Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Consideration of mutagenicity to somatic cells and the need for further information

Summary of available in vivo data for 3-(2,3-epoxypropoxypropyl]trimethoxysilane

Method

Route of administration

Result

Toxicity to bone marrow

Systemic effects

Reference

Micronucleus

Intraperitoneal injection

Positive

Yes Moderate to severe reductions of 22 to 59% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in the test article-treated groups relative to the vehicle control animals

Clinical signs included lethargy and piloerection at 1000 and 2000 mg/kg.

BioReliance, 1999

Micronucleus

Intraperitoneal injection

Negative

Toxicity to bone marrow was observed in female mice at 24 h observation.

All animals showed clinical signs following dosing (1600 mg/kg bw as a suspension in corn oil)

Hüls, 1994

Micronucleus

Oral gavage

Negative

No data

No data

Dow Corning Corporation, 1982a

Sister chromatid exchange

Intraperitoneal injection

Negative

No data; a high degree of bioavailability is expected after ip administration

No data

Dow Corning Corporation, 1982d

Sister chromatid exchange

Inhalation

Negative

No data

No data

Dow Corning Corporation, 1982d

There is insufficient information on the potential for mutagenicity to somatic cells to be able to draw a conclusion, for the following reasons:

1. Toxicokinetics: no toxicokinetic data are available for the substance. There is evidence of exposure to the bone marrow following oral administration from an in vivo bioavailability study for another trialkoxysilane (see section 7.1 of IUCLID and 5.1 of the CSR). In addition, systemic toxicity was observed in two of the three micronucleus assays; no information was available for the third study.. Lethargy and piloerection were reported following intraperitoneal exposure in one of the micronucleus studies (BioReliance, 1999) at 1000 and 2000 mg/kg bw. In the more recent of the negative micronucleus assay, severe clinical symptoms were observed following intraperitoneal (ip) application (Hüls 1994). The observations from these studies indicate that the substance is bioavailable and the negative micronucleus study (oral exposure) is valid and relevant.

2. The positive in vivo micronucleus assay is considered not relevant as intraperitoneal administration it is not physiologically relevant route for this substance, taking into account expected routes of exposure for the substance. There is no clear explanation for the positive result but it is likely that route of exposure is important.

3. The weight of evidence from all studies (micronucleus and sister chromatid exchange), and the agreement of results from studies using a physiologically relevant route of exposure, is that the substance is not mutagenic.

4. There is no information for in vitro cytogenicity for the substance itself, but data are available for the structural analogue [3 -(2,3 -epoxypropoxy)propyl]triethoxysilane which was reported to be mutagenic to bacteria but did not induce micronuclei in HepG2 cells in an in vitro assay conducted according to OECD 487 (Lionti et al., 2014).

5. In vivo micronucleus assays are not considered appropriate to investigate the mutagenic effects of substances that are mutagenic to bacterial and mammalian cells. This is implied in the REACH Regulation in the column 2 adaptations for in vitro mammalian cell cytogenicity and mutagenicity testing, and is stated in ECHA Guidance R07a. Appropriate assays are, according to the ECHA Guidance (p 364), the transgenic rodent assay (OECD 488) and the in vivo Comet assay (OECD 489) or, if justified, Unscheduled DNA Synthesis, in vivo (OECD 486). There are no in vitro effects that indicate that a micronucleus assay is the appropriate in vivo assay to conclude on somatic cell mutagenicity.

6. Carcinogenicity: the substance was not considered tumorigenic when applied to the clipped skin of mice (25 μl dose of 25% TMSPGE in acetone) three times per week for approximately 78 weeks (BRRC, 1982). The ECHA Guidance (ECHA, 2014, R07a p 337) states: “There is considerable evidence of a positive correlation between the mutagenicity of substances in vivo and their carcinogenicity in long-term studies with animals.” As the substance has been shown not to be carcinogenic, it is unlikely that it is mutagenic.

To summarise, the negative data from the in vitro micronucleus test on the structural analogue and the negative in vivo micronucleus results following a physiologically relevant route of exposure, with additional evidence of bioavailability indicate that there is not sufficient evidence to classify as Mutagen Category 2, and so a new proposal to conduct an in vivo comet assay is included. The comet assay can detect evidence of DNA damage that may lead to both gene mutation and chromosome mutation events and is therefore the ideal test method for the follow-up of the in vitro / in vivo genotoxicity profile of the substance (ECHA guidance on mutagenicity (R.7a, 19 August 2014, p 343) states: “[…] the alkaline comet assay recognises primary DNA damage that would lead to gene mutations and/or chromosome aberrations) but will also detect DNA damage that may be effectively repaired or lead to cell death". The comet assay can therefore be considered a conservative estimate of genetic toxicity to somatic cells. The proposed assay will comply with OECD guideline 489.

Strategy for identification of key studies.

Where there was more than one result for an endpoint the most reliable study available was chosen as key study. Where there was more than one reliable study, the most recent study was selected. If the results were not in agreement, studies giving positive results were chosen, unless the weight of evidence indicated the result was not indicative of potential for mutagenicity. The results of all the bacterial studies were in agreement, and showed evidence of mutagenicity. In mammalian mutagenicity studies, positive results were obtained when tested in L5178Y mouse lymphoma cells (Litton Bionetics, 1983), but negative results when Chinese hamster ovary cells were used (similar to OECD 476, Reliability 4), (Allied Corporation, 1979).

No data are available for the registered substance for in vitro cytogenicity, however, data are available for the structural analogue [3-(2,3-epoxypropoxy)propyl]triethoxysilane from an in vitro micronucleus assay. [3-(2,3-epoxypropoxy)propyl]triethoxysilane has been tested for ability to induce formation of micronuclei in a study conducted according to OECD 487 (Lionti et al., 2014). No evidence of test-substance induced micronucleus formation was observed in HepG2 cells in either the presence or the absence of metabolic activation. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance in negative for cytogenicity under the conditions of the test.

Read-across hypothesis

The hypothesis is that both the registered substance and the read-across substance have equivalent potential for genetic toxicity as they hydrolyse to form identical silicon-containing hydrolysis products, and the other products of hydrolysis are not genotoxic.

Read-across justification

a) Structural similarity

Both substances have the same epoxy side chain attached to a silicon. The only difference is the other groups attached to the silicon which are either methoxy or ethoxy.

b) Structural alert for genetic toxicity

Both substances, and the hydrolysis product, include an epoxy functional group, which may give rise to genetic toxicity in some substances (Benigni et al., 2008).

c) Hydrolysis

3-(2,3-epoxypropoxypropyl]trimethoxysilane and [3-(2,3-epoxypropoxy)propyl]triethoxysilane share a common hydrolysis product, [3-(2,3-epoxypropoxy)propyl]silanetriol.

[3-(2,3-epoxypropoxy)propyl]trimethoxysilane hydrolyses fairly rapidly (hydrolysis half-life of 6.5 hours at pH 7); the read-across substance, [3-(2,3-epoxypropoxy)propyl]triethoxysilane hydrolyses more slowly (hydrolysis half-life 12 – 36 hours (predicted)). It is considered that read-across between these substances is appropriate as hydrolysis is likely to occur during testing and following exposure.

d) Bacterial mutagenicity

Both substances give positive results in bacterial mutagenicity assays.

e) Non-silanol products of hydrolysis are not genotoxic.

The other products of hydrolysis, methanol and ethanol for the registered and read-across substances respectively, are not genotoxic (OECD, 2004a) and OECD (2004b).

f) Other epoxy-functional silicon containing substances

Data are available for five epoxy-functional silicon-containing substances; positive results were obtained in one or more in vitro test for most of the silicon-containing epoxy groups. These data are shown in the supporting report (PFA, 2013aa) attached in Section 13 of the IUCLID 5 dossier.

Benigni et al (2008). The Benigni/Bossa rule base for mutagenicity and carcinogenicity JR Scientific report EUR 23241 EN.

OECD (2004a). SIDS Initial Assessment Report for SIAM 19, Berlin, Germany, 18-20 October 2004, Methanol, CAS 67-56-1.

OECD (2004b). SIDS Initial Assessment Report for SIAM 19, Berlin, Germany, 19-22 October 2004, Ethanol, CAS 64-17-5.

Short description of key information:
In vitro:
Gene mutation (Bacterial reverse mutation assay / Ames test): positive with and without activation in TA97 and TA100 (similar to OECD TG 471) (Microtest Research Ltd., 1988).
In vitro micronucleus: read-across from structural analogue [3-(2,3-epoxypropoxy)propyl]triethoxysilane (CAS 2602-34-8): negative with and without metabolic activation in HepG2 cells (OECD 487) (Lionti et al., 2014).
Mutagenicity in mammalian cells: positive in L5178Y mouse lymphoma L5178Ycells (similar to OECD TG 476) (Litton Bionetics, 1983); negative in Chinese hamster Ovary (CHO) cells (similar to OECD 476, Reliability 4), (Allied Corporation, 1979).
In vivo:
There are three in vivo micronucleus assays available with conflicting results. Therefore an in vivo alkaline comet assay is proposed to clarify the potential for mutagenicity to somatic cells.

Endpoint Conclusion: No study available (further information necessary)

Justification for classification or non-classification

A further in vivo study is proposed to give enough information to come to a conclusion on whether the in vitro positive results are a true indication of genetic toxicity to somatic cells. It is not possible to come to a conclusion about somatic cell mutagenicity until the results of further testing are known.