Difluoromethane

Endpoint summary

Description of key information

The NOAEL for repeated dose toxicity is higher than about 105000 mg/m3 (50000 ppm v/v) in rats (highest tested concentration) exposed for a period of 90 days.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, near-guideline study, available as unpublished report, no restrictions, fully adequate for assessment (SIDS score: 1b).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Alpk:APfSD (Wistar-derived)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS:
- Source: Alderley Park (Cheshire, UK)
- Age at stuyd initiation: 5-6 weeks
- Mean body weight at study initiation: 141-169 g for males, 130-151 g  for females
- Satellite groups: 10 additional males and 10 additional females were  included with high and control groups in order to study reversibility for  4 weeks after the end of exposure.
- Housing: 5 per cage (sexes seperately)
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-20
- Humidity (%): 35-65
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure generation: atmosphere generation: test atmospheres were generated by passing  liquid HFC 32 through a copper coil immersed in a water bath maintained  at 45°C. The resultant vapour was then passed through an equilibration coil to flowmeters via a copper distribution plenum.
- Expsure chamber volume: 300 l
- Air flow rate: 70 l/min

TEST ATMOSPHERE
- Brief description of analytical method used: test atmospheres were sampled using an automated air sampling system and analysed automatically using a gas chromatograph (Hewlett Packard HP5880A) including a Porapak P-S column (80/100 mesh, 1.8m x 4mm ID stainless steel, Waters Ltd.), fitted with a gas sampling valve (Hewlett Packard) and a flame ionisation detector.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test atmospheres were sampled using an  automated air sampling system and analysed automatically using a gas  chromatograph (Hewlett Packard HP5880A) including a Porapak P-S column  (80/100 mesh, 1.8m x 4mm ID stainless steel, Waters Ltd.), fitted with a  gas sampling valve (Hewlett Packard) and a flame ionisation detector. The  peak area attributable to HFC 32 was used to calculate the atmosphere  concentration in parts-permillion (ppm v/v), after suitable calibration  (Hewlett Packard HP3396A integrator, Waters 860 Networked Vax Data  System).

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
5000, 15000, 50000 ppm (v/v)
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
4940 ± 160, 14600 ±  470 and 49100 ± 1600 ppm (v/v)
Basis:
analytical conc.

No. of animals per sex per dose:

10 males and 10 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: 4 weeks (only for control and high-dose groups)

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY:
- Clinical signs: observed frequently during exposure and then once a day
- Mortality: daily noted
- Body weight: recorded every weeks (on the same day)
- Food consumption: weekly measured

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
- Weighted organs: adrenals, brain, heart, kidneys, liver, lungs (with  trachea attached but larynx removed), spleen and testes.
- Macroscopic and microscopic examination: Cardio-vascular and hematopoietic system: heart, aorta, lymph node (mesenteric), thymus, spleen, bone marrow . Digestive system: oral cavity, salivary glands, oesophagus, stomach,  liver, pancreas, duodenum, jejunum, ileum, caecum, colon, rectum. Glandular system: pituitary, thyroid, parathyroids, adrenal, mammary  gland, Harderian gland. Nervous system:  brain, spinal cord, sciatic nerve, eye. Respiratory system: nasal turbinates, trachea, lungs, larynx. Uro-genital system: kidney, bladder, ovary, uterus, testes,  epididymides, seminal vesicle, prostate, cervix. Other: skin, muscles, femur, sternum.

Other examinations:

- Haematology: blood samples were taken by tail bleeding from 5 males and 5 females on week 5 and 14 (every main study groups) and on weeks 18 (satellite groups).
Parameters measured by a using a Technicon H1 analyser (Bayer Diagnostics): haemoglobin, haematocrit, red blood cell count, white blood cell count, differential leukocyte count (only for control and high-dose groups), platelet count, mean cell volume, mean cell haemoglobin concentration, mean cell haemoglobin
Parameters measured on a "Coag-a-mate" (Organon-Teknika): prothrombin time, kaolin-cephalin time.

