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This study was conducted to assess the test article for its ability to cause point (gene) mutation in Salmonella typhimurium and E. coli. The study was conducted in compliance with OECD test guideline 471; EC Directive 2000/32/EC Annex 4D-B.13/14 No. L 136/57; OPPTS 870.5100; JMAFF no. 8147; ICH guideline S2A and S2B. Histidine dependent auxotrophic mutants of Salmonella typhimurium strains TA 1535; TA 1537; TA98; and TA100 and a tryptophan dependent mutant of E. coli strain WP2 uvrA (pKM101) were exposed to the test article in vapor phase. Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with Aroclor following preliminary tests to determine the maximum achievable vapor concentration and toxicity towards tester bacteria. All tests were modified plate incorporation assays. Nominal concentrations of the test article up to 70% volume v/v (nominal) were tested in the main tests. This was the maximum achievable concentration at 37C under reduced atmospheric pressure. Other concentrations used were 52.5; 35; 26.25; and 17.5% v/v (nominal). Toxicity (observed as a reduction in revertant colony numbers) was clearly seen in strains TA98 and TA100 following exposure to the test article at 5000ug/plate (70% v/v) in the second main test. Based on the results of the study, the test article is not mutagenic in the Ames assay with or without metabolic activation.

 

The study was conducted to assess the ability of the test article (Lot 4F-11379) to induce chromosomal aberrations in human lymphocytes cultured in vitro. Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance in the presence and absence of S-9 mix derived from rat livers. 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 from chromosomal damage. The mitotic index was calculated for all cultures treated with the test substance and solvent control. 1st test: 3 hour treatment, 17 hour recovery: without S9 mix = 15, 17, and 19 % v/v; with S9 mix = 15, 17, and 19% v/v. No statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at any dose level as compared to the solvent control. No statistically significant increases in polyploid metaphases were observed during metaphase analysis. Based on the results of the study, the test article showed no evidence of clastogenic activity in the presence or absence of metabolic activation.

 

The test article was evaluated for mutagenic potential in the L5178Y mouse Lymphoma Mutagenesis assay in the presence and absence of a metabolic activation system (S9 -mix). The study was performed in compliance with OECD GLP regulations. The test method was based on OECD 476 (1997). The cells were exposed to the test article as a vapor at 2.5, 5, 10, 20, or 30 % (v/v). Two independent tests were conducted. In the first test, five duplicate cultures were treated for 4 hours in the presence and absence of S9 -mix, and 24 hours in the absence of S9 -mix. In the second test (repeat of the 4 hour treatment group), five duplicate cultures were treated in the absence of S9 -mix. Methyl methanesulphonate (MMS) and 3 -methylcholanthrene (MCA) were used as positive control substances in the absence and presence of S9 -mix, respectively and clean air served as the negative control in all tests. In the first test in the 4 hour treatment group in the absence of S9 -mix, the criteria for the positive control were not met. Therefore, this part of the test was repeated. All criteria were met in the second test. In the presence of S9-mix, the initial cell yield and/or relative suspension growth and/or relative total growth were not reduced when compared to the negative control. In the absence of S9-mix after 4 hour treatment, test substance was slightly toxic to the cells resulting in a mean RTG at the highest concentration evaluated (30%) of 84% (first experiment) or 88% (repeat experiment). In the absence of S9-mix after 24 hour treatment however, the initial cell yield and/or relative suspension growth and/or relative total growth were reduced by more than 10% at and above 10% test article (v/v). The three highest dose levels of the test substance evaluated for mutagenicity were 10, 20, and 30% (v/v); the RTG at these doses were 27%, 3%, and 0.2%, respectively. In the first and second experiment, in the absence and presence of S9 -mix after 4 hour treatment, no increase in the mutant frequency by more than 88 or 126 mutants per 1 million clonable cells (no equivocal or positive response) compared to the negative control was observed at any dose level. In the absence of S9 -mix, after 24 hours treatment, at the highest concentration of 30% (v/v), an increase in the mean mutant frequency by more than 88 but less than 126 mutants per 1 million clonable cells was observed. The increase in mutant frequency was, however only observed at a concentration causing more than 99% cytotoxicity (RTG value <1%). Therefore, the increase is considered to be not biologically relevant. At concentrations up to 99% cytotoxicity no increase in mutant frequency was observed. Based on the results of the study, the test article is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the presence or absence of metabolic activation (S9 -mix).


Short description of key information:
In vitro genetic toxicity studies have been conducted on HFP Kinetic Dimer. The results of the studies are:

Bacterial Reverse Mutation Assay: Negative when tested according to OECD 471.

Chromosome Aberration Study in Cultured Peripheral Human Lymphocytes: Not clastogenic when tested according to OECD 473:

Mouse Lymphoma Mutagenesis Assay: Negative when tested according to OECD 476.

Endpoint Conclusion:

Justification for classification or non-classification

Criteria for classifying the test article as mutagenic are not met.