Potassium methylsilanetriolate

Endpoint summary

Additional information

Reliable information is available for potassium methylsilanetriolate for bacterial mutagenicity and mutagenicity to mammalian cells. The results of these assays were negative. Cytogenicity data are available for the related substance trimethoxy(methyl)silane, (CAS 1185 -55 -3): positive results were obtained in in vitro tests on mammalian cells (cytogenicity and mutagenicity). These results were not confirmed in an in vivo micronucleus assay, and are thought to be misleading, so a mammalian mutagenicity study has been conducted on the registered substance. The result of this study was negative, supporting the conclusion that the positive mammalian gene cell mutation result for trimethoxy(methyl)silane is misleading, and an in vitro cytogenicity assay on the registered substance is being conducted. Depending on the outcome of the in vitro cytogenicity study, read across from trimethoxy(methyl)silane may be reconsidered.

Read-across justification

Non-testing methods including read-across from surrogate substances are able to provide information on mutagenic toxicity (REACH Guidance part 07a, R.7.7.3). In the case of genetic toxicity the presence or absence of functional groups that are known to be related to genetic toxicity is considered important, as the presence or absence of reactive groups and molecular substructures is associated with mutagenic and carcinogenic properties of chemicals (Benigni et. al., 2008). Consideration is therefore given to the structural similarity, particularly presence or absence of structural alerts for genetic toxicity, when selecting surrogate substances for genetic toxicity endpoints. Additional information is given in a supporting report (PFA, 2013aa) attached in Section 13 of the IUCLID 5 dossier.

Analogue approach justification

(a) Structural similarity

Trimethoxy(methyl)silane is a close structural analogue of potassium methylsilanetriolate, which hydrolyses rapidly (hydrolysis half-life 2.2 h at 20°C) giving methylsilanetriol and methanol. The registered substance dissociates under aqueous conditions producing methylsilanetriolate and free potassium ions. Under comparable conditions of concentration and pH, methylsilanetriolate is equivalent to the parent acid methylsilanetriol. Potassium ions are ubiquitous in nature and important in metabolism and are not of concern for genetic toxicity in vivo.

Methanol has been tested in vitro in bacterial and mammalian mutagenicity assays and in micronucleus and chromosome aberration assays. The majority of the results were negative (OECD, 2004a). In the ECHA disseminated dossier for methanol, the conclusions of all the key in vitro studies and the weight of evidence of the in vivo assays are negative. Further discussion of read-across can be found in the data matrix attached to Section 13 of the IUCLID.

(b) Structural alerts for genotoxicity

Potassium methylsilanetriolate and trimethoxy(methyl)silane do not include structural alerts for genotoxicity (Benigni et al, 2008).

(c) Lack of genetic toxicity (other than pH effects) of non-silanol products of hydrolysis.

Potassium methylsilanetriolate has been tested in a valid and reliable study conducted in accordance with OECD 471 and under GLP (Laboratory of Pharmacology and Toxicology, 2002). No mutagenic effect was observed for the test substance tested up to cytotoxic concentration in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in a plate incorporation study with and without metabolic activation. The result was confirmed in a pre-incubation study. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

The surrogate substance trimethoxy(methyl)silane has been tested in a valid study conducted according to OECD 473 and in compliance with GLP (Bioreliance, 2004). The test substance induced a statistically significant dose related increase in the number of structural aberrations in Chinese hamster ovary (CHO-K1) cells in the presence of activation. No test-substance related genotoxicity was observed in the absence of metabolic activation. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance is positive for the induction of chromosome aberrations in the presence of activation under the conditions of the study.

Evidence for mutagenicity to mammalian cells is available for the surrogate substance trimethoxy(methyl)silane, which has been tested using mouse lymphoma L5178Y cells in a valid study conducted according to OECD 476 (1997) and in compliance with GLP (TNO, 2002). The results without metabolic activation were considered ambiguous: an increase of the mutant frequency was observed at a concentration of 26 µg/ml test substance, but the result would not be considered a positive response if assessed using the criteria set out in the guideline for in vitro mammalian cell gene mutation assays using the thymidine kinase gene (OECD 490, 2015). In the presence of metabolic activation, a dose-related increase in the mutant frequency was observed at concentrations above 560 µg/ml. Only the highest concentration induced an increase in mutant frequency which exceeded the global evaluation factor for the microwell method of 160 E-06. A relative increase in smaller colonies relative to larger colonies was observed at the two highest concentrations. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the substance is positive for mutagenicity to mammalian cells under the conditions of the test.

