5,7-di-tert-butyl-3-[4-(2-hydroxyethoxy)phenyl]-1-heteropolycycl-2(3H)-one

Endpoint summary

Additional information

IN VITRO BACTERIAL GENE MUTATION

The potential for the test material to cause in vitro bacterial gene mutation was determined in an Ames test performed under GLP conditions and in accordance with the standardised guidelines OECD 471, EU Method B.13/14 and other EPA and Japanese regulatory guidelines.

S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A were exposed to the test material at concentration ranging from 0.15 to 500 µg/plate with and without metabolic activation (S9 mix) using the plate incorporation method.

Under the conditions of the test there were 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. A small, statistically significant increase in TA1537 revertant colony frequency was observed in the presence of S9-mix in the main test at 5 µg/plate. This increase was 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 level (5 µg/plate) were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.48 times the concurrent vehicle control.

The test material was therefore considered to be non-mutagenic under the conditions of the test.

IN VITRO MAMMALIAN CHROMOSOME ABERRATION

The potential for the test material to cause in vitro mammalian chromosome aberration was determined using human lymphocytes in a study performed under GLP conditions and in accordance with the standardised guidelines OECD 473, EU Method B.10, EPA OPPTS 870.5375, 40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test, Japanese Ministry of Economy, Trade and Industry (METI) guidlines and UKDoH Guidelines for the Testing of Chemicals for Mutagenicity as detailed in the UKEMS Recommended Procedures for Basic Mutagenicity Tests (1990).

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. in Experiment 1, 4 hours 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 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours 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 (µg/mL): 4(20)-hour without S9: 2, 4, 8, 12, 16, 20, 24, 32; 4(20)-hour with S9 (2 %): 2, 4, 8, 12, 16, 20, 24, 32; 24-hour without S9: 1, 2, 4, 6, 8, 12, 16, 32; and 4(20)-hour with S9 (1 %): 1, 2, 4, 6, 8, 12, 16, 32.

All vehicle (DMSO) 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 material 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 induced approximately 50 % mitotic inhibition or greater.

The test material was therefore considered to be non-clastogenic under the conditions of the test.

IN VITRO MAMMALIAN GENE MUTATION

The potential for the test material to cause in vitro mammalian gene mutations was determined in a mouse lymphoma study performed under GLP conditions and in accordance with the standardised guidelines OECD 476, EU Method B.17, EPA OPPTS 870.5300 and Japanese METI/MHLW guidelines for testing of new chemical substances.

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 material 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 material at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (2 % S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and was 0.03 to 2 µg/mL in absence of metabolic activation and 0.25 to 6 µg/mL in the presence of metabolic activation in Experiment 1. In Experiment 2 the dose range was 0.125 to 5 µg/mL in the absence of metabolic activation, and 0.25 to 6 µg/mL in the presence of metabolic activation.

The maximum dose levels used in the Mutagenicity Test were limited by test material-induced cytotoxicity. No precipitate of the test material was observed at any dose level 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 material 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.

The test material was therefore considered to be non-mutagenic under the conditions of the test.

All three key studies were performed in line with GLP and accepted standardised guidelines with a high standard of reporting. The studies were assigned a reliability score of 1 in accordance with the criteria outlined by Klimisch (1997) and considered suitable for assessment as an accurate reflection of the test material.

Justification for selection of genetic toxicity endpoint

All studies were considered together. A single study could not be selected as key since they address different types of mutagenicity and are all required in order to sufficiently address the genetic toxicity endpoint.

Short description of key information:

IN VITRO BACTERIAL GENE MUTATION

Ames test: negative in the presence and absence of metabolic activation, OECD 471, EU Method B.13/14, Bowles & Thompson 2012.

IN VITRO MAMMALIAN CHROMOSOME ABERRATION

Chromosome Aberration: negative in the presence and absence of metabolic activation, OECD 473, EU Method B.10, EPA OPPTS 870.5375, Morris 2013

IN VITRO MAMMALIAN GENE MUTATION

Mouse Lymphoma Assay: negative in the presence and absence of metabolic activation, OECD 476, EU Method B.17, EPA OPPTS 798.5300, Brown 2013

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the test material does not require classification for genetic toxicity based on the overall negative response noted in the available in vitro genetic toxicity studies.