(2E)-1,1,1,4,4,4-hexafluoro-2-butene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect at the time of study conduct.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect dated 2008.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect dated 1998.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

66711-86-2

Cas Number:

66711-86-2

IUPAC Name:

66711-86-2

Test material form:

gas under pressure: liquefied gas

Details on test material:

- Purity: 99.79%

Method

Target gene:

The hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F12 medium supplemented with 3 mM L-glutamine and 5% (v/v) heat-inactivated and dialyzed fetal bovine serum (F12FCM5)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Dose Range-Finding Assay: 0.195, 0.391, 0.781, 1.56, 3.13, 6.25, 12.5, 25.0, 50.0 and 100% (v/v, in air)

CHO/HPRT Mutagenicity Assay: 7.91, 15.8, 31.6, 42.2, 56.3, 75.0 and 100% (v/v, in air) with and without S9.

Vehicle / solvent:

- Vehicle used: air

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

other: ethylmethanesulphonate (-S9); benzo(a)pyrene (+S9)

Remarks:

both controls were diluted in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: plated in medium

DURATION
- Preincubation period: overnight
- Exposure duration: 5 (±0.5) hours in the presence and absence of S9
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): After the 5-hour treatment, the treatment media were removed, the cultures were washed twice with 10 mL CMF-HBSS and then were trypsinized and counted. Cells were subcultured at ~2.4E6 cells/225-cm2 flask in 30 mL F12FCM5 in duplicate for phenotypic expression and incubated under standard conditions

SELECTION AGENT (mutation assays): Cultures treated at concentrations of 15.8, 31.6, 56.3, 75.0 and 100% (v/v, in air) were chosen for mutant selection (cultures treated at concentrations of 7.91 and 42.2% were discarded prior to selection because a sufficient number of other concentrations was available).

NUMBER OF REPLICATIONS: All test substance, negative and positive control concentrations were evaluated in duplicate cultures.

NUMBER OF CELLS EVALUATED: 6E5 cells/150-mm plate (4 plates total)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The test substance was considered to have produced a positive response if it induced a statistically significant and dose-dependent increase in mutant frequency (p≤ 0.05) that exceeded the 95% confidence limit of the historical vehicle control data from the testing laboratory. If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95% confidence interval), the results were considered equivocal. If none of these criteria were met, the results were considered to be negative.

Statistics:

Statistical analyses were performed using the method of Snee and Irr.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

HPRT

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
These results indicate that the test substance was negative in the In Vitro Mammalian Cell Gene Mutation (CHO/HPRT) Assay, in the presence and absence of metabolic activation, under the conditions and according to the criteria of the test protocol.

Executive summary:

The test substance was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary(CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). The test substance was evaluated in a preliminary dose range-finding assay at concentrations of 0.195, 0.391, 0.781, 1.56, 3.13, 6.25, 12.5, 25.0, 50.0 and 100% (v/v, in air) with and without an exogenous S9 metabolic activation system (the highest concentration evaluated was the limit dose for this assay). Ambient air was used as the negative (untreated) control. No visible precipitate was observed at the beginning or end of treatment, and the test substance did not have an adverse impact on the pH of the cultures. Adjusted relative survival at a concentration of 100% (v/v, in air) was 44.37 and 13.52% with and without S9, respectively.

Based on these results the test substance was evaluated in the definitive mutagenicity assay at concentrations of 7.91, 15.8, 31.6, 42.2, 56.3, 75.0 and 100% (v/v, in air) with and without S9. No visible precipitate was observed at the beginning or end of treatment. The average adjusted relative survival at a concentration of 100% (v/v, in air) was 96.25 and 72.04% with and without S9, respectively. Cultures treated at concentrations of 15.8, 31.6, 56.3, 75.0 and 100% (v/v, in air) were chosen for mutant selection (cultures treated at concentrations of 7.91 and 42.2% were discarded prior to selection because a sufficient number of other concentrations was available). No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p> 0.05). In contrast, the positive controls induced a significant increase in mutant frequency (p< 0.01).

These results indicate that the test substance was negative in the In Vitro Mammalian Cell Gene Mutation (CHO/HPRT) Assay, in the presence and absence of metabolic activation, under the conditions and according to the criteria of the test protocol.