2,2,2-trichloro-1-phenylethyl acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

The registered substance, 2,2,2-Trichloro-1-phenylethyl acetate (CAS No. 90-17-5) was tested non-mutagenic (negative) both in the presence and absence of S9 metabolic activation in an bacterial gene mutation study using Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 strains. The test was performed according to OECD TG 471 and GLP.

In vitro mammalian chromosome aberration study:

An in vitro cytogenicity study with the registered substance (CAS 90-17-5) according to OECD 473 is ongoing.

 

In vitro mammalian cell gene mutation assay:

The registered substance; (2,2,2-trichloro-1-phenylethyl) acetate (CAS No. 90-17-5), was tested non-mutagenic in an in vitro mammalian cell gene mutation test in the presence and absence of S9 metabolic activation system. The test was performed according to OECD 476 and in compliance with the OECD principles of Good Laboratory Practise (GLP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data available for the test chemicals was reviewed to determine the mutagenic nature. The studies are as mentioned below:

Bacterial reverse mutation test:

The mutagenic potential of the test substance, 2,2,2-Trichloro-1-phenylethyl acetate (CAS No. 90 -17 -5, E.C. no.: 201-972-0) has been tested on Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102. The test was performed according to OECD 471, adopted on July 21, 1997. The test was performed using the plate incorporation method (Trial I, Experiment I-II) and the preincubation method (Trial II, Experiment I-II) both in the presence and absence of S9 metabolic activation system. Test concentrations were selected based on the result of a preliminary cytotoxicity test with strains TA98 and TA100. In the pre-test, eight concentrations of the test substance, i.e., 0.0 (NC), 0.0 (VC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were tested in triplicates. Complete cytotoxicity was observed at 5 and 1.582 mg/plate in strains TA98 and TA100, both in the presence and absence of S9 metabolic activation system. At 0.501mg/plate, cytotoxicity demonstrated as a reduction in colony count and diminution in background lawn was noted with and without metabolic activation in TA98 and TA100 strains. There was no reduction in colony count or background lawn growth inhibition from 0.002 mg/plate to 0.158 mg/plate in both TA98 and TA100 in the presence and absence of S9 metabolic activation system. Based on the results of the pre-experiment, the following doses were selected for the main study trials: Experiment 1: 0.0(NC), 0.0(VC), 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate, both in the presence (+S9) and absence (-S9) of metabolic activation. In Experiment 2: Additional testing at 0.501mg/plate along with 0.0(NC), 00(VC) and positive controls, both in the presence (+S9) and absence (-S9) metabolic activation. The concentrations used in the experiment were spaced with (√10) half-log intervals. The bacterial strains were exposed to the test substance for 48 hours at 37 ± 2⁰C. The colonies were counted manually. The mean values of plates for each concentration, together with the standard deviation, were compared to the spontaneous reversion rates. Microsoft Office Excel-based calculation was used for descriptive statistical analysis. No significant increase in the revertant colony numbers of any of the tester strains was observed after the treatment with 2,2,2 -Trichloro-1 -phenylethyl acetate (CAS No. 90 -17 -5) at different dose concentrations in Experiment 1 and Experiment 2, both in the presence and absence of metabolic activation (S9 mix). The positive controls produced an expected increase in the number of revertant colonies, thus confirming the validity of the assay. Hence, the test substance 2,2,2 -Trichloro-1 -phenylethyl acetate (CAS No. 90 -17 -5) did not induce gene mutation within the histidine operon in Salmonella Typhimurium tester strains in the presence and absence of S9 metabolic activation system. The test was performed according to OECD TG 471 and GLP. The study was considered reliable without restrictions (Klimisch 1).

