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Bacterial mutation (Ames) test

The test substance was evaluated in the Ames/Salmonella-E.coli Liquid Pre-incubation Assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of salmonella typhimurium (TA1535, TA1537, TA1538, TA98 and TA100), and at the tryptophan locus in one Escherichia coli tester strain (WP2uvrA-), in the presence and absence of an exogenous metabolic activation system (S9). Toxicity of the test item was first evaluated in a pre-screen by treating duplicate cultures of strains TA1538, TA100 and WP2uvrA- with the test article at doses of 50, 167, 500, 1670 and 5000 ug/plate in the absence of S9. Results of the pre-screen indicated the test item was not toxic to tester strain WP2uvrA- at doses of 50, 167 and 500 ug/plate. Inhibited growth (characterised by the absence of a confluent bacterial lawn and/or the presence of pindot colonies) or complete toxicity was observed in both Salmonella strains at all doses evaluated, and in strain WP2uvrA- at dose =>1670ug/plate. In addition, the test article precipitated from solution at doses => 1670 ug/plate.

Based upon these findings, the test item was evaluated in triplicate cultures in all five strains of Salmonella at doses of 0.167, 0.5, 1.67, 5, 16.7 and 50 ug/plate, and in strain WP2uvrA- at dose of 16.7, 50, 167, 500, 1670 and 5000 ug/plate, in the presence and absence of S9. Six dose levels of the test substance were evaluated with and without S9 in the event of unacceptable toxicity and/or insolubility at the highest dose levels evaluated in the mutation assay. The S9 mixture included 6% (v/v) Aroclor 1254 -induced male Sprague-Dawley rat liver homogenate with the appropriate buffer and cofactors. The test article again precipitated from solution =>1670 ug/plate. Inhibited growth again was observed in all Salmonella strains at doses of 5, 16.7, and/or 50 ug/plate with and/or without S9, and in tester strain WP2uvrA- at doses of 1670 and/or 5000 ug/plate with and/or without S9. Revertant frequencies for all doses, in all tester strains with and without S9 approximated or were less than those observed in the concurrent negative control cultures.

The test substance as re-evaluated in the confirmatory assay under identical conditions, and similar results were observed. The test substance again precipitated from solution at doses =>1670 ug/plate. Inhibited growth again was observed in all Salmonella strains at doses of 5, 16.7 and/or 50 ug/plate with and/or without S9, and in tester strains WP2uvrA- at doses of 1670 and/or 5000 ug/plate with and/or without S9. Revertant frequencies for all dose of the test substance in all tester strains with and without S9 approximated or were less than control value. All positive and negative control values in both assays were within acceptable limits. Therefore, the results were negative in the Ames/Salmonella-E.coli Liquid Pre-incubation Assay under the conditions, and according to the criteria, of the test protocol.

Chromosome aberration test

Introduction: This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations.

Methods : Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. in Experiment 1 , 4 -hour in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1 % final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels used in the main experiments were selected using data from the preliminary toxicity test.

Results: All vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item was moderately toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that achieved or exceeded optimum toxicity in some exposure groups.

Conclusion. The test item was considered to be non-clastogenic to human lymphocytes in vitro.

Mammalian Cell Gene Mutation Assay

Introduction : The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

Method : Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24 hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The maximum dose level used in the main test was limited by test item induced toxicity.

Results : A precipitate of test item was observed at and above 320 ug/mL. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.

Conclusion : The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.


Justification for selection of genetic toxicity endpoint
GLP quality studies performed according to the latest OECD test guidelines.

Short description of key information:
A GLP bacterial reverse mutation assay (Ames test) according to OECD 471. A GLP chromosome aberration test in human lymphocytes according to OECD 473. A GLP gene mutation assay in mouse lymphoma L5178Y cells TK locus according to OECD 476.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for mutagenicity according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available data, no additional classification for mutagenicity is proposed according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).