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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria: non mutagenic

Gene mutation in mammalian cells: non mutagenic

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

According to Annex VII of the REACH Regulation (EC 1907/2006) and ECHA Guidance Chapter R.7a: Endpoint specific guidance, Version 5.0 – December 2016, 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 target substance was available, thus a read across approach was followed using available data on Similar Substance 01 and Similar Substance 02. Details on the read across are available in section 13 .

In particular, the assessment was based on available data on mutagenic potential of Similar Substance 01 and 02 in Ames assay (OECD 471) as well as on mutagenic potential of Similar Substance 01 in mammalian cells (OECD 476).

Both Ames assays on Similar Substance 01 included 4 Salmonella typhimurium strains instead of the 5 recommended by the current version of OECD guideline 471. However, significance and reliability of the result was confirmed by an available study on Similar Substance 02, which included the 5 required strains.

In an Ames assay from 1995 on Similar Substance 01, a sample with high purity (90 % dye, described as "purified") was tested at concentration up to 5000 µg/plate using water as vehicle. The study included a plate incorporation test (experiment I) and a pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100.

Slight toxic effects occurred in strains TA 1535 and TA 1537 without S9 mix.

Slight increases in revertant colony numbers were observed in strain TA 1537 at 5000.0 µg/plate without S9 mix and at 2500.0 µg/plate with S9 mix as well as in strain TA 98 at 5000.0 µg/plate with S9 mix in experiment II. These effects were considered as biologically irrelevant, due to the relatively low level of of the corresponding solvent control (strain TA 1537) and could not be reproduced in the independent experiment (strain TA 1537 and TA 98).

On these bases, test substance was considered as non-mutagenic in this assay.

In an Ames assay from 1994 on Similar Substance 01, a sample with 80 % purity, described as "raw", was tested at concentration up to 5000 µg/plate using DMSO as vehicle. Two independent experiments were run using the plate incorporation method.

In the original and confirmatory experiment, strain TA 1537 without metabolic activation showed a slight increase in the number of revertant colonies at the concentration of 5000 µg/plate. No effect was seen with TA 1537 with metabolic activation. Moreover, no effect was seen in the other strains both with and without metabolic activation.

As no mutagenic effect was seen in the study from 1995, despite

- the content of test substance was higher than in the test sample that used for the study from 1994,

- both plate incorporation and preincubation method were used,

the slight mutagenic response in the study from 1994 was reasonably attributed to the impurities content of test sample. Therefore, such study was disregarded, while the study from 1995 was selected as key study.

In addition, an Ames assay was conducted on Similar Substance 02 using 5 Salmonella typhimurium strains:

- TA 1535, TA 100 and TA 102 to detect base-pair substitutions,

- TA 98 and TA 1537 to detect frame-shifts.

No cytotoxicity, evident as reduction in the number of revertants, was reported. Moreover, no mutagenic effects were noted, in both the original and the confirmatory experiment; in particular, positive findings with TA 102 strain in the confirmatory experiment were considered as incidental.

Based on these experimental evidences, a mutagenic potential was reasonably excluded.

A gene mutation assay in mammalian cells with Similar Substance 01 was run using a sample of 80 % purity, i.e. same sample as that exhibiting a mutagenic effect in the Ames assay from 1994. Two independent experiments were run and each concentration was tested in duplicate. Test concentrations were selected in a preliminary test, where cytotoxic effects were noted. In both experiments, no relevant increase of mutant frequencies compared to controls was noted. Accordingly, a mutagenic potential in this assay was excluded.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), a mutation means a permanent change in the amount or structure of the genetic material in a cell. The term ‘mutation’ applies both to heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known.

The more general terms ‘genotoxic’ and ‘genotoxicity’ apply to agents or processes which alter the structure, information content, or segregation of DNA, including those which cause DNA damage by interfering with normal replication processes, or which in a non- physiological manner (temporarily) alter its replication.

For the purpose of classification for germ cell mutagenicity, substances are allocated to one of two categories:

 

Category 1: substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans.

Category 1A: based on positive evidence from human epidemiological studies.

Category 1B: based on:

- positive result(s) from in vivo heritable germ cell mutagenicity tests in mammals; or

- positive result(s) from in vivo somatic cell mutagenicity tests in mammals, in combination with some evidence that the substance has potential to cause mutations to germ cells.

- positive results from tests showing mutagenic effects in the germ cells of humans, without demonstration of transmission to progeny; for example, an increase in the frequency of aneuploidy in sperm cells of exposed people.

 

Category 2: substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans, based on positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

- 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

 

Based on negative result in available experimental studies, a mutagenic potential was excluded and the substance was not classified under the CLP Regulation (EC 1272/2008).

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