9-Octadecenoic acid (Z)-, zinc salt, basic

Endpoint summary

Additional information

Ames test Gene Mutation Assay In Vitro

Introduction

The method used is similar or equivalent to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, and meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods

The following bacterial strains were used: Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA-. Each strain was treated with solutions of the test material using the Ames plate incorporation method at five to seven dose levels, in triplicate, both with and without metabolic activation. The dose range for the range-finding test was 1.5 to 5000 μg/plate. The dose levels used for Experiment 2 were 50 to 5000 μg/plate, which was performed on a separate day.

Results The vehicle (tetrahydrofuran) 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 material caused no visible reduction in the growth of the bacterial background lawn at any dose level. A precipitate was observed at and above 1500 μg/plate, however, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose of the test material, either with or without metabolic activation. Conclusion The test material was considered to be non-mutagenic under the conditions of this test.

Mammalian Cell Gene Mutation Assay In Vitro

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.

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 (acetone) 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

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity and the onset of precipitate. Precipitate of the test item was not observed at any of the dose levels in the Mutagenicity Test. The vehicle controls (acetone) had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system. In Experiment 1 (tested dose 4-hour without S9 : 4, 8, 16, 32, 40, 48 ; 4-hour With S9 (2%) : 10, 20, 25, 30, 35, 40) the test item did induce statistically significant dose-related (linear-trend) increases in the mutant frequency at 32 µg/mL (optimum toxicity) in the absence of metabolic activation and at 40 µg/mL (near optimum toxicity) in the presence of metabolic activation.

However the GEF value was not exceeded in the analyzable dose levels. In both exposure groups the response observed was within the acceptable range for a vehicle control and was therefore considered to be of no toxicological significance. It should be noted that the largest increases in mutant frequency were observed to dose levels exhibiting excessive toxicity.

In Experiment 2 (tested dose : 24-hour Without S9 : 8, 16, 24, 32, 40, 48, 56 ; 4-hour with S9 (2%) : 16, 24, 32, 40, 48, 56) the test item did not induce any statistically significant or dose related (linear- trend) increases in the mutant frequency in either exposure group. Optimum toxicity was achieved in the absence of metabolic activation and near to optimum toxicity was achieved in the presence of metabolic activation. The highest increases in mutant frequency can be observed at dose levels exhibiting excessive toxicity.

Conclusion The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.

Mammalian Cell Chromosome Aberration Test In Vitro

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.

Method

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 (acetone) 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. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item was toxic but 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 generally induced approximately 50% mitotic inhibition.

Conclusion

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

Justification for selection of genetic toxicity endpoint
All three studies provided clear evidence of the absence of genotoxic potential.

Short description of key information:
Three Klimisch grade 1, GLP compliant in vitro assays; an in vitro gene mutation assay in bacteria (Ames test); an in vitro gene mutation assay in mammalian cells; an in vitro clastogenicity assay in mammalian cells.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Three in vitro assays designed to detect the mutagenicity and clastogenicity potential of the substance were all negative and therefore there is no justification for classification.