The new OECD guideline for mutagenicity at the thymidine kinase locus (OECD 490), and a new version of OECD 476 for the test for mutation using the hprt and xprt genes, have been introduced in an attempt to reduce false (or misleading) positive results, where a false positive is a positive result in an in vitro study which does not correlate with results which would be obtained from in vivo mutagenicity or carcinogenicity studies. The problem of false positives from in vitro testing has been under consideration for a long time, and is discussed in the literature (for example, Kirkland et al., 2007; Kirkland et al., 2005). Cytotoxicity can make in vitro data difficult to interpret, and accurate measurement of cytotoxicity in genetic toxicity studies is important (Fowler et al., 2012). As it is thought that the positive result reported for trimethoxy(methyl)silane is a misleading positive result and does not reflect potential for mutagenicity in vivo, the registered substance potassium methylsilanetriolate has been tested for mutagenicity to mammalian cells in a study conducted in accordance with OECD 476 (1997) and OECD 490. The result of this study, which is described below (Eurofins, 2015), was negative, which suggests that the positive result obtained with trimethoxy(methyl)silane was misleading. An in vitro cytogenicity study is being conducted according to OECD 473 with the registered substance. When the result of this study is available, depending on the outcome of the study, read across from trimethoxy(methyl)silane may be reconsidered..

Potassium methylsilanetriolate has been tested for mutagenicity to mammalian cells in a study conducted in accordance with OECD 476 (1997) and OECD 490 (In vitro Mammalian Cell Gene Mutation Test, 2015) and in compliance with GLP (Eurofins, 2015). No evidence of test substance induced increase in mutant frequency (in excess of the global evaluation frequency) or incidence of small colonies was observed when tested with and without metabolic activation up to limit concentration (10 mM) in mouse lymphoma L5178Y cells. Solvent and positive controls were included and gave expected results. The initial experiment (exposure period 4 hours) was repeated (exposure period 4 hours with metabolic activation, 24 hours without metabolic activation). It is concluded that the test substance is negative for the induction of mutations under the conditions of the test.

The potential for genetic toxicity which was observed in the mammalian cytogenicity study on the surrogate substance was not confirmed when trimethoxy(methyl)silane was tested in a valid in vivo mouse micronucleus study conducted according to OECD 474 and in compliance with GLP (Research Toxicology Centre, 2002). No increase in the incidence of micronucleated PCE was observed resulting from exposure to the test substance by oral gavage up to limit concentrations. Appropriate positive and vehicle controls were included and gave expected results. It was concluded that the test substance is negative for the induction of micronuclei under the conditions of the test.

In the light of the negative results in bacterial and mammalian in vitro mutagenicity studies with potassium methylsilanetriolate, and the negative result from the in vivo micronucleus study on trimethoxy(methyl)silane, it is concluded that the registered substance is not mutagenic to germ cells.

References

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

Fowler, P. et al., (2012). Reduction of misleading (“false”) positive results in mammalian cell genotoxicity assays. II. Importance of accurate toxicity measurement. Mut Res; 747(1):104-117.

Kirkland D., Aardema M., Henderson L., Müller L. (2005). Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens I. Sensitivity, specificity and relative predictivity. Mut Res;584(1-2):1-256.

Kirkland, D. et al., (2007). How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: Report of an ECVAM Workshop. Mutat Res.30;628(1):31-55.

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

Justification for selection of genetic toxicity endpoint
Conclusion based on the following assays: Bacterial reverse mutation assay (Ames test) and Mammalian cell gene mutation assay using the target substance; assays on the surrogate substance: In vitro mammalian chromosome aberration test; In vivo micronucleus assay. The data were conducted according to appropriate OECD guidelines and in compliance with GLP.

Short description of key information:
In vitro
Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without activation in all strains tested (OECD TG 471) (Laboratory of Pharmacology and Toxicology, 2002).
Cytogenicity in mammalian cells: read-across from analogous substance trimethoxy(methyl)silane: positive in CHO cells (with metabolic activation), negative without metabolic activation (OECD TG 473) (Bioreliance, 2004).
Cytogenicity in mammalian cells: a study is being conducted with the registered substance (OECD TG 473).
Mammalian gene cell mutation: negative with and without metabolic activation in mouse L5178Y cells (OECD TG 476 (1997) and OECD TG 490 (2015)) (Eurofins, 2015).
In vivo:
Micronucleus assay oral study in mouse: the related substance trimethoxy(methyl)silane was negative for induction of micronuclei (OECD TG 474) (Research Toxicology Centre, 2002).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available information for the substance indicates that when tested in vitro, potassium methylsilanetriolate (CAS 31795-24-1) did not induce mutations in bacterial or mammalian cells. The analogous substance trimethoxy(methyl)silane CAS number (CAS 1185-55-3), although producing an increase in the percentage of cells with aberrations in the presence of activation in an in vitro cytogenicity study, did not cause induction of micronuclei in vivo in the mouse micronucleus assay. Therefore it is considered that classification for mutagenicity is not required.