Registration Dossier

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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Hexafluoropropylene (HFP) has been tested in a variety of genotoxicity tests in vitro and in vivo. This includes several in vitro tests for point mutation (AMES test; HGPRT test) an in vitro test on structural chromosomal aberrations. Two micronucleus tests were performed with CD1 mice and a dominant lethal test in Charles River CD rats. In addition, a DNA Repair study has been performed in male rats to investigate whether HFP will cause unscheduled DNA synthesis. An overview of previously existing studies is available in the JACC Report No 48 (hexafluoropropylene) Table 4 and 5 (page 4041). An assessment of the available in vitro and in vivo studies is provided in the following paragraphs.

Summary and evaluation (JACC Report No. 48)

The genotoxic potential of HFP has been assessed in a number of studies for gene mutation and chromosome aberrations. HFP did not induce gene mutations in bacteria or in mammalian cells in vitro. It exhibited weak clastogenic activity in CHO cells in vitro and in males in one mouse micronucleus test, but only at 1200 ppm and when data from all sampling times (24, 48 and 72 hours) were pooled and analysed. HFP was not mutagenic in the rat dominant lethal test (a germcell chromosome aberration assay). HFP was negative in an in vivo assay for UDS (unscheduled DNA synthesis) in rat hepatocytes. The cysteine conjugates of HFP were also without mutagenic activity in the Ames test.(Section 7.3). It was concluded that the mutagenicity endpoint is of very low concern for HFP.

At the time of the JACC report data review, a second Micronucleus test (report no Hoechst 93.0298) performed with HFP had been identified but was not available for analysis. [Note: the results of this Micronucleus Test No.2 are provided in the next paragraph]

Micronucleus Test No. 2:Hoechst Report No 93.0298 (1993) Result: “not mutagenic”

The second in vivo micronucleus test with HFP used 4 different HFP exposure levels (300, 600, 800, 1200 ppm) and covered the same exposure range as the previous micronucleus test, but using an additional higher exposure level of 800 ppm in addition to the 1200 ppm level. 2000 PCE per exposed group were assessed in this second study. HFP in this study did not lead to a statistically significant increase in micronucleated polychromatic erythrocytes in male or female mice It was concluded that HFP was not genotoxic in this in vivo test.

This second micronucleus report is included now in the reevaluation of all existing data for chromosomal aberrations below.

The small [weakly positive] effect observed in the first micronucleus test was not confirmed in the second, more extended in vivo study (4 exposure levels, see above).

Both micronucleus studies have demonstrated that systemic toxicity seems to be higher in male mice than in females. A higher degree of body weight reduction in the group exposed at the highest concentration in the first micronucleus report has been documented. As reported in the second micronucleus test (report no 2), 1200 ppm was a significantly toxic exposure level, leading to lethality in some of the animals (males and females). In particular, the higher degree of PCE depletion (60 %) in male mice to the highest concentration of HFP (micronucleus test no 1) can be interpreted as a sign of acute anemia. (”In males, significant depression of the PCE:NCE ratios was seen in all dose groups…”). Hemolytic anemia has been identified as a cause of an unspecific induction of micronuclei, better known under the term of “HowellJolly bodies,” not related to a specific genotoxic mechanism. Therefore, the unique occurrence of a small induction of micronuclei in one of two in vivo studies, and also being limited to male animals is not considered to be indicative of a relevant genotoxic response in these tests, in light of two additional negative in vivo studies with HFP (see below).

Further support of the conclusion that HFP is not overtly clastogenic in vivo is supported by another in vivo test with HFP. HFP was tested in the dominant lethal test in rats. As per OECD 478 (2016) “The purpose of the Dominant Lethal (DL) test is to investigate whether chemical agents produce mutations resulting from chromosomal aberrations in germ cells”. In this test with repeated exposure to HFP (rats were exposed to HFP at concentrations of 25, 100 or 400 ppm for 5 consecutive days), HFP did not show any evidence of chromosomal aberrations in terms of dominant lethality in rats.

