reactions were observed in test animals when the test substance was applied at the concentration of 25% in PEG 300 but not at the item concentration of 15% in PEG 300. The presence of skin reactions of grade 1 in 30% and 20% of the test animals after 24 and 48 hours respectively in the second challenge and absence of any evidence of irritation in the second control group support the conclusion that the skin reactions in the test animals are indicative of sensitisation. The equivocal first challenge data followed by positive re-challenge data, lead the study authors to the overall conclusion that the test material is sensitising. However, the reviewer considers the result ambiguous due to the methodological issues in the study. However, the reviewer considers the result ambiguous due to the methodological issues in the studyinconsistent irritation potential reported between the experiments and the positive skin reactions seen in the negative controls. The study authors give no explanation for the irritation seen in the negative control animals at challenge despite the negative screening results at 25% for irritation. It is possible that this concentration is slightly irritating and may not be a non-irritating dose. In addition, at the time of the rechallenge, the 25% and 15% test patches were used on the same animals, though on different sides of the animal. This could have led to possible confounding effects of dosing in this manner.

 

It is also noted that the key study by Charles River (2021) and the supporting study by Toxikon (2013) both found the test material not sensitising, when a higher test material concentration (50 % w/w) was used, than the study by Harlan Laboratories (2009) which concluded the test material to be skin sensitising. This supports the conclusion that the results from the key study (Charles River, 2021) and the supporting study (Toxikon, 2013) are a more accurate representation of the true sensitisation potential of the registered substance.

 

In addition to the in vivo experimental animal data, two producers of trimethoxy(methyl)silane have provided statements which confirm that there have been no cases of skin sensitisation amongst workers during more than 20 years of manufacture (Wacker, 2017; Momentive, 2017).

 

These data included internal information from: (1) relevant plants (including the number of employees and exposure descriptions as well as medical surveillance and regular health checks (especially concerning skin status) already performed on employees; (2) information from the Network of Departments of Dermatology for the surveillance and scientific evaluation of contact allergies; (3) information from an Employer's liability insurance association (BG Bau); (4) information from customers; and (5) a comprehensive literature search. This information demonstrates that there have been no cases of skin sensitisation during more than 20 years of production, handling and use of trimethoxy(methyl)silane.

 

There is therefore no evidence that this substance causes skin sensitisation under relevant exposures in workers.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available skin sensitisation data, together with over 20 years of occupational medical data, literature search results and data from an insurance association, trimethoxy(methyl)silane does not require classification for skin sensitisation according to Regulation (EC) No. 1272/2008.