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Toxicology Letters 209 (2012) 21– 29

Contents lists available at SciVerse ScienceDirect

Toxicology Letters

jou rn al h om epage: www.elsev ier .com/ locate / tox le t

ptake and disposition of 1,1-difluoroethane (HFC-152a) in humans

ena Ernstgårda,∗, Bengt Sjögrena, Wolfgang Dekantb, Tobias Schmidtb, Gunnar Johansona

Work Environment Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, SwedenUniversity of Wuerzburg, Wuerzburg, Germany

r t i c l e i n f o

rticle history:eceived 28 October 2011eceived in revised form4 November 2011ccepted 25 November 2011vailable online 4 December 2011

eywords:,1-Difluoroethane

a b s t r a c t

The aim of this study was to determine the toxicokinetics of inhaled 1,1-difluoroethane (HFC-152a)in humans. Healthy volunteers were exposed to 0, 200 or 1000 ppm 1,1-difluoroethane for 2 h at lightexercise in an exposure chamber. Capillary blood, urine and exhaled air were sampled up to 22 h post-exposure and analyzed for 1,1-difluoroethane. Fluoride and other potential metabolites were analyzed inurine. Symptoms of irritation and central nervous system effects were rated and inflammatory markerswere analyzed in blood. Within a few minutes of exposure to 200 and 1000 ppm, 1,1-difluoroethaneincreased rapidly in blood and reached average levels of 7.4 and 34.3 �M, respectively. The post-exposuredecreases in blood were fast and parallel to those in exhaled air. The observed time courses in blood

FC-152axperimental exposureoxicokineticsnflammatory markersuman

and breath agreed well with those obtained with the PBPK model. The PBPK simulations indicate a netuptake during exposure to 1000 ppm of 6.6 mmol (6.7%) which corresponds to the amount exhaled post-exposure. About 20 �mol excess fluoride (0.013% of inhaled 1,1-difluoroethane on a molar basis) wasexcreted in urine after exposure to 1000 ppm, compared to control. No fluorine-containing metaboliteswere detected in urine. Symptom ratings and changes in inflammatory markers revealed no exposure-

related effects.

. Introduction

The hydrofluorocarbon (HFC) 1,1-difluoroethane (HFC-152a,ereafter called difluoroethane) is a colorless, flammable gas atoom temperature and normal atmospheric pressure. It has a slightthereal odor and low toxicity. Difluoroethane has a low globalarming potential and is one of the major HFCs that have replaced

hlorofluorocarbons (CFC) and hydrochlorofluorocarbons (HCFC)n refrigeration and foam applications. In addition to serving as aefrigerant, difluoroethane is also commonly found in electronicleaning products, and many consumer aerosol products (ECETOC,004).

Difluoroethane has become a substance of abuse due to its acces-ibility. When inhaled (huffed), difluoroethane causes euphoriaBroussard et al., 1997). Fatal cardiac arrhythmias due to intoxi-ation with difluoroethane have been reported (Avella et al., 2006).his intoxication has been associated with sudden death involv-ng cardiac arrhythmias termed “sudden sniffing death syndrome”

Bass, 1970; Groppi et al., 1994; Xiong et al., 2004).

In a study by Keller et al. (1996) inhalation exposure to 3000 ppmor 4 h of 1,1-difluoroethane showed no effect in rats. The substance

∗ Corresponding author at: Work Environment Toxicology, Institute of Environ-ental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden.

el.: +46 8 524 82226; fax: +46 8 33 69 81.E-mail address: Lena.Ernstgard@ki.se (L. Ernstgård).

378-4274/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.toxlet.2011.11.028

© 2011 Elsevier Ireland Ltd. All rights reserved.

may, however, induce cardiac sensitization at higher exposurelevels (150 000 ppm, 405 000 mg/m3). No evidence of toxicity orcarcinogenicity was found in a 2-year rat inhalation study withexposures up to 25 000 ppm (67 500 mg/m3) (ECETOC, 2004). Nomaternal or developmental toxicity was noted after exposure ofrats at 50 000 ppm (135 000 mg/m3), the highest level tested (Emaet al., 2010).

The low toxicity of diflouroethane contrasts that of the struc-turally similar substance 1,2-diflouroethane which was highly toxicto rats upon inhalation for 4 h. The toxicity is thought to be medi-ated by fluoroacetate, a metabolite of 1,2-difluoroethane but not of1,1-difluoroethane. Because of its toxicity, fluoroacetate has beenused as a rodenticide for over 40 years (Keller et al., 1996).

No toxicokinetic data were found in the scientific literature,except one paper where the time course (up to 15 min) of difluo-roethane was studied in blood, brain, heart, liver, and kidney in 30rats exposed at levels corresponding to abuse (Avella et al., 2010).No concentration of difluoroethane in air was reported in thisstudy.

The main objective of this study was to determine uptake, dis-tribution and elimination of difluoroethane in humans during andafter short-term inhalation exposure. To this end we analyzed diflu-oroethane in blood, urine and breath. We also looked for potential

urinary metabolites, including fluoride ion and fluoroacetate. Irri-tative and central nervous system (CNS) symptoms were assessed.Since inflammatory diseases were previously reported in relationto occupational exposure to HFC (Hagberg and Lillienberg, 1999;

22 L. Ernstgård et al. / Toxicology

Table 1Body composition of the volunteers in the study (average and range).

Women Men

Number of subjects 6 7Age (y) 23 (20–28) 23 (20–29)Height (cm) 164 (157–169) 183 (180–188)Weight (kg) 56.6 (49.5–66.2) 68.3 (58.1–82.2)Fatmass (kg)a 13.6 (6.2–23.0) 4.9 (1.2–11.6)Musclemass (kg)a 40.9 (35.4–46.1) 60.2 (53.7–67.4)

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a Estimates from a combined scale and impedance meter.

illienberg et al., 2002), inflammatory markers in plasma or serumere sampled before and after exposure to difluoroethane.

