Difluoromethane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

According to the information requirements specified in Annex X, information concerning the potential to induce gene mutations and chromosomal aberrations needs to provided. One gene mutation assay in bacteria, two in vitro cytogenicity studies are available, all with negative results. A read-across with 1,1,1-trifluoroethane (HFC-245fa) is also performed regarding to the gene mutation assay in mammalian cells. The substance did not demonstrate mutagenic potential in an in vitro cell mutation assay using the Thymidine Kinase Gene.

Link to relevant study records

Reference

Reference 1

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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment (SIDS score: 1b).

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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

Target gene:

his

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

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.

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'.

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

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).

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

None

Species / strain / cell type:

other: human lymphocytes

Details on mammalian cell type (if applicable):

- Source: two healthy non-smoking human volunteers (a man and a woman)
- Cell cycle length: no data
- Number of passages: no data
- Method of maintenance of cell cultures: RPMI 1640 medium supplemented with 10% FBS, 1 IU/ml heparin, 100 IU/ml penicillin, 100 µg/ml streptomycin and 0.5 ml phytohaemagglutinin for appoximately 48h at 37°C.
- Absence of mycoplasma: no data

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, prepared by male Wistar rat liver enzyme induction by Aroclor 1254

Test concentrations with justification for top dose:

10, 50 and 100% (v/v in air)

Vehicle / solvent:

air

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylene oxide, mitomycin C, cyclophosphamide

Details on test system and experimental conditions:

PRELIMINARY STUDY
No cytotoxicity was observed for the 6 tested concentrations from 5 to 100% v/v in air, harvested 72 h, with and without S-9 mix.

MAIN STUDY:
- Incubation temperature: 37°C
- Exposure / recovery:
without S9 mix: 72/0 and 96/0 h
with S9 mix: 72/0 and 96/0 h
The 96h-treatment was only performed on lymphocytes of the female donor (the only tested dose was 100%).

NUMBER OF REPLICATES: 2

NUMBER OF CELLS EVALUATED: 100

DETERMINATION OF CYTOTOXICITY
Method: mitotic index

METAPHASE ARREST
- Spindle poison used: colcemid or demeocolcine (0.4 µg/ml)
- Duration of exposure: 2 h (at 70 or 94 hours after culture initiation for a period of 2 hours at 37°C)
- Time before harvest: 21 h

CELL PROCESSING:
- Harvesting:
Cells are centrifuged at 400 G for 5 minutes and resuspended in 5 ml of 0.075 M KCl at room temperature for approximately 10 minutes to allow swelling to occur.
- Fixation:
After centrifugation at 400 G for 5 minutes, supernatant was discarded and cells were fixed by dropping fresh methanol/glacial acetic acid (3:1, v/v). The volume was then made up to 10 ml. The fixative was changed by centrifugation and resuspension twice.
- Slide preparation:
Several drops of cells suspension were spread on slides. Cells are then stained in 10% (v/v) filtered solution of buffered Giemsa stain (Gurr's R66) in double-deionised water for 7 minutes. Slides are rinsed, dried and mounted in DPX.

CONTROLS
- Positive controls: ethylene oxide (1200 ppm), mitomycin C (1µg/ml), cyclophosphamide (50 µg/ml)
- Negative controls: air and 100% nitrogen atmospheres.

Evaluation criteria:

- Cytotoxicity test:
Cytotoxicity was evaluated using the mitotic index (which indicates whether an item induces mitotic inhibition).

- Chromosomal aberration test:
Number of metaphases analyzed: 100 cells from each selected culture.
Types of sought aberrations: gaps, chromatid and chromosome breaks and exchanges, and others (multiple aberrations and interchanges).

Statistics:

Fisher's Exact Test

Species / strain:

other: human lymphocytes

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 obvious reductions in mitotic activity were observed in any treatment group.

