Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non-mutagenic

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

Genetic toxicity in vivo

Description of key information

Non-mutagenic (erythrocyte micronucleus)

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

Additional information

GENETIC TOXICITY IN VITRO

in vitro gene mutation study in bacteria

The mutagenic potential of the test substance was evaluated according to OECD Guideline 471 and ICH genotoxicity Guideline S2A and S2B. Strains TA98, TA100, TA1535 and TA1537 of S.typhimurium and strain WP2uvrA of E.coli in the presence and absence of S9 were exposed to the test substance. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance. In the initial toxicity-mutation assay, the dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate. In the retest of the initial toxicity-mutation assay, the dose levels tested were 50, 150, 500, 1500 and 5000 μg/plate. No positive mutagenic responses were observed with any of the tester strains in the absence and presence of S9. Neither precipitate nor appreciable toxicity was observed. In the confirmatory mutagenicity assay, no positive mutagenic responses were observed. The dose levels tested were 50, 150, 500, 1500 and 5000 μg/plate. Neither precipitate nor appreciable toxicity was observed. Negative with and without metabolic activation.

Another available study was conducted to determine the mutagenic potential of Similar substance 01, according to the OECD Guideline 471, ICH genotoxicity Guideline S2A and S2B in compliance with GLP; justification for Read Across is given in Section 13 of IUCLID. Tester strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium and strain WP2uvrA of Escherichia coli were exposed to test substance in the presence and absence of S9 . The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance. In the initial toxicity-mutation assay, the dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate. In the retest of the initial toxicity-mutation assay, the dose levels tested were 50, 150, 500, 1500 and 5,000 μg/plate. No mutagenic responses were observed with any of the tester strains in the absence and presence of S9. Neither precipitate nor appreciable toxicity was observed. In the confirmatory mutagenicity assay, no mutagenic responses were observed. The dose levels tested were 50, 150, 500, 1500 and 5,000 μg/plate. Neither precipitate nor appreciable toxicity was observed.

Under the test conditions, the test substance was concluded to be non-mutagenic in the bacterial reverse mutation assay.

in vitro cytogenicity / chromosome aberration study in mammalian cells

The ability of the test substance to induce chromosome aberrations was evaluated by considering data on Similar Substance 01, due to the absence of data on the substance itself; justification for Read Across is given in Section 13 of IUCLID.

In a first experiment, the potential of the similar substance to induce chromosome aberrations in cultured peripheral human lymphocytes, both in the presence and absence of a metabolic activation system, was evaluated according to the OECD Guideline 473 and EU Method B.10. The possible clastogenicity of the substance was tested in two independent experiments. In the first cytogenetic assay, the substance was tested up to 4000 and 3750 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8 % (v/v) S9-fraction, respectively. Appropriate toxicity was reached at these dose levels. In the second cytogenetic assay, the substance was tested up to 1500 µg/ml for a 24 and 48 h continuous exposure time with a 24 and 48 h fixation time in the absence of S9-mix. In the presence of S9, the highest test concentration was 4500 µg/ml for a 3 h exposure time with a 48 h fixation time. Appropriate toxicity was reached at these dose levels.

In the first cytogenetic assay the substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence of S9-mix. In the presence of S9-mix the number of cells with chromosome aberrations was not statistically significant increased, however, exchange figures were observed at a concentration of 3750 µg/ml.

Whilst the results from this study cannot be easily dismissed, there are reasons to question the reliability of the results. Since the only findings worthy of study director comment were at concentrations that exceed the current recommended upper limit, they may in any case not be biologically relevant.

Due to the questions regarding the reliability and interpretation of the above study, and in particular the use of concentrations exceeding the maximum recommended in OECD guideline 473 (OECD, 2016) a new study was conducted at a different laboratory. Additional information on the reason for disregarding this study is reported in the document attached in the relative end-point study record.

The test item was therefore tested in an in vitro chromosome aberration assay using human lymphocyte cultures prepared from the pooled blood of three female donors. Treatments covering a broad range of concentrations were performed both in the absence and presence of metabolic activation (S9) from β-Naphthoflavone/Phenobarbital-induced rats. The test article was formulated in dimethyl sulphoxide (DMSO) and the highest concentration tested in the Chromosome Aberration Experiment was 2000 µg/mL. Treatments were conducted 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for chromosome analysis were selected by evaluating the effect of the test item on mitotic index. DMSO (vehicle) was included as negative control, while Mitomycin C (MMC) and Cyclophosphamide (CPA) were employed as positive control chemicals in the absence and presence of rat liver S-9 respectively. All acceptance criteria were considered met and the study was therefore accepted as valid.

Treatment of cells with the test item in the absence and presence of S-9 resulted in frequencies of cells with structural chromosome aberrations (excluding gaps) that were similar to and not significantly (p ≤ 0.05) higher than those observed in concurrent vehicle control cultures for the majority of concentrations analysed (all treatment regimens). A single exception to this was observed at the highest concentration analysed (2000 µg/mL) following 3+17 hour treatment in the absence of S9 where a weak but statistically significant increase was apparent. However, this weak increase was not reproduced at the same concentration and higher levels of mitotic inhibition following 20 hours treatment in the absence of S-9. Therefore, the isolated weak statistical increase observed following 3+17 hour treatment in the absence of S-9 was considered to be due to chance and of no biological importance. It is concluded that the test item did not induce biologically relevant increases in chromosome aberrations in cultured human peripheral blood lymphocytes following treatments in the absence and presence of a β-Naphthoflavone/Phenobarbital induced rat liver metabolic activation system (S9).

