Difluoromethane

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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other: GLP compliant, comparable to guideline study, available as unpublished report, no restrictions, fully adequate for assessment (SIDS score: 1b).

Objective of study:

toxicokinetics

Principles of method if other than guideline:

Comparable to guideline study regarding pharmacokinetics and metabolism.

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

[14C]-HFC32

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

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)

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.

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.

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.

Reference 2

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).

Objective of study:

toxicokinetics

Principles of method if other than guideline:

Comparable to guideline study regarding pharmacokinetics and metabolism.

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

[14C]-HFC32

Species:

rat

Strain:

other: Alpk:APfSD Wistar-derived

Sex:

male

Details on test animals or test system and environmental conditions:

- Source: Barriered Animal Breeding Unit, ICI Plc (Alderley Park,  Cheshire, UK)
- Weight at study initiation: 247 ± 23 g
- Diet: ad libitum
- Water: ad libitum

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

Route of administration:

inhalation

Vehicle:

unchanged (no vehicle)

Details on exposure:

ADMINISTRATION:
- Type of inhalation study: whole body
- Atmosphere generation: The vial containing the radio-labelled material  was opened inside an evacuated and sealed 10 litre Tedlar gas bag (SKC,  Dorset, UK). To this bag was added 5 litres of unlabeled HFC32 giving a  specific activity of 5.5µCi/mmol. Aliquots of [14C]-HFC32 from this  radiolabeled stock bag were diluted with unlabelled HFC32 to give  [14C]-HFC32 at the required specific activity for the individual  exposures (0.57-0.99 µCi/mmol for 10000 ppm exposures, 0.25-0.37 µCi/mmol  for 25000 ppm exposures and 0.12-0.15 µCi/mmol for 50000 ppm exposures.  The [14C]-HFC32/HFC32 was mixed with silica gel-dried laboratory air to  give the desired concentration of fluorocarbon which was drawn through  the chamber at a flow rate of 1 litre/min.
- Atmosphere analysis: The atmosphere concentration of HFC32 within the chamber was monitored by a PYE GCD gas-chromatograph equipped with a  Poropack PS column (80-100 mesh, glass, 1.5m x 2mm) [detector not  mentionned] at approximately 20 minute intervals throughout the exposure.

Duration and frequency of treatment / exposure:

6 hour(s)

Remarks:

Doses / Concentrations:
10000, 25000 and 50000 ppm

No. of animals per sex per dose / concentration:

males: 9

Control animals:

no

Details on dosing and sampling:

- Blood: Blood samples were obtained after 2, 4 or 6 hours of exposure by killing rats by CO2 asphyxiation and cardiac puncture. Samples were immediately analysed by two independent methods:
Vial equilibration method:
Blood samples were transferred to heparinised Reacti-vials, sealed with teflon-coated rubber septa. Those vials placed on a blood roller at room temperature for at least two hours to equilibrate the fluorocarbon between the blood and head-space. The head-space HFC32 concentration was then analysed in duplicate from each vial by gas-chromatography against standard gas sample bags containing either 5000 ppm or 10000 ppm HFC32 in laboratory air. The amount of HFC32 in the original blood sample is the sum of the amounts of fluorocarbon partitioned in the head-space and blood at equilibrium. Thus, from knowledge of the blood:air partition coefficient at the equilibration temperature, determined in a preliminary study (1.44 ± 0.08 at 21°C) and the amount of HFC32 in the head-space at equilibrium the blood concentration of HFC32 can be determined.
Liquid scintillation analysis method:
Blood samples were transferred to heparinised Reacti-vials, corked with teflon-coated rubber septa and containing 3 ml tetrahydrofuran (THF). Vials were shaken to aid partitioning of the HFC32 from blood to THF prior to radiolabel determination by liquid scintillation counting.

- Exhaled air: radio-labelled carbon dioxide was collected during and up to 11.5 hours after exposure by trapping it in 2N NaOH solutions. Every 30 minutes, solutions were changed and stored at 4°C before being purged with nitrogen, allowing thus to harvest the dissolved [14C]-CO2 for counting them in Hionic fluor with a liquid scintillation counter.

