Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial mutagenicity: does not induce point mutations in the Ames test

Mammalian gene mutation: does not induce micronuclei in human lymphocytes

Mammalian mutagenicity: does not induce point mutations in the HPRT assay

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro bacterial reverse mutation (Ames) assay (OECD Guideline 471, 1997): The potential for the test item to induce gene mutations was evaluated using tester strains of Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Escherichia coli WP2 uvrA, both in the presence and absence of metabolic activation (S9), according to the both the plate incorporation and pre-incubation methods, at 313-5000 µg/plate. No toxicity, precipitation or relevant increses in reventant numbers were observed at any dose level, with any tester strain, neither in the absence nor presence of S9 metabolic activation, in either test. Therefore, the test item does not induce reverse mutations in bacterial cells under the reported experimental conditions.

In vitro micronucleus assay on human lymphocytes (OECD Guideline 487, 2016): The potential for the test item to induce micronuclei in human lymphocytes in vitro was evaluated in (1) a short treatment of 3 hours in the absence of metabolic activation, (2) in the presence of metabolic activation, harvested after approximately 32 hours, and (3) a long term (continuous) treatment in the absence of metabolic activation until harvest at approximately 31 hours. In both experiment (1) and (2), no relevant toxicity was observed at any dose level, in the absence or presence of metabolic activation, while a dose related cytotoxic effect was noticed. Based on the results obtained in experiment (3), the concentrations for the scoring of micronuclei were selected. One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells. No statistically significant increase in the incidence of micronucleated cells over the control value was observed at any dose level, in any treatment series. Therefore, the test item does not induce micronuclei in human lymphocytes in vitro, under the reported experimental conditions.

In vitro mammalian cell gene mutation assay (OECD Guideline 476, 2016): The in vitro gene mutation potential of the test item was evaluated in an HPRT assay. Chinese hamster lung fibroblasts (V79) were exposed to test item (128 to 5000 µg/mL) with and without metabolic activation for 3 hours. Up to the highest dose tested, no relevant increases in mutant frequencies, precipitation or cytotoxicity was observed either with or without metabolic activation. The test item does not induce point mutations under the experimental conditions at the HPRT locus in Chinese hamster V79 cells, and is considered non-mutagenic in this HPRT assay.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), the term ‘mutation’ refers a permanent change in the amount or structure of the genetic material in a cell, both to heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known (including specific base pair changes and chromosomal translocations). The terms ‘mutagenic’ and ‘mutagen’ are used for agents giving rise to an increased occurrence of mutations in populations of cells and/or organisms. For the purpose of the classification for germ cell mutagenicity, substances may be allocated to one of two categories:

- Category 1: substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans. Further sub-classification can be made into the following:

- Sub-category 1A: in the presence of positive evidence from human epidemiological studies; or

- Sub-category 1B: in the presence of positive result(s) from (i) in vivo heritable germ cell mutagenicity tests in mammals; (ii) in vivo somatic cell mutagenicity tests in mammals, in combination with some evidence that the substance has the potential to cause germ cells mutations (derived from mutagenicity/genotoxicity tests in germ cells in vivo, or by demonstrating the ability of the substance or its metabolite(s) to interact with the genetic material of germ cells); or (iii) tests showing mutagenic effects in the germ cells of humans, without demonstration of transmission to progeny; for example, an increase in the frequency of aneuploidy in sperm cells of exposed people.

- Category 2: substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans. Classification in Category 2 is based on positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from either (i) somatic cell mutagenicity tests in vivo, in mammals; or (ii) other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

According to Table R.7.7-5 of the ECHA Guidance R7.a, in the presence of a positive result in a bacterial Gene Mutation test, further in vitro testing should be performed on mammalian cells before classification. This is because the unique positive response observed in a bacterial gene mutation test could be due to the specific bacterial metabolism of a test item. Provided the further in vitro mutagenicity testing on mammalian cells provide negative results, the test item can be classified as non-mutagenic and no in vivo tests would normally be required. However, in the presence of a positive in vitro test on mammalian cells, further in vivo testing would be required to determine the true mutagenicity of the test item.

Regarding the test item in question, evidence for the absence of mutagenic potential was demonstrated in three separate tests: an AMES test using both the plate incorporation method and pre-incubation method (specific to azo-dyes), which used bacterial cell lines in vitro; an in vitro Chinese hamster V79 cell mutagenicity assay; and an in vitro Micronucleus Assay on human lymphocytes. Considering the negative test results, the substance is considered not mutagenic. Therefore, no classification for genotoxicity is warranted according to the CLP Regulation (EC 1272/2008).