Difluoromethane

Specific investigations: other studies

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

specific investigations: other studies

Type of information:

experimental study

Adequacy of study:

supporting 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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Principles of method if other than guideline:

The anaesthetic potential of HFC-32 was assessed by exposing 4 groups of mice, each for a period of 10 minutes, to atmospheres of the test substance at target concentrations of 7.5, 15, 25 and 50%. The study design was non-standard and no specific test guideline was available.

GLP compliance:

yes

Type of method:

in vivo

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CD-1, Charles River Ltd.
- Housing: individually
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5-22.5
- Humidity (%): 30-66
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

each single group of mice was exposed continuously for 10 minutes (whole-body exposure)

Frequency of treatment:

once

Post exposure period:

1 and 2 hours

Remarks:

Doses / Concentrations:
7.5, 15, 25 and 50%
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
7.6, 15, 26, 50.5%
Basis:
analytical conc.

No. of animals per sex per dose:

5

Control animals:

no

Examinations:

- Mortality: twice daily
- Clinical signs: during exposure, 1 and 2 hours after exposure
- Body weight: daily (during acclimation period and day of exposure)

Details on results:

Mortality:
No mortality was observed.

Clinical signs:
No anaesthesia was seen during exposure of mice up to 50% (v/v) of HFC-32.
Higher grooming/cleaning activity seen at higher concentrations may be consistent with an excitatory phase prior to attaining an anaesthetised state.
Signs consistent with a definite pre-anaesthetic effect were seen during exposure at 15%, evidenced by reduced locomotory activity in one mouse and slight ataxia in all mice towards in the second half end of exposure. These signs got more and more prominent as the concentrations increased.
At up to 50% HFC-32 in air (with added oxygen to maintain 21% O2) effects were seen earlier than previous concentrations and were more pronounced. Signs consistent with CNS compromise (paddling of limbs, general unsteadiness and lack of balance) were evident during exposure, and may be considered consistent with a pre-anaesthetic phase, although no animals became unconscious.

Bodyweight:
There were no treatment-related effects.

Conclusions:

No anaesthesia resulted from exposure to 7.5, 15, 25 or 50% HFC-32 for 10 minutes. The effects seen lead to the conclusion that the EC50 (10 minutes) is greater than 50%.

Reference 2

Endpoint:

specific investigations: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Principles of method if other than guideline:

Method accoding to Reinhardt et al.; Arch. Env. 1971; 22: 265-279

GLP compliance:

yes

Type of method:

in vivo

Species:

dog

Strain:

Beagle

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ORGANISMS:
- Source: Interfauna UK (Abbots Ripton Road, Wyton, Huntingdon,  Cambridgeshire)
- Age: 6-7 months
- Weight at study initiation: 11.4-14.9 kg
- Diet: 400 g/daily
- Water: ad libitum

Route of administration:

inhalation

Vehicle:

other: clean air

Details on exposure:

ADMINISTRATION:
- Adrenaline dose: 2, 4 or 8 µg/kg (chosen animal to animal)
- Type of exposure: snout only
- Exposure schedule: On M0 (minute 0) an ECG is launched, with a  subsequent adrenaline injection on M2. The exposure to the test substance starts on M7 and still runs during a second adrenaline injection on M12  and afterwards. Exposure and ECG recording are both stopped on M17. Each animal is exposed to every concentrations in a dose-increasing order but two consecutive experiments are not performed on the same day, allowing  thus the animal to recover.
- Atmosphere generation: the test substance is a gas and was therefore  directly diluted with air and oxygen before being administered to dogs by using a respiratory mask at an air flow of 40 l/min. In contrast, positive control substance was a liquid and required therefore a  vaporiser.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations monitoring: The test atmosphere was sampled continuously  using a metal bellows pump and analysed using a Miran lA CVF infra-red  gas analyser. The output of the analyser was recorded continuously on a  Philips PM 8252 chan recorder. The measuring wavelength for CFC 11 was  12.1 µm and for HFC 32 was 9.0 µm.

Duration of treatment / exposure:

Exposure period: 10 minute(s)

Frequency of treatment:

Each animal is exposed once to every concentrations in a dose-increasing order but two consecutive experiments are not performed on the same day, allowing  thus the animal to recover.

Remarks:

Doses / Concentrations:
15, 20, 25, 30 and 35 % v/v in air
Basis:
nominal conc.

