5-amino-3-[[4-[[4-[[4-anilino-2-hydroxyphenyl]azo]phenyl]amino]-3-sulphophenyl]azo]-4-hydroxynaphthalene-2,7-disulphonic acid, sodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES: point mutations by frameshifts in the genome of strain TA1538 and indication of a possible mutagenic response in strain TA1537.

CA: no induction of structural chromosome aberrations in the V79 Chinese hamster cell line.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

MA: no induction of micronuclei in the bone marrow cells of the mouse and

UDS: no induction of DNA-damage leading to repair synthesis in the hepatocytes of the treated rats

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

No experimental data from in vitro mutagencity tests on the substance are available. The following data were obtained for Similar Substance 01. It is expected that the Target Substance will present a similar mutagenicity profile. Justification for the use of a read-across approach is provided in Section 13 of IUCLID.

Investigation on the genetic toxicity was performed with the integrated evaluation of the following studies: in vitro Ames tests, in vitro and in vivo chromosomal aberration assay and in vivo DNA damage and/or repair study on mammalian cell.

IN VITRO GENE MUTATION ASSAY IN BACTERIA

Two tests have been performed on Similar Substance 01 assessing the potential to induce gene mutations in bacteria. Both tests were conducted using Salmonella typhimurium strains.

A plate incorporation test was performed on S. typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100, in two independent experiments, both with and without rat liver microsomal activation. In addition, a third experiment (III) was performed only with TA1535 and TA1537 with and without metabolic activation.

Toxic effects occurred in strain TA1535 at 100.0 μg/plate (exp. I) and in strain TA1537 at 1000.0 μg/plate (exp. III), both without S9 mix. Also in strain TA 98 at 5000.0 μg/plate with metabolic activation in both experiments. The plates incubated with the test article showed normal background growth up to 5000.0 ug/plate with and without S9 mix in all strains used. In strain TA1537 (with S9 mix) a dose-dependent increase in the number of revertants was observed in the concentration range of 333.3 up to 5000.0 ug/plate. This increase was less significant in exp. II. In the experiment III no relevant increase in the number of revertants was observed. Based on these results a mutagenic potential of the test article in strain TA1537 cannot be excluded, but this the effect was not reproduced in all three experiments. In strain TA1538 (with S9 mix) a dose-dependent increase was obtained up to 333.3 μg/plate (exp. I) and up to 1000.0 μg/plate (exp. ΙI). No indication of mutagenic response occurred in strains TA1535, TA 98, and TA 100 as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists, with and without metabolic activation. The substance induced point mutations by frameshifts in the genome of the strain TA1538 and there is an indication of a possible mutagenic response in strain TA1537.

The second test was conducted on S. typhimurium strains TA 98, TA100, TA1535, TA1537, with and without the addition of a metabolic activation system; two experiments were carried out.

The substance showed no toxicity for any of the strains at any of the concentrations tested, in both experiments I and II. In experiment I, no mutagenic response was observed for any of the strains tested, with or without the addition of S9; strain TA98 showed a statistically significant response but it cannot be considered to be mutagenic as the highest revertants count never doubled that of the control. During the experiment II, no mutagenic response was observed for any of the strains tested, with or without the addition of S9.

IN VITRO CHROMOSOMAL ABERRATION ASSAY and IN VIVO MICRONUCLEUS

The Similar substance 01 potential to induce structural chromosome aberrations was assayed both in in vitro and in in vivo test systems.

The in vitro test was performed using V79 cells of the Chinese hamster, in the absence and presence of metabolic activation. Treatment with concentrations higher than 0.001 mg/ml (without S9 mix) and 0.03 mg/ml (with S9 mix) reduced distinctly the plating efficiency of the V79 cells. In the cytogenetic experiment the mitotic index was reduced in the samples treated with the highest scorable dose level at each fixation interval except at interval 7 (with S9 mix). With higher dose levels not enough scorable cells could be found. Both, in the absence and presence of S9 mix the test article did not increase the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (1.00 % - 5.00 %) were in or near the range of the control values: 0.00 % - 4.50 %.

The in vivo experiment was a micronucleus test, which assessed the potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. 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. The mean values of micronuclei observed after treatment with the substance were in the same range as compared to the negative control groups.

UNSCHEDULED DNA SYNTHESIS

The substance was assessed in the in vivo UDS assay for the potential to induce DNA repair (UDS) in the hepatocytes of rats. The test procedures followed can be considered as comparable to those described in the OECD guideline 486, which was published later. No toxic reactions of the animals occurred at any of the treatment periods or dose group. The viability and the in vitro attachment of the hepatocytes was not affected due to the in vivo pre-treatment with the test article. The interindividual variations obtained for the numbers of isolated hepatocytes as well as for the attachment efficiency were in the range of the historical laboratory control. No dose level of the test article revealed UDS induction in the hepatocytes of the treated animals as compared to the concurrent negative controls. Neither the nuclear grains nor the resulting net grains were enhanced due to the in vivo treatment of the animals with the test article for 4 or 16 hours.

CONCLUSION

Investigation on the genetic toxicity has been performed with the integrated evaluation of the following studies: in vitro Ames tests, in vitro and in vivo chromosomal aberration assay and in vivo DNA damage and/or repair study on mammalian cell.

The preliminary assessment of mutagenicity in S. typhimurium bacteria showed that the test article is able to induce point mutations by frameshifts in the genome of strain TA1538; indication of a possible mutagenic response has also been recorded in strain TA1537.

The investigations in vitro of chromosomal aberration in mammalian cells and the in vivo micronucleous assay in bone marrow cells of the mouse did not show any potential for genetic toxicity. Also the in vivo experiment performed to assess the potential for DNA repair synthesis induction in the hepatocyte of rats gave negative results.

In conclusion, the substance did not showed any potential for genetic toxicity in mammalian cells.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 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 available information suggest that test substance did not show any reasons of concern from the genotoxicity point of view.

 

In conclusion, the substance does not meet the criteria to be classified for genetic toxicity, according to the CLP Regulation (EC) No 1272/2008.