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BACTERIAL MUTATION TEST

Introduction. The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline- Bacterial Reverse Mutation Test.

Methods. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of Experiment 1, and ranged between 5 and 5000 µg/plate, depending on presence or absence of S9-mix.

Up to seven test item dose levels were selected in Experiment 2 in arder to achieve both four non-toxic dose levels and the toxic limit of the test item.

Results. The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. Results from the first mutation test showed that the test item induced toxicity to all of the bacterial tester strains, at 5000 µg/plate in the absence and presence of S9-mix respectively. Results from the second mutation test were identical to Experiment 1 with weakened bacterial background lawns at 5000 µg/plate in the absence and presence of S9-mix respectively. A test item precipitate was observed on the plates at the maximum doses tested in both the presence or absence of S9-mix.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the first mutation test. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2.

Conclusion. 0S11211AP was considered to be non-mutagenic under the conditions of this test.

MAMMALIAN CELL GENE MUTATION TEST

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.MethodTwo 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.ResultsThe 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.

IN VITRO MICRONUCLEUS ASSAY

Introduction.This study describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.

Methods.Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions were used for the study. experiment 1 used a 4 -hour exposure in the presence and absence of a standard metabolizing system (S9, at a 2% final concentration). Experiment 2 used a 24 -hour exposure in the absence of metabolic activation and was performed concurrently with Experiment 1. At the end of the exposure period, the cell cultures were washed and then incubated for a further 28 hours in the presence of cytochalasin B. The dose levels were selected using the data from the preliminary toxicity test.

Results.All vehicle (dimethyl sulphoxide) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.The positive control items induced statistically significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test method was operating as expected. The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with micronuclei, in either of the two experiments, using a dose range that included a dose level that approached the 50% redcution in CBPI.

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


Short description of key information:
A modern OECD 473 compliant, GLP bacterial mutation assay, Klimisch grade 1.
A modern OECD 476 compliant, GLP mammalian cell gene mutation assay, Klimisch grade 1.
A modern OECD 487 compliant, GLP mammalian cell gene mutation assay, Klimisch grade 1.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

All the studies are negative for mutagenicity and no classification is required.