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Key value for chemical safety assessment

Additional information

In vitro: Reverse mutation assay

In a reverse mutation assay, performed according to OECD Guideline 471 and GLP, salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material using the desiccator methodology, a modification of the Ames plate incorporation methodology, in the presence and absence of S9 -mix (BioReliance 2011). The assay was performed in three phases: a preliminary toxicity assay, a mutagenicity assay and a confirmatory mutagenicity assay. Based on the findings of the preliminary toxicity assay, the maximum dose plated in the confirmatory mutagenicity assay was 37 mmoles/L (905,000 ppm). In the mutagenicity and confirmation assay, no precipitation was observed and toxicity was observed beginning at 11, 21 or at 37 mmoles/L. In the mutagenicity and confirmation assay, no positive mutagenic response was observed. In conclusion, under the conditions of this study, the test article did not cause a positive mutagenic response in either the presence or absence of Aroclor-induced rat liver S9.

In vitro: Chromosome aberration test

An in vitro chromosome aberration study according to OECD Guideline 473 and GLP was performed (Huntington Life Sciences, 2011). Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Solvent and positive control cultures were also prepared. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. In the first test, cells were treated with 0, 60.89, 101.48, 169.13, 281.88, 469.80, 783, and 1305 ug/mL and in the second test with 0, 169.13, 281.88, 469.80, 783, and 1305 ug/mL. In the first test, cells were treated for three hours followed by 18 hour recovery. In the second test, cells were treated for three hours followed by 18 hour recovery in the presence of S9 mix, and in the absence of S9 mix cells were continuously treated for 21 hours.The dose levels selected for the metaphase analysis were 469.80, 783, and 1305 μg/mL. In both the absence and presence of S9 mix, the test substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the solvent control, in either test. No statistically significant increases in the proportion of polyploid cells were observed during metaphase analysis, in either test. All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix. It is concluded that the test substance has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

In vivo: Micronucleus assay (mouse)

In a GLP-compliant erythrocyte micronucleus test, performed according to OECD guideline 474, two groups of 10 male CD-1 mice were exposed to the test substance at concentrations of 0 or 50000 ppm via inhalation for 4 hours (TNO, 2009c). 24 and 48 hours after exposure 5 mice per group were sacrificed. A positive control group consisted of 5 males treated with a single intraperitoneal dose of mitomycin C (0.75 mg/kg bw) and sacrificed 24 hours after injection. The group mean numbers of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) in the positive control group showed a statistically significant increase compared to the negative control group. The mean number of MPE per 2000 PE in the negative control group was within the historical control range. Therefore, the study was considered valid. Both at 24 and 48 hours after inhalation exposure, no statistically significant differences between the test substance and the negative control group were found with respect to the mean number of PE per 200 erythrocytes (E) and the mean number of MPE per 2000 PE. It was therefore concluded that, under the conditions of this study, inhalation exposure to the test substance for 4 hours at a concentration of 50000 ppm, resulting in a dose of about 65800 mg/kg body weight, which far exceeded the limit dose of 2000 mg/kg body weight, did not induce damage to the chromosomes and/or mitotic spindle apparatus (micronuclei) in the bone marrow target cells of male mice.

In vivo: Micronucleus assay (rat)

In a GLP-compliant erythrocyte micronucleus test, performed according to OECD guideline 474, five groups of Sprague-Dawley rats were exposed to the test substance at concentrations of 0, 2000, 4500, 7500, 10000 ppm via inhalation 6 hours/day on five days/week for four weeks TNO, 2009b). The group mean numbers of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) in the positive control group (Mitomycin C; 1.5 mg/kg bw, administered once intraperitoneally) showed a statistically significant increase compared to the negative control group. The mean numbers of MPE per 2000 PE in the negative control group were within the historical control range. Therefore, the study was considered valid. The results showed no statistically significant differences in the number of MPE per 2000 PE between the test substance treated groups and the negative control group (clean air). The number of PE per 200 erythrocytes in the positive control group was statistically significantly lower than that in the negative control group, indicating that the positive control substance induced cytotoxicity to the bone marrow. Though the mean numbers of PE per 200 erythrocytes in the groups exposed to the test substance showed some marginal decreases compared to negative controls (at 4500 and 10000 ppm), a clear treatment-related cytotoxicity to the bone marrow cells could not be demonstrated. It was concluded that, under the conditions of this study, the test substance at exposure levels up to 10000 ppm, resulting in a dose of 12932 mg/kg-bw/day, which far exceeded the limit dose of 1000 mg/kg-bw/day, did not induce damage to the chromosomes and/or mitotic spindle apparatus (micronuclei) in the bone marrow target cells of male rats.

In vivo: Unscheduled DNA synthesis test

In a GLP compliant unscheduled DNA synthesis-test performed according to OECD Guideline 486, the test substance was examined for its potential to damage DNA as reflected by induced DNA synthesis to repair the damage using hepatocytes harvested from exposed male Sprague-Dawley rats (TNO, 2009b). Groups of five male rats were exposed by inhalation at concentrations of 0 (clean air, negative control), 7500 or 10000 ppm 6 hours/day on 5 days per week for four weeks. An additional group of the same size was treated once by oral gavage with the positive control substance 2-Acetylaminofluorene (2-AAF). The mean net nuclear grains (NNG) in the 7500 and 10000 ppm groups (-8.33 and -7.47, respectively) were comparable to the mean NNG in the negative control group (-8.30) and clearly lower than the mean NNG in the positive control group (-3.63). The percentage ‘cells in repair’ in the 7500 and 10000 ppm groups (1.00% and 0.60%) was comparable to that in the negative control group (1.80 % ‘cells in repair’) and clearly lower than that in the positive control group (24.51 %). It is concluded that under the conditions of this study the test substance did not induce unscheduled DNA synthesis (UDS) in hepatocytes of male rats.


Short description of key information:
The test substance gave negative results in the Ames test with Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and Escherichia coli strain WP2 uvrA, with and without metabolic activation. Negative results were also observed in the chromosome aberration test with human lymphocytes in the presence and absence of metabolic activation. In vivo, the test substance was negative in a micronucleus test in mouse and rat and in an unscheduled DNA synthesis test in rat.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data and in accordance with Directive 67/548/EEC (DSD) and EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not necessary for mutagenicity.