Cobaltate(3-), bis[6-amino-5-[(2-hydroxy-3,5-dinitrophenyl)azo]-1-naphthalenesulfonato(3-)]-, sodium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES assay: negative

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 Acid Geen 104 was available. Therefore, a read across approach was followed, using data on Similar Substance 01. In particular, Acid Green 104 and Similar Substance 01 are cobalt complexes with 2 phenyl-azo-naphtyl ligands. Details on the read-across process are reported in section 13.

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 preincubation 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. Test substance was applied at doses of 33 to 5200 μg/plate, both with and without metabolic activation.

Precipitation of the test substance 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 was taken as not mutagenic.

Further studies were available on Similar Substance 02, i.e. a cobalt complex with the same ligands as those in Acid Green 104, with minor differences in functional groups. Such studies were taken as a further confirmation to the lack of genotoxic potential of Acid Green 104.

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 4.0 – July 2015.

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. Such experiments were run as follows:

- with S9 mix, highest dose 500 µg/ml, exposure duration of 4 h,

- 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, by using V79 Chinese hamster cells. Upon 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 as follows:

- without S9 mix, highest dose 4000 µg/ml, exposure period of 18 h, preparation interval at 18 h

- 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. Indeed, further in vitro studies, i.e. a gene mutation assay and a chromosomal aberration assay, gave negative responses. On these bases, even in case a positive response in Ames assay on Acid Green 104 were available, a genotoxic potential would not likely be confirmed by further studies.

Justification for classification or non-classification

According to the CLP Regulation (EC 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 (2015): 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, Acid Green 104 is not classified for genetic toxicity within the CLP Regulation (EC 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 inin vitromammalian 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

-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.