Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation in Ames assay: negative

Gene mutation in mammalian cells in mouse lymphoma assay: negative

Chromosome aberrations assay in Chinese hamster lung fibroblasts: weakly positive

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus assay in mouse: negative

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Mutagenicity testing strategy is reported in ECHA Guidance Chapter R.7a: Endpoint specific guidance, Version 5.0 – December 2016.

A preliminary assessment of mutagenicity normally includes data from a gene mutation test in bacteria unless existing data for analogous substances indicates this would be inappropriate. To complete the assessement as for requirements in Annex VIII of the REACH Regulation (EC 1907/2006), in vitro studies in mammalian cells should then be performed.

As a first step, an in vitro cytogenicity study is usually carried out to assess the potential for test substance to induce chromosomal aberrations. In case of a positive result, an in vivo cytogenicity assay is generally required to prove the in vivo relevance of in vitro results.

An in vitro gene mutation assay usually completes the assessment, especially in case of negative results in the bacterial gene mutation test and the first study in mammalian cells.

The substance was tested to assess its potential to induce: bacteria reverse mutation, gene mutation and chromosomal aberration in vitro as well as micronucleus in vivo.

A part from an Ames assay, for which a study report was available, all data used in the assessment derive from literature publications. Details on studies performance and result were available and were found to be consistent generally accepted scientific principles.

It should be noted that literature data was also a main part of the assessment on Acid Red 018 made by authorities, such as SCCNFP (SCCNFP/0792/04) and EFSA (EFSA Journal 7(11) 1328, 2009), and toxicological consultancy company (bibra ltd.).

As for bacteria reverse mutation, an experimental study report (2005) was selected as key study as it was performed on all strains as recommended by the OECD guideline 471 and it was well documented. The target substance resulted as non mutagenic in this assay.

Available literature data confirmed the lack of mutagenic potential in such type of assay, generally carried out on some, but not all, strains required by the guideline. In particular, a study from 1987 was included as supporting evidence.

In a chromosomal aberration assay on Chinese hamster lung fibroblasts without metabolic activation, the test item showed an incidence of structural aberrations of 13 and 12 % after 24 and 48 hr, respectively. At the dose of 3.06 mg/ml, 20 % of observed metaphases showed structural aberrations. Overall, the result was considered as positive (threshold value 10 %). However, as the CA assay was only conducted without metabolic activation, the relevance of such result with respect to mammalian in vivo conditions could not be proved.

In addition, the potential of the substance to induce chromosomal aberrations was assayed by an in vivo micronucleus test in bone marrow cells of male mice. Test animals were dosed intraperitoneally at concentrations ranging for 300 to 2400 mg/kg as single injections and at concentration of 300 mg/kg , repeated 4 times at 24 -hour intervals. Vehicle and positive controls were adequate. Overall, there was no evidence of increased micronucleus formation in the bone marrow upon exposure either as single dose or as four separate aliquots.

A mouse lymphoma assay on L5178Y TK+/- cells was carried out to assess the potential of the target substance to induce gene mutations in mammalian cells. In this assay, an increase in mutant frequency comparable to spontaneous control frequency was seen, thus the response was considered as negative.

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.

The overall assessement on the genotoxic potential of test substance was based on: negative outcome in bacterial reverse mutation assay (AMES test), weakly positive outcome in in vitro chromosome aberration assay without metabolic activation and negative result in in vivo micronucleus test in bone marrow cells of mouse. A further negative result was obtained in an in vitro gene mutation assay.

Based on these results, test susbstance was considered as non genotoxic and it was not classified within the CLP Regulation (EC 1272/2008).