Difluoromethane

Genetic toxicity: in vivo

Administrative data

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other: GLP near-guideline study with acceptable restriction, adequate for assessment, unpublished report (SIDS score: 2c).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

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

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: LN21646-33
CTL reference number: Y01259/007
Purity: 99.95%

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Remarks:

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

Doses / concentrations 2

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

Examinations

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.

Results and discussion

Test results

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.

Applicant's summary and conclusion

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.