 

in vitro gene mutation study in mammalian cells

The ability of the test substance to induce gene mutations in mammalian cells was evaluated by considering data on Similar Substance 01, due to the absence of data on the substance itself; justification for Read Across is given in Section 13 of IUCLID.

The study was performed according to the OECD Guideline 476 and EU Method B.17. The test was performed in two independent experiments in the absence and presence of S9-mix. In the first experiment, the substance was tested up to a concentration of 5000 μg/ml in the absence and presence of 8 % (v/v) S9-mix. The incubation time was 3 hours. The test substance was tested up to cytotoxic levels of 65 and 89 % in the absence and presence of S9-mix, respectively. In the second experiment, the test substance was tested up to concentrations of 4000 and 5000 μg/ml in the absence and presence of 12 % (v/v) S9-mix, respectively. The incubation times were 24 hours for incubations without S9-mix and 3 hours for incubations with S9-mix. The substance was tested up to cytotoxic levels of 87 and 79 % without and with S9-mix, respectively.

With metabolic activation, the test substance did not induce a significant increase in the mutation frequency in the first and the repeat (1A). The substance induced a 4.6-fold increase in the mutation frequency in the first mutation experiment at 4000 μg/ml, with S9 -mix. The mutation frequency of 367 x 10^-6 was above the (GEF + MF(controls): 206 x 10^-6) of the negative controls and outside the historical control data range. This response fulfilled the criteria for a positive response. At this concentration, the substance increased mutation frequency of both the small and large colonies; this indicates increases in both chromosome aberrations and gene mutations. However, the relative total growth (RTG) at this dose was 11 % of controls and the next higher dose (5000 μg/ml) lacked a positive response even with an RTG of 24 %. In the second experiment, no significant increase in the mutation frequency at the TK locus was observed after treatment in the presence of S9-mix. The numbers of small and large colonies in the substance treated cultures were comparable to the numbers of these colonies of the solvent controls. Since no dose-related increase was observed in the first experiment and the increase was observed at one cytotoxic concentration only and could not be repeated in the independent repeat experiment, the biological relevance of this finding is doubtful. The results in the presence of S9-mix are considered equivocal. The test substance was not mutagenic in the absence of S9-mix.

GENETIC TOXICITY IN VIVO

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

The toxicity of Similar substance 01 and its ability to induce bone marrow suppression was evaluated according to the OECD Guideline 474. Test and control substance formulations were administered at a dose volume of 10 ml/kg/day by oral gavage. In the dose range finding assay (DRF), the maximum dose tested was 2000 mg/kg/day. The dose levels tested were 500, 1000 or 2000 mg/kg/day in 3 animals/sex. Following scheduled euthanasia times, femoral bone marrow was collected; bone marrow slides were prepared and stained with acridine orange. Bone marrow cells were examined microscopically. The ratio of polychromatic erythrocytes (PCEs) to total erythrocytes (EC) in the test substance groups relative to the vehicle control groups was evaluated to reflect the test substance’s cytotoxicity. No bone marrow suppression was observed in any of the groups. No appreciable reductions in the PCEs/EC ratio in the test substance groups compared to the vehicle control group was observed indicating the test article did not induce cytotoxicity. Thus, under the conditions of this study, the test article is non-toxic at and up to 2000 mg/kg in male and female rats.

Justification for classification or non-classification

The substance is not mutagenic in the in vitro gene mutation study in bacteria, in the in vitro chromosome aberration and in the in vitro gene mutation study in mammalian cells without metabolic activation. However, some effect was observed in the latest study with metabolic activation: in the first experiment the substance induced a 4.6-fold increase in the mutation frequency at 4000 µg/ml (cytotoxicity concentration). In the second experiment, no significant increase in the mutation frequency at the TK locus was observed after treatment with the substance in the presence of S9-mix. Since no dose-related increase was observed in the first experiment and the increase was observed at one cytotoxic concentration only and could not be repeated in the independent repeat experiment, the biological relevance of this finding is doubtful and the results are considered as equivocal. In the in vivo mammalian erythrocyte micronucleus test, the substance did not induce bone marrow suppression nor cytotoxicity. Thus, under the conditions of this study, the test article is non-toxic at and up to 2000 mg/kg in male and female rats.

According to the CLP Regulation (EC) No. 1272/2008 substances can be allocated in two categories for germ cell mutagenicity: Category 1- subcategories 1A and 1B and Category 2. This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

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

Category 1A: The classification in Category 1A is based on positive evidence from human epidemiological studies. Substances to be regarded as if they induce heritable mutations in the germ cells of humans

Category 1B: The classification in Category 1B is 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. It is possible to derive this supporting evidence from mutagenicity/genotoxicity tests in germ cells in vivo, or by demonstrating the ability of the substance or its metabolite(s) to interact with the genetic material of germ cells; or – 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. The classification in Category 2 is 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. 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.

According to the Guidance on the Application of CLP criteria (version 5.0, July 2017): 'in vitro results can only lead to a Category 2 mutagen classification in a case where there is support by chemical structure activity relationship to known germ cell mutagens. In the case where there are also negative or equivocal data, a weight of evidence approach using expert judgement has to be applied.

The substance does not show genotoxic effects in none of the performed experiments, for this reason it is not classified for mutagenicity according to the CLP Regulation (EC) No.1272/2008.