CHROMOSOMAL ABERRATION ANALYSIS:
No statistically or biologically significant increases in chromosomal aberration frequencies (including or excluding gap-type aberrations) were observed in either donor, in the presence or absence of metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Executive summary:

Human lymphocytes were exposed to 10, 50 and 100% (v/v in air) difluoromethane in atmosphere for 96 hrs, in the presence and in the absence of S-9 mix. The highest doses were chosen by a preliminary toxicity test. Positive controls containing ethylene oxide, mitomycin C, cyclophosphamide were incorporated in the main tests. 2 hrs prior to the end of the exposure, cell division was arrested by incubation with 0.4 µg/ml colcemid. Clastogenic potential was assessed by the analysis of 100 cells/slide in metaphase. 2 slides /culture were analysed. Cytotoxicity was evaluated by using the mitotic index (% cells in metaphase) calculated by examination of 1000 cells/culture.

No obvious reductions in mitotic activity were observed in any treatment group.

No statistically or biologically significant increases in chromosomal aberration frequencies (including or excluding gap-type aberrations) were observed in either donor, in the presence or absence of metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

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: 1).

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

None

Species / strain / cell type:

other: Chinese Hamster Lung (CHL) cells

Details on mammalian cell type (if applicable):

- Source: National Institute of Hygienic Science (Tokyo)
- Cell cycle length: 15 hours
- Number of passages: no data
- Method of maintenance of cell cultures:
Eagle's medium supplemented with 10% heat inactivated calf serum
- Absence of mycoplasma: no data

Metabolic activation:

with and without

Metabolic activation system:

post-mitochondrial supernatant fraction of liver homogenates (S9)

Test concentrations with justification for top dose:

0, 10, 20, 40 and 80%

Vehicle / solvent:

high purity air

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylchloride (without S9) and vinyl chloride (with S9), mitomycin C, benzo(a)pyrene

Details on test system and experimental conditions:

ADMINISTRATION:
- Number of replicates: 2
- Cell density at seeding: 4000 cells/ml
- Positive controls: Ethylchloride (45% [24h-treatment] and 28% [48h-treatment]), vinylchloride (2.5%), benzo(a)pyrene (0.01 mg/ml) and mitomycin C (40 µg/l)

EXPOSURE/RECOVERY DURATION:
- Exposure duration without S9: 24/0 and 48/0 hours
- Exposure duration with S9: 6/16 hours

METAPHASE ARREST:
- Spindle poison used: colcemid (0.2 µg/ml)
- Duration of exposure: 2 h
- Time before harvest: 2 h

CELL PROCESSING:
- Harvesting:
Cells are exposed to trypsin and incubated in 0.075 M KCl at 37°C for 20 minutes to allow swelling to occur.
- Fixation:
Cells are fixed with ethanol/acetic acid (3:1, v/v).
- Slide preparation:
Cells are then spread on clean glass slides, air-dried and stained with a 2.5% Giemsa solution for 12 minutes.

EXAMINATION:
- Cytotoxicity tests:
Cytotoxicity was evaluated at 0, 5, 10, 20, 40 and 80%, by using the following parameters:
cell growth index (calculated as the percentage of live cells)
mitotic index (which indicates whether an item induces mitotic inhibition, calculated as the percentage of cells in mitosis).
A minimum of 1000 cells per culture were examined for determining the mitotic index.
This cytotoxicity test was also performed as a preliminary test for chromosomal aberrations observation.

- Chromosomal aberration test
Number of metaphases analyzed: 100/duration/concentration.
Types of sought aberrations: gaps, chromatid and chromosome breaks and exchanges, polyploidy, fragmentation.
Microscope: no data

Evaluation criteria:

Acceptance criteria: negative control to be within the historical values  of the lab and statistical increase in chromosomal aberrations to be observed with the positive controls.
Evaluation criteria: no data.

Statistics:

Chi-square test

Species / strain:

other: Chinese Hamster Lung (CHL) cells

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:
- Cell growth index:
A slight but not dose-related effect was observed without metabolic activation. No effect was seen with metabolic activation.
- Mitotic index:
A slight but not dose-related effect was observed without metabolic activation. No effect was seen with metabolic activation.

