Pentasodium bis{7-[4-(1-butyl-5-cyano-1,2-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridylazo)phenylsulfonylamino]-5'-nitro-3,3'-disulfonatonaphthalene-2-azobenzene-1,2'-diolato} chromate (III)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro Bacterial Reverse Mutation, negative

in vitro Mammalian Chromosomal Aberration -V79 cells, negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

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 92/69. Two experiments were performed using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100: the plate incorporation test with Aroclor 1254-induced rat liver S9 mix (experiment I) and the pre-incubation test with uninduced hamster liver S9 mix (experiment II). The two independent experiments were performed 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 water and tested at six concentrations ranging from 33.33 to 5000.0 μg/plate. Previously, a pre-experiment for toxicity was carried out with strainsS. typhimuriumTA 100 and TA 98 to determine the highest concentration to be used in the mutagenicity assay.

Toxic effects, evidenced by a reduction in the number of revertants, occurred at the higher concentrations with and without metabolic activation in the strains TA 1537 and TA 98 in experiment II. The plates incubated with the test item showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with test item at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

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 hamsterin vitroin the absence and presence of metabolic activation (S9 mix) in two independent experiments, according to the OECD 473 (1983) and the method B.10 of the EEC Directive 92/69. The substance was dissolved in MEM. The chromosomes were prepared 18 h and 28 h after start of treatment with the test item. The treatment interval was 4 h with metabolic activation, 18 h and 28 h without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations. The concentration range of the test item was determined in a pre-test using the XTT-assay and a qualitative evaluation of cell density and morphology as indicator for toxicity response. Dose selection was influenced by solubility of the test item. Precipitation was observed at concentrations higher than 600 µg/ml. Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosomal aberrations.

In the pre-test toxic effects could be observed within a concentration range of 1000.0- 5000.0 µg/ml. In the absence of S9 mix, in both experiments the mitotic index was reduced after treatment with the highest evaluated concentration at each fixation interval. In the presence of S9 mix in both experiments no reduction of the mitotic Indices occurred. Evaluation of cultures treated with higher concentrations could not be performed due to cytotoxic effects in the absence of S9 mix or precipitation in the presence of S9 mix. In both independent experiments, there were no biologically relevant increases of structural aberrations and polyploid cells after treatment with the test item at fixation intervals 18 h (with S9 mix) and 28 h (with and without S9 mix).

The test substance did not induce structural chromosomal aberrations as determined by the chromosomal aberration test in V79 cells (Chinese hamster cell line), under the experimental conditions. Therefore, the substance is considered to be non-mutagenic in this chromosome aberration test.

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).