In vitro mammalian cell gene mutation assay:

An In vitro Mammalian Cell Gene Mutation Test of (2,2,2-trichloro-1-phenylethyl) acetate (CAS No. 90-17-5) was carried out according to OECD 476 to determine its ability to induce mutations at the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) locus in the genome of Chinese hamster ovary (CHO) cells. Based upon the preliminary tests conducted to assess the solubility/precipitation and cytotoxicity of test chemical, CHO cells were exposed to the test item in duplicate cultures at 500, 250, 125 and 62.5 μg/ml of culture medium, in the absence and presence of metabolic activation system (S9). Liver S9 fraction, prepared from; sodium phenobarbitone and b-naphthoflavone-induced Wistar rats, were used. The CHO cells were exposed to the test chemical for 3 hours at 37 ± 1°C, with approximately 5% CO₂ supply. Concurrent vehicle (acetone), untreated (distilled water) and positive control groups were also included in the experiment, as specified by the test guideline. Acetone was used as a vehicle. After treatment, cells were immediately subcultured to determine cytotoxicity and initiate phenotypic expression prior to mutant selection. Treatment-related cytotoxicity was determined by the relative survival (RS, i.e. cloning efficiency measured immediately after treatment and adjusted for any cell loss during treatment as compared to the negative control). The culture flasks were incubated at 37 ± 1oC with approximately 5% CO2 supply during experimental periods. The cultures were maintained in a growth medium and subcultured during the expression period at suitable intervals to allow near-optimal phenotypic expression of the induced mutation. After phenotypic expression, about 2 million cells were seeded in a medium containing the selective agent (6-Thioguanine) to detect mutant copies, and 150 cells were seeded in medium without a selective agent to determine the cloning efficiency (viability). These plates were incubated at 37 ± 1oC with approximately 5% CO2 supply for 9 days. After incubation, the medium was discarded, and the plates were stained and observed for the number of clones. The relative survival (RS) and mutant frequency (MF) were calculated for all treatment, vehicle control, untreated control, and positive control groups. In the preliminary cytotoxicity test, the treatment with 500 μg/ml of (2,2,2-trichloro-1-phenylethyl) acetate (CAS No. 90-17-5) induced 10 to 20% RS (relative survival) both in the presence and absence of S9 metabolic activation system. The % relative survival (% RS) for the cloned cultures were 14.26, 42.76, 52.80 and 83.82% and 19.41, 41.56, 58.37, and 78.15% at 500, 250, 125 and 62.5 μg/ml for cultures treated without and with S9 metabolic activation, respectively. The concentration that yielded 10-20% RS was chosen as the highest test concentration in the main test and hence, 500 μg/ml was selected as the highest test concentration to assess mutagenicity. Applying spacing factor 2 the following concentrations were tested in the mutagenicity test with and without S9 metabolic activation: 0 (NC), 0 (VC), 62.5, 125, 250 and 500 μg/ml. In the mutagenicity test, the mutant frequency for cultures treated with (2,2,2-trichloro-1- phenylethyl) acetate (CAS No. 90-17-5) in the absence of the S9 metabolic activation was 7.65, 13.67, 10.44 and 11.01 mutants/106 cells at 62.5, 125, 250 and 500 μg/ml, respectively. The mutant frequency of untreated control, vehicle control and positive control in the absence of metabolic activation was 10.12, 9.39 and 102.63 mutants/106 cells, respectively. The mutant frequency for cultures treated with (2,2,2-trichloro-1-phenylethyl) acetate (CAS No. 90-17-5) in the presence of the metabolic activation 7.31, 5.73, 10.09 and 8.51 mutants/106 cells at 62.5, 125, 250 and 500 μg/ml respectively. The mutant frequency of control, vehicle control and positive control in the presence of metabolic activation was 6.43, 8.14 and 164.38 and mutants/106 cells, respectively. Thus, the present study results indicated that; there was no significant difference in the mutant frequencies of cultures treated with (2,2,2-trichloro-1-phenylethyl) acetate (CAS No. 90-17-5) as compared to the vehicle control cultures, either in the absence or presence of metabolic activation. The mutant frequency, relative survival (cytotoxicity) in the vehicle and untreated control groups were comparable with the range reported in the literature. An increase in the mutant frequency of concurrent positive control(s), demonstrated the sensitivity of the assay in the absence and presence of metabolic activation. Based on the above results, it was concluded that the test chemical, (2,2,2-trichloro-1- phenylethyl) acetate (CAS No. 90-17-5) did not induce gene mutation at the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) locus in cultured Chinese hamster ovary (CHO) cells neither in the presence nor in the absence of S9 metabolic activation system.

Justification for classification or non-classification

The registered substance, 2,2,2-Trichloro-1-phenylethyl acetate (CAS No. 90-17-5) is tested non-mutagenic (negative) in bacterial and mammalian cells in vitro. An in vitro cytogenicity test according to OECD 473 is ongoing.