No. of animals per sex per dose:

8

Control animals:

yes, concurrent vehicle

Details on study design:

PRINCIPLE OF THE TEST:
This test aims at detecting potential for cardiac sensitisation to  adrenaline of a test substance.  Cardiac sensitisation can be defined as the ability of the test substance  to induce multiple mutifocal ectopic beats following adrenaline  intravenous injection. The study is divided into three stages:
- Stage 1: Prior to the exposure to the test substance, animals are first  exposed to increasing intravenous adrenaline doses until ectopic beats  are detected by electrocardiogram (ECG). This dose is then selected for  being used in the following stages and will not exceed 12 µg/kg.
- Stage 2: Animals are exposed to a positive control substance after an  adrenaline injection.
- Stage 3: Animals are exposed to the test substance after an adrenaline injection.

Examinations:

EXAMINATIONS: 
- Clinical signs: yes
- Electrocardiogram: The standard Lead II electrocardiogram was applied throughout the study. Appropriate areas on the dog limbs were shaved and electrode gel applied.  Standard ECG limb leads were then connected to the prepared areas on the  dog with blunt clips. The electrocardiograph (Devices 3442 ECG amplifier  with a two-channel chan recorder) was calibrated with 1 mV peaks and then  switched over to Lead II settings.
- Evaluation criteria:  The criterion for a positive effect was the appearance of a burst of  multifocal ventricular ectopic activity (MVEA) or ventricular  fibrillation (VF). Ventricular tachycardia alone is not always definitive  evidence of a positive response.

Positive control:

CFC11 (2%)

Details on results:

CLINICAL SIGNS:
Head tremors and limb tensing were observed in most of dogs at 30 and 35%  HFC 32 and at 2% CFC 11.

CARDIAC FINDINGS:
In stage 1, once a siutable dose was chosen for a dog, the response to  that dose was found to be consistent throughout the study. The results of stage 2 indicate that the experimental model is sensitive  to cardiac sensitising agents. The results of stage 3 show that HFC 32 does not induce cardiac  sensitisation (no positive responses) at the concentrations tested (15 %,  20 %, 25 %, 30 %, 35 %).  Two dogs 409 & 417 each showed a short burst of ectopic activity at 15 %  and 35 % HFC 32 respectively. Dog 409 did not show a response at higher  concentrations and was therefore considered negative. Dog 417 was not  exposed above 35% HFC 32 but data from stage 1 shows that spontaneous  ectopic activity is possible without exposure to the test gas.  It is therefore concluded that HFC 32 does not induce cardiac  sensitisation to adrenaline in the dog at or below concentrations of 35%  HFC 32 in air. In contrast, exposure to positive control substance (CFC  11) showed a positive response in 62.5% of dogs.

Conclusions:

Whatever the tested concentration up to 35% in air, HFC 32 does not induce cardiac sensitisation to adrenaline in the dog.

Reference 3

Endpoint:

specific investigations: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Principles of method if other than guideline:

Method according to Lazarow's method (1981)

GLP compliance:

no

Type of method:

in vitro

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ORGANISMS: 
- Source: Charles River France (Saint-Aubin-les-Elbeuf, 76410 Cléon,  France) 
- Cell type: hepatocytes

Route of administration:

other: in vitro

Vehicle:

other: air

Details on exposure:

in vitro method

Details on analytical verification of doses or concentrations:

not applicable

Duration of treatment / exposure:

Exposure period: 4 day(s)

Frequency of treatment:

once

Post exposure period:

None

Remarks:

Doses / Concentrations:
10, 30, 40, 50, 60, 70 80 and 90% v/v in air
Basis:
nominal conc.

No. of animals per sex per dose:

not applicable (in vitro method)

Control animals:

other: untreated and DMF (dimethylformamide)

Details on study design:

PRELIMINARY STUDY:
A cytotoxicity study was performed in a dose-finding purpose. The following concentrations were tested: 10, 30, 40, 50, 60, 70, 80 and 90%.