- Blood chemistry: blood samples were taken by tail bleeding from 5 males and 5 females on week 5 and 14 (every main study groups) and on weeks 18 (satellite groups). The following parameters were measured by using a Kone specific analyzer:
Electrolytes: calcium, chloride, phosphorous (as phosphate), potassium, sodium
Enzymes: alkaline phosphatase, alanine-aminotransferase, aspartate-aminotransferase, gamma-glutamyl-transferase, creatinine kinase
Other: albumin, blood creatinine, blood urea nitrogen, glucose, total bilirubin, total cholesterol, total serum protein, triglycerides

- Urinanalysis: urine samples were collected overnight on the last day of weeks 4 and 12 from 5 males and 5 females from each main study group and on the last day of weeks 13 and 16 from 5 males and 5 females per satellite group.
Parameters measured with a Jenway 3040 Ion Analyser: colour, urine volume and pH
Parameter measured with an ATAGO refractometer: specific gravity
Parameters measured with ames Multistix SG strips (Bayer Diagnostics): proteins, glucose, ketones, blood, urobilinogen
Parameter measured with a Russell Ion Selective Electrode in conjunction with the Jenway 3040 Ion Analyser: fluoride.

Statistics:

Bodyweights were considered by analysis of covariance and food  consumption by analysis of variance on initial bodyweight, separately for  males and females.
Haematology, blood and urine clinical chemistry were considered by  analysis of variance. With the exception of urine protein, male and  female data were analysed together and the results examined to determine  whether any differences between control and treated groups were  consistent between sexes.
Organ weights were considered by analysis of variance and analysis of  covariance on final bodyweight, separately for males and females. Differences from control were tested statistically by comparing each  treatment group least-squares mean with control group least-squares mean  using a two-sided Student's T-test, based on the error mean square of the  analysis.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS: There were no clinical abnormalities observed during exposure and none  observed during the study which were attributable to exposure to HFC 32.  Those findings recorded during the study were few in number and were of a  type and incidence normally expected in rats of this age and strain  (chromodacryorrhea, damaged tails, trimmed teeth, one upper incisor  missing).

MORTALITY: No deaths occurred during the study.

BODY WEIGHT: There were no effects of exposure to HFC 32 on male or female body  weights. Minor differences in bodyweight, some of which achieved statistical  significance, were seen but these were small (from -2.7% to +3.76% when  compared to control) and showed no coherent dose response relationship.

FOOD CONSUMPTION: There were no effects of exposure to HFC 32 on male or female food  consumption. Some variation between the groups/times was seen but this is expected in  studies where there are only 2 cages/sex/group, resulting in mean levels  that are highly sensitive to any unusual values. A number of statistically significant differences were seen sporadically  between treatment groups and control. However, the authors consider them  as small (from -16.4% on week 12 in females exposed to 49100 ppm to  +10.3% on week 11 in males exposed to 49100 ppm) and possibly attributed  to the factors described above. Furthermore, the lack of  dose-relationship make them of no toxicological relevance.

OPHTHALMOSCOPY: A small incidence of ocular changes (stained eyelid, hazy corneal  opacity, increased lacrymation) was observed in treated and control  animals. None was considered to be treatment related and the nature and  incidence of these changes were consistent with expected background for  this strain of rat.

HAEMATOLOGY: There were no effects of exposure to HFC 32 on male or female haematology  parameters. Tere was an increase of platelet count in all treated males  on week 5 (+41.0% at 4940 ppm, +45.4% at 14600 ppm and +38.8% at 49100  ppm). This difference was considered attributable to individual control  values which were more variable than expected, was not evident at week 14  and is therefore conidered unrelated to treatment. The other occasional statistically significant changes were small, did  not follow a coherent dose or time relationship and consequently are not  considered to be related to treatment. They consist in:
- a decrease in mean cell haemoglobin concentration (-1.8%) in  exposed  males and a slight increase in monocyte count in females from the  satellite group, both groups exposed to 49100-ppm on week 14,
- a decrease in white blood cell count (-13.1%) and lymphocyte count  (-16.6%) on week 18 in males from the satellite group exposed to 49100  ppm, - a decrease in red blood cell count (-3.5%) and an increase in mean cell  haemoglobin (+2.8%) on week 18 in females from the satellite group  exposed to 49100 ppm.

