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In vitro Mammalian Cell Gene Mutation Test

The potential of the test material to cause gene mutation in Chinese hamster lung fibroblasts (V79) at the Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus was investigated in accordance with the standardised guideline OECD 476 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The Chinese hamster lung fibroblasts (V79) were exposed to the test material in ethanol in both the presence and absence of metabolic activation at the following concentrations:

- Experiment I: 10.0, 30.0, 100.0 and 300.0 µg/mL (without S9 mix); 30.0, 200.0, 300.0 and 450.0 µg/mL (with S9 mix).

- Experiment II: 30.0, 300.0, 350.0 and 400.0 µg/mL (without S9 mix); 30.0, 100.0, 200.0, 300.0 and 600.0 µg/mL (with S9 mix).

The mutation rates found in the groups treated with the test material were comparable to the negative and solvent controls. It was concluded that no relevant increase of gene mutation was observed. 

Under the conditions of this study, 2,6-xylenol did not induce gene mutations at the HPRT locus in V79 cells and was considered non-mutagenic in both the presence and absence of metabolic activation.  

 

In vitro Mammalian Chromosome Aberration Test

The potential of the test material to cause chromosome aberration in Chinese hamster lung fibroblasts (V79) was investigated in accordance with the standardised guideline OECD 473 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The Chinese hamster lung fibroblasts (V79) were exposed to the test material in ethanol in both the presence and absence of metabolic activation at the following concentrations which were evaluated for cytogenetic damage:

- Experiment I and II: 18 hours at 10, 30 and 100 µg/mL, 28 hours at 100 µg/mL (without metabolic activation).

- Experiment I: 18 hours at 30, 100 and 300 µg/mL, 28 hours at 600 µg/mL (with metabolic activation).

- Experiment II: 18 hours at 100, 300 and 400 µg/mL, 28 hours at 300 µg/mL (with metabolic activation).

In both experiments in the absence of S9 mix, 2,6-xylenol did not induce biologically relevant increases of the frequency of cells with aberrations. The aberration frequencies were in or near to the range of solvent control values and in or near to the range of the laboratory's historical control data. The statistical evaluation showed a significant difference between the treatment group 100 µg/mL (28 hours) in experiment II versus the corresponding solvent control; however, this was regarded as being not biologically relevant, since the % aberrant cells in this treatment group was within the laboratory's historical control data range.

In the presence of S9 mix in both experiments, statistically significant and biologically relevant increases in the aberration frequencies were observed after treatment with 2,6-xylenol (Exp. I: 28 hours at 600 µg/mL, 8.5 %; Exp. II: 18 hours at 400 µg/mL, 14.5 %; 28 hours at 300 µg/mL, 6.0 %). In addition, a distinct increase in cells carrying exchanges was found (Exp. I: 28 hours at 600 µg/mL, 3.0 %; Exp. II: 18 hours at 400 µg/mL, 7.5 %) as compared to 1.0 and 0.0 %, respectively, in the corresponding solvent controls.

Under the conditions of this study, the test material was found to be negative for chromosome aberration in the absence of metabolic activation and positive for chromosome aberration in the presence of metabolic activation.

  

In vitro Bacterial Reverse Mutation Assay

The potential of the test material to cause mutation in bacteria was investigate in accordance with the standardised guideline OECD 471 under GLP conditions. As only S. typhimurium strains (TA1535, TA1537, TA98 & TA100) were used, the study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The S. typhimurium strains were exposed to the test material in ethanol in both the presence and absence of metabolic activation at concentrations of 10.0, 33.3, 100.0, 333.3, 1000.0, 2500.0, and 5000.0 µg/plate. Both the plate incorporation and preincubation methods were used.

The test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Under the conditions of this study, 2,6-xylenol is considered to be non-mutagenic in both the presence and absence of metabolic activation.

 

In vivo Mammalian Erythrocyte Micronucleus Test

The potential of 2,4,6 trimethyl phenol, a structural analogue of the registered material, to exhibit mutagenic properties in the mouse was investigated in accordance with the standardised guideline OECD 474 under GLP conditions. 2,6-Xylenol and the structural analogue were determined to have sufficiently similar properties, such that available data on the structural analogue is considered to be suitable to address this endpoint. Due to the use of read across, the study was awarded a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Male and female ICR mice were exposed to the test material in corn oil via the intraperitoneal route at dose levels of 125, 250, or 500 mg/kg. Five animals per sex were dosed in the lower dose groups and 10 animals per sex were dosed with the highest dose level tested.

Mice were sacrificed after 24 hours (and 48 hours in the highest dose group) and evaluated for micronuclei in the bone marrow. Bone marrow cells, polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) were analysed for the presence of micronuclei.

No appreciable reductions in the ratio of PCEs to total erythrocytes was observed in the test material-treated groups relative to the vehicle control groups, suggesting that the test material did not inhibit erythropoiesis.

Under the conditions of this study, the test material did not exhibit any mutagenic activity in mice in the in vivo micronucleus assay.

 

In vivo Mammalian Bone Marrow Chromosome Aberration Test

The potential of the test material to cause chromosome aberrations in the rat was investigated using methodology comparable to that outlined in the standardised guideline OECD 475 under GLP conditions. However, only 50 (instead of 100) cells were scored per animal in the cytogenetics assay and a minimum of 500 (instead of 1000) cells were counted to determine the mitotic index. Therefore the study was awarded a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Male and female Sprague-Dawley rats were exposed to the test material via gavage in corn oil at concentrations of 350, 700 and 1400 mg/kg and 300, 600 and 1200 mg/kg, respectively (5 animals per sex per dose per exposure time). Bone marrow was collected 12, 24 and 36 hours after treatment.

The percentage of cells with structural chromosomal aberrations was not increased in the 2,6-xylenol treated animals, regardless of sex, dose or sacrifice time.

Under the conditions of this study, the test material did not exhibit any mutagenic activity in rats in the in vivo chromosome aberration test.

Discussion

The available in vitro bacterial and mammalian gene mutation data indicate that 2,6-xylenol is non-genotoxic both with and without metabolic activation. Although an in vitro chromosome aberration study showed a mutagenic response in the presence of metabolic activation, both an in vivo chromosome aberration study and an in vivo micronucleus study (using a structural analogue) indicate that 2,6-xylenol is non-mutagenic.

The weight of evidence therefore indicates that 2,6-xylenol is non-mutagenic.


Justification for selection of genetic toxicity endpoint
No single key study was selected on the basis that the available studies all address different aspects of genetic toxicity and the data should be considered as a whole. All studies were conducted in accordance with standardised guidelines under GLP conditions, with the exception of the mammalian bone marrow chromosome aberration test which was carried out under GLP conditions using methodology comparable to that of the standardised guideline.

Short description of key information:
IN VITRO
In a mammalian cell gene mutation test, 2,6-xylenol did not induce gene mutations at the HPRT locus in V79 cells and was considered non-mutagenic in both the presence and absence of metabolic activation.
In a mammalian chromosome aberration test the test material was found to be negative for chromosome aberration in the absence of metabolic activation and positive for chromosome aberration in the presence of metabolic activation in V79 cells.
In a bacterial reverse mutation assay 2,6-xylenol was non-mutagenic in both the presence and absence of metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100.

IN VIVO
In a mammalian erythrocyte micronucleus test a structural analogue of the registered material did not exhibit any mutagenic activity in mice.
In a mammalian bone marrow chromosome aberration test, the test material did not exhibit any mutagenic activity in rats.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to mutagenicity.