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Bacterial Mutation Assay

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 (TSCA) OPPTS harmonised guidelines.

Methods. Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrAwere treated with the test item,OS48604M, using the Ames plate incorporation method at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 0.5 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. The experiment was repeated on a separate day using an amended dose range (ranging from 0.5 to 1500 µg/plate), depending on strain type and presence or absence of S9-mix. Fresh cultures of the bacterial strains and fresh test item formulations were employed.

Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

Results. The vehicle (dimethyl sulphoxide) 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 test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains initially from 500 µg/plate both with and without metabolic activation (S9-mix). The test item was tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit depending on bacterial strain type and presence or absence of S9-mix. A test item film (opaque in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No toxicologically 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 either the range-finding or main tests. Small, statistically significant increases in revertant colony frequency were observed in the range-finding test at 150 µg/plate (TA1537 in the absence of S9-mix) and 1.5, 5, 15 and 150 µg/plate (TA98 in the presence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.9 times the concurrent vehicle controls.

Conclusion. The test item,OS48604M, was considered to be non-mutagenic under the conditions of this test.

In Vitro Chromosome Aberration Test

Introduction. This report describes the results of anin vitrostudy 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 (Scott et al, 1990). The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008 and the US EPA OPPTS 870.5375 Guideline and is acceptable to the Japanese New Chemical Substance Law (METI).

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, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising 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 and were as follows:

Experiment

Group

Final concentration ofTest Item(µg/ml)

1

4(20)-hour without S9

6.25, 12.5, 25, 50, 100, 125

4(20)-hour with S9 (2%)

25, 50, 100, 125, 150, 200

2

24-hour without S9

6.25, 12.5, 25, 50, 100, 150

4(20)-hour with S9 (1%)

6.25, 12.5, 25, 50, 100, 150

 

Results. All vehicle (dimethyl sulphoxide) control groups 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 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 generally included a dose level that induced approximately 50% mitotic inhibition.

Conclusion. The test item was considered to be non-clastogenic to human lymphocytes in vitro. In Vitro mammalian Cell 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 VitroMammalian 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 acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

Methods. 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 was selected following the results of a preliminary toxicity test, and for Experiment 1 was 2.5 to 80 µg/ml in both the absence and presence of metabolic activation. In Experiment 2 the dose range was 1.25 to 80 µg/ml in the absence of metabolic activation, and 2.5 to 100 µg/ml in the presence of metabolic activation.

Results. The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. No precipitate of test item was observed at any of the dose levels in the Mutagenicity Test. 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 metabolising system.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion. The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.


Justification for selection of genetic toxicity endpoint
Three in vitro assays in bacteria and mammalian cells were negative and showed no evidence of genotoxic potential.

Short description of key information:
Bacterial gene mutation assay (Ames test) in vitro
Mammalian cell mutation assay in vitro
Mammalian cell chromosome aberration test in vitro

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification