Difluoromethane

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, comparable to guideline study, available as unpublished report, no restrictions, fully adequate for assessment (SIDS score: 1b).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

Objective of study:

toxicokinetics

Principles of method if other than guideline:

Comparable to guideline study regarding pharmacokinetics and metabolism.

GLP compliance:

yes

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

COLD SUBSTANCE:
Source: ICI Chemicals and Polymers
CTL reference number: Y02105/010/001
Purity: 99.94% (checked by GC/MS)

RADIO-LABELLED SUBSTANCE:
Source: Imperial Chemical Industries PLC
CTL reference number: Y02105/012/001
Purity: > 97% (radiochemical purity), >95% (chemical purity, radio GC)
Specific activity: 19 mCi/mmol

Radiolabelling:

yes

Remarks:

[14C]-HFC32

Test animals

Species:

other: rat and mouse

Strain:

other: Alpk:APfSD-1 (rat), Alpk:APfCD-1 (mouse)

Sex:

male

Details on test animals or test system and environmental conditions:

Rat:
- Strain: Alpk:APfSD Wistar-derived
- Source: Barriered Animal Breeding Unit, ICI Plc (Alderley Park, Cheshire, UK)
- Weight at study initiation: 204-220 g
- Diet: ad libitum
- Water: ad libitum

Mouse:
- Strain: Alpk:APfCD-1
- Source: Barriered Animal Breeding Unit, ICI Plc (Alderley Park, Cheshire, UK)
- Weight at study initiation: 33-34 g
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

inhalation

Vehicle:

unchanged (no vehicle)

Details on exposure:

ADMINISTRATION:
- Type of inhalation study: whole body
- Atmosphere generation: HFC32 was mixed with radio-labeled material using the following procedure to give a specific activity in the range 5.27-7.38 µCi/mmol. The vial containing the radio-labeled material was opened inside an evacuated and sealed 10 litre Tedlar gas bag (SKC, Dorset, UK). The bag was then filled with 5 litres of unlabelled HFC32 followed by 5 litres of silica gel-dried laboratory air. The contents were drawn from the bag and mixed with silica gel-dried laboratory air to give a concentration of 10000 ppm HFC32 which was drawn through the chamber at a flow rate of 1l/min.
- Atmosphere analysis: The atmosphere concentration of HFC32 within the chamber was monitored by gas-chromatography at approximately 20 minute intervals throughout the exposure. During the exposure period samples of the atmosphere (1 ml) were removed every 60 minutes to determine the specific activity of the [14C]-HFC32 within the chamber.

Duration and frequency of treatment / exposure:

6 hour(s)

Doses / concentrations

Remarks:

Doses / Concentrations:
10000 ppm

No. of animals per sex per dose / concentration:

males: 4 rats and 4 mice

Control animals:

no

Details on dosing and sampling:

EXAMINATION:
- Before exposure:
During the 24h acclimation period, urine was collected over dry ice for fluoride ion determination.

- During exposure:
Urine and faeces were collected over dry ice.

- After exposure:
Urine and faeces were collected over dry ice at 6h-intervals up to 4 days and stored at -20°C.
Expired organic material was collected by dissolution into dry ice cooled acetone (100ml).
Carbon dioxide was collected by dissolution into 2M sodium hydroxide.
Carbon monoxide was collected by passing through a catalyst (Hopcalit, 10g) to convert it to carbon dioxide which was then trapped in 2M sodium hydroxide solution.

Rats:
At termination (4 days), the animals were killed by terminal anaesthesia followed by cervical dislocation (rat 1) or cardiac puncture (rats 2-4). The blood was collected in heparin tubes and part of each blood sample (rats 2-4) was centrifuged at 1500g for 15 minutes at 4°C to obtain plasma. The plasma and whole blood samples were stored at -20°C until analysed for radioactivity and carboxyhaemoglobin. Rat 1 was assayed for total carcass radioactivity. Rats 2-4 were dissected and the following organs and tissues were removed and stored at -20°C until they were assayed for radioactivity: liver, kidneys, lungs, heart, brain, testes, muscle, renal fat, spleen and bone (femur).

Mice:
At termination (4 days), the animals were killed by terminal anaesthesia followed by cervical dislocation (mouse 5) or cardiac puncture (mice 6-8). The blood was collected in heparin tubes and part of each blood sample (mice 6-8) was centrifuged at 1500g for 15 minutes at 4°C to obtain plasma. The plasma and whole blood samples were stored at -20°C until analysed for radioactivity and carboxyhaemoglobin. Mouse 5 was assayed for total carcass radioactivity. Mice 6-8 were dissected and the following organs and tissues were removed and stored at -20°C until they were assayed for radioactivity: liver, kidneys, lungs, heart, brain, testes, muscle, renal fat, spleen and bone (femur).