. Materials and methods

.1. Subjects

Volunteers of both sexes were recruited by advertisement among students atarolinska Institutet, (Stockholm), Stockholm University, and Institute of Technol-gy (Stockholm). Inclusion criteria were: age 20–50 years, healthy, non-smoker andithout chronic diseases. A medical examination, including clinical blood chem-

stry tests, was performed prior to inclusion in the study. To avoid fetal exposure,emales performed a pregnancy test (HCG One Step Pregnancy Test, Acon Labora-ories, Inc., San Diego, USA) immediately before each exposure. The subjects werenformed about the design of the study, possible hazards, and their right to imme-iately and unconditionally interrupt the exposure. Each participant was informedrally and in writing and signed a written consent. The study was performed accord-ng to the Helsinki declaration and approved by the Regional Ethical Review Boardn Stockholm.

Six women and four men completed all exposure sessions (control, 200 ppmnd 1000 ppm difluoroethane). In addition, one male underwent the control and the00 ppm condition, one male only 200 ppm and one male performed only the controlondition (n = 12 for control, n = 12 for 200 ppm, and n = 10 for 1000 ppm). Bodyomposition, including fat mass, total body water and muscle mass, was estimatedy a combined scale and impedance meter (Tanita BC-418 ma, Stetoskop, Denmark).ges and anthropometric data are given in Table 1.

.2. Experimental design

Two subjects at a time were exposed for 2 h at three separate occasions to vaporsf difluoroethane (98%, provided from AGA Gas AB, Sundbyberg, Sweden) at 200 and000 ppm and to clean air as control exposure. The volunteers followed differentxposure sequences, according to a balanced design. They were informed about thexperimental design, including the exposure concentrations, but were blinded toheir personal exposure sequence. The exposures of each volunteer were separatedy at least one week.

The exposures were carried out during light exercise (50 W) on computer-ontrolled ergometer bicycles (Monark Ergomedic 829 E, Monark Exercise AB,ansbro, Sweden) in an exposure chamber (20 m3) with a controlled climate.eart rate was recorded by automatic electrocardiographic (ECG) telemetry (PolarE 3000, Finland). The target temperature and relative humidity were 18 ◦C and0%, respectively. The temperature was chosen to achieve a pleasant environ-ent for light exercise while remaining at a plausible indoor air temperature

t the work place. The actual climate conditions were continuously recordedVaisala HMP 36, ETM, Sweden), and logged on a personal computer in 1-minntervals.

Difluoroethane was introduced to the chamber via a preheated glass tube con-ected to the inlet air stream via a pressure regulator (AGA Gas AB, Sundbyberg,weden) and a mass flow controller (DFC 2600 digital mass flow controller with DFCtilization software, v. 1.04, Aalborg Instruments & Controls, Inc., NY). The difluo-oethane vapor was mixed with clean air and dispersed into the entire exposurehamber through the ceiling. A fan situated within the chamber further ensuredn even distribution. In order to prevent leakage of difluoroethane vapor fromhe chamber to the surrounding laboratory, the inlet airflow rate was kept lowerhan the outlet rate, thus causing a slightly lower atmospheric pressure inside thehamber.

.3. Sampling and chemical analyses

.3.1. Chamber airThe concentration of difluoroethane in chamber air was checked by gas chro-

atography (GC) at 5-min intervals throughout the exposure sessions. Air wasucked from the upper central part of the exposure chamber via a Teflon®-coatedube to the air-sampling loop of the GC (Auto system, PerkinElmer) by means of an airump. The GC was equipped with a Poraplot Q (10.0 m, 0.32 i.d., 10 �m, Chrompaco. 7550, Middelburg, The Netherlands) capillary column and a flame ionization

Letters 209 (2012) 21– 29

detector. Nitrogen was used as a carrier gas at a column pressure of 10 psi. The tem-perature of the injector was 225 ◦C and that of the detector was 250 ◦C. The columntemperature started at 140 ◦C and was increased by 25 ◦C/min to 190 ◦C, and thenkept isothermal for 0.35 min. The retention time of difluoroethane was 1.1 min. TheTotalchrome (v.6.2, PerkinElmer) software was used for peak integration. Calibra-tion standards (56–1124 ppm) were prepared by filling Tedlar bags (1–3 L, SKC Inc.,Eighty Four, PA, USA) with known volumes of clean air by means of a calibratedpump (AirCheck sampler, Model 224-PCXR8, SKC Inc., Eighty Four, USA) and knownamounts of difluoroethane added by gastight micro syringes (Hamilton, USA). Themethod error, determined from all prepared standards (n = 102) was 5.9%.

2.3.2. Exhaled airMixed exhaled air was collected once before the exposure, at five times during

(at 9, 26, 55, 85, and 106 min) and seven times after the exposure (at 133, 146, 157,173, 215, 360 min, and 22 h from the onset of exposure). The pulmonary ventila-tion was recorded with an electric spirometer (K.L. Engineering, USA) during everybreath sampling period. The procedure is described in detail in Nihlén et al. (1998).The breath samples were analyzed with the same equipment and settings as usedfor chamber air (see above), except for the last three breath samples from eachexposure session which were concentrated on adsorption tubes in order to achievesufficient analytical sensitivity. In these cases, about 0.3 L of mixed exhaled air wassampled on stainless steel adsorption tubes, filled with Carbotrap (20/40 Mesh),Carbotrap C (20/40), and Carbosieve SIII (60/80) (all from Supelco Park, Bellefonte,USA) for 2 min by means of an air pump (Aircheck 224-PCXR8, SKC Inc., EightyFour, USA). The exact volume of sampled air was noted for use in the final cal-culation of concentrations. Duplicate samples were collected and analyzed in theafternoon after the end of exposure. Samples were desorbed (Automated ThermalDesorption system ATD-400, PerkinElmer Ltd., Beaconsfield, England) and analyzedusing the same GC system as described previously by Nihlén et al. (1998). The ther-mal desorption settings were: desorption oven 300 ◦C, desorption time 5 min, valve200 ◦C, trap low −30 ◦C, high 300 ◦C, isothermal 5.0 min, line 200 ◦C and vial pressure3 psi. A 25-m Poraplot Q capillary column (i.d. 0.53 mm, coating 0.2 �m, no 7353,Chrompack, Middelburg, The Netherlands) was used in the GC analysis. Nitrogenwas used as carrier gas at a pressure of 10.0 psi. The injector and detector temper-atures were 200 ◦C and 250 ◦C, respectively. The column temperature was initiallykept at 40 ◦C for 2 min, then raised to 150 ◦C by 10 ◦C/min and finally kept isothermalfor 4 min. The retention time of difluoroethane was 10.1 min. Calibration standards(0.06–8.6 ppm) were prepared in Tedlar bags as described above. The method error,determined from all prepared standards (n = 57) was 10.5%. The limit of detection(LOD), calculated from the minimum peak area by the GC integrator, was about0.03 ppm.

