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Indigo was neither mutagenic nor clastogenic in respective in vitro or in vivo genotoxicity tests. Several tests with synthetic Indigo reported a positive test result with metabolic activation in frameshift Salmonella strains. However, this positive effect could clearly be attributed to mutagenic or comutagenic impurities. The study was designed to investigate the mutagenic properties of distinct known impurities of synthetic Indigo and to determine possible comutagenic effects of combinations of these impurities.These impurities' combinations are not contained in the registered substance. Hence, Indigo as registered is not mutagenic in the bacterial reverse mutation assay.

In the SIDS Initial assessment report (March 1994) on Indigo a positive in-vitro chromosomal aberration study with a commercial Indigo (purity stated: 97.2%) is cited. The study has been conducted by MHW, Japan and is cited as MHW, Japan (1993), unpublished report on mutagenicity test of Indigo blue. The study was done on CHL cells with and without metabolic activation in a concentration range of 124, 500, 1000, 2500 µg/mL and the result was positive. The same document stated in chapter 2.6 water solubility: substance of very low solubility; the solubility is cited as 0.99mg/L, i.e. 0.99 µg/mL. Hence, the concentrations tested lie well above the limit of solubility. However, there is no information on any precipitation of the test substance in the medium. As we know from our own in-vitro chromosome aberration study, macroscopic precipitation of the test compound was observed in a dose range of 327.9 µg/ml and above and microscopic precipitation of the test compound in a dose range of 20.5 µg/ml and above. Hence, this study was conducted at test substance concentrations above solubility where macroscopically visible precipitations should have occurred, which would lead to a false positive test result.

Based on the data available, the MHW study is not reliable.

The in-vitro chromosome aberration assay of DyStar was performed on a well-defined Indigo batch at concentration were only microscopic precipitation, but no macroscopic precipitation was visible.

First experiment with 3/20 h treatment/sampling time: without S9-mix: 20.5, 41.0,82.0, 164.0, 327.9, 655.7, 1311.4 and 2622.7 µg/mL; with S9-mix: 20.5, 41.0,82.0, 164.0, 327.9, 655.7, 1311.4 and 2622.7 µg/mL

Second experiment with 20/20 h treatment/sampling time: without S9-mix: 20.5,41.0, 82.0, 164.0, 327.9, 655.7, 1311.4 and 2622.7 µg/mL

Second experiment with 3/28 h treatment/sampling time: with S9-mix: 41.0,82.0, 164.0, 327.9, 655.7 and 1311.4 µg/mL

Second experiment with 28/28 h treatment/sampling time: without S9-mix: 20.5,41.0, 82.0, 164.0 and 327.9 µg/mL

bold= concentrations at which metaphase analysis was conducted

The highest concentration of 2622.7 µg/mL equals 10 mM, which is the upper limit for testing according to the OECD guideline; hence, higher concentrations were not applied. After treatment time, macroscopic precipitation of the test compound was observed in a dose range of 327.9 µg/mL and above and microscopic precipitation of the test compound in a dose range of 20.5 µg/mL and above.

The evaluated concentrations showed no lowering of the mitotic index. Cell survival was reduced below 50 % in the highest evaluated concentration groups in the absence and in the presence of S9‑mix in both experiments.

Up to the highest investigated dose the test compound induced no toxicologically relevant or dose-dependent increase in the number of aberrant metaphases.

Indigo is hence not clastogenic in a valid and reliable in-vitro chromosome aberration assay with the relevant test substance.

For the only classification relevant impurity aniline, it is well known that no clear genotoxic effects can be produced in in-vitro or in-vivo tests. As stated in the evaluation of the BAuA (October 2002) for aniline, the tests are either negative or equivocal. Clastogenic effects can only be observed at very high, cytotoxic concentrations. All genotoxic or carcinogenic effects observed for aniline were due to haemotoxic effects, which led secondary to micronuclei or spleen tumours, respectively.

Due to the fact that the registered Indigo with a specified composition was unequivocally negative in the in-vitro chromosome assay and the in-vivo micronucleus study was negative in literature, it is concluded that Indigo does not have a clastogenic effect.


Short description of key information:
Genetic toxicity negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Indigo is not genotoxic, therefore, no classification is necessary.