pentasodium 4-hydroxy-7-[(sulfonatomethyl)amino]-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]-8-[(2-sulfonato-4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-2-sulfonate

Endpoint summary

Additional information

Mutagenicity:

in-vitro

A study was conducted to determine the mutagenic potential of test substance according to OECD Guideline 471, EPA OPPTS 870.5100, EU method B.13/14.and Japanese Substance Control Law (JSCL) Test Guideline III.1.

 

Strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA were used in the mutagenicity assay.

Two independent mutagenicity studies were conducted, one as the standard plate test with the plate incorporation method and the other as a modified preincubation test (Prival test). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate or a hamster liver homogenate. For both studies, the compound was dissolved in deionized water, and each bacterial strain was exposed to 5 dose levels.

Doses for both studies ranged from 50 to 5000µg/plate.

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range. All the positive control compounds gave the expected increase in the number of revertant colonies.

In the preincubation test the number of revertant colonies of the solvent controls in the presence of

S9-mix and the number of revertants colonies of the positive controls in the absence of S9-mix

with the strain WP2uvrA were out of the historical control data range, but the criteria for the

negative/positive responses were fulfilled.

Toxicity: In the mutagenicity experiments toxicity was not observed either with or without metabolic activation.

Plate incorporation test: Both in the absence and in the presence of rat liver (10 % (v/v)) metabolic activation system the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains.

In the absence and in the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival, test substance did not result in relevant increases in the number of revertant colonies with any of the tester strains.

The test substance is not mutagenic in the absence and presence of exogenous metabolic activation in the standard plate incorporation and the preincubation tests (Dr. Kauffmann HM, 2002).

 

Clastogenicity

 

 in-vivo

 

A study was conducted to assess the potential of the test substance to cause chromosomal damage (clastogenicity) in a rat bone marrow micronucleus test according to OECD Guideline 474, EPA OPPTS 870.5395 and EU method B.2.

The test substance was dissolved in sesame oil and was given twice at an interval of 24 h as an oral dose of 2000 mg/kg bw to male and female rats (Hsd:Sprague Dawley). At study start the animals were 6 wk of age and had mean body weights of 187.9 g (M) and 146.5 g (F). According to the test procedure the animals were killed 24 h after administration.

Cyclophosphamide was used as positive control substance and was administered once orally at a dose of 40 mg/kg bw.

The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with test substance and differed less than 20 % of the control value.

Cyclophosphamide induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

 

The test substance is considered not clastogenic in the micronucleus test in SD rats.

 

in-vitro

A study was conducted to investigate the potential of test substance to induce chromosome aberrations in V 79 cells of the Chinese hamster lung in vitro according to OECD Guideline 473, EU Method B.10., EPA OPPTS 870.5375 and Japanese Substance Control Law (JSCL) Test Guideline III.1.

The test substance was dissolved in cell culture medium and tested at the following concentrations:

First experiment with 3/20 h treatment/sampling time:

without S9-mix: 39.1#, 78.1#, 156.3#, 312.5, 625#,1250$, 2500.0$and 5000$µg/mL

with S9-mix: 39.1#, 78.1#, 156.3#, 312.5, 625#, 1250$, 2500$and 5000$µg/mL

#= not used because higher concentrations were evaluated

S=concentrations at which metaphase analysis was conducted

 Second experiment with 20 h treatment time:

Because of mutagenicity in the first experiment metaphase analysis of the second experiment was not conducted.

 The maximum concentration based on the regulations of the corresponding guidelines. The highest concentration produced a mild lowering of the mitotic index 3 h after treatment in the absence of S9-mix.

Precipitation of the test compound was not observed.

There was an enhancement of the aberration rates at the 3 h treatment time with 2500 and 5000 µg/mL without S9-mix and with S9-mix at 5000 µg/mL. These data were found significantly enhanced in the Fisher's exact-test. This is an indication of heavy chromosomal damage. In addition, an increased number of aberrations with test substance was found after 3 h treatment with 2500 and 5000 µg/mL with and without S9-mix. On the basis of these findings the evaluation of slides of the longer treatment time (20 h) is not necessary and the 20 h treatment was not performed.

Appropriate reference mutagens used as positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay, and the efficacy of the S9-mix.

The test substance did induce chromosome aberrations both in the presence and in the absence of a metabolic activation system and it is considered to be clastogenic in this in vitro chromosome aberration assay with V79 Chinese hamster lung cells (Dr. Kauffmann HM, 2002).

Short description of key information:
The test substance was found to be non-genotoxic both in in-vitro bacterial reverse mutation and in-vivo micronucleus test in mice. However, it was positive for genotoxicity in in-vitro chromosomal aberration chromosomal aberration test.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test substance was found to be non-genotoxic both in in-vitro bacterial reverse mutation and in-vivo micronucleus test in mice. However, it was positive for genotoxicity in in-vitro chromosomal aberration chromosomal aberration test. This false positive outcome of vinyl sulfone dyes is a well-known effect in in-vitro chromosome aberration assays. The vinyl sulfone ester reacts in a Michael-Addition and leads to a depletion of the glutathione transferase in an in-vitro assay which per se lacks of phase II enzymes. The in-vivo very effective de-toxification system is hence not working ptoperly and leads to false positive effects in the in-vitro assay for this class of substances.

Based on the overall weight of evidence test substance, is expected not to have any genotoxic potential. Therefore no classification is required for genotoxicity according to EC criteria (67/548/EEC) and according to CLP criteria (EC 1272/2008).