2.3.3. Blood samplingVenous blood (10 ml) was collected from the brachial vein in heparinized

tubes (Venosafe, Terumo Europe, Leuwen, Belgium) prior to exposure. These bloodsamples were used to prepare analytical calibration standards. Venous blood(approximately 20 ml) was further collected in four tubes (2 citrate, 1 lithium-heparin, and 1 for serum) before, at 3 h and 22 h after exposure for analysis ofinflammatory markers as described later. The citrate tubes were centrifuged at2000 × g for 15 min and plasma was frozen to −70 ◦C for later assays. The lithium-heparin tube and the tube for serum were centrifuged at 2700 × g for 10 min andplasma/serum was stored at −20 ◦C until analyzed.

Arterialized capillary blood was sampled from the subjects’ finger tips before,during (at 4, 7, 11, 15, 20, 32, 61, 92, and 117 min) and after exposure (at 122, 127,130, 136, 142, 150, 178, 214, 271, 356 min, and 22 h), 21 samples in total. To allowsampling, the subject extended his or her hand through a hole in the chamber wall.About 1 min prior to sampling, the subject immersed his hand in hot water (approxi-mately 40 ◦C) to increase the blood flow and reduce the risk of sample contaminationby difluoroethane deposited on the skin. Capillary blood (200 �L) was collected intwo heparinized glass capillaries of 100 �L each (Kebo AB, Sweden). The samplewas quickly transferred to a gas-tight head-space vial (21.4 ml) which was instan-taneously capped with a Teflon-lined rubber septum. Blood sampling was carriedout inside a box flushed with air to avoid sample contamination. The blood sampleswere analyzed the same day by head-space GC.

2.3.4. Urine samplingUrine was sampled in glass bottles once before exposure and at 2, 4, and 6 h

after the onset of exposure, as well as twice in the evening and once the followingmorning. The volunteers were instructed to completely void the bladder at specifiedtime points (at 9, 13 and 22 h post-exposure). Any urine that had to be voided inbetween was saved and added to the sample collected at the next time point. Theurine volume was recorded and pH was measured (Beckman 295 pH/temp/mV/ISEmeter, Beckman Instruments INC, Fullerton, USA), thereafter several portions of eachsample were transferred to glass vials and stored at −20 ◦C for later analysis offluoride and other possible urine metabolites. Urine sampled prior to exposure was

used to prepare calibration standards for difluoroethane.

2.3.5. Difluoroethane in blood and urineDifluoroethane in blood and urine was analyzed by head-space GC (Hewlett

Packard GC System, HP 6890 Series, automated headspace sampler, HP 7694, flame

cology Letters 209 (2012) 21– 29 23

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Table 2Human blood:air and tissue:blood partition coefficients used in the PBPK model.

Tissue Partitioncoefficient

Reference

Blood:air 1.08 Ernstgård et al. (2010b)Fat:blood 3.94 Ernstgård et al. (2010b)Liver:blood 0.40 Beliveau et al. (2003)Muscle:blood 0.77 Meulenberg and Vijverberg (2000)Lung:blood 0.74a Gunnare et al., 2006Vessel-rich group:blood 0.74a Meulenberg and Vijverberg (2000)

L. Ernstgård et al. / Toxi

onization detector, HP ChemStation integrator). The vials with blood (200 �L) andrine (2 ml) were thermostated for 15 min at 40 ◦C prior to GC analyses. A 25-

Poraplot Q capillary column (i.d. 0.53 mm, coating 20 �m, Chrompac no. 7554,iddelburg, The Netherlands) was used with nitrogen at a pressure of 14.0 psi (about

3 ml/min) as carrier. The temperatures were: injector 80 ◦C, detector 250 ◦C andolumn initial 40 ◦C. After 2 min the column temperature was increased by 4 ◦C/mino 60 ◦C, and finally by 40 ◦C/min to 200 ◦C. The retention time was 4.0 min. The

ethod error, including sampling as well as GC analysis, was 2.6%, as measuredrom 96 duplicates collected during exposure to difluoroethane. The LOD, calcu-ated from the minimum peak area by the GC integrator, was about 0.001 �M foroth blood and urine. Samples with undetectable amounts of difluoroethane weressigned half the LOD in the statistical analyses.

.3.6. Metabolites in urineFluoride in urine was analyzed with an ion selective electrode (Beckman 295

H/temp/mV/ISE meter, USA with Combined Fluoride Electrode, no 51141 Beckmannstruments, Inc., USA). The LOD (according to the manufacturer) was 1.1 mM andhe analytical error was 2.5%.

