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Table 7.6/1: Summary of genotoxicity tests

 

Test n°

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration

Statement

(Envigo, 2016)

Ames Test

(OECD 471)

K, rel. 1

Gene mutation

TA 1535,

TA 1537,

TA 98,

TA 100

E.coli WP2 uvrA

-S9

+S9

Up to cytotoxic concentration

-S9 : weak mutagenic

+S9 : weak mutagenic

2

(Envigo, 2016)

L5178YTK+/- MLA test (OECD 490)

K, rel. 1

Gene mutation

L5178Y tk+/-(3.7.2C) mouse lymphoma cells

-S9

+S9

Up to cytotoxic concentration

-S9 : non mutagenic

+S9 : non mutagenic

3

(Envigo, 2016)

MNT vitro

(OECD 487)

K, rel.1

Chromosomal aberration

Human lymphocytes

-S9

+S9

Up to cytotoxic concentration

-S9 and +S9 Not clastogenic

-S9 and +S9 Not aneugenic

Gene mutation Assays (Tests n° 1 and 2):

- A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD test guideline No 471 with the substance (See Table 7.6/1). In two experiments (repeated), small but toxicologically significant increases in the revertant colony frequency of TA100, TA1535 (absence and presence of S9-mix) and WP2uvrA (absence of S9-mix only) were observed at the upper test item dose levels. In the second experiment, a dose-related response in WP2uvrA frequency between 1000 and 5000 μg/plate in the absence of S9-mix was noted. The increases over the concurrent vehicle control for this strain were 2.5 times at 1500 μg/plate, 4.1 times at 3000 μg/plate and 4.7 times at 5000 μg/plate (mean numbers). Furthermore, the individual revertant colony counts at 3000 and 5000 μg/plate exceeded the in-house historical control range for the bacterial tester strain. Any excursions outside the maxima ranges, particularly when reproducibility is apparent (5000 μg/plate in Experiments 1 and 2), must be considered to be evidence of a biological response. There was no evidence of a response with WP2uvrA dosed in the presence of S9-mix, this lack of activity may be due to partial inhibition following metabolic activation. Smaller responses were also noted to TA100 (1.5 times at 5000 μg/plate in the absence of S9-mix and 1.4 times at 5000 μg/plate in the presence of S9-mix) ) and TA1535 (1.6 times at 1000 μg/plate and 3.0 times at 1500 μg/plate in the absence of S9-mix. 1.8 times at 500 and 1500 μg/plate, 2.0 at 1000 μg/plate - in the presence of S9-mix), although there was a slight shift in toxicity to TA1535 in the second mutation test with weakened lawns initially noted at 1500 μg/plate compared to 5000 μg/plate in the first mutation test. A small increase in TA98 revertant colony frequency was also noted at and above 3000 μg/plate (absence of S9-mix) in the second mutation test, however this increase was non-reproducible over the two experiments. The test item was cytotoxic in Salmonella strains TA1535 and TA1537 at 5000 μg/plate and from 1500 μg/plate in Experiments 1 and 2, respectively, in both the presence and absence of metabolic activation (S9-mix). No toxicity was noted to Salmonella strains TA100 and TA98 and Escherichia coli strain WP2uvrA.

The Ames test was a weak positive response (TA100, TA1535 and E.coli) and the response was attenuated to a certain extent by the presence of S9 which suggests that the test item is a direct acting mutagen that may be partly inactivated by S9. The substance was also toxic to the Salmonella bacteria TA1535 but in this case the toxicity was much less reduced by S9.

- Considering the weak positive results observed in the Ames assay, further testing was conducted to investigate the potential for gene mutations in mammalian cells in vitro. An inability to produce gene mutation was observed in mammalian cells using an in vitro gene mutation assay in L5178Y tk+/-(3.7.2C) mouse lymphoma cells (L5178Y TK+/- MLA test) (Test n°2). None of the dose levels up to the cytotoxicity limit, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat experiments whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. Therefore the substance are considered as negative for inducing gene mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non-activation conditions used in this assay.

Therefore this result does not confirm the results of the Ames test but it demonstrates the non-mutagenic effect of the substance to mammalian cells.

 

Chromosomal aberration (Test n°3)

Based on the contradictory results between weak mutagenic potential observed in the Bacterial Reverse mutation Assay and no mutagenicity observed in mammalian cells in vitro,further testing was conducted to investigate the potential for chromosome aberrations in vitro. The clastogenic potential of the substance was determined using an in vitro micronucleus test in human lymphocytes (OECD 487), which measures the potential of a substance to increase the incidence of micronuclei in cultured human lymphocytes. The test item was toxic to human lymphocytes but it did not induce any statistically significant increases in the frequency of cells with micronuclei, in any of the exposure groups, using a dose range that included a dose level that induced a sufficient reduction in the cytokinesis block proliferation index (CBPI). Both positive and negative controls validated the sensitivity of the assay.

Under the conditions of this test, the test substance is therefore considered as non-clastogenic and non-aneugenic in vitro.

 

The Ames response was weak and was attenuated or inactivated to a certain extent by metabolic activation. A TIMES (Q)SAR analysis (Reliability 2) revealed no alert for mutagenicity of the five main constituents of this substance and the predictions for all the assessed constituents fall within the applicability domain (See the attached QMRF and QPRFdocuments for CAS no. 25966 -79, 25966 -79 -4 or 68916 -71 -2, 28387 -62 -4, 25966 -81 -8 and 79768 -26 -6). Furthermore, the substance was negative in two mammalian cell assays (MLA and MNT in vitro), which can detect point mutations (the same as in the Ames test), chromosome aberrations and aneuploidy. The possibility exists that the weak positive results observed in the Ames assay could be microbial specific and of no relevance to humans. The attenuation of the weak activity by the presence of mammalian enzymes also indicates that mammalian metabolism can inactivate the weak mutagenic activity to bacteria. Therefore, the substance is predicted to be negative in any follow-up in vivo assay and thus no additional testing has been undertaken and no testing proposal will be submitted.

Justification for selection of genetic toxicity endpoint
No study was selected, since all in vitro studies performed on the substance itself were negative and of high quality. A weak positive response was observed in a bacterial mutation assay but when tested in mammalian cells for mutagenicity and clastogenicity the results were negative. Furthermore, the activity in the bacterial assay was attenuated by the presence of mammalian metabolic enzymes, which suggests that a negative result can be predicted for any follow-up test in vivo.

Short description of key information:
- Ames Test (OECD 471, GLP, K, rel. 1): weak mutagenic up to cytotoxic concentrations in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.
- L5178Y/MLA Mammalian Cell Gene Mutation Assay (OECD 490, GLP, K, rel. 1): non mutagenic.
- Micronucleus test in vitro (OECD 487, GLP, K, Rel.1): not clastogenic and not aneugenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Harmonized classification:

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

Self classification:

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