reaction mass of: pentasodium bis[6-anilino-3,5'-disulfonatonaphthalene-2-azobenzene-1,2'-diolato]cobaltate(III);tetrasodium [6-anilino-3,5'-disulfonatonaphthalene-2-azobenzene-1,2'-diolato][6-anilino-5'-sulfamoyl-3-sulfonatonaphthalene-2-azobenzene-1,2'-diolato]cobaltate(III);trisodium bis[6-anilino-5'-sulfamoyl-3-sulfonatonaphthalene-2-azobenzene-1,2'-diolato]cobaltate(III)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro Bacterial Reverse Mutation, OECD 471 (1997), negative

in vitro Mammalian Chromosomal Aberration, OECD 473 (1998) -V79 cells, negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

BACTERIAL REVERSE MUTATION TEST

The test item was assessed for its potential to induce gene mutations, according to the OECD Guideline 471 (1983) and method B.14 EEC-Directive 2000/32/EC. The plate incorporation test (first experiment with and without metabolic activation, second experiment without activation) and the pre-incubation test (second experiment with metabolic activation) were performed using Salmonella typhimurium strains TA 100, TA 1535, TA 98, TA 1537, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Negative (solvent) and positive control treatments were included for all strains. Each concentration and the controls were tested in triplicate. The test item was dissolved in dimethyl sulfoxide (DMSO) and tested at five concentrations from 312.5 to 5000 µg/plate. Each strain was additionally tested In the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control. Previously, a pre-experiment for toxicity was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay.From the results obtained, the highest concentration suitable for the first mutagenicity test was selected. Both in the first and the second mutagenicity main tests, with and without metabolic activation, no substantial increase in the number of revertant colonies was observed after treatment with substance at any concentrations. In both mutagenicity tests normal background growth was observed with all strains at all concentrations. The number of revertant colonies was not reduced. The test item did not precipitate on the surface of the agar plates. The mean numbers of revertant colonies on negative control plates were found to be within acceptable ranges. The positive controls induced appropriate increases in the number of revertant colonies in all experiments, thus demonstrating the correct strain functioning and the activity of the S9-mix.

The test item did not induce gene mutations by base-pair changes or frameshifts in the genome of the strains used.

IN VITRO MAMMALIAN CHROMOSOMAL ABERRATION TEST

The test item was assessed for its potential to induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation (S9 mix), according to the OECD 473 (1998). Two independent experiments were performed. In experiment I, the exposure period was 4 hrs with and without metabolic activation. In experiment II the exposure period was 4 hrs with S9 mix and 18 hrs and 28 hrs without S9 mix. The chromosomes were prepared 18 hrs (exp. I and II) and 28 hrs (exp. II) after start of treatment with the test item. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

No clear toxic effects indicated by reduced cell numbers and mitotic indices were observed at the concentrations evaluated, except in experiment at interval 28 hrs in the presence of S9mix and in experiment after 18 hrs continuous treatment at interval 18 hrs in the absence of S9 mix, where the cell numbers or the mitotic indices were clearly reduced, respectively.

In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in experiment I, in the absence of S9 mix a single significant increase (3.5 %) was observed but was within our historical control range (0.0 - 4.0 % aberrant cells, exclusive gaps) and is regarded as being biologically irrelevant.

No increase in the frequencies of polypoid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

The test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008) a mutation means a permanent change in the amount or structure of the genetic material in a cell. The term ‘mutation’ applies 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 term ‘mutagenic’ and ‘mutagen’ will be 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 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.

The test substance did not show any reasons of concern in the tests performed.

In conclusion, the substance does not meet the criteria to be classified for genetic toxicity according to the CLP Regulation (EC 1272/2008).