CHROMOSOMAL ABERRATION ANALYSIS:
In both the 24 and 48 hours exposures of the test substance to CHL without metabolic activation, no statistical increases in structural aberrations including gaps and excluding gaps or in polyploidy induction were observed at any concentration level of the test substance.
In the 6 hours exposure of the test substance to CHL with the metabolic activation with and without addition of the S9 mix, no statistical increases of chromosomal aberrations was observed.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Executive summary:

Chinese Hamster Lung (CHL) cells were exposed to 0, 10, 20, 40 and 80% (v/v in air) difluoromethane in atmosphere until 16 hours without S-9 mix 96 hrs, or until 48 hours in the presence S-9 mix. The highest doses were chosen by a preliminary toxicity test. Positive controls containing Ethylchloride (45% [24h-treatment] and 28% [48h-treatment]), vinylchloride (2.5%), benzo(a)pyrene (0.01 mg/ml) and mitomycin C (40 µg/l) were incorporated in the main tests. 2 hrs prior to the end of the exposure, cell division was arrested by incubation with 0.2 µg/ml colcemid. Clastogenic potential was assessed by the analysis of 100 cells/slide in metaphase. 2 slides /culture were analysed. Cytotoxicity was evaluated by using the mitotic index (% cells in metaphase) calculated by examination of 1000 cells/culture. In both the 24 and 48 hours exposures of the test substance to CHL without metabolic activation, no statistical increases in structural aberrations including gaps and excluding gaps or in polyploidy induction were observed at any concentration level of the test substance. In the 6 hours exposure of the test substance to CHL with the metabolic activation with and without addition of the S9 mix, no statistical increases of chromosomal aberrations was observed. A slight but not dose-related effect on cell growth and mitotic index was observed without metabolic activation. No effect was seen with metabolic activation. No statistically or biologically significant increases in chromosomal aberration frequencies (including or excluding gap-type aberrations) were observed in either donor, in the presence or absence of metabolic activation. In both the 24 and 48 hours exposures of the test substance to CHL without metabolic activation, no statistical increases in structural aberrations including gaps and excluding gaps or in polyploidy induction were observed at any concentration level of the test substance. In the 6 hours exposure of the test substance to CHL with the metabolic activation with and without addition of the S9 mix, no statistical increases of chromosomal aberrations was observed.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June 2018 - 24 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver was stored at -90 to -70°C.

Test concentrations with justification for top dose:

The highest concentration selected (70% v/v) was the maximum achievable concentration in the test system without detrimental effects on the cell culture.

Vehicle / solvent:

Sterile air

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

L5178Y mouse lymphoma (3.7.2c) cells heterozygous at the thymidine kinase locus, TK +/- were used. Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24-hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196 to -150°C, in heat inactivated donor horse serum (HiDHS) containing 10% DMSO. Cultures were used within ten days of recovery from frozen stock. Cell stocks are periodically checked for freedom from mycoplasma contamination.

The following media were used:
R0 RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 ug/mL gentamicin.
R10p R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v.
R10p medium was used for cell culture unless otherwise specified.
Selective medium consisted of R10p containing 4 ug/mL trifluorothymidine (TFT).

Positive Controls
In the absence of S9 mix, Methyl methanesulphonate (MMS) in DMSO at concentration of 10 ug/mL (3-hour exposure).
In the presence of S9 mix, Benzo[a]pyrene (BaP) in DMSO at concentration of 1.5 ug/mL (3-hour exposure).

S9 Metabolizing System
S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver and stored at -90 to -70°C. S9 fraction (5% v/v), glucose-6-phosphate (6.9 mM), NADP (1.4 mM) in R0 was used. The co-factors were prepared, neutralised with 1N NaOH and filter sterilised before use.

The highest concentration selected (70% v/v) was the maximum achievable concentration in the test system without detrimental effects on the cell culture. Concentrations within the range of 5 to 30% was avoided due to the known lower and upper flammability levels of the test item, 7 and 19% respectively.
All concentrations cited in this report are expressed in terms of pure HFC-143a as received.

