Difluoromethane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

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:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment (SIDS score: 1b).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Difluoromethane

EC Number:

200-839-4

EC Name:

Difluoromethane

Cas Number:

75-10-5

Molecular formula:

CH2F2

IUPAC Name:

difluoromethane

Details on test material:

Name of test material: difluoromethane
Source: ICI Chemicals and Polymers
Batch number: 20764/1
CTL reference number: Y02105/004/001
Purity: 99.96%

Method

Target gene:

his

Species / strain

Species / strain / cell type:

other: S. typhimurium TA1535, TA1537, TA98, TA100 and E.coli WP2P and WP2P uvrA

Metabolic activation:

with and without

Metabolic activation system:

liver S9-mix prepared from Aroclor 1254-induced Alderley Park rats

Test concentrations with justification for top dose:

0, 5, 10, 25, 50, 75 and 100% (v/v in air)

Vehicle / solvent:

air

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: acridine mutagen, 2-aminoanthracene, daunorubicine, N-methyl-N'-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION
The plate incorporation protocol was adapted for exposure of the test plates to a gaseous compound. This exposure regimen was validated by vinyl chloride as appropriate positive control showing that mutagenic activity was adequately detected in case of a gaseous compound.

DURATION
- Incubation time: 3 days
- Incubation temperature: 37°C

NUMBER OF REPLICATIONS: 3 (except 5 for negative controls and 2 for positive controls)

DETERMINATION OF CYTOTOXICITY
- determined by examination of background lawn growth

ANALYTICAL DEVICE
Colonies were counted electronically using an automatic colony counter AMS 40-10 Image Analyser.

POSITIVE CONTROLS
- Positive controls: prepared in DMSO
without S9-mix
. for TA1535: N-methyl-N'-nitro-N-nitrosoguanidine (5 µg/plate)
. for TA1537: acridine mutagen ICR191 (2 µg/plate)
. for TA98: daunorubicine (1 µg/plate)
. for TA100: N-methyl-N'-nitro-N-nitrosoguanidine (5 µg/plate)
. for WP2P: N-methyl-N'-nitro-N-nitrosoguanidine (2 µg/plate)
. for WP2P uvra: N-methyl-N'-nitro-N-nitrosoguanidine (2 µg/plate)
with S9-mix
. for TA1535: 2-aminoanthracene (2 µg/plate)
. for TA1537: 2-aminoanthracene (2 µg/plate)
. for TA98: (2 µg/plate) (1 µg/plate)
. for TA100: 2-aminoanthracene (1 µg/plate)
. for WP2P: 2-aminoanthracene (20 µg/plate)
. for WP2P uvra: 2-aminoanthracene (5 µg/plate)

Evaluation criteria:

Result is considered as positive when the following criteria are observed:
- a statistically significant dose-related increase in revertant colony  mean count
- the mean number of revertant colonies per plate with the test substance  is at least more than twice that of the concurrent negative control.

Statistics:

One-tailed Student's t-test.

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA1535, TA1537, TA98, TA100 and E.coli WP2P and WP2P uvrA

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

Additional information on results:

CYTOTOXICITY:
No significant cytotoxicity was evidenced (no significant loss of background growth and/or reduction in colony numbers).

GENOTOXICITY:
No significant increases in the number of revertant colonies were observed in any experiment, whatever the strain and dose with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

HFC-32 did not show a mutagenic response under the conditions of the test.

Executive summary:

Mutagenic activity of HFC-32 was evaluated by using 4 histididine dependent strains of Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537) and two tryptophan-dependent strains of Escherichia coli (WP2 and WP2 uvrA). Preliminary toxicity tests were carried out to select the maximal concentration for the main test. Tests were carried out in presence and in absence of Rat liver derived S-9 mix activation system. Cells were incubated with 0, 5, 10, 25, 50, 75 and 100% (v/v in air)  HFC-32 for 3 days at 37°C.Suitable positive controls were used in the presence (2 -aminoanthracene) and in the absence (nitrosoguanidine, acridine, daunorubicine and aminoanthracene) of S-9 mix. No significant cytotoxicity was evidenced and no significant increases in the number of revertant colonies were observed in any experiment, whatever the strain and dose with and without metabolic activation.