Disodium 7-[[4,6-bis[(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not genotoxic

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Not genotoxic

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

BACTERIA GENE MUTATION ASSAY

Direct Red 253 (DR253) was tested for detecting its potential gene mutagenic activity according to the plate incorporation method of Ames et al. 1975, using the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. The tests were performed with and without metabolic activation. The compound was examined in triplicate at 8 concentrations from 1.53 to 5000 µg/plate. Neither quantitative nor qualitative evidence of a toxic effect of the compound was observed. In the experiments performed, no relevant increase of the revertant colony numbers was observed in any Salmonella typhimuriumstrain tested, in the presence and in the absence of S-9 mix (Guenard, 1982). It has to be note that the combination of strains recommended in the currently adopted OECD guideline was not assayed: none of the E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 was tested.

Further investigations on bacterial reverse mutation assay (e.g. Ames test) were conducted on DR253 sodium/triethanolammmonium salt (DR253 Na:TEA). The usage of information on DR253 Na:TEA, which has the same main component and with a different counter ion, can be considered as suitable and appropriated because the difference in salification is expected to not influence the characteristics related to the specific end-point.

In most of the experiments, under the test conditions, the test article did not induced point mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98 and TA 100 of Salmonella typhimurium used.

Toxic effects were recorded in two cases: evidenced by a reduction in the number of revertants, occurred in strain TA 1537 at 5000.0 µg/plate in the absence of S9 mix in experiment I (Poth, 1991). In strain TA 100, a slight increase of revertant colonies was found at 10.0 and 100.0 µg/plate in the presence of metabolic activation in experiment II; however these effects have been considered as not being relevant because they were not reproduced in the independent experiment (Poth, 1990).

In one test, a significant and reproducible dose-dependent increase in revertant colony numbers was obtained in the strains TA 1535, TA 1537 and TA 98 with and without metabolic activation in experiment I and II, except in strain TA 98 in experiment Il with and without S9 mix, where only a dose-dependent but no significant increase was obtained. In strain TA 100, only at the highest investigated dose was a mutagenic response observed without S9 mix in experiment I and with and without S9 mix in experiment II. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies (Poth, 1990).

MAMMALIAN CELL GENE MUTATION ASSAY

A study was performed to investigate the potential of DR253 Na:TEA to induce gene mutations at the HGPRT locus in V79 cells of the Chinese hamster in vitro. The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. The mutation rates found in the groups treated with the test article were considered not to be a biologically relevant increase compared with the negative and solvent controls. The test article did not induce a reproducible concentration-related increase in mutant colony numbers. The mutant values of the groups treated with the test article were in the range of the negative controls. Up to the highest investigated dose no relevant increase in mutant colony numbers was obtained in two independent experiments. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies (Heidemann, 1990).

The usage of information on DR253 Na:TEA, which has the same main component and with a different counter ion, can be considered as suitable and appropriated because the difference in salification is expected to not influence the characteristics related to the specific end-point.

CHROMOSOMAL ABERRATION ASSAY

Chromosomal aberration potential has been investigated in in vivo and in vitro experiments, testing DR253 Na:TEA. The difference in salification is expected to not influence the characteristics related to the specific end-point.

DR253 Na:TEA salt was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation by S9 mix. Treatment with 2.90 and 2.76 mg/ml, respectively, reduced the plating efficiency of the V79 cells. The mitotic index was not reduced even after treatment with the highest concentration at any fixation interval either in the presence or absence of S9 mix. Higher concentrations of the test article than 2.90 mg/ml could not be dissolved in the culture medium. There was no relevant increase in cells with structural aberrations after treatment with the test article at any fixation interval either without or with metabolic activation by S9 mix. Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. Under the experimental conditions reported, it has been concluded that the test article did not induce structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line (Heidemann, 1991).

The in vivo genetic toxicity potential of DR253 Na:TEA salt to induce micronuclei in polychromatic erythrocytes was investigated in mouse bone marrow. The animals expressed slight toxic reactions. After treatment with the test article, the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects. In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article (Völkner, 1990)

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 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:

- 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

- 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).