MAIN STUDY:
Hepatocytes harvesting: an anesthetized rat was perfused in its portal  vein with 200 ml buffered Hank's saline solution containing EGTA  (ethylene glycol-bis(beta-aminoethyl)N-N'-tetracetic acid) followed by  250 ml of Williams medium E (WME) supplemented with 5 mM Ca2+ and  containing collagenase. Liver was then removed and the capsule was  opened. Cells were collected in WME+collagenase medium and washed once  with Leibowitz L15 medium supplemented with 9% calf serum.
- Treatment: After being inoculated in round, flat-bottomed sterile glass  bottles (with screw-capped lids), cells were exposed to various  atmospheric concentrations of test compounds.
- Vehicle: sterile air
- Incubation temperature: 37°C
- Number of replicates: 3

Examinations:

EXAMINATION:
Palmitoyl-CoA-oxidase activity: Cells were incubated at 37°C for 11 minutes with radio-labelled  palmitoyl-CoA (20 nCi/ml) and cofactors.  Reaction was stopped by the addition of 250 µl of perchloric acid at  18°C.  After one hour on crushed ice, the tubes were centrifugated at 4°C and  3000 rpm for 10 minutes. 250 µl of the supernatant were collected and added to 4.75 ml of  scintillant liquid (Ready-safe, Beckmann) in counting vials. Counting was performed using a Beckmann LS 1800 scintillation counter. - Protein content: determined using a Hitachi 717 analyzer, by the Pierce BCA (bicinchoninic  acid) method, according to Smith (Anal. Biochem. 1985 ; 150 : 76-85) Results were expressed as nanomol oxidized palmitoyl-CoA per minute and  per milligram protein (PCO).

STATISTICAL TEST:  No data

Positive control:

clofibric acid (0.5 M in DMF)

Details on results:

PRELIMINARY STUDY:
During the preliminary cytotoxicity study performed at 10, 30, 50, 70 and  90%, using two bottles per concentration, the test substance was found  cytotoxic at the concentration of 90% (great number of dead cells). The  toxic effect was less pronounced at 50 and 70, and no cytotoxic effect  was observed at lower concentrations. Consequently, the concentrations selected for the peroxisome  proliferation study were 30, 40, 50, 60, 70 and 80%.

MAIN STUDY:
After a 96-hour culture period, no toxic effect was noted whatever the  concentration.  Evaluation of palmitoyl-CoA-oxidase activity was performed at the five  highest concentrations: 40, 50, 60, 70 and 80%. No increase in the palmitoyl-CoA-oxidase activity was observed whatever  the concentration of Forane 32 tested (cf. Attached Document). The positive control (clofibric acid) induced an increase in the PCO  activity at the concentration of 0.5 mM (induction factor in relation to  the DMF solvent control: 5.5), which confirms the sensitivity of the  cultures.

Conclusions:

Forane 32 is not an in vitro peroxisome inducer in rat hepatocytes.

Description of key information

HFC 32 did not show any cardiac sensitisation potential dogs exposed for 10 minutes at concentrations from 150,000 to 350,000 ppm v/v (315000 to 735000 mg/m3).
No increase in the palmitoyl-CoA-oxidase activity was observed at concentrations of 40, 50, 60, 70 and 80% difluoromethane (exposure for 4 days) in hepatocytes from a Sprague-Dawley male rat in a peroxisome proliferation assay.
No anaesthetic effects were seen in CD-1 mice exposed to 0-50% HFC-32 for 10 minutes.

Additional information

HFC 32 did not show any cardiac sensitisation potential to adrenaline in the Beagle dog exposed for 10 minutes at concentrations from 150,000 to 350,000 ppm v/v (315000 to 735000 mg/m3). The substance did not induce any multiple multifocal ectopic beats following adrenaline intravenous injection.

A peroxisome proliferation assay was performed in hepatocytes from a Sprague-Dawley male rat. The cells were cultured in vitro and exposed to concentrations of 40, 50, 60, 70 and 80% of difluoromethane for 4 days. No increase in the palmitoyl-CoA-oxidase activity was observed at any tested concentrations.

No anaesthetic effects were seen in CD-1 mice exposed to 0-50% HFC-32 for 10 minutes. Higher grooming/cleaning activity seen at higher concentrations may be consistent with an excitatory phase prior to attaining an anaesthetised state. Signs consistent with a definite pre-anaesthetic effect were seen during exposure at 15%, evidenced by reduced locomotory activity in one mouse and slight ataxia in all mice towards in the second half end of exposure. These signs got more and more prominent as the concentrations increased. At up to 50% HFC-32 in air (with added oxygen to maintain 21% O2) effects were seen earlier than previous concentrations and were more pronounced. Signs consistent with CNS compromise (paddling of limbs, general unsteadiness and lack of balance) were evident during exposure, and may be considered consistent with a pre-anaesthetic phase, although no animals became unconscious.