BLOOD CHEMISTRY: There were no effects of exposure to HFC 32 on male or female blood  chemistry parameters.   Statistically significant differences were found in some parameters:
- a decrease in creatinine in males on week 5 (-30.3% at 14600 ppm and  -24.2% at 49100 ppm) and on week 14 (-14.8% at 4940 ppm and -19.7% at  49100 ppm), and in females on week 14 too (-15.5% at both 4940 ppm and  14600 ppm),
- an increase in triglycerides in males on week 5 (+48.7% at 49100 ppm)  and on week 14 (+32.2% at 49100 ppm) but a decrease in females on week 14  (-26.8% at 4940 ppm),
- a decrease in bilirubin in females on week 14 (-37.8%, -35.1% and- 32.4% at 4940 ppm, 14600 ppm and 49100 ppm respectively). All of these differences are considered attributable to high individual  control values (with the exception of triglycerides in males at 49100 ppm  which is attributable to high individual values in this group), showed no  coherent dose/time relationship and are considered to be unrelated to  treatment.

Other statistically significant differences consist in:
- an apparent increase in alanine-aminotransferase (ALAT) in males on  week 5 (+23.3% at 4940 ppm) ; a dose-related increase in ALAT in females  on the same week (+26.0%, +30.7% and +34.4% at 4940 ppm, 14600 ppm and  49100 ppm respectively). 
- an increase in aspartate-aminotransferase (ASAT) in females on week 14  (+25.3% at 4940 ppm),
- a decrease in gamma-glutamyl-transferase in females on week 14 (-52.0%  at 4940 ppm),
- an increase in alkaline phosphatase in females on week 14 (+28.0% at  49100 ppm),
- a decrease in cholesterol in males on week 5 (-14.2% and -22.3% at 4940  ppm and 14600 ppm, respectively),
- a decrease in glucose in females on week 5 (-10.8% at 14600 ppm),
- an increase in calcium in males on week 14 (+5.6% at 14600 ppm),
- a decrease in phosphorus in the male satellite group on week 18 (-7.1%).
However, these differences were small, and/or attributable to high  individual values, showed no relationship with dose/time and were  considered to be unrelated to treatment.

URINALYSIS: There were no effects of exposure to HFC 32 on male or female urinalysis.  There was an apparent dose related increase in urine volumes in both  sexes at week 5 (in males  +38.2% and statistically significant at 49100  ppm only when compared to controls ; in females +73.3% and statistically  significant only at 14600 ppm) with a concommittant reduction in specific  gravity. These differences were not  evidenced at week 13 and considered  to be of no toxicological significance.  Other occasional statistically significant changes showed no consistent  relationship to exposure concentration or time and are considered to be  unrelated to treatment. They consist in:
- a decrease in proteinuria on week 5 in females at 4940 ppm (-50.7%) and  on week 17 in the satellite male group exposed to 49100 ppm (-20.2%),
- a decrease in fluoride in males at 4940 ppm on weeks 5 and 13 (-22.5%  and -27.3% respectively) but an increase in females on week 5 at 14600  ppm and 49100 ppm (+34.8% and +54.5% respectively),
- a small decrease in pH in males and/or females occasionally on week 5,  13 or 17.

