Polyhaloalkene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

468-710-7

EC Name:

-

Cas Number:

754-12-1

Molecular formula:

C3H2F4

IUPAC Name:

2,3,3,3-tetrafluoroprop-1-ene

Test material form:

other: gas

Details on test material:

- Name of test material (as cited in study report): 1-propene,2,3,3,3-tetrafluoro- (HFO 1234yf)
- Storage condition of test material: ambient temperature

Method

Target gene:

Thymidine kinase locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium (with HEPES and Glutamax-I) supplemented with heat-inactivated horse serum (10% v/v for growing in flasks, and 20% for growing in microtiter plates), sodium pyruvate and penicillin/streptomycin.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: S9 liver homogenate

Test concentrations with justification for top dose:

76, 60, 40, 20 and 10% (v/v) (all ± 10%)

Vehicle / solvent:

Since the test material is a gas, cells in culture flasks were exposed in modular incubator chambers (Billups-Rothenburg, USA) to various concentrations. The atmosphere in the chamber consisted of 19% O2; 5% CO2; and the test material supplemented with N2. Air without the test material was used as negative control.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Sham exposed

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: The test substance is a gas therefore a test atmosphere was generated by mixing a mass flow controlled amount of gaseous test material with a mass flow controlled stream of nitrogen, together accounting for 76% of the air. A mass flow controlled stream of oxygen (19%) and a mass flow controlled stream of carbon dioxide (5%) were added. The resulting test atmosphere was directed to the inlet of the exposure boxes. The boxes were flushed with test atmosphere for a sufficient amount of time to achieve at least 99% of the steady state target concentration

DURATION
- Exposure duration: 4 hours and 24 hours in the absence of S9-mix, and for 4 hours in the presence of S9-mix
- Selection time: 10-14 days

SELECTION AGENT: Trifluorothymidine

NUMBER OF REPLICATIONS: Duplicate

DETERMINATION OF CYTOTOXICITY
- Method: The cytotoxicity of the test material was determined by measuring the relative initial cell yield, the relative suspension growth (RSG) and the relative total growth (RTG).

Evaluation criteria:

See: "Any other information on materials and methods incl. tables"

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

In the presence of S9-mix. See: "additional information on results"

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see: "additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Positive and negative controls:
The negative controls were within the acceptance criteria and treatment with the positive controls yielded the expected significant increase in mutant frequency compared to the negative controls for all treatment conditions tested. Therefore, the test was considered valid.

Concentration levels and visual observations before and after treatment:
The actual concentrations were measured at start and end of exposure. Based on the results of these measurements it can be concluded that at the start of the exposure, all target concentrations were met taking into account the margin of 10% mentioned in the study plan. In one case (lowest concentration of the 24 hour exposure) the concentration seemed just below target, but as the concentration at the end of exposure was higher, this was considered to be an error in the measurement. At the end of the exposure the target concentration was met in all but one case (taking into account the margin of 10%), which was the lowest concentration of the 4 hour exposure. In this case the deviation was -25%. It should be noted that the second sample is taken to demonstrate that the container/incubator was not leaking. In general, the lower values obtained from the second sample may also indicate that the test material was taken up in the culture medium or metabolized during exposure. As the four higher concentrations were all within the 10% margin of the target concentrations, and the guideline requires four concentrations tested in duplicate for a valid assay, the test was considered valid. At the start and end of both the 4 hours and 24 hours treatment in the absence and presence of S9-mix, no visual abnormalities were observed. At the end of the treatment period, the viability of the cells at 76% of the test substance was at or above 90% for all treatment conditions tested.

Cytotoxicity:
In the presence of S9-mix, the test material was toxic to the cells resulting in a reduction in the RTG. The mean RTG at the highest three concentrations evaluated in the absence of S9-mix (76%, 60% and 40%) was 61%. In the absence of S9-mix after 4h treatment no toxicity was observed, but after 24h treatment the test material was slightly toxic to the cells resulting in a mean RTG at the highest concentration evaluated (76%) of 83%.

Mutagenicity:
In the presence of S9-mix (4 hours exposure), a dose related increase in the mean MF by more than 88 or 126 mutants per 1,000,000 clonable cells compared to the negative control was observed from a concentration of 20% onwards. In the absence of S9-mix, both after 4 and 24 hours, no increase in the mean MF by more than 88 or 126 mutants per 1,000,000 clonable cells, i.e. no equivocal or positive response, compared to the negative control was observed at any dose level.

Colony sizing:
At concentrations causing an increase in MF the mutant colonies were scored using the criteria of small and large colonies. In the presence of S9-mix an induction in both the number of small and large colonies was observed. The mean percentage of small colonies at 20, 40, 60 and 76 % (positive responses in MF) was 57%, 57%, 57% and 54% compared to 44%, 44%, 43% and 46% of large colonies. Although in general slightly more small than large colonies were formed, the amount of both small and large colonies were increased, and therefore, none of the mechanisms (clastogenicity and mutagenicity) can be excluded.

Any other information on results incl. tables

Table 1. Summary of the results in the absence of S9-mix

Conc. (%)	absence of S9-mix (24h)			Conc. (%)	absence of S9-mix (4h)
	MF*	¿MF*	RTG*		MF*	¿MF*	RTG*
76	89	13	83	76	68	15	118
60	101	25	90	60	76	24	130
40	73	-3	92	40	46	-7	127
20	90	14	91	20	65	12	101
10	55	-20	112	10	52	0	106
0	76	0	100	0	53	0	100

*Mean of duplicate cultures

Table 2. Summary of the results in the presence of S9-mix

Conc. (%)	presence of S9-mix (4h)
	MF*	¿MF*	RTG*
76	447	376	61
60	427	356	61
40	306	235	61
20	228	157	94
10	134	63	96
0	72	0	100

* Mean of duplicate cultures

Values were increased by more than 126 mutants per 1,000,000 clonable cells compared to the negative control.

MF = mutant frequency; ¿MF = increase in mutant frequency; RTG = relative total; growth; conc. = concentration of the test material

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material is mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the presence of metabolic activation (S9-mix).

Executive summary:

According to OECD guideline 476 and GLP the test substance was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix). A single test was conducted. In the 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.

The highest nominal concentration of the test substance evaluated for mutagenicity was 76%. As the atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test material (supplemented with N2 to achieve lower concentrations), the highest achievable concentration was 76%. Additional concentrations of 60, 40, 20 and 10% (v/v) (all ± 10%) were used in the chambers to expose the cells. Air without the test material was used as negative control. Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and in the presence of S9-mix, respectively and clear air served as negative control in both tests. As all acceptance criteria were met, the test was considered valid. In the presence of S9-mix the test material was toxic to the cells resulting in a reduction in the relative total growth (RTG). The mean RTG at the highest three concentrations evaluated in the absence of S9-mix (76%, 60% and 40%) was 61%. In the absence of S9-mix after 4h treatment no toxicity was observed, but after 24h treatment the test material was slightly toxic to the cells resulting in a mean RTG at the highest concentration evaluated (76%) of 83%. In the presence of S9-mix, a dose related increase in the mean mutant frequency (MF) by more than 88 or 126 mutants per 1,000,000 clonable cells compared to the negative control was observed as from a concentration of 20% onwards. In the absence of S9-mix, both after 4 and 24 hours, no increase in the mean MF by more than 88 or 126 mutants per 1,000,000 clonable cells, i.e. no equivocal or positive response, compared to the negative control was observed at any dose level.

It is concluded that under the conditions used in this study, the test substance is mutagenic at the TK-locus of mouse lymphoma L5178Y cells, in the presence of metabolic activation (S9-mix).