An in vitro chromosomal aberration assay showed significant inductions of chromosomal changes in Chinese hamster ovary cells and was considered positive in this assay both with and without metabolic activation. Due to the intrinsic properties of the test compound, however, there are some limitations reported in this study which need careful consideration. It is stated in the study report that the clastogenic activity was seen only “at concentrations which were markedly cytotoxic, as measured by cell cycle delay.” “Cytotoxicity studies showed significant cell cycle delay at HFP concentrations ≥0.13 % (in air) under non-activated conditions and ≥0.49 % the lowest concentration tested with activation.“ It is therefore not possible to assess to which degree the overall cell toxicity contributed to the clastogenic effect, which was independent of metabolic activation.

Cytotoxicity leading to false positive results in in vitro cytogenicity studies have been repeatedly reported in the scientific literature: a) Galloway (2000) Environ. Mol. Mutagen., 35 pp191ff “…aberrations can occur secondary to toxicity, with compounds that do not react with DNA and are not genotoxic in vivo. Thus, some positive results in the in vitro aberration assay are not relevant to human risk.; b) Hilliard et al (1998) Environ Mol. Mutagen.,31 pp 316ff: Title: Chromosome aberrations in vitro related to cytotoxicity of non-mutagenic chemicals and metabolic poisons. “Chromosome aberrations can occur by secondary mechanism(s) associated with cytotoxicity, induced by chemicals that do not attack DNA. Clearly, chemicals can give “false-positive” results in the chromosome aberration assay at cytotoxic levels, though cytotoxicity does not always produce aberrations.” Even for proven non carcinogenic chemicals, false positive results have been reported in in vitro cytogenic assays. For the reasons above, the positive finding in the in vitro assay for chromosomal aberrations provides relatively little weight in the overall weight-of-evidence assessment of HFP genotoxicity.


A 2017 REACH substance (CoRAP) evaluation resulted in a request to conduct an in vitro micronucleus test, with H FP gas, (OECD test guideline 487) in human lymphblastoid TK6 cells to clarify the concern for genotoxicity which could also inform on the potential mode of action for HFP. HFP was negative in this GLP-compliant OECD 487 study. HFP did not induce micronuclei in human lymphoblastoid TK6 cells during 4-hour incubations both, with or without metabolic activation nor in a 27-hour incubation without metabolic activation. Therefore, HFP should not be considered to have a genotoxic mode of action as it did not induce micronuclei in TK6 cells in the 27 hour treatment without metabolic activation or in the 4-hour treatments, both with and without metabolic activation, under the conditions of this test system. The test system met all criteria for validity.

Endpoint Conclusion:No adverse effect observed (negative for inducing micronuclei in human TK6 cells)

Justification for classification or non-classification

Based on a weight-of-evidence evaluation of available data from in vivo and in vitro genotoxicity studies with HFP, there is strong, scientifically-defensible support to conclude that HFP does not have a genotoxic mechanism of action in mammalian systems.

The one positive finding in the rodent in vitro assay for chromosomal aberrations provides relatively little weight in the overall weight-of-evidence assessment of HFP genotoxicity (i.e. clastogenic activity was observed at concentrations which were markedly cytotoxic, as measured by cell cycle delay). Furthermore, this single positive finding in the in vitro assay for chromosomal aberrations was not confirmed by the most recent OECD 487 in vitro cytotoxicity-micronucleus assay conducted with HPF gas in human lymphoblastoid TK6 cells, which was negative for genotoxicity both with and without metabolic activation.

The conclusion that HFP does not have a genotoxic mechanism of action is further supported by the results of an in vivo DNA repair test where rats were exposed to 1000 and 1500 ppm HFP and did not induce unscheduled DNA repair synthesis in the hepatocytes of exposed animals. Additionally, HFP also did not cause damage to germ cell genetic material as evidenced by the negative results in a rodent dominant lethal study.

It is concluded that based on a comprehensive weight-of-evidence evaluation of these in vitro and in vivo data there is no concern for a genotoxic mode of action with HFP. Therefore, HFP does not need to be classified for mutagenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.