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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity- in vitro:

- Bacterial reverse mutation assay: C.M. Verspeek-Rip (2003) tested the substance in the Salmonella typhimurium reverse mutation assay with four histidine requiring strains of Salmonella typhimurium and in Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. Based on the results of this K1 study, performed according to OECD guideline 471 and EEC Directive 2000/23/EC, Part B: Methods for the Determination of Toxicity; B.13/14, it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
- Chromosome aberration assay: C.A.F. Buskens (2003) evaluated the substance for its ability to induce structural chromosome aberrations in cultured human lymphocytes. The study, performed according OECD Guideline 473 and EEC Directive 2000/32/EC, Part B, concluded that the substance is not clastogenic in human lymphocytes under the experimental conditions tested.

- Gene mutation study in mammalian cells: The substance was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transfrase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay (Dutta, 2015; OECD 476).

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

Genetic toxicity in vivo

Description of key information

Genetic toxicity - in vivo:

- Mouse bone marrow erythrocytes micronucleus test: Krsmanovic (2011) evaluated the substance for its genotoxic potential (clastogenicity/aneugenicity) as measured by its ability to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female ICR mice. The study, performed according to OECD Guideline 474 and ICH S2A, ICH S2B, concluded that a single oral administration of the substance at doses up to and including a dose of 500 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of male or female ICR mice. Therefore, the substance was concluded to be negative in the mouse micronucleus assay.

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

Additional information

Genetic toxicity in vitro:

Bacterial reverse mutation assay:

The substance was tested in Salmonella typhimurium resverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA 1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9 -mix (Aroclor-1254 induced rat liver S9 -mix).

In the dose range finding test, the substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 -mix in the strains TA100 and WP2uvrA. The substance did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the first and in the second mutation assay, the substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 -mix. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed. The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings. The substance did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9 -metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study, it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Chromosome aberration assay:

Buskens (2003) investigated the effect of the substance on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9 -mix). The possible clastogenicity of the substance was tested in two independent experiments. In the first cytogenetic assay,

the substance wa

s tested up to 1000 µg/mL for a 3h exposure time with a 24h fixation time in the absence and presence of S9 -mix. Appropriate toxicity was reached at this dose level.

In the second cytogenetic assay, the substance was tested up to 800 µg/mL for a 24h continuous exposure time with a 24 h fixation time and up to 875 µg/mL for a 48 h continuous exposure time with a 48h fixation time in the absence of S9 -mix. In the presence of 1.8% (v/v) S9 -fraction the substance was tested up to 1250 µg/mL for a 3h exposure time with a 48h fixation time. Appropriate toxicity was reached at these dose levels.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9 -mix) functioned properly.

The substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9 -mix, in two independently repeated experiments.

In the first cytogenetic assay the substance increased the number of polyploid cells both in the absence and presence of S9 -mix. This may indicate that the substance has the potential to disturb mitotic processes and cell cycle progression, thereby inducing numerical chromosome aberrations.

Finally, it is concluded that the substance is not clastogenic in human lymphocytes under the experimental conditions of this test.

In vitro gene mutation study in mammalian cells:

Dutta (2015) performed an in vitro gene mutation study in mammalian cells according to OECD guideline 476. The substance was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay.

Genetic toxicity in vivo:

Mouse Bone Marrow Erythrocyte Micronucleus Test Following Oral Administration of XTJ-568:

Krsmanovic (2011) evaluated the substance for its genotoxic potential (clastogenicity/aneugenicity) as measured by its ability to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female ICR mice. The micronucleus test was conducted in two phases: a dose range finding (DRF) study and a definitive micronucleus study/assay.

In order to evaluated toxicity of the test article and set the dose levels for the definitive micronucleus assay, the dose range finding study (DRF study) was conducted initially exposing five male and five female mice to the substance at 2000 mg/kg. As mortality was observed at this dose, 5 animals/sex were orally exposed to the substance at 1000 mg/kg. Since mortality was also observed at this dose, five animals/sex/group were exposed to the substance at 500 and 750 mg/kg.

Based on the mortality and clinical signs observed in the DRF study, a dose of 500 mg/kg was determined to be the maximum tolerated dose and was used as high dose in the definitive micronucleus study. Two lower doses, one fourth and one half of the highe dose, 125 and 150 mg/kg were also tested.

The definitive study consisted of 7 groups each containing 5 animals/sex. Mice in five of these groups were exposed to either the control articles (vehicle or positive) or to dose levels of 125, 250 or 500 mg/kg the substance and were euthanized at 24 hours post-dose. Mice in the remaining two groups were dosed either with vehicle control or the substance at a dose of 500 mg/kg and were euthanized at 48 hours post-dose. In addition, 5 animals/sex were dosed with the substance at 500 mg/kg to be used as replacement group, in the event of mortality at the high dose. As no mortality was observed, these animals were euthanized without bone marrow collection. Following scheduled euthanasia, bone marrow smears (slides) were prepared and stained with acridine orange stain (nucleic acid specific). Bone marrow cells (polychromatic erythrocytes (2000 PCEs/animal)] were examined microscopically for the presence of micronuclei (micronucleated PCEs; MPCEs). A statistical analysis of data was performed using the Kastenbaum-Bowman tables (binomial distribution, p< or = 0.05).

Under the conditions of the study a single oral administration of the substance at doses up to and including a dose of 500 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of male or female ICR mice. Therefore, the substance was concluded to be negative in the mouse micronucleus assay.

Justification for classification or non-classification

Based on the results of the above mentioned studies, the substance does not need to be classified for genetic toxicity (germ cell mutagenicity).