Trisodium, bis [2-((E)-(4-hydroxy-2-oxido-3-((E)-(5-sulfonatonaphthalen-1-yl)diazenyl)phenyl)diazenyl)benzoate] chromate(3-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagenic

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

AMES Plate Incorporation: potential to induce point mutations by base pair substitutions or frameshifts was evaluated in an experimental study comparable to OECD Guideline 471 (1983). Salmonella typhimurium strains TA 97, TA 98, TA 100 and TA 1535 were exposed to 5 test item concentrations (50, 100, 500, 1000 and 5000 µg/plate) in triplicate both with and without S9 metabolic activation. A negative control (DMSO only) and positive controls (TA 97: benzo(a)pyrene (with S-9) and 4 -nitro-ortho-phenylenediamine (without S-9); TA 98: 3,3'-dichlorobenzidine; TA 100: 3-methylcholanthrene; TA 1535: cyclophosphamide) were tested in parallel. A result was considered positive (mutagenic) if two consecutive doses had p-value < 0.05 and a dose-response relationship was evident.

AMES Pre-Incubation: potential to induce point mutations by base pair substitutions or frameshifts was evaluated in an experimental study according to the OECD Guideline 471 (1983) and the EU Method B.14 (1984) adapted for azo dyes. Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 were exposed to 5 test item concentrations (10, 100, 333.3, 1000 and 5000 µg/plate) in triplicate both with and without S9 metabolic activation, and was incubated for 30 minutes at 30 °C before plating. A solvent control (aqua bidest.) and positive controls (without S9: sodium azide (TA 1535 and TA 100) and 4 -nitro-o-phenylene-diamine (TA 1537 and TA 98); with S9: Congo Red) were tested in parallel. A result was considered positive (mutagenic) if either a significant dose-related increase in the number of revertants (a mean reversion count of two (TA 100) or three (TA 1535, TA 1537 and TA 98) times higher than the known spontaneous reversion rates) or a significant and reproducible increase for at least one test concentration was induced.

Chromosome Aberration Assay: evaluated in an experimental study using Chinese hamster ovary (CHO) cells, comparable to the OECD Guideline 473 (1983). CHO cell cultures in the exponential growth phase were exposed to test item concentrations of 25, 50, 250, 500 and 2500 µg/ml in DMSO for 24 hours in triplicate, both with and without S9 metabolic activation (initial 2 hours), and with colchicine added to the flasks in the last 3 hours. A solvent control (DMSO) and positive controls (without S9: methyl methanesulfonate; with S9: cyclophosphamide) were tested in parallel. Cells were scored for gaps, breaks, fragments, reunion figures and aberrations, and scored for chromosome aberration frequencies and mitotic index.

Mouse Lymphoma Assay: gene mutation, clastogenic and aneugenic effects via 5-trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells were evaluated in a fluctuation method study according to the OECD Guideline 490 (2015). Cell cultures in complete medium (5 %) and test item concentrations (39.1, 78.1, 156, 313, 625, 833, 1040 and 1250 µg/ml) in RPMI minimal medium were incubated at 37 °C for 3 hours in a 5 % CO2 atmosphere both with and without metabolic activation, alongside a solvent control and a positive control (benzo(a)pyrene). Cells were then plated to test for 5-trifluorothymidine resistance and for viability. A positive (mutagenic) result was considered a statistically significant, dose-dependent increase in mutant frequencies, and higher induced mutation frequency (IMF) above Global Evaluation Factor (GEF) values, outside of historical control range for negative controls.

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, no experimental studies demonstrated positive results; the evidence of the absence of mutagenic potential was demonstrated in three separate tests (an AMES test using the Plate Incubation method and a modified AMES test using a Pre-Incubation method designed for Azo Dyes, which both used bacterial cell lines in vitro; as well as a Chromosome Aberration Assay which utilised mammalian cells in vitro). The Mouse Lymphoma Assay provided equivocal results. Considering that the substance does not evoke gene mutation or chromosomal aberrations in bacteria and mammalian cells respectively, it could be expected that the substance is not mutagenic. Therefore, no classification for genotoxicity is warranted according to the CLP Regulation (EC 1272/2008).