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Key value for chemical safety assessment

Additional information

in vitro:

- Gene mutation in bacteria: 2,6 -dichloro-4 -triflouromethylaniline (DCPTFMA) was tested for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA102 at concentrations ranging from 6.25 to 500 µg per plate (Rhône-Poulenc 1993, Val. 1). The tests were conducted using the pre-incubation and direct plate incorporation method, on agar plates in the presence and absence of an Aroclor 1254 induced rat liver preparation and co-factors (S9 mix) required for mixed-function oxidase activity. Positive control compounds demonstrated the sensitivity of the assay and the metabolising potential of the S9 mix. The negative and solvent control results were equivalent compared to historical data. The number of revertants induced by the positive controls was higher than the controls, indicating the sensitivity of the test system. The test substance 2,6 DCPTFMA did not induce any significant increase in the number of revertants, with S9 mix, in any of the 5 strains and without S9 mix, in the TA 1535 strain. However, for the 4 strains: TA 1537, TA 98, TA 100 and TA 102, without S9 mix, it induced a reproducible statistically significant dose-related increase in the number of revertants. Concluding that under these experimental conditions, the test substance 2,6 DCPTFMA is a direct mutagen.

- Chromosome Aberration: The objective of this in vitro study (Rhône-Poulenc, 1993, Val. 1) was to evaluate the potential of the test substance 2,6 DCPTFMA to induce chromosome breakage (clastogenesis) in cultured human lymphocytes, according to OECD/EEC guidelines (test 473). This test enables the detection of any chromosomal and chromatid structural aberrations

in the cells blocked at the metaphase stage. The test substance was tested with or without a metabolic activation system, the S9 mix prepared from a liver microsomal fraction (S9) of rats induced with Aroclor 1254. The conditions of treatment were as follows, using 2 cultures/experimental points: - without S9 mix: the test or control substances remained in the culture medium until the appropriate harvest times: 24 and 48 hours. - with S9 mix: the test or control substances remained in a culture medium containing 15% S9 mix for 2 hours. The cells were then rinsed and fresh medium added. The cultures were incubated until the appropriate harvest times: 24 and 48 hours. Two hours before harvesting, the cells were treated with a colcemid solution to block them at the metaphase-stage of mitosis. The chromosomal preparations were stained and screened microscopically for mitotic index and for aberrations: 200 well-spread metaphases per concentration were read, whenever possible. The concentrations of 2,6 DCPTFMA were: 3.125, 6.25, 12.5, 25, 50 and 100 µg/ml, 100 µg/ml being the limit of solubility of the test substance in the culture medium. The aberrant cells frequency in the negative and solvent controls was within the range of the historical data. The aberrant cells frequency in the positive controls was significantly higher (p< 0.001) than that of the negative controls, indicating the sensitivity of the test system. The test substance did not induce any significant increase in the aberrant cells frequency, with or without S9 mix for the 2 harvest times.

Concluding that under these experimental conditions, the test substance 2,6 DCPTFMA did not show clastogenic activity in this chromosomal aberration test performed in cultured human lymphocytes.

- Mammalian gene mutation: 2,6 DCPTFMA was assayed for its ability to induce mutations at the tk locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol (Rhône-Poulenc, 1995, OECD Guideline 476). The study consisted of two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9). The highest surviving dose was 80 µg/mL which yielded 4.0% and 3.5% relative survival

in the absence and presence of S-9 respectively. Negative (solvent) and positive control treatments were included in each mutation

experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline-1-oxide (without S-9) and benzo(a)pyrene (with

S-9). Therefore the study was accepted as valid. No statistically significant increases in mutant frequency were observed following

treatment with 2,6 DCPTFMA at any dose level in the absence or presence of S-9 in experiment 1 or 2. It is concluded that, under the conditions employed in this study, 2,6 DCPTFMA is not mutagenic in this test system.

in vivo: The work described was conducted to investigate the mutagenic potential of 2,6 -dichloro-4 -triflouromethylaniline (DCPTFMA) by assessment of its effect on the formation of micronuclei in bone marrow erythrocytes. The procedures and experimental design employed complied with the recommendations of the relevant toxicity testing guideline of the OECD (Guideline 474, 1983). The effect of 2,6 DCPTFMA on chromosome structure in bone marrow cells was investigated following acute oral administration to mice. Chromosome damage was measured indirectly by counting micronuclei. Adverse reactions to treatment were observed in mice dosed at 2000 mg/kg and 1000 mg/kg. No real indication of bone rnarrow toxicity, as evidenced by depression of bone marrow proliferation, was noted in any group treated with 2,6 DCPTFMA. Frequencies of micronucleated polychromatic erythrocytes in animals killed 24, 48 or 72 hours after administration of 2,6 DCPTFMA were similar to those in concurrent vehicle controls. This lack of treatment-related effect was apparent in both sexes, and was confirmed by statistical analysis. The sensitivity of the test was shown by statistically significant increases in the frequency of micronucleated polychromatic erythrocytes over control values in positive control group animals given chlorambucil at 30 mg/kg.

Conclusion:

The test substance was only genotoxic in an in vitro experiment using bacterial cells but not genotoxic in in vitro experiments using mammalian cells and in an in-vivo mouse micronucleus assay using bone marrow cells. Taking all results together DCPTFMA is considered not to be mutagenic.


Short description of key information:
in vitro: Ames Test: OECD guideline: positive
Chromosome Aberration Test in human cells: OECD guideline: negative
Mammalian gene mutation: OECD guideline: negative
in vivo: MNT-Test: OECD guideline: negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test substance was genotoxic in an in vitro experiment using bacterial cells but not genotoxic using mammalian cells and in an in-vivo mouse micronucleus assay using bone marrow cells.

As in the majority of the tests no genotoxic effects were observed, 2,6 -dichloro-4 -trifluoromethylaniline does not need to be classified according to Directive 67/548/EEC and according to Regulation (EC) No. 1272/2008.