To identify potential metabolites of difluoroethane excreted with urine (diflu-roacetic acid, difluoroacetaldehyde, difluoroethanol, and conjugates), 12 urineamples from three subjects collected before, 2, 4, and 6 h after exposure to000 ppm of difluroethane were analyzed by nuclear magnetic resonance (NMR)pectroscopy, as described by Schuster et al. (2008). After thawing at 4 ◦C, 1 ml of auman urine sample was vortexed and then centrifuged for 10 min at 14 000 rpmnd 4 ◦C. An aliquot (720 �L) of the obtained supernatant was added to 80 �L ofeuterium oxide. No further sample workup was performed before 19F NMR anal-sis. 19F NMR spectra were recorded with a Bruker DRX 400 NMR spectrometerith a 5 mm fluoride probe operating at 376 MHz. 19F chemical shifts were refer-

nced to external CFCl3. Spectra were recorded with a 30◦ pulse, a pulse length of0 �s and a cycle delay of 10 �s. The acquisition time was 1.9 s and 8192 scansere recorded to obtain a good signal to noise ratio (S/N 16). The 19F spectraere acquired with proton coupling and a spectral with of 100 ppm (50–150 ppm).

OD of the method for fluoroacetic acid is in the low micromolar range inrine (e.g. approximately 3.7 �M for 3,3,3-trifluorolactic acid (S/N 8.6)). Since noetabolites were found in the investigated samples, no additional samples were

nalyzed.

.3.7. Inflammatory markers in bloodHigh sensitivity C-reactive protein (CRP), serum amyloid A protein (SAA),

nterleukin-6 (IL-6), fibrinogen, D-dimer, factor VIII, plasminogen activatornhibitor-1 (PAI-1), and von Willebrand-factor were analyzed in venous blood col-ected before and at 3 h and 22 h post-exposure. The analyses were carried out byhe standard clinical chemistry laboratory at the Karolinska University Hospital,tockholm, Sweden.

.4. Symptom ratings

Symptom ratings were performed using a 0–100 mm visual analogue scaleVAS) graded from “not at all” to “almost unbearable”. The ten symptoms to beated in the questionnaire were: “discomfort in the eyes: burning, irritated, orunning eyes”, “discomfort in the nose: burning, irritated, or runny nose”, “discom-ort in the throat or airways”, “breathing difficulty”, “solvent smell”, “headache”,fatigue”, “nausea”, “dizziness” and “feeling of intoxication”. The questionnaire waslaborated for vapor exposure and has been used in several similar inhalation stud-es performed in our laboratory (Ernstgård et al., 2006a,b,c; Iregren et al., 1993;undblad et al., 2004). Symptom ratings were performed immediately before, dur-ng exposure (at 62, and 110 min), and after exposure (at 340 min from onset ofxposure).

.5. Calculations

.5.1. Toxicokinetic analysesThe inhaled amount was calculated as the pulmonary ventilation multiplied by

he difluoroethane concentration in inhaled air. The area under the concentrationime curve (AUC) for blood, urine and exhaled air was calculated by the trapezoidal

ethod. The half-time of the difluoroethane decrease in urine was calculated byeast-square fitting of a mono exponential decay function using Microsoft Excelolver (v. 2007). Pooled data were used in the toxicokinetic analysis of urine sinceost samples had non-detectable difluoroethane levels.

.5.2. PBPK modelA perfusion-limited physiological based pharmacokinetic (PBPK) model was

sed to further examine the experimental data and to calculate the systemic uptakef difluoroethane. The model has previously been used in a similar fashion to sim-late the time-courses of 1,1,1-trifluroethane, 1,1,1,2-tetrafluoroethane (Gunnare

t al., 2006, 2007) and 1,1,1,3,3-pentafluoropropane (Ernstgård et al., 2010a) inlood and exhaled air. The PBPK model consists of six compartments, namely; lungsnd arterial blood combined, rapidly perfused tissues, fat tissues, working muscles,esting muscles and liver. Metabolism (if any) is assumed to occur only in the liver.uscles tissues were divided into two compartments, representing working and

a Calculated as average values of the brain:blood and kidney:blood partition coef-ficients.

resting muscles, respectively. This division was initially introduced in a PBPK modeldeveloped by Johanson and Näslund (1988) and could well explain the experimen-tally observed concentration–time curves of various solvents in arterial and brachialvenous blood during simultaneous inhalation exposure and bicycle exercise. Equa-tions and parameters (except partition coefficients) are given by Gunnare et al.(2006). The PBPK parameters (compartmental volumes, blood flows and cardiac out-put) were calculated for each individual by allometric scaling based on body weightand height and to 50-W workload and rest during and after exposure, respectively.Scaling equations are given by Nihlen and Johanson (1999). Furthermore, individu-ally measured pulmonary ventilation rates and chamber air concentrations duringexposure were used in the simulations. A local sensitivity analysis provided similarresults as presented by Gunnare et al. (2006), with the blood:air partition coefficientbeing the most sensitive parameter. For additional details, see Nihlen and Johanson(1999).

Human blood:air, water:air (physiological saline:air) and oil:air partition coef-ficients for difluoroethane were determined in a separate study (Ernstgård et al.,2010b). The liver:blood partition coefficient was calculated according to quantita-tive structure–property relationships given by Beliveau et al. (2003). The adiposetissue:blood, brain:blood and kidney:blood partition coefficients were calculatedfrom the partition coefficients of blood:air, water:air and olive oil:air, taking intoaccount the amount of fat and water in the respective tissues (Meulenberg andVijverberg, 2000). The tissue:blood coefficients for lung and vessel-rich group weresubsequently calculated as average values of the brain:blood and kidney:blood par-tition coefficients (Gunnare et al., 2006). The resulting partition coefficients arepresented in Table 2.

2.5.3. Statistical analysesThe non-parametric Wilcoxon’s test was used to test for gender differences in

physiological and toxicokinetic parameters. Due to the skewed distribution of theVAS ratings, differences between the exposure conditions were analyzed by theFriedman test using the Dunn’s multiple comparisons test for post hoc analyses.The same tests were used to analyze differences in fluoride excretion. Statisticalcomparisons of inflammatory markers measurements between exposure conditionsand gender were made with repeated measures analysis of variance (ANOVA). Thevalues of the inflammatory markers SAA, IL-6, CRP, fibrinogen, factor VIII, and vonWillebrand-factor were log transformed prior to the statistical analyses in order toachieve normality. No statistical analysis was performed on D-dimer and PAI-1 sincemost of the samples were below the detection limit. The significance level was setat 0.05.