- Urine: Urine was collected during the exposure and 11.5 hours onwards. Samples were stored at -70°C before being purged with nitrogen, in order to remove dissolved [14C]-HFC32 and counting them in Optiphase MP with a liquid scintillation counter.

Details on absorption:

BLOOD CONCENTRATIONS:
Upon whole body exposure of rats to [14C]-HFC32 at atmosphere concentrations of 10000, 25000 and 50000 ppm the partitioning of fluorocarbon between the alveolar air space of the lung and blood had reached equilibrium at 2 hours.

The dose-concentration relationship was linear. Actually at 10000 ppm HFC32 the blood concentration of this fluorocarbon measured over a 2-6 hour exposure period was 23.7 ± 1.4 µg/ml. Increasing the atmosphere concentration to 25000 ppm gave a blood concentration over the same exposure period of 62.5 ± 2.6 µg/ml. Thus, for a 2.5-fold increase in the atmosphere concentration of HFC32 there is a corresponding increase in the blood concentration. Similarly, at an atmosphere concentration of 50000 ppm the blood concentration again increased linearly with dose to 120.2 ± 5.2 µg/ml.

Comparable blood concentrations were measured following extraction of radiolabel from blood into THF and quantification following liquid scintillation counting, suggesting that the bulk of the material present in blood was unchanged HFC32.

Details on excretion:

EXHALED AIR:
At atmospheric concentrations of 10000 ppm, 25000 ppm and 50000 ppm the amount of [14C]-HFC32 metabolised and exhaled as [14C]-carbon dioxide increased linearly during the exposure period to maximum levels of around 125 µmol/kg/hr (10000 ppm), 355 µmol/kg/hr (25000 ppm) and 667 µmol/kg/hr (50000 ppm) by 4-5 hours. This level was maintained up to 6 hours. Upon cessation of exposure the amount of radiolabel exhaled as 14-CO2 declined rapidly at all doses during the first two hours to between 25-30% of maximum levels then more slowly up to 11.5 hours post-exposure.
Exhalation of carbon dioxide was estimated to account for approximately 90% of total metabolism.

URINE:
The amount of radiolabel excreted in urine during and following a six hour exposure at an atmosphere concentration of 10000 ppm and collected at 11.5 hours post-exposure was 96.3 µmol/kg.
At atmosphere concentrations of 25000 ppm and 50000 ppm the amount of radiolabel excreted in urine increased to 252.8 µmol/kg and 358.0 µmol/kg bwt, respectively.

Assuming these values to be equivalent to 80% of the radiolabel eliminated by this route, the total amounts of radiolabel excreted in urine resulting from a six hour exposure to HFC32 at atmosphere concentrations of 10000, 25000 and 50000 ppm are estimated to be 120.4 µmol/kg, 316.0 µmol/kg and 447.5 µmol/kg respectively.

Metabolites identified:

yes

Details on metabolites:

Exhalation of carbon dioxide was estimated to account for approximately 90% of total metabolism.

Conclusions:

As expected for a fluorinated hydrocarbon, the liquid:air and tissue:air partition coefficients measured for HFC32 are small. Consequently the uptake of HFC32 from the alveolar air space of the lung into blood is limited. HFC32 blood levels increased linearily with exposure concentrations between 10000 and 50000 ppm.
The steady state is reached within the first 2 hours of exposure and remaining at that level over the time period of the study (2-6 hours).
The test substance is mainly transformed in carbon dioxide and therefore pulmonarily eliminated (it is assumed that this route accounts for approximately 90% of the total metabolism).

Reference 3

Endpoint:

basic toxicokinetics

Type of information:

calculation (if not (Q)SAR)

Remarks:

Migrated phrase: estimated by calculation

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: PBPK modelling study, available as unpublished report, adequate for assessment.