The final nominal concentrations to which cells were exposed initially are given below:
Preliminary toxicity test:: 1, 5, 30, 40, 50, 60 and 70% v/v
Mutation tests:
-S9 mix (3 hours) 30, 40, 50, 60 and 70% v/v
+S9 mix (3 hours) 30, 40, 50, 60 and 70% v/v (also assessed for determination of the mutant phenotype)
Additional Mutationtests:
-S9 mix (3 hours) 22.3*, 40, 50, 60 and 70% v/v (also assessed for determination of the mutant phenotype)
* Actual concentration

Osmolality and pH
The effects of HFC-143a on the osmolality and pH of the culture medium were measured by analyzing samples of R10p media treated with either the vehicle (sterile air) or test item at 70% v/v. Precipitate was assessed by eye at the end of the exposure period in treated R10p media-only cultures as part of the preliminary toxicity test.


Preliminary Toxicity Test Procedure: Cells were exposed to the substance for 3 hours in the absence and presence of S9 mix. For 3-hour exposures, cultures contained a total of 1.2 x 10^7 cells. The final volume of the cultures was 10 mL and the final concentration of the S9 fraction was 2% v/v. One culture was prepared for each concentration of the substance for each test condition. Vehicle controls were tested in duplicate for each test condition.

Mutation Test Procedure: 3-hour Treatment in the Absence and Presence of S9 Mix. The procedure for preparing the cell suspension was the same as for the preliminary toxicity test. Cultures contained a total of 1.2 x 10^7 cells in a final volume of 10 mL. The final concentration of the S9 fraction was 2% v/v. Duplicate cultures were prepared throughout for each concentration of substance and positive control. Quadruplicate cultures were prepared for vehicle controls. Aliquots of 100 µL of positive control were added to the relevant cultures, and then all cultures were incubated, for 3 hours at 34 to 39°C. Five dilutions of the substance were tested.

Evaluation criteria:

Acceptance criteria for substance:
The highest concentration tested was one that allowed the maximum exposure up to 70% v/v for freely soluble compounds, or the limit of toxicity (i.e. relative total growth reduced to approximately 10 to 20% of the concurrent vehicle control) or the limit of solubility. For a toxic substance, at least 4 analysable concentrations should have been achieved which ideally spanned the toxicity range of 100 to 10% RTG.

Acceptance criteria for vehicle controls:
The mean vehicle control value for mutant frequency was between 50 to 170 x 10^-6.
The mean cloning efficiency was between 65 to 120%.
The mean suspension growth was between 8 to 32 on Day 2 following 3-hour treatments.

Obvious outliers were excluded. However, there were at least 2 vehicle control cultures remaining.

Acceptance criteria for positive controls:
Positive controls showed an absolute increase in mean total MF above the mean concurrent vehicle control MF of at least 300 x 10^-6. At least 40% of this was due to the number of small mutant colonies.
Mean RTG’s for the positive controls were greater than 10%.
There was an absence of confounding technical problems such as contamination, excessive numbers of outliers and excessive toxicity.

Criteria for Assessing Mutagenic Potential
The following criteria were applied for assessment of individual assay results using data for MF where the RTG normally exceeded 10%:
Definitions: GEF = Global Evaluation Factor. For microwell assays this is 126 x 10-6.
Providing that all acceptability criteria were fulfilled, the test item was considered to be clearly positive if, in any of the experimental conditions examined the increase in MF above the concurrent background exceeded the GEF and the increase was concentration related (i.e., there is a significant positive linear trend). The test item is then considered able to induce mutation in this test system.

Statistics:

The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users). Statistics were only reported if the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was exceeded, and this was accompanied by a significant positive linear trend (p<0.05).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The osmolality and pH of the substance in medium were measured by analysing samples of R10p media, dosed with either the vehicle (sterile air) or a substance formulation at 70% v/v. For medium dosed with substance at 70% v/v; no fluctuations in osmolality of the medium of more than 50 mOsmol/kg and no fluctuations in pH of more than 1.0 unit were observed compared with the vehicle control. The maximum final concentration tested in the preliminary toxicity test was 70% v/v as this is the maximum achievable concentration in the test system without detrimental effects on the cell culture.