3. Results

3.1. Exposure chamber conditions

The measured mean concentrations during exposure to difluo-roethane were 200 and 958 ppm, respectively (Table 3). Thus, theaverage concentrations were close to the target levels of 200 and1000 ppm. The chamber air temperature was close to the target of18 ◦C, with little variation between exposure conditions (18.3 ◦C,18.3 ◦C, and 18.1 ◦C at 0, 200, and 1000 ppm, respectively) as wellas within experiments (SD 0.2 ◦C). The relative humidity was alsoclose to the target of 30% at all three exposure conditions (30.0%,31.3%, and 28.9%), with modest variability within experiments (SDbetween 1.2% and 3.9%).

3.2. Physiological parameters

The average pulmonary ventilation during and after exposureare presented in Table 3. As expected, due to their smaller body sizewomen had somewhat higher heart rate during all three exposure

24 L. Ernstgård et al. / Toxicology Letters 209 (2012) 21– 29

Table 3Physiological and toxicokinetic parameters in 13 subjects exposed to 200 and 1000 ppm difluroethane for 2 h during light physical exercise (50 W). Values are given asarithmetic means with standard deviations in parentheses.

Measured values PBPK model

200 ppm 1000 ppm 200 ppm 1000 ppm

ExposureMeasured exposure concentration 199.9 ± 11.6 957.6 ± 34.0 199.9 ± 11.6a 957.6 ± 34.0a

Men 194.9 ± 14.3 955.8 ± 46.8Women 205.0 ± 2.22 946.3 ± 29.3

Pulmonary ventilation(l/min)During exposure 20.5 ± 1.72 20.9 ± 1.96

Men 20.9 ± 0.99 21.1 ± 7.16Women 20.2 ± 2.36 20.7 ± 1.89

After exposure 10.2 ± 2.42b 9.54 ± 2.29Men 11.6 ± 2.41 11.2 ± 2.36Women 8.73 ± 1.60 8.43 ± 1.33

Heart rate, during exposure (bpm) 107 ± 8.6 104 ± 7.3Men 99 ± 9.0 93 ± 12Women 111 ± 8.6 116 ± 7.0

Respiratory uptake (0–2 h)Inhaled amount (mmol) 20.6 ± 1.9 99.6 ± 9.0 20.3 ± 2.09 98.2 ± 9.52

Men 20.7 ± 1.2 101.0 ± 12.5 20.1 ± 1.85 94.1 ± 6.67Women 20.5 ± 2.5 98.7 ± 6.9 20.5 ± 2.46 100.9 ± 10.7

Net uptake (mmol) Ndc Ndc 1.38 ± 0.33b 6.6 ± 1.9Men Ndc Ndc 1.67 ± 0.15 7.5 ± 2.0Women Ndc Ndc 1.15 ± 0.18 5.3 ± 0.91

Relative uptake during exposure (% of inhaled amount) – - 7.0 ± 1.8b 6.7 ± 1.7Men – – 8.3 ± 1.2 7.5 ± 1.8Women – – 5.7 ± 1.2 5.6 ± 0.80

Respiratory excretion (2–23 h)Exhaled amount after exposure (mmol) 0.47 ± 0.15 2.2 ± 0.57 0.92 ± 0.11 4.4 ± 0.60

Men 0.52 ± 0.19 2.5 ± 0.62 0.89 ± 0.094 4.6 ± 0.66Women 0.41 ± 0.06 1.8 ± 0.32 0.95 ± 0.12 4.3 ± 0.53

Exhaled amount after exposure (% of inhaled amount) 2.3 ± 0.8 2.1 ± 0.5 4.6 ± 0.69 4.5 ± 0.52Men 2.5 ± 1.0 2.5 ± 0.31 4.5 ± 0.63 4.5 ± 0.56Women 2.0 ± 0.36 1.9 ± 0.41 4.7 ± 0.80 4.6 ± 0.53

Urine parameters (2–23 h)Excreted amount after exposure (�mol) 0.81 ± 0.69 8.4 ± 7.2

Men 1.00 ± 0.92 9.8 ± 8.0Women 0.76 ± 0.69 7.3 ± 7.2

Excreted amount after exposure (% of inhaled amount) 0.0039 ± 0.0031 0.0086 ± 0.0075Men 0.0041 ± 0.0033 0.010 ± 0.0088Women 0.0038 ± 0.0033 0.0075 ± 0.0072

AUC (�M min) 190 ± 62d 1271 ± 828d

Men 206 ± 74 1861 ± 1042Women 171 ± 44 895 ± 383Half-time (min) 40e 45e

Blood parametersC120

f (�M) 7.4 ± 0.49 34.3 ± 3.2 8.2 ± 0.67b 39.3 ± 1.9Men 7.2 ± 0.52 36.3 ± 2.1 7.8 ± 0.70 38.5 ± 2.0Women 7.6 ± 0.36 33.0 ± 3.1 8.7 ± 0.22 40.6 ± 0.8

AUC (�M min) (0–23 h) 1042 ± 58d 4572.3 ± 437.5d 1103 ± 117b 5317 ± 361Men 1049 ± 71 4525.4 ± 602.8 1010 ± 92 5155 ± 362Women 1037 ± 60 4603.5 ± 351.6 1196 ± 34 5560 ± 198

Half-time (min)Vessel-rich group 0.48 0.48Liver 0.36 0.36Working muscle 23.4 23.4Resting muscle 23.4 23.4Fat tissue 179 179

a Individually measured exposure concentrations were used in the PBPK simulations.b Significant difference between females and males, p < 0.05.c Not determined.d Calculated by the trapezoidal method.

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e Calculated from pooled data assuming a mono-exponential decrease.f C120, concentration at 120 min of exposure (end of exposure).

onditions (ergometer bicycle work, p = 0.004, Wilcoxon test), andower pulmonary ventilation after the exposures (resting condition,

= 0.04) (Table 3).