Objective of study:

other: PBPK model for uptake and metabolism of HFC32 in the rat

Qualifier:

no guideline available

GLP compliance:

yes

Radiolabelling:

no

Species:

rat

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

No data as the study is a PBPK model

Route of administration:

inhalation: gas

Vehicle:

unchanged (no vehicle)

Details on exposure:

No data as the study is a PBPK model

Duration and frequency of treatment / exposure:

6 hours

Remarks:

Doses / Concentrations:
10000, 25000 and 50000 ppm

No. of animals per sex per dose / concentration:

No data as the study is a PBPK model

Control animals:

no

Positive control reference chemical:

No data as the study is a PBPK model

Details on study design:

The model, which was based on that described by Ramsey and Andersen (1984) and Andersen et al. (1987), describes the uptake of inhaled HFC-32 from the lung into blood and its distribution to body compartments combined in groups displaying similar uptake and perfusion characteristics. Metabolism occurs only in the liver compartment. The model was constructed using the ACSL (advanced continuous simulation language) computer programme.
The model parameters (partition coefficients and physiological constants) were taken from literature.
The biochemical constants were measured.

Details on dosing and sampling:

No data as the study is a PBPK model

Preliminary studies:

Not performed

Details on absorption:

The HFC-32 rat blood:air partition coefficient of 1.25, is significantly lower than that of any of the other dihalomethanes (cf dichloromethane = 19). Consequently uptake of HFC-32 from the alveolar airspace into blood is minimal and is the rate limiting factor for all other processes, including metabolism.
A comparison of the blood levels obtained from the model with those measured experimentally by Ellis et al. (1994) confirms that the PB-PK model provides an accurate representation of the uptake process. Sensitivity analysis showed that the blood levels were controlled almost entirely by the blood:air partition coefficient and were not significantly affected by any other physiological or metabolic parameter. Based on the inhaled doses calculated according to Elles et al. (1992), approximately 2.1% of the HFC-32 entering the airways is absorbed into the systemic circulation and the tissues.

Specific modelled absorption figures:
10529 ppm > 2.1%
24131 ppm > 2.2%
49506 ppm > 2.1%

Details on distribution in tissues:

HFC-32 is not significantly soluble in fat and is not stored in the fat compartment during or post-exposure. The partition coefficients for the other body compartments were also comparable to the blood:air partition coefficient.

Metabolites identified:

not measured

The metabolism of HFC-32 in the liver is flow limited, that is, the rate of substrate reaching the liver is limited by the low blood:air partition coefficient. As can be expected for a chemical with a limited uptake from the alveolar airspace into blood, maximal metabolic rates are not achieved up to the highest dose level of 50000 ppm used in the study. As a result metabolism is linear throughout the dose range and is adequately described by a linear rate constant Kf. From the model it can be determined that 63% of the absorbed dose is metabolised throughout the dosing range. Thus, metabolism expressed as a percentage of the absorbed dose is high, but when expressed as a percentage of the exposed dose is much lower, being approximately 1.4% of the dose entering the airways.

Conclusions:

In conclusion, the blood levels of HFC-32 are determined solely by the inhaled concentration blood/air partition coefficient, and metabolism, which is flow limited, can be adequately described by a first-order rate constant over the range of dose levels studied. This is also likely to apply in other species, including humans. Species differences in metabolism and pharmacokinetics between rodents and between rodents and humans generally arise from differences in the size and perfusion rates of tissue compartments, particularly fat, and from differences in metabolic rates. HFC-32 is not significantly soluble in fat and it is unlikely that the blood:air partition coefficient will vary significantly between species. A comparison of the metabolism of HFC-32 between rats and mice also failed to identify and major species differences (Ellis et al. 1992). Thus it is probable that uptake will also be limited in humans and that metabolism will be linear over a wide range of doses. The exposure level at which metabolism is saturated in either animals or humans cannot be predicted from the data currently available.

Description of key information

HFC 32 is poorly absorbed by inhalation (about 1%) ; mainly eliminated via exhalation as unchanged substance. Metabolised at low rates. Carbon dioxide was identified as the major metabolite in rats and mice.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

0

Absorption rate - dermal (%):

0

Absorption rate - inhalation (%):

1

Additional information

The requirements of REACH are to provide an assessment of the toxicokinetic behaviour of the substance to the extent that can be derived from the relevant available information.

 

Kinetic and metabolic studies in rats and mice indicate that during inhalation exposure difluoromethane is poorly absorbed (about 1%), eliminated mainly via exhalation as unchanged compound and as carbon dioxide, the major metabolite, the remaining being excreted in urine as formic acid presumably. The equilibrium in blood is reached after 2 hours of exposure.