Reaction mass of Cuprate(4-), [4,5-dihydro-4-[[8-hydroxy-7-[[2-hydroxy-5-methoxy-4-[[2-(sulfooxy)Vinyl]phenyl]azo]-6-sulfo-2-naphthalenyl]azo]-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylato(6-)], trisodium salt and Cuprate(4-), [4,5-dihydro-4-[[8-hydroxy-7-[[2-hydroxy-5-methoxy-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-6-sulfo-2-naphthalenyl]azo]-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylato(6-)]-, sodium and Cuprate(4-), [4,5-dihydro-4-[[8-hydroxy-7-[[2-hydroxy-5-methoxy-4-[2-(sulfooxy)Ethanol]phenyl]azo]-6-sulfo-2-naphthalenyl]azo]-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylato(6-)], trisodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

RA Substance 01:

- gene mutation assays in bacteria, using strains of Salmonella typhimurium and E.coli: negative

- chromosomal aberration assay: negative

RA Substance 02:

- gene mutation assay in bacteria, using strains of Salmonella typhimurium and E. coli: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

RA Substance 01:

- micronucleus test in mouse: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

According to Annex VII of the REACH Regulation (EC 1907/2006) and ECHA Guidance Chapter R.7a: Endpoint specific guidance, Version 4.0 – July 2015, a preliminary assessment of mutagenicity should normally include data from a gene mutation test in bacteria unless existing data for analogous substances indicates that this would be inappropriate. When the result of the bacterial test is positive, it is important to consider the possibility of the substance being genotoxic in mammalian cells, whereas a negative response would generally not require any further testing. However, if results from further studies of genotoxicity are available, it is recommended to take them into account in the assessement.

No data on genotoxic potential of test substance was available, thus the assessment was based on data of RA Substance 01 and RA Substance 02. In particular, RA Substance 01 is a copper complex and shares most of its structure with target substance (all 3 constituents) except for a small portion, which may possibly be released upon cleavage of an azo bond. RA Substance 02 was used to account for possible effects caused by this portion, based on its structural similarity. A detailed description of the read across approach is attached in section 13.

Genotoxic potential of RA Substance 01 was examined in a standard battery of tests, which allowed to trace its genotoxic profile: in vitro gene mutation assays in bacteria (OECD 471), in vitro chromosomal aberrations assay (OECD 473) and in vivo micronucleus assay (OECD 474).

As for bacterial gene mutation, in the first study (1983), the potential of inducing gene mutations in bacteria strains, namely Salmonella typhimurium strains and Escherichia coli strain, was tested in the Ames assay, both in presence and in absence of rat liver S9 mix metabolic activation. No evidence of mutagenicity was noted.

The second study (1989) was conducted on Salmonella Typhimurium strains, using:

- standard plate test (Ames test), with or without rat liver activation system (10 %)

- modified preincubation test (Prival test), with rat liver S9 mix (30 %) or hamster liver S9 mix (30 %) as metabolic activation.

In all cases, no evidence of mutagenicity was noted.

Additional studies on RA Substance 01 were available and confirmed the lack of concern. Specifically, in vitro chromosomal aberration assay was conducted in V79 Chinese hamster cells with and without metabolic activation by S9 mix. No evidence of mutagenicity was noted up to the highest concentration of 5000 µg/ml, chosen upon a preliminary test on cytotoxicity at concentrations from 100 to 8580 µg/ml.

In vivo micronucleus test in 5/sex mice was carried out at dose of 5000 µg/kg bw. No increase in the frequency of micronucleated polychromatic erythrocytes in treated animals was noted, thus excluding a mutagenic potential of RA Substance 01 in this assay.

In order to complete the assessment, mutagenic potential of RA Substance 02 was examined. Available data included an in vitro gene mutation assays in bacteria (OECD 471) using 5 Salmonella typhimurium strains and one Escherichia coli strain, using direct incorporation method (both experiments without S9 mix and one experiment with S9 mix) and preincubation method (with S9 mix). No evidence of mutagenicity was noted.

Moreover, genotoxic potential of RA Substance 02 was reviewed in EFSA Journal 7(11): 1331, 2009.

Studies on micronucleus induction in vitro and in vivo Sister Chromatid Exchange (SCE), micronucleus and chromosome aberration tests were negative. Data from an unscheduled DNA synthesis (UDS) assay conducted in vitro and ex vivo on mammalian cells were also negative.

The substance induced chromosomal aberrations in Chinese hamster fibroblast cell line and showed a significant increase in SCE and chromosomal aberrations in mouse and rat bone marrow cells, following acute and chronic exposure to high doses via the diet. Using the Comet assay, RA Substance 02 induced DNA damage in the colon of mice starting at dose of 10 mg/kg bw (Sasaki 2002). However, the transient DNA damage in the colon of mice was unable to be fixed in stable genotoxic lesions and might be partly explained by local cytotoxicity of the substance. In contrast, in a more recent study, RA Substance 02 did not reveal genotoxic effect in the micronucleus gut assay in mice at doses up to 2000 mg/kg bw (Poul 2009).

RA Substance 02 was also assessed for genotoxicity of its metabolites, mainly sulphonated aromatic amines produced upon reduction of the azo bond. Since sulphonated aromatic amines, in contrast with unsulphonated analogues, have no or very low genotoxic potential, metabolites of RA Substance 02 are unlikely to cause any concern.

The available carcinogenicity studies on RA Substance 02 included six carcinogenicity studies reviewed by JECFA, as well as three more recent ones described by TemaNord, namely the publications of Maekawa 1987, Borzelleca and Hallagan from 1988, plus the most recent study by Moutinho 2007. These studies demonstrated that RA Substance 02 does not have a potential to induce benign or malignant neoplasias.

In light of the negative carcinogenicity studies and negative results in standard genotoxicity studies, the biological significance of the positive genotoxicity results in other studies was uncertain. Therefore it was concluded that the effects reported in these studies were not expected to result in carcinogenicity.

Based on available findings on RA Substance 01 and RA Substance 02, relying on a read-across approach, it was concluded that target substance is non genotoxic in these test systems.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), Annex I, Part 3, substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans are classified in Category 2. This classification is based on positive evidence obtained in:

— somatic cell mutagenicity tests in vivo, in mammals; or

— other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

Note: substances which are positive in in vitro mammalian mutagenicity assays, and which also show chemical structure activity relationship to known germ cell mutagens, shall be considered for classification as Category 2 mutagens.

In vitro mutagenicity tests are the following:

—in vitro mammalian chromosome aberration test;

—in vitro mammalian cell gene mutation test;

— bacterial reverse mutation tests.

Based on negative responses in all available experimental studies, namely:

- in vitro bacterial reverse mutation tests on RA Substance 01 and RA Substance 02,

- in vitro mammalian chromosome aberration test on RA Substance 01,

- in vivo micronucleus test in mice on RA Substance 01,

along with the conclusion on the genotoxic potential of RA Substance 02 reported in the EFSA Journal (2009), relying on a read-across approach, a genotoxic potential for the substance was excluded. Therefore, it was not classified according to the CPL Regulation (EC 1272/2008).