.3. Difluoroethane in blood

The initial increase in difluoroethane in blood was fast and aver-ge concentrations of 7.4 and 34.3 �M were reached within a few

minutes of exposure to 200 and 1000 ppm, respectively (Fig. 1A).The first phase of the post-exposure decrease in blood was fastas well; this was followed by a slower phase. Within 4 h post-exposure the concentration was less than 1% of the steady state

level. The blood concentrations were below the detection limit 22 hpost-exposure. The AUCs of difluoroethane in blood were 1042, and4572 �M min at 200, and 1000 ppm of difluoroethane, respectively,indicating linear (i.e. dose-proportional) kinetics.

L. Ernstgård et al. / Toxicology

A

B

C

Fig. 1. Time courses of difluoroethane in blood (A), exhaled air (B) and urine (C) from12 volunteers exposed for 2 h to 200 and 1000 ppm of difluoroethane during lightp(g

3

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3

b1

hysical exercise (50 W). Geometric means (�, ©) and 5–95% confidence intervals|) were calculated assuming lognormal distribution. Times for urine samples areiven as mid-times.

.4. Difluoroethane in exhaled air

The total inhaled amount of difluoroethane was approximately0.6 and 99.6 mmol during 200 and 1000 ppm exposure, respec-ively (Table 3). The net respiratory uptake could not be determinedxperimentally with any accuracy, since almost identical concen-rations of difluoroethane in exhaled air and inhaled (chamber) airere measured. The post-exposure decrease in exhaled air wasarallel to that in blood (Fig. 1B). On average, 0.47 and 2.2 mmolifluoroethane were exhaled unchanged after exposure at 200 ppmnd 1000 ppm, respectively, corresponding to 2.3% and 2.1% of thenhaled amount (Table 3). It should be noted that these figures areuite variable as they were obtained by interpolation from seven 2-in breath samples collected during the 2 h post-exposure period.

hey may be further underestimated since the initial rapid decayhase in breath was not captured.

.5. Difluoroethane in urine

The post-exposure decrease in urine was also parallel to that oflood (Fig. 1C). The AUCs of difluoroethane in urine were 190, and271 �M min at 200, and 1000 ppm of difluoroethane, respectively,

Letters 209 (2012) 21– 29 25

again, indicating linear kinetics. About 0.004% and 0.009%, of thetotal amount inhaled was excreted in the urine within 23 h afterexposure to 200 and 1000 ppm, respectively (Table 3).

3.6. PBPK modeling

The time courses of difluoroethane in blood and exhaled airwere further described using a previously developed PBPK model(Gunnare et al., 2006). The observed time courses agreed wellwith the exhaled air and are consistent with a very low or zerometabolism (Fig. 2). Assuming zero metabolism, the absorbedamount of difluoroethane during exposure was estimated to 1.38and 6.6 mmol at 200 and 1000 ppm, respectively (Table 3). This cor-responds to 6.7% and 7.0% of the inhaled amount. Three phases ofclearance may be identified from the half-times of the individualPBPK compartments; one fast phase of less than 1 min correspond-ing to the vessel-rich and liver compartments, one intermediate of23 min for the muscles and one slow phase of about 3 h for the fattissues (Table 3).

3.7. Metabolites in urine

About 20 �mol excess fluoride (0.013% of inhaled) was excretedin urine after exposure to 1000 ppm, compared to control. Thisexcess was significantly higher compared to both the control andthe 200 ppm condition (p = 0.008, Friedman test) and remained sig-nificant in the post hoc test (p < 0.05, Dunn’s multiple comparisontest), (Fig. 3). The excretion rate of fluoride varied a lot, how-ever, it was significantly higher in the two first urine samples afterexposure to 1000 ppm compared to control and 200 ppm exposureconditions (p = 0.0004, Friedman test, p < 0.05, and p < 0.01, respec-tively, Dunn’s multiple comparison test). Fluorine-containingmetabolites could not be demonstrated in the urine even by ahighly sensitive 19F NMR-analysis. This suggests that the extentof difluoroethane biotransformation in humans is very low underthe exposure conditions applied.

The pH in urine was in the normal range of 4.9–7.4.

3.8. Symptom ratings

The median rating of smell, irritation and CNS symptoms dur-ing exposure to difluoroethane were low, and did not exceed theverbal label “somewhat” (26 mm) at any time-point. There wereno significant differences in ratings during exposure to 200 ppm or1000 ppm difluoroethane compared to the control condition andno difference between the two exposure levels.

3.9. Inflammatory and biochemical markers

The repeated measures ANOVA analyses of inflammatorymarkers in plasma revealed no exposure-related effects of diflu-oroethane (Table 4). The D-dimer and PAI-1 concentrations wasbelow the detection limit (0.25 mg/L, and 5 E/L, respectively) inalmost all samples, hence no statistical analyses could be per-formed.

3.10. Gender differences

No gender differences were detected for any of the experimentaltoxicokinetic parameters of difluoroethane (Table 3) although thePBPK simulations suggested gender differences in the uptake and

blood levels. Thus, the modeled relative uptake as well as the netuptake was about 30% higher in men, whereas the AUC and max-imum concentration in blood (C120) were between 7% and 16%higher in women.

26 L. Ernstgård et al. / Toxicology Letters 209 (2012) 21– 29

Fig. 2. Comparison of experimentally observed (dots) and PBPK simulated (lines) time courses of difluoroethane in blood (�, �, ) and exhaled air (♦, ©, —) in volunteersexposed to 200 (�, ♦) and 1000 (�, ©) ppm difluoroethane for 2 h during light physical exercise (50 W). Data are shown for the 10 subjects who completed both exposureconditions.

L. Ernstgård et al. / Toxicology

Fig. 3. Cumulative excretion of fluorides in urine from 12 volunteers exposed for 2 htmd

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dc

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o 0, 200 and 1000 ppm of difluoroethane during light physical exercise (50 W). Geo-etric means and 5–95% confidence intervals were calculated assuming lognormal

istribution. Times are given as mid-times.

