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Administrative data

Key value for chemical safety assessment

Additional information

Genetic toxicity in bacteria:

3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl 2-chloropropenoic acid ester (CAS 96383-55-0) was tested for its mutagenic properties in bacteria in a study according to OECD 471 and in compliance with GLP (Verspeek-Rip, 2013). S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli strain WP2 uvrA were treated with the test substance diluted in DMSO up to 5000 µg/plate in 2 independent experiments (standard plate test) in the presence and absence of metabolic activation (S9-mix prepared from the livers of rats treated with Phenobarbital and Naphthoflavone). No precipitation was observed up to 5000 µg/plate and no cytotoxicity (reduction of bacterial background lawn, number of revertants) was observed in any strain at any concentration. No increase in the number of revertants was observed with the test substance in any strain at any concentration. The solvent control and the positive controls were valid. In conclusion, the test substance was not mutagenic in bacteria in the presence and absence of metabolic activation under the experimantal conditions of the study.

Chromosome aberration in mammalian cells:

A chromosome aberration test according to OECD 473 and in compliance with GLP was performed with 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl 2-chloropropenoic acid ester in Chinese hamster lung cells (CHL/IU) (Yasunga, 2013). In an initial call growth inhibition test the cells were treated with 3.91 , 7.81, 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL test item dissolved in dehydrated acetone for 6 h followed by a substance-free post incubation period of 18 h and at 0.488, 0.977, 1.95, 3.91, 7.81, 15.6, 31.3 and 62.5 µg/mL test item for a duration of 24 h. The experiment was performed in the presence and absence of a cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital and 5,6-benzoflavone. As a result of the cell growth inhibition test, the selected concentrations for the chromosomal aberration test were 5, 10, 15, 20, 25, 30 and 50 µg/mL in the presence of S9 mix and 25, 50, 100, 150, 200 and 250 µg/mL in the absence of S9 mix.

The treatment period in the first experiment was 6 h. 22 h after begin of treatment colcemid (0.1 µg/mL) was added to the test culture to inhibit spindle formation. 24 h after begin of treatment cells were fixed and stained with Giemsa, whereby 2 replicates per concentration were made. For assessment of chromosome aberration and cytotoxicity (mitotic index) 200 metaphases and 1000 cells per concentration were evaluated, respectively. Based on the results of the cell growth assay and the preliminary observations, the concentrations chosen for the metaphase analysis were 15, 20 and 25 µg/mL without S9 mix and 100, 150 and 200 µg/mL with S9 mix. In the absence of S9-mix an increase of aberrant cells was observed from 1% (solvent control) to 4.5%, 5.5%, and 27.5% after treatment with 15, 20, and 25 µg/mL test item, respectively. The mitotic index was in the range from 40.1 to 66.4%. Based on the evaluation criteria given a positive response is available as a dose-related response was observed and the ratio of aberrant cells was >10% at 25 µg/mL. In the presence of S9-mix an increase of aberrant cells was observed from 1% (solvent control) to 6.5%, 4.0%, and 4.5% after treatment with 100, 150, and 200 µg/mL test item, respectively. As no dose-response was observed and the increase of aberrant cells was <10% at all concentrations the result was evaluated as inconclusive, as described in the evaluation criteria applied. The mitotic index varied between 36.6 and 81.3% in the experiments with metabolic activation. The solvent and positive controls were valid and were within the historical control data. No substance precipitation was observed at any dose level, and no other confounding factors have been identified. Because the result of the short term treatment (6 h) in the first experiment was positive, the continuous treatment assay for 24 h to identify any chromosomal aberrations was not conducted in a second experiment. In conclusion, the test item did induce chromosome aberrations in the absence of metabolic activation under the experimental conditions of the study chosen.

Micronucleus test in vivo:

For further assessment of the structural effects on chromosomes, as seen in the in vitro chromosome aberration test, a micronucleus test in vivo was performed according to OECD guideline 474 and in compliance with GLP. Five male CD-1 mice per dose group were treated intraperitoneally with the test substance (25, 50, and 100 mg/kg bw) twice at a 24-h interval (Kawabata, 2013). The concentrations were based on a range-finding test were mortality and clinical signs of toxicity were observed at 300 and 1000 mg/kg bw. 24 h after the last treatment the bone marrow was collected, the cells were fixed and the cell suspension was stained with acridine orange just before microscopic observation, and was spread on a slide. For assessment of cytotoxicity the ratio of immature erythrocytes (IMEs) within 1000 erythrocytes (mature + immature) was determined. For assessment of mutagenic properties 2000 IMEs were scored and the number of micronucleated IMEs (MNIMEs) was determined. Treatment with the test item did not change the incidence of MNIMEs (0.10%, 0.13%, 0.13% at 25, 50, 100 mg/kg bw/day), when compared to the solvent control (0.10%). The ratio of IMEs among total erythrocytes was 54.2%, 53.1%, 53.2%, and 48.5% for the solvent control, 25, 50, and 100 mg/kg bw/day dose group, respectively. At 100 mg/kg bw/day the ratio was significantly reduced indicating a test substance induced effect on the erythrocytes and thus indicating systemic availability of the test substance. The positive control substance cyclophosphamide (20 mg/kg bw/day) increased the incidence of MNIMEs to 0.71%. The results were within the historical control data. In conclusion, the test substance did not induce mutagenic effects in the micronucleus test in vivo while showing systemic availability indicated by a decrease of the ratio IMEs/total erythrocytes under the experimental conditions of the study.

Gene mutation in mammalian cells:

According to the Reach regulation, Annex VIII, column 1, 8.4.3, an in vitro gene mutation study in mammalian cells is only required, if a negative result in Annex VII, Section 8.4.1. (here OECD 471, Verspeek-Rip, 2013), and Annex VIII, Section 8.4.2. (here OECD 473, Yasunaga, 2013) is available. Since the OECD 473 study (chromosomal aberration assay in vitro) provided a positive result, the in vitro gene mutation study in mammalian cells does not need to be conducted. Instead, appropriate in vivo studies are required. This conclusion is also confirmed in the respective technical guidance document: According to the ECHA Guidance on information requirements and Chemical Safety Assessment, Chapter R.7a: Endpoint specific guidance, Version 2.4 (February 2014), Table R.7.7-5 (p. 360), #9, available and reliable data from an OECD 471 (negative), OECD 473 (positive) and OECD 474 (negative) are sufficient to fulfil the standard data requirements accrding to Annexes VII-X of the Reach regulation. Further testing is not required, because the quality and relevance of the available data is considered adequate and sufficient. Moreover, the available data are sufficient in order to take a conclusion (i.e. not genotoxic) with regards to classification and labelling according to Regulation (EC) 1272/2008 and Directive 67/548/EEC, respectively.


Justification for selection of genetic toxicity endpoint
No study was selected as the endpoint conclusion is based on an assessment including several key studies. There was one study available showing positive results for in vitro chromosome aberration. The available studies on in vitro gene mutation in bacteria, in vitro gene mutation in mammalian cells, and in vivo micronucleus test were negative.

Short description of key information:
Gene mutation in bacteria (OECD 471, Ames test): S. typhimurium TA 100, TA 1535, TA 98, TA 1537, TA 1538 and E. coli WP2: negative with and without metabolic activation
Chromosome aberration, in vitro (OECD 473): CHL/IU cells: positive without metabolic activation, inconclusive with metabolic activation
Chromosome aberration, in vivo (OECD 474): mouse bone marrow cells: negative with and without metabolic activation

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of the test item do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.