Preliminary Toxicity Test:
No precipitate (observed by eye at the end of treatment) was observed at concentrations of 70% v/v in the absence and presence of S9 mix, respectively, following a 3-hour exposure. Exposure to the substance at concentrations from 1 to 70% v/v in the absence and presence of S9 mix resulted in no reduction in the relative suspension growth (RSG) values. Concentrations used in the main test were based upon these data.

Main Mutation Test - 3-hour Treatment in the Absence of S9 Mix:
Cultures were exposed to the substance at concentrations from 25.2 to 45.7% v/v. No precipitate was observed by eye at the end of treatment. The results of the achieved concentration analysis showed that the highest concentration was -34.7% from the nominal concentration of 70% v/v, this was considered to be unacceptable. The test was therefore terminated and an additional test was performed.

Additional main Mutation Test - 3-hour Treatment in the Absence of S9 Mix:
Cultures were exposed to the substance at concentrations from 22.3 to 70% v/v. No precipitate was observed by eye at the end of treatment. Cultures exposed to the substance at concentrations from 22.3 to 70% v/v were assessed for determination of mutation frequency. Mean relative total growth (RTG) values from 84 to 65% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity.

The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Main Mutation Test - 3-hour Treatment in the Presence of S9 Mix:
Cultures were exposed to the substance at concentrations from 30 to 70% v/v. No precipitate was observed by eye at the end of treatment. Cultures exposed to the substance at concentrations from 30 to 70% v/v were assessed for determination of mutation frequency. Mean RTG values from 107 to 92% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.

The positive control, benzo[a]pyrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Formulation Analysis:
The results of formulation analysis showed that the achieved concentrations of HFC-143a were within the target range 100% +/- 20%, with the exception of the highest concentration in the main test –S9 mix, and the lowest concentration in the additional main test –S9 mix, which were -34.7% and -25.7%, respectively.

Remarks on result:

other: No mutagenic potential.

Remarks:

Negative study.

Table 1: Preliminary toxicity test, relative suspension growth

i) In the absence of S9 mix – 3-hour exposure

Treatment / Nominal concentration (% v/v)	Replicate ID	Cell concentration (x10^5/mL) 24h/48h	Suspension Growth Day 2	Relative Suspension Growth (%)
Vehicle Control*	A B	4.37/12.17 5.24/10.53	13.30 13.80	100
HFC-143a (1)	A	4.80/10.72	12.87	95
HFC-143a (5)	A	4.55/11.34	12.90	95
HFC-143a (30)	A	5.28/9.87	13.04	96
HFC-143a (40)	A	5.92/9.31	13.79	102
HFC-143a (50)	A	4.69/10.50	12.30	91
HFC-143a (60)	A	4.81/10.25	12.32	91
HFC-143a (70)	A	5.14/9.97	12.81	95
*Vehicle control = Sterile air

ii) In the presence of S9 mix – 3-hour exposure

Treatment / Nominal concentration (% v/v)	Replicate ID	Cell concentration (x10^5/mL) 24h/48h	Suspension Growth Day 2	Relative Suspension Growth (%)
Vehicle Control*	A B	5.98/11.14 5.80/10.80	16.64 15.65	100
HFC-143a (1)	A	6.12/10.91	16.68	103
HFC-143a (5)	A	6.28/11.07	17.36	108
HFC-143a (30)	A	6.10/10.89	16.60	103
HFC-143a (40)	A	5.87/11.59	17.01	105
HFC-143a (50)	A	6.26/11.15	17.46	108
HFC-143a (60)	A	5.85/11.68	17.09	106
HFC-143a (70)	A	5.92/11.49	17.01	105
*Vehicle control = Sterile air

Table 2: Additional main mutation test – 3-hour treatment in the absence of S9 mix, relative total growth