There were no exposure-related gender differences in symp-om ratings (data not shown) or inflammatory markers (data nothown). Women had a significantly higher levels (2.4 g/L, SD 0.5)f fibrinogen compared to men (2.0 g/L, SD 0.2, p = 0.04, repeatedeasures ANOVA), but this difference was unrelated to the difluo-

oethane exposure.

. Discussion

To our knowledge this is the first study of uptake and dispo-ition of difluoroethane in humans. Avella et al. (2010) reportedoncentrations of difluoroethane in blood and various organs inats exposed at levels corresponding to abuse. As the concentra-ion of difluoroethane in air was not reported by Avella et al., no

omparisons can be done with our study.

The concentration–time profiles at 200 and 1000 ppm ofifluoroethane were parallel and proportional to the exposure con-entration in all three media (blood, exhaled air, and urine) (Fig. 1).

able 4nflammatory markers in plasma of 13 volunteers, before and after exposure to cleanir (control), 1,1-difluoroethane at 200 ppm and 1000 ppm for 2 h. Values are givens geometric means with 5–95% confidence intervals in parentheses.

Exposure condition

Sampling time Clean air 200 ppm 1000 ppm

Interleukin-6 (ng/ml)Before 0.8 (0.6–1.0) 0.6 (0.4–0.9) 0.7 (0.4–1.0)3 h after 0.7 (0.5–1.0) 0.8 (0.5–1.1) 1.0 (0.7–1.6)22 h after 0.6 (0.4–0.9) 0.6 (0.4–1.1) 0.8 (0.5–1.2)

C-reactive protein (mg/l)Before 0.4 (0.2–0.9) 0.6 (0.3–1.1) 0.3 (0.1–1.0)3 h after 0.4 (0.2–0.8) 0.6 (0.4–1.1) 0.3 (0.1–0.8)22 h after 0.4 (0.2–0.9) 0.6 (0.3–1.2) 0.4 (0.1–1.0)

Serum amyloid A protein (mg/l)Before 2.8 (1.5–5.3) 2.9 (1.6–5.2) 2.1 (1.1–3.9)3 h after 2.6 (1.4–4.8) 2.7 (1.5–4.8) 1.9 (1.0–3.7)22 h after 2.9 (1.6–5.4) 3.0 (1.7–5.4) 2.4 (1.3–4.5)

Fibrinogen (g/l)Before 2.0 (1.9–2.2) 2.2 (2.0–2.5) 2.1 (1.8–2.4)3 h after 2.0 (1.8–2.2) 2.2 (2.0–2.5) 2.1 (1.8–2.4)22 h after 2.1 (2.0–2.2) 2.2 (2.0–2.5) 2.2 (1.9–2.5)

Factor VIIIBefore 1.1 (0.9–1.3) 1.3 (1.1–1.5) 1.1 (1.0–1.3)3 h after 1.1 (0.9–1.3) 1.3 (1.1–1.5) 1.1 (0.9–1.2)22 h after 1.2 (1.0–1.3) 1.2 (1.1–1.4) 1.2 (1.0–1.3)

Von-Willebrand factorBefore 0.9 (0.8–1.0) 1.0 (0.9–1.2) 0.9 (0.8–1.0)3 h after 0.9 (0.8–1.1) 1.0 (0.9–1.2) 0.9 (0.8–1.0)22 h after 0.9 (0.8–1.1) 0.9 (0.8–1.1) 1.0 (0.9–1.1)

Letters 209 (2012) 21– 29 27

In addition, the AUCs in blood and urine were proportional to expo-sure, i.e. approximately five-fold higher at 1000 ppm comparedto 200 ppm (Table 3). This indicates linear kinetics at least up to1000 ppm.

The PBPK predictions for blood and exhaled air agree well withthe observed time courses with most individual experimental data,both at 200 and 1000 ppm (Fig. 2). The predicted post-exposuredecreases depend to a large extent on the value the blood:airand tissue:air partition coefficients. The former was determinedexperimentally using pooled blood from other subjects than thoseparticipating in the present study, whereas the latter coefficientswere obtained indirectly by empirical equations or QSAR analyses.Use of individual blood:air partition coefficients and experimen-tally determined tissue:air partition coefficients would probablygive an even better fit to the experimental toxicokinetic data. Inaddition to interindividual differences in partition coefficients, itis likely that the individuals differ more with respect to e.g. pul-monary ventilation, blood flows, and body composition, than whatis estimated by the allometric scaling. All these factors may con-tribute to the divergences between observed and predicted data.

The concentration–time profiles of difluoroethane in blood,exhaled air and urine are similar to those of 1,1,1-trifluoroethane(Gunnare et al., 2006), 1,1,1,2-tetrafluoroethane (Gunnare et al.,2007), and 1,1,1,3,3-pentafluoropropane (Ernstgård et al., 2010a).As with the hydrofluorocarbons in the mentioned studies, PBPKmodeling made it possible to calculate the net respiratory uptakeof difluoroethane. The use of a PBPK model is advantageous sinceit circumvents the difficulty to experimentally measure the netuptake of poorly blood soluble chemical vapors such as difluo-roethane (blood:air partition coefficient 1.08, Table 2). For suchchemicals, the net uptake is so low that it cannot, due to experimen-tal variability and analytical error, be obtained as the difference inconcentration between inhaled and exhaled air.

No major gender differences in the toxicokinetics of difluo-roethane were detected. The PBPK simulations indicate a 30%higher uptake in men compared to women (Table 3). This differenceis consistent with the gender differences in body size and body com-position. On the other hand, women reached slightly higher bloodlevels of difluoroethane. This may be explained by the higher rela-tive workload (50 W for both sexes) in women, resulting in a higherpulmonary ventilation and a higher total uptake when expressedper kg body weight. The higher relative workload in women is alsoreflected as a higher heart rate (Table 3). The small gender dif-ferences indicated by PBPK modeling could not be experimentallyverified, probably because of experimental variability not capturedby the model, such inter individual variability in body composi-tion, inter and intra individual variability in breathing patterns andblood flows, and method errors in sampling, chemical analyses andlung ventilation measurements.

