Diammonio(ethyl)[4-[[4-[ethyl(3-sulphonatobenzyl)amino]phenyl](2-sulphonatophenyl)methylene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Results of available in vitro studies:

- AMES tests: negative;

- Gene mutation study in mammalian cells: negative

- Transformation study in mammalian cells: negative

- DNA damage and/or repair study: negative

- Cytogenicity /Chromosome aberration study in mammalian cells: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Results of available in vitro studies:

- Mammalian somatic cell study: cytogenicity /erythrocyte micronucleus: negative

- Comet Assay: negative

- Mammalian cell study: DNA damage and/or repair: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In this summary a bacterial reverse mutation study on Target Substance has been reported as key study. Furthermore, a series of different genotoxicity studies on Similar Substance 1 are reported to complete the assessment for the target substance.

 

IN VITRO STUDIES

The substance was assayed for the mutagenicity by the Bacterial Reverse Mutation Testtill concentration 5000μg/plate. Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coliWP2 uvrA strain were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats. The results showed that the test item was non-mutagenic for all the used tester strains without as well as with metabolic activation.

 

Similar Substance 1 was tested in a valid GLP compliant Ames test following OECD guideline 471. Tester strains Salmonella typhimurium TA 98, 100, 1535, 1537 and 102 as well as E. coli WP2 uvrA were used. Water was used as a vehicle. A moderate to marked coloration was observed in the Petri plates when scoring the revertants at all dose-levels. This coloration made the plates unreadable at dose-levels > 1 mg/plate. No noteworthy toxicity was noted towards the strains used, with and without S9 mix. No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. No increase in the mutant frequency was observed. Positive and negative control incubations confirmed the quality of the study performance.

Similar Substance 1 was tested in a valid GLP compliant mammalian cell culture assay (Mouse lymphoma assay) according to OECD guideline 476 (RCC 2000). The assay was performed with two independent experiments, each with two parallel cultures. For the first experiment, an exposure period of 4h was used both with and without metabolic activation. The second experiment was performed in the absence of metabolic activation with an exposure period of 24h. Doses of up to 5 mg per plate were applied. There was no precipitation and no cytotoxicity. No mutagenicity was observed. There was no shift in the ratio of small to large colonies. Positive and negative control incubations confirmed the quality of the study performance.

In addition to the information of the above described studies, a results of in-vitro genotoxicity tests have been published in the literature since 1977. No genotoxic hazard was identified. In the Ishidate et al. study (1984), the chromosomal aberration test (Chinese hamster fibroblast cell line, CHL) was reported as positive. However, these results were accompanied by excessive variations in the osmolality of the culture medium and were therefore not considered relevant.

 

IN VIVO STUDIES

Similar Substance 1 was tested in a micronucleus assay in male mice published by Hayashi 1988, the substance was administered by one to four intraperitoneal injections (0, 500, 1000, 2000 mg/kg bw). It was part of a project of the Japanese Food Chemistry Division, Environmental Health Bureau, Ministry of Health and Welfare. The procedure is identical to that of the current OECD testing guideline 474 with the exception that only 1000 instead of 2000 femoral marrow cells per animal were scored. This is however acceptable, as the study consists of two separate experiments, which means that in sum, a sufficiently high number of cells were scored. In the first experiment, groups of each 6 rats were injected with a single dose of 500, 1000 or 2000 mg/kg bw and the animals were sacrificed 26h after dosing. One animal of the high dose group died. In the second study, animals were given one injection of 1010 mg/kg bw per day for four consecutive days and the animals were sacrified 24h after the last application. Physiological saline served as vehicle. MMC was used as positive control. No increase in micronuclei was observed as a result of treatment with Similar Substance 1.

Results of a comet assay in male ddY mice were published by Sasaki in 2002. Groups of four animals were treated by gavage once with the limit dose of 2000 mg/kg bw. Three and 24h after dosing, the comet assays were performed on glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow. Treatment did not yield a statistically significant increase in DNA damage in any organs. Although in this study, several deviations from the standard protocol can be observed, the study seems to be sufficiently reliable.

Another study is reported on Similar Substance 1 for unscheduled DNA synthesis (OECD 486) showing absence of an in-vivo genotoxic potential. This study is reported in EFSA expert panel (EFSA, 2010).

 

As it can be evinced from the results of the reported studies, Target Substance shows a similar behaviour in the bacterial reverse mutation assay (AMES test) to Similar Substance 1. It is therefore expected a similar behaviour in other tests too. A detailed justification for Read Across is given in Section 13 of IUCLID. Further studies on Target Substance are in conclusion not considered necessary to complete the assessment of genetic toxicity.

References:

- Ishidate et al. 1984. Primary mutagenicity screening of food additives currently used in Japan.Fd Chem. Toxic22: 623-636.

- Hayashi et al. 1988.Micronucleus tests in mice on 39 food additives and eight miscellaneous chemicals.Fd. Chem. Toxicol.26, 487-500.

- Sasaki et al. 2002. The comet assay with 8 mouse organs: results with 39 currently used food additives.Mutation Research,519: 103-119

- EFSA 2010. Scientific Opinion on the re-evaluation of Brilliant Blue FCF (E 133) as a food additive1, EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS).

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No hazard for genotoxicity was identified. As a result, according to the CLP Regulation (EC 1272/2008), the substance is not considered to be classified for genetic toxicity.