Treatment / Nominal concentration (% v/v)	Replicate ID	Cell concentration (x10^5/mL) 24h/48h	Mean Suspension Growth	Viability Plate Count* Day 2	Mean Cloning Efficiency (%)	RTG (%)	Mean RTG (%)
Vehicle Control**	A B C D	4.39/12.85 4.91/11.88 5.18/14.55 4.63/11.43	15.19	47 (192) 32 (192) 33 (192) 38 (192)	102	100	100
HFC-143a (22.3#)	A B	3.89/11.94 4.32/12.54	12.57	39 (192) 56 (192)	87	75 67	71
HFC-143a (40)	A B	4.92/11.58 4.40/14.10	14.87	42 (192) 55 (192)	86	87 78	83
HFC-143a (50)	A B	4.27/13.27 4.71/13.23	14.88	43 (192) 52 (192)	87	85 82	84
HFC-143a (60)	A B	4.47/11.58 3.63/11.47	11.68	30 (192) 39 (192)	107	97 67	82
HFC-143a (70)	A B	4.41/11.53 3.87/11.72	12.03	52 (192) 48 (192	84	67 63	65
MMS (10 µg/mL)	A B	5.20/10.89 3.92/11.78	12.85	61 (192) 49 (192)	78	65 64	65
* Number of non-colony bearing wells (total number of wells) ** control = Sterile air # Actual concentration reported MMS - Methyl methanesulphonate RTG - Relative Total Growth

Table 3: Additional main mutation test – 3-hour treatment in the absence of S9mix, mutation frequency

Treatment / Nominal concentration (% v/v)	Replicate ID	Mutant Plate Count* Day 2			Mean RTG (%)		Mean MF (x10 ^-6)
Vehicle Control**	A B C D	160 (192) 157 (192) 159 (192) 159 (192)			100		93
HFC-143a (22.3#)	A B	159 (192) 159 (192)			71		108
HFC-143a (40)	A B	163 (192) 160 (192)			83		101
HFC-143a (50)	A B	159 (192) 163 (192)			82		101
HFC-143a (60)	A B	157 (192) 162 (192)			82		86
HFC-143a (70)	A B	160 (192) 156 (192			65		116
MMS (10 µg/mL)	A B	52 (192) 55 (192)			65		818
* Number of non-colony bearing wells (total number of wells) ** control = Sterile air # Actual concentration reported MMS - Methyl methanesulphonate RTG - Relative Total Growth MF - Mutant Frequency

Table 4: Additional main mutation test – 3-hour treatment in the absence of S9mix, colony size analysis

Treatment / Nominal concentration (% v/v)	Replicate ID	Small Mutant Plate Count* Day 2			Mean Small Colony MF (x10^-6)		Large Mutant Plate Count* Day 2	Mean Large Colony MF   (x10^-6)
Vehicle Control**	A B C D	175 (192) 176 (192) 176 (192) 174 (192)			45		173 171 173 173	52
MMS (10 µg/mL)	A B	69 (192) 72 (192)			641		146 151	164
* Number of non-colony bearing wells (total number of wells) ** control = Sterile air MMS - Methyl methanesulphonate RTG - Relative Total Growth MF - Mutant Frequency

Conclusions:

The substance did not demonstrate mutagenic potential in this in vitro cell mutation assay in the presence or absence of a metabolic activation system, under the experimental conditions described.

Executive summary:

The mutagenic potential of the substance was evaluated by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix) according to the most recent OECD guideline in compliance with GLP. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK^+/-) to the thymidine kinase deficient genotype (TK^-/-).

The study consisted of a preliminary toxicity test and three independent mutagenicity assays. The cells were exposed for either 3 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix. The substance was tested up to a maximum final concentration of 70% v/v in the preliminary toxicity test, in order to test up to the maximum achievable concentration in the test system without detrimental effects on the cell culture. 

Following a 3-hour exposure to the substance at concentrations from 1 to 70% v/v, no reduction in the relative suspension growth (RSG) was observed in the absence or presence of S9 mix. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to test up to the maximum achievable concentration in the test system without detrimental effects on the cell culture.