Every samples were analysed for radioactivity by using:
- a Tri-carb 2000 CD Liquid Scintillation system (Packard Ltd) for urine and expired air
- a Hionic Fluor for carbon dioxide, carbon monoxide, tissues and carcasses
- a Optiphase MP for expired organic material and plasma
- a Packard Sample Oxidiser Model 307 for transforming faeces to radio-labelled carbon dioxide.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Rats:
Absorption was low: approximately 1% of the inhaled dose.

Mice:
Absorption was low: approximately 1 % of the inhaled dose.

Details on distribution in tissues:

Rats:
The distribution of radioactivity was relatively uniform. The highest concentrations, expressed as nmol of HFC32 per g of tissue, were as follows: lung (286 nmol/g), liver (152 nmol/g), kidney (151 nmol/g), fat (149 nmol/g), spleen (111 nmol/g) and heart (103 nmol/g). Blood and other organs, such as muscle, brain, bone, testes, exhibited concentrations below 100 nmol/g.

Mice:
The distribution of radioactivity was relatively uniform. The highest concentrations, expressed as nmol of HFC32 per g of tissue, were as follows: lung (601 nmol/g), liver (346 nmol/g), kidney (323 nmol/g), spleen (274 nmol/g), fat (235 nmol/g) and heart (221 nmol/g). Blood and other organs, such as muscle, brain, bone, testes, exhibited concentrations below 200 nmol/g.

Details on excretion:

Rats:
- Pulmonary elimination:
Radio-labelled organic substance was found in exhaled air (up to 0.5% of the inhaled dose) and reasonably assumed to be unchanged HFC32.
Exhalation of carbon dioxide was the second major route for excretion of HFC32 metabolites and accounted for about 0.23% of the inhaled dose. It is postulated to result from oxydative metabolism mediated by cyt P450.
Carbon monoxide could not be detected as a metabolite in exhaled air. Besides, carboxyhaemoglobin values in treated and control animal were similar (2.5% vs 2.0% respectively). It can be concluded therefore that carbon monoxide, if formed, is an extremely minor metabolite of HFC32.

- Urinary elimination:
Urinary excretion of HFC32 metabolites was found to be the second most favoured route. Those metabolites accounted for 0.13% of the inhaled dose.

- Fecal elimination:
Elimination in faeces was minimal and accounted only for 0.03% of the inhaled dose.

Mice:
- Pulmonary elimination:
. Radio-labelled organic substance was found in exhaled air (up to 0.45% of the inhaled dose) and reasonably assumed to be unchanged HFC32.
. Exhalation of carbon dioxide was the second major route for excretion of HFC32 metabolites and accounted for about 0.27% of the inhaled dose. It is postulated to result from oxydative metabolism mediated by cytochrome P450.
. Carbon monoxide could not be detected as a metabolite in exhaled air. Besides, carboxyhaemoglobin values in treated and control animal were similar (1.3% vs 1.2% respectively). It can be concluded therefore that carbon monoxide, if formed, is an extremely minor metabolite of HFC32.

- Urinary elimination:
Urinary excretion of HFC32 metabolites was found to be the most favoured route. Those metabolites accounted for 0.34% of the inhaled dose.

- Fecal elimination:
Elimination in faeces was minimal and accounted for only 0.07% of the inhaled dose.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Rats:
The metabolism of HFC32 was low since metabolites accounted for approximately 0.51% of the inhaled dose.
Carbon dioxide was found to be the main metabolite inasmuch radio-labelled carbon dioxide accounted for 0.23% of the inhaled dose.
Fluoride ions were expected to be released but due to the low metabolism, fluoride levels in urine in exposed rats were similar and even lower than levels observed in unexposed animals (96 vs 114 nmol/h).
The assumed metabolism is described in attached document.
From the known routes of metabolism of dihalomethanes, it is postulated that HFC 32 is biotransformed by oxidation, mediated presumably by cytochrome P450, leading to formic acid (postulated as the major urine metabolite) and then to carbone dioxide.

Mice:
The metabolism of HFC32 was low since metabolites accounted for approximately 0.80% of the inhaled dose.
Carbon dioxide was found to be the main metabolite inasmuch radio-labelled carbon dioxide accounted for 0.27% of the inhaled dose.
The assumed metabolism is the same that presented for rats.