Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

non-mutagenic with and without metabolic activation (Ames)

clastogenic with metabolic activation, non-mutagenic without metabolic activation (Chromosome aberration)

ambiguous with metabolic activation, non-mutagenic without metabolic activation (Gene mutation-mammalian cells)

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 in vitro gene mutation study in bacteria was evaluated based on the data on the substance itself and on Similar Substance 01. Justification for Read Across is given in Section 13 of IUCLID.

The study was conducted according to the OECD Guideline 471 and ICH genotoxicity Guideline S2B in compliance with GLP. Tester strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium and strain WP2uvrA of Escherichia coli in the presence and absence of S9 were exposed to the test substance at 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate during the initial toxicity-mutation assay and at 50, 150, 500, 1500 and 5000 μg/plate during the confirmatory mutagenicity assay. The assay was performed in two phases, using the plate incorporation method. No mutagenic responses were observed with any of the tester strains in the absence and presence of S9 in any of the two phases. Neither precipitate nor appreciable toxicity was observed. Non mutagenic with and without metabolic activation.

A 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. 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 5000 μg/plate. No mutagenic responses were observed with any of the tester strains in the absence and presence of S9. In the confirmatory mutagenicity assay, no mutagenic responses were observed. The dose levels tested were 50, 150, 500, 1500 and 5000 μg/plate. 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 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.

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. Although both in the absence and presence of S9-mix the number of cells with chromosome aberrations was within the historical control data (per 200 metaphases) it should be noted that chromosomal exchanges are comparatively rare spontaneous events and greater significance should be attached to the observation of exchange figures. Therefore, the increases observed after treatment with 3750 µg in the presence of S9-mix are considered relevant. In the second cytogenetic assay the test material 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, one exchange figure was observed in one culture at a concentration of 4500 µg/ml. It was noted that the substance increased the number of polyploid cells in the presence of S9-mix in a dose dependent manner. The test substance is clastogenic in human lymphocytes only in the presence of S9-mix under the experimental conditions; the substance may have the potential to disturb mitotic processes.

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 the test substance and its ability to induce micronuclei in polychromatic erythrocytes in rat bone marrow following 3 consecutive days of treatment administered by intraperitoneal injectionwas evaluated according to the OECD Guideline 474 and the ICH guideline S2 (R1). The animals were acclimatised for 7 days. In the range-finding phase, the test substance in glycerol formal, was injected peritoneally once daily for 3 consecutive days at dosage levels 500, 1000 and 2000 mg/kg/day for Groups 2-4 respectively, each consisting of 3 animals/sex. All surviving animals were euthanised and discarded without necropsy, 18-24 hours after the last dose administration (study day 3). In the definitive phase, test substance was administered in the same dosage levels and same frequency to Groups 2-4 and a concurrent vehicle group (Group 1) received the vehicle on a comparable regime. A positive control group (Group 5) received a single oral dose of 60 mg/kg/day of cyclophosphamide on study day 2. All animals were observed twice daily for mortality and morbidity. Mortality, body weight loss and several clinical observations seen when PEG 400 was used as the vehicle. During the range-finding phase following study restart, mortality was noted in the test substance-treated group and 1 male in the 2000 mg/kg/day group was found dead. During the definitive phase, all animals survived until scheduled euthanasia on study day 3. Decreased activity, reddened left ear (males only) and impaired muscle coordination seen in the 2000 mg/kg/day male and female group. Clinical observations of flushed extremities noted for 3 animals in the 2000 mg/kg/day group. The test substance did not increase the mean number of micronucleated polychromatic erythrocytes compared to the vehicle control group and no bone marrow cytotoxicity noted in the test substance-treated group. The test substance met the criteria for a negative response for bone marrow cytotoxicity and clastogenicity under the conditions of the assay.

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 was found to be non-mutagenic in the in-vitro gene mutation study in bacteria, in the in-vitro chromosome aberration study without metabolic activation and in the in-vitro gene mutation study in mammalian cells also without metabolic activation. However, effects in the latest two studies were observed only in the presence of metabolic activation. In the in-vitro chromosome aberration study the number of cells with chromosome aberrations was not statistically significant increased, however, exchange figures were observed at a concentration of 3750 µg/ml (in the first experiment) and one exchange figure was observed in one culture at a concentration of 4500 µg/ml (in the second experiment). The substance increased the number of polyploid cells in the presence of S9 mix in a dose dependent manner and this may indicate that it has the potential to disturb mitotic processes. In the in-vitro gene mutation study in mammalian cells, 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 increase the mean number of micronucleated polychromatic erythrocytes compared to the vehicle control group and no bone marrow cytotoxicity noted in the test substance-treated group. The test substance met the criteria for a negative response for bone marrow cytotoxicity and clastogenicity under the conditions of the assay.

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 was found positive only in the presence of metabolic activation, in the chromosome aberration study in mammalian cells. However, the substance resulted as not mutagenic in the in vivo mammalian erythrocyte micronucleus test. The substance gave a negative response for bone marrow cytotoxicity and clastogenicity.

The substance is not classified for mutagenicity according to the CLP Regulation (EC) No.1272/2008.