Following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentration assessed for mutant frequency in the 3-hour treatment in the absence and presence of S9 mix was 70% v/v. In the both the absence and presence of S9 mix there was no significant reduction in RTG.

In all tests the concurrent vehicle and positive control were within acceptable ranges.

It was concluded that the substance did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

According to the information requirements specified in Annex X, information concerning the potential to induce gene mutations and chromosomal aberrations needs to provided. One in vivo micronucleus study is available with negative results.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP near-guideline study with acceptable restriction, adequate for assessment, unpublished report (SIDS score: 2c).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

The total number of examined polychromatic erythrocytes (1000 PCE per animal) is insufficient. OECD guideline 474 advises 2000 PCE per animal.

Principles of method if other than guideline:

Schmid W (1976). The micronucleus test fot cytogenetic analysis. In: Hollender A (Ed). Chemical Mutagens: Principles and methods for their detection. Vol. 4, Plenum, New York 31-43.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS:
- Source: Charles River UK Ltd, Margate, UK 
- Age: 6-8 weeks
- Body weight at study initiation: no clear data
- Housing: 5/cage (sexes (seperately)
- Diet: ad libitum
- Water: ad libitum)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 50 +/- 15
- Air changes (per hr): 20-30
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation

Vehicle:

clean, dry and filtered laboratory air

Details on exposure:

TYPE OF INHALATION EXPOSURE
- Type of exposure: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Atmosphere generation: HFC 32 atmospheres were generated by passing  liquid HFC 32 into a copper coil which was placed in a Techne TE-7 water  bath at approximately 45°C. The resultant HFC 32 vapeur was then passed  through a copper equilibrium coil at room temperature to flowmeters via a copper distribution plenum. From the flowmeters HFC 32 vapeur was metered  to individual exposure chambers and then diluted by addition of clean, dry air [dried and filtered using equipment supplied by Atlas Copco (Sweden)] into the top of each chamber at a flow rate of 15 litres/minute. Air flow rates were monitored continuously using  flowmeters (KDG Flowmeters, Burgess Hill, Sussex, UK) and were recorded  at approximately 30 minute intervals during the exposure period.
- Exposure chamber: 17 l volume all-glass desiccators, one chamber per sex per concentration.

TEST ATMOSPHERE
- Atmosphere analysis: Samples of the test atmospheres for analysis of  HFC 32 were taken approximately every 30 minutes using agas tight  syringe. Samples were analysed using a gas chromatograph (Pye Unicam GCD  gas chromatograph equipped with a gas sampling valve (Pye), a Porapak P-S  (80/100 mesh) 1.5m x 2mm ID Glass column (Waters) and flame ionisation  detector). The peak area attributable to HFC 32 was used to calculate the  atmospheric concentration in parts-per-million (ppm v/v). Air control  atmospheres and room atmospheres were also sampled and analysed.

Duration of treatment / exposure:

6 hours

Frequency of treatment:

single exposure

Post exposure period:

24 and 48 h

Remarks:

Doses / Concentrations:
150000 ppm
Basis:
other: target concentration

Remarks:

Doses / Concentrations:
150000 ± 2877 (males) and 149300 ± 2215 (females) ppm
Basis:
other: achieved concentration

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes

Positive control(s):

cyclophosphamide
- justification for choice of positive control: The main study (Phase II) was conducted using vinyl chloride as a positive control. Due to unexpected toxicity and technical problems with vinyl chloride, the main study was subsequently conducted as phase III with cyclophosphamide as a positive control.
- Route of administration: oral
- Dose: single administration of 65 mg/kg bw

Tissues and cell types examined:

bone marrow

Clinical observations (every 30 minutes during the exposure and at least twice a day thereafter)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Range-finding study performed with groups of 5 males and 5 females, which were exposed to a target concentration of 150000 ppm for 6 hours and observed for 4 consecutive days in order to determine the Maximum Tolerated Concentration (MTC). The concentration of 150000 ppm was considered to represent the maximum tolerable concentration (MTC) as no death occurred. 