NECROPSY:
- Organ weight: There were no effects of exposure to HFC 32 on male or female organ  weight.
There was a statistically significant decrease in relative kidney weight  in all treated female groups at week 14 (-6.1%  in all groups). This  change was small and consistent accross all groups, showed no  relationship to exposure concentration and considered not to be related  to exposure. In addition, there was a statistically significant increase in relative  liver weight in males at week 14 (+8.6% at 4940 ppm, +9.2% at 49100 ppm).  These diferences were small, showed no coherent dose relationship, did  not correlate with any clinical chemistry or histopathological changes  and are considered to be unrelated to treatment.
- Macroscopic findings: There were no gross findings which were considered to be treatment  related.
- Microscopic findings: At termination of the study in week 14, there was a slight increase over  controls of unilateral hydronephrosis in the kidneys of male rats exposed  to 49100 ppm (5 cases vs 1 in control). The incidence of this finding in  male control rats from the satellite group (30%) was higher than in male  controls at 14 weeks (10%) and similar to the incidence in males exposed  to 49100 ppm terminated after 14 weeks (50%). This was, therefore,  considered to be an incidental finding. Other changes were either present to the same extent in controls or were  considered to be part of the normal spectrum of changes seen in this  strain of rat.

Dose descriptor:

NOAEL

Effect level:

49 100 ppm

Sex:

male/female

Basis for effect level:

other: no toxic effects observed

Critical effects observed:

not specified

Conclusions:

Inhalation exposure of rats to measured concentrations of 0, 4940, 14600 and 49100 ppm (v/v) HFC 32 (6 hours/day, 5 days/week) for 13 weeks produced no effects considered attributable to exposure to HFC 32.

Executive summary:

Groups of 10 male and 10 female Alpk:APfSD (Wistar-derived) rats were exposed to nominal values of 0, 5,000, 15,000 and 50,000 ppm (0, 10.5,31.5 and 105 g/m3) HFC-32 (6 hrs/day, 5 days/week).  Additional groups of 10 male and 10 female rats were designated for a 4-week recovery period. The highest exposure concentration tested (50000 ppm) is considered to be a limit concentration for studies of this type on low toxicity, highly volatile fluorocarbons. No deaths, no treatment-related effects on clinical signs, bodyweights, food consumption, haematological and clinical chemistry parameters, and urine analyses were observed during the treatment and treatment-free periods. There were no changes attributable to HFC 32 exposure on group mean organ weights (including testes) or in the incidence of macroscopic or microscopic pathology findings (including reproductive organs) in any exposed group. Therefore, the NOAEC in rats was considered to be 50000 ppm v/v (105 g/m3).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

105 000 mg/m³

Study duration:

subchronic

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

The study was assessed for its reliability and was concluded to be reliable without restrictions.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In accordance with section 2 of REACH Annex XI, studies via the oral and dermal route do not need to be conducted as the substance is a gas.

Regarding inhalation exposure, the most reliable data are provided from a 28-day and a 13-week inhalation studies in Alpk:ApfSD (Wistar-derived) rats (GLP, near-guideline studies).

Animals were whole body exposed 6 hours/day, 5 days/week to HFC 32 up to nominal concentration of 50000 ppm v/v (105000 mg/m3) for 28 days or 13 weeks respectively. The highest exposure concentration tested (50000 ppm, 105000 mg/m3) is considered to be a limit concentration for studies of this type on low toxicity, highly volatile fluorocarbons. In both studies, there were no deaths, no treatment-related effects on clinical signs, bodyweights, food consumption, haematological and clinical chemistry parameters, and urine analyses. There were no changes attributable to HFC 32 exposure on group mean organ weights (including testes) or in the incidence of macroscopic or microscopic pathology findings (including reproductive organs) in any exposed group. Therefore, the NOAEC in rats is 50000 ppm v/v (105000 mg/m3) based on the 13 week study.

Justification for classification or non-classification

Based on the NOAEL of 105000 mg/m³ in a 13 week subchronic toxicity study with rats, classification of difluoromethane for repeated dose toxicity is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2000.