A local sensitivity analysis of the PBPK model for difluoroethaneyielded sensitivity coefficients (not shown) of the different modelparameters that were very close to those obtained for 1,1,1-trifluoroethane in our previous study (Gunnare et al., 2006). Thus,both blood and breath levels were insensitive to metabolic clear-ance, indicating that the model alone is not able to determine theextent of metabolism. With no metabolism, the only eliminationpathway is via exhalation, as the excretion via skin, urine and fecesis very close to zero. Thus, the net uptake will decrease toward zeroas steady-state is approached, According to the PBPK model, thereis still an uptake of a few percent of the inhaled amount after 2 hof exposure. This is explained by accumulation in the fat tissues. Inother words, a steady-state has not yet been reached after 2 h in

spite of the apparent plateau of the concentration–time curve.

About 0.004% and 0.009%, after exposure to 200 and100 ppm, respectively, of the inhaled difluoroethane was recoveredunchanged in urine. Similarly, in our previous studies, the urinary

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8 L. Ernstgård et al. / Toxi

ecovery of 1,1,1-trifluoroethane, 1,1,1,2-tetrafluoroethane, and,1,1,3,3-pentafluoropropane was 0.002%, 0.0007%, and 0.001%,espectively (Gunnare et al., 2006, 2007; Ernstgård et al., 2010a).he low urinary recovery of difluoroethane is hardly explained byvaporation losses, since all voided urine samples were immedi-tely stored in gas tight glass bottles until being analyzed. On theontrary, the low recovery was expected and in agreement with theow water:air partition coefficient of 1.11 (Ernstgård et al., 2010b).hus, the concentration of difluoroethane in blood and urine areery similar and parallel (Fig. 1) indicating a blood:urine partitionoefficient close to one. This is in agreement with the experi-entally measured blood:air (1.08) and saline:air (1.11) partition

oefficients and supports the notion that difluoroethane diffusesapidly across the cell linings in the kidney glomeruli and tubuli andhat, therefore, a diffusion equilibrium between blood and urine isttained. Due to the low urinary recovery of difluoroethane, thisathway was not included in the PBPK model.

Although the excretion of fluoride varied widely in unex-osed subjects (S.D. about 0.04 �mol/min), the excretion rate wasignificantly higher in the first two urine samples after expo-ure to 1000 ppm difluoroethane compared to after control and00 ppm exposure. The excretion of fluoride at both 200 and000 ppm exposure corresponds to about 2% of the inhaled amountf difluoroethane on a molar basis. This indicates that theres some metabolism of difluoroethane. Potential metabolites ofifluroethane are difluoroacetic acid, difluoroacetaldehyde, difluo-oethanol, and their conjugates. Previously acetyl fluoride has beenetected in rodents after exposure to 3000 ppm of difluoroethaneKeller et al., 1996). A rodenticide; fluoroacetate is a metabolitef the isomer 1,2-difluoroethane. However, no fluorine-containingetabolites could be demonstrated in the urine samples. Assum-

ng a LOD of 3.7 �M for fluoro metabolites in urine, as wasbtained for 3,3,3-trifluorolactic acid (S/N 8.6), we should be able toetect approximately 0.02% of the inhaled difluoroethane as fluoroetabolites in the urine. In previous human inhalation exper-

ments with 1,1,1-trifluoroethane, 1,1,1,2-tetrafluoroethane, and,1,1,3,3-pentafluoropropane zero or negligible metabolism wasound (Gunnare et al., 2006, 2007; Ernstgård et al., 2010a). How-ver, in the present study the exposure level was higher, 1000 ppmompared to 500 and 300 ppm, respectively.

Besides toxicokinetic characterization, the potential for irrita-ion of the airways and eyes, inflammatory responses of the airwaysnd central nervous system effects were investigated by symptomuestionnaires and analyses of inflammatory markers in plasma.ery low ratings of irritation and CNS symptoms were obtained.urthermore, the ratings did not increase significantly during expo-ure and were not higher at 200 ppm than at 1000 ppm, suggestinghat difluoroethane does not cause irritation, nor CNS effects, up toxposure of 1000 ppm. This is similar to the finding in our previoustudy with 1,1,1,3,3-pentafluoropropane which caused no or onlyinor symptoms up to 500 ppm (Ernstgård et al., 2010a). Exposure

o 1,1,1-trifluroethane and 1,1,1,2-tetrafluoroethane on the otherand, resulted in low but yet increased symptom ratings (Gunnaret al., 2006, 2007).

There was no relationship between inflammatory markers andxposure to difluoroethane. Since the number of participants wasmall, this negative finding does not exclude that difluoroethaneay cause an inflammatory response, however, a major inflamma-

ory response to difluoroethane is unlikely at the tested exposureevels.

.1. Conclusion

This study describes for the first time the toxicokineticehavior of inhaled 1,1-difluoroethane in humans. The kineticrofile is similar to what we have previously described for

Letters 209 (2012) 21– 29

three related hydrofluorocarbons, 1,1,1,2-tetrafluoroethane, 1,1,1-trifluoroethane and 1,1,1,3,3-pentafluoropropane (Gunnare et al.,2006, 2007; Ernstgård et al., 2010a). The characteristics are: avery low respiratory uptake, a fast increase in blood levels withinthe first few minutes and, correspondingly, a fast post-exposuredecrease. The profiles are proportional to the exposure concen-trations, suggesting linear, first-order kinetics at least up to thehighest tested level of 1000 ppm. According to the PBPK model, theprofile is consistent with a low metabolism where the low uptakeduring exposure can be explained by temporary storage of difluo-roethane in adipose tissues. No increased symptom rating and noinflammatory responses to difluoroethane were seen.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgement

We are grateful to Mr. Birger Lind for skillful technical assistance.

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