TREATMENT AND SAMPLING TIMES: Bone marrow smears were prepared at 24 and 48 h for the air control and HFC 32 treated animals, and at 24 h after exposure to cyclophosphamide.

DETAILS OF SLIDE PREPARATION: At the scheduled sacrifice times (24 or 48 hours after the end of the test substance exposure and 24h after the end of the cyclophosphamide exposure), mice were sacrificed by CO2 asphyxiation (subsequently ensured by cervical dislocation). Immediately following sacrifice, the femurs were removed and the bone marrow was taken out by dipping a fine paint brush in the marrow canal. The bone marrow cells were transferred to clean glass slides. The slides were allowed to air dry then stained with polychrome methylene blue and eosin using an Ames Hema-Tek staining machine.

METHOD OF ANALYSIS:
- PCE/NCE ratio was determined until a total of at least 1000 cells (PCE+NCE) were counted;
- 1000 polychromatic erythrocytes were examined for the presence of micronuclei using x10 or x12.5 eye pieces and a x100 oil immersion objective lens for each animal.

Evaluation criteria:

The positive control should induce a significant increase in  micronucleated polychromatic erythrocytes compared to the control values  and the test material should be tested at a level that causes a decrease  in the percentage of polychromatic erythrocytes (indicating a cytotoxic effect on the bone marrow) or at the maximum tolerated concentration.

POSITIVITY CRITERIA: No data

Statistics:

Analysis of variance followed by a one-sided Student's t-test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY STUDY:
No lethalities or adverse reactions were observed over a 4 day observation period after a 6h-exposure to 150000 ppm (males) and 149300 ppm (females) of test substance. Therefore the tested concentration was selected as MTC for the main study.

MAIN STUDY:
- Clinical findings:
No significant adverse reactions to treatment were observed for either males and females exposed to HFC 32.

- Medullar toxicity (PCE/NCE):
No statistically or biologically significant decreases in the percentage of polychromatic erythrocytes, compared to the air control values, were observed at either sampling time in either males or females exposed to HFC 32.

- Genotoxicity (percentage of micronuclei):
No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, over the air control values, were observed at the MTC at either sampling time in either males or females exposed to HFC 32.
In contrast, positive control presents a significant increase in micronucleated erythrocytes.

Conclusions:

No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, over the air control values, were observed at the MTC at either sampling time in either males or females exposed to HFC 32.

Executive summary:

Groups of male and female CD-1 mice were exposed to 150000 ppm difluoromethane in the atmosphere for 6 hours. 5 male and 5 female mice per group were killed 24 hrs and 48 hrs after the exposure. A preliminary toxicity test was carried out. Concurrent negative and positive control groups were exposed to air or administered 65 mg/kg cyclophosphamide, respectively. Bone marrow smears on glass slides were made from each animal. A total of at least 1000 erythrocytes/animal was examined for the presence of micronuclei. Calculated number of micronuclei per 1000 polychromatic erythrocytes were analysed. The ratio of polychromated/mature cells was also determined as an indicator of cytotoxicity.

No significant adverse reactions to treatment were observed for either males and females exposed to HFC 32. No statistically or biologically significant decreases in the percentage of polychromatic erythrocytes, compared to the air control values, were observed at either sampling time in either males or females exposed to HFC 32. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, over the air control values, were observed at the Maximal Tolerated Concentration at either sampling time in either males or females exposed to HFC 32.

In contrast, positive control presents a significant increase in micronucleated erythrocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

According to the information requirements specified in Annex X, information concerning the potential to induce gene mutations and chromosomal aberrations needs to provided. One gene mutation assay in bacteria, two in vitro cytogenicity studies and one in vivo micronucleus study are available, all with negative results. Read-across with structural analogs indicate also no concerns for gene mutation in mammalian cells.

Justification for classification or non-classification

In view of the lack of mutagenic properties of HFC 32, there is no concern for mutagenic properties of difluoromethane based on the available data. Classification according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2000 is not warranted.