Heptasodium bis[4-hydroxy-5-[(2-hydroxy-1-naphthyl)azo]-3-[(2-hydroxy-3-nitro-5-sulphophenyl)azo]naphthalene-2,7-disulphonato(5-)]cobaltate(7-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

non mutagenic

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No data on the test item was available, therefore, a read-across approach was followed using data on Similar Substance 01. Details on the read-across approach are reported in section 13 of IUCLID.

The Ames test was conducted in 2015 using 5 bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (Ames standard plate test and Prival pre-incubation test, according to OECD 471). The modified bacterial reverse mutation test according to Prival facilitates azo reduction and is therefore the most approriate method for the investigation of azo-dyes and diazo compounds. Five strains were used, namely Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The test item was applied at doses between 33 and 5200 μg/plate, both with and without metabolic activation. Precipitation of the test item was found from about 2600 μg/plate onward, with and without S9 mix. Bacteriotoxicity was occasionally noted, depending on the strain and test conditions, from about 1000 μg/plate onward. No mutagenic effects in terms of a relevant increase in the number of his+ or trp+ revertants was seen under experimental conditions. Therefore, Similar Substance 01 is considered not mutagenic.

Further genotoxicity studies were available on Similar Substance 02. Such studies were taken as a further confirmation to the lack of genotoxic potential of the test item. Details on the read-across approach are reported in section 13 of IUCLID.

In particular, Acid Black 076 and Similar Substances 01 and 02 are all cobalt complexes with 2 phenyl-azo-naphtyhl ligands, but Acid Black 076 and Similar Substance 02 show the presence of nitroaromatic (NO₂-Ar) groups. Therefore, in order to confirm the absence of the genotoxic potential of Acid Black 076, the available studies on Similar Substance 02 are reported below:

An AMES test (1994) was carried out on 4 strains, namely S. typhimurium TA 1535, TA 1537, TA 100 and TA98, and mutagenic acivity was evident in TA 1537 and TA 98 strains. Due to the positive result, further studies were carried out to complete the assessment according to the mutagenicity testing strategy as reported in ECHA Guidance Chapter R.7a: Endpoint specific guidance, Version 6.0 – July 2017.

A gene mutation assay in mammalian cells (OECD 476) was available on Similar Substance 02. A pre-test on cytotoxicity was run to select the concentrations. The main assay consisted of an original and a confirmatory experiment, using V79 cells of Chinese hamster. The test was run with S9 mix (highest dose 500 µg/ml, exposure duration of 4 h), and without S9 mix (highest dose 100 µg/ml, exposure duration of 21 h). Toxicity was evaluated in terms of growth inhibition. Mutagenicity was evaluated by comparing the number of mutant colonies in the cultures treated by test substance and in the controls. Toxic effects on cells were noted upon treatment and expression time, with decreasing intensity. However, no mutagenic activity was noted.

A chromosome aberration test (OECD 473) was available on Similar Substance 02, using V79 Chinese hamster cells. In a pre-test on toxicity with 5000 µg/ml as highest tested concentration, top concentrations for the main experiment were chosen based on growth inhibition effects on cells in treated cultures. The main study was run a) without S9 mix, highest dose 4000 µg/ml, exposure period of 18 h, preparation interval at 18 h; and b) with S9 mix, highest dose 1000 µg/ml, exposure period of 4 h, preparation interval at 18 h. In all cases, no mutagenic effects were reported in terms of reduction of the mitotic indices, increase in the number of cells with chromosomal aberrations or increase in the frequency of polyploid metaphases.

Overall, Similar Substance 02 was proved to be non-genotoxic, despite the positive outcome in the Ames assay, as in further in vitro studies, i.e. a gene mutation assay and a chromosomal aberration assay, the test item was negative for genotoxicity. Based on these findings, even in case a positive response in Ames assay on Acid Black 076 were to become available, a genotoxic potential would not likely be confirmed by further studies.

Justification for classification or non-classification

According to the CLP Regulation (EC) no. 1272/2008, for the purpose of classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- 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 or;

- category 2: substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

Based on Chapter R.7a (2017): Endpoint specific guidance, at the REACH Annex VII level, the absence of genotoxicity in a study on bacteria (Ames assay) allows to conclude that the substance is not genotoxic.

Relying on a read across approach with Similar Substance 01, the test item is not classified for genetic toxicity within the CLP Regulation (EC) no. 1272/2008. A further confirmation to this conclusion was derived from the assessment on Similar Substance 02.

According to the CLP Regulation (EC) 1272/2008, Annex I, Part 3, substances which cause concern for humans, owing to the possibility that they may induce heritable mutations in the germ cells of humans, are classified in Category 2. This classification is based on positive evidence obtained in:

- somatic cell mutagenicity tests in vivo, in mammals; or

- other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

Note: substances which are positive in in vitro mammalian mutagenicity assays, and which also show chemical structure activity relationship to known germ cell mutagens, shall be considered for classification as Category 2 mutagens.

In vitro mutagenicity tests are the following:

- in vitro mammalian chromosome aberration test;

- in vitro mammalian cell gene mutation test;

- bacterial reverse mutation tests.

Based on responses obtained in available experimental studies on Similar Substance 02, namely:

- in vitro bacterial reverse mutation test: positive;

- in vitro mammalian chromosome aberration test: negative; and

- in vitro mammalian cell gene mutation test: negative;

Therefore, despite the mutagenic potential in bacteria, different studies considered of higher significance, as conducted on mammalian cells, gave no indication of mutagenicity up to cytotoxic levels.