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Controlled Ethyltert-Butyl Ether (ETBE) Exposure of Male Volunteers
TOXICOLOGICAL SCIENCES ARTICLE NO.
46, 1–10 (1998)
TX982516
Controlled Ethyl tert-Butyl Ether (ETBE) Exposure of Male Volunteers I. Toxicokinetics Annsofi Nihle´n,*,†,1 Agneta Lo¨f,* and Gunnar Johanson*,‡ *Department of Occupational Medicine, National Institute for Working Life, SE-171 84 Solna, Sweden; †Institute of Environmental Medicine, Division of Toxicology, Karolinska Institute, Solna, Sweden; and ‡Department of Occupational and Environmental Medicine, University Hospital, Uppsala, Sweden Received December 31, 1997; accepted June 3, 1998
emissions to the atmosphere. Today, MTBE is widely used in gasoline all over the world. One reason for the interest in ETBE is the potential to increase the market for bioethanol, as ETBE can be manufactured from ethanol and isobutene. ETBE has additional advantages compared to MTBE, in terms of lower vapor pressure and higher octane number (Iborra et al., 1988). Examples of occupational exposure to oxygenates are during manufacturing, blending, and transportation of gasoline and in gasoline stations (Hartle, 1993; U.S. EPA, 1993). The general public is exposed to oxygenates mainly during gasoline filling. Furthermore, groundwater could be contaminated with MTBE from leaking underground gasoline storage tanks, and the general public is today exposed to low levels of MTBE from the drinking water in parts of several states in the United States (HEI, 1996). Biotransformation of ETBE appears to be similar to that of MTBE. Thus, O-dealkylation of MTBE and ETBE will result in the same metabolite, tert-butyl alcohol (TBA) (Brady et al., 1990; White et al., 1995b; Miller et al., 1997; Bernauer et al., 1998). After MTBE exposure TBA was detected in blood and urine in animals (Savolainen et al., 1985; Brady et al., 1990; Miller et al., 1997; Bernauer et al., 1998) and in humans (Moolenaar et al., 1994; Prah et al., 1994; White et al., 1995a; Cain et al., 1996; Pekari et al., 1996; Buckley et al., 1997; Nihle´n et al., 1998b). In MTBE-exposed rats, 2-methyl-1,2propanediol, a-hydroxyisobutyric acid, and TBA conjugates were detected in the urine (Miller et al., 1997; Bernauer et al., 1998), which indicates further oxidation of TBA. In addition, other studies show formation of formaldehyde, acetone and carbon dioxide in TBA-treated rats and rat liver microsomes (Cederbaum et al., 1980; Baker et al., 1982). The methyl and ethyl parts of MTBE and ETBE are biotransformed to formaldehyde and acetaldehyde, respectively (White et al., 1995b; HEI, 1996). To our knowledge, no metabolites other than TBA have been identified in biological samples from humans exposed to MTBE, and no studies of the uptake and disposition of ETBE in man have been performed. It is important to further
Controlled Ethyl tert-Butyl Ether (ETBE) Exposure of Male Volunteers. I. Toxicokinetics. Nihle´n, A., Lo¨f, A., and Johanson, G. (1998). Toxicol. Sci. 46, 1–10. Ethyl tert-butyl ether (ETBE) might replace methyl tert-butyl ether (MTBE), a widely used additive in unleaded gasoline. The aim of this study was to evaluate uptake and disposition of ETBE, and eight healthy male volunteers were exposed to ETBE vapor (0, 5, 25, and 50 ppm) during 2 h of light physical exercise. ETBE and the proposed metabolites tert-butyl alcohol (TBA) and acetone were analyzed in exhaled air, blood, and urine. Compared to a previous MTBE study (A. Nihle´n et al., 1998b, Toxicol. Appl. Pharmacol. 148, 274 –280) lower respiratory uptake of ETBE (32– 34%) was seen as well as a slightly higher respiratory exhalation (45–50% of absorbed ETBE). The kinetic profile of ETBE could be described by four phases in blood (average half-times of 2 min, 18 min, 1.7 h, and 28 h) and two phases in urine (8 min and 8.6 h). Postexposure half-times of TBA in blood and urine were on average 12 and 8 h, respectively. The 48-h pulmonary excretion of TBA accounted for 1.4 –3.8% of the absorbed ETBE, on an equimolar basis. Urinary excretion of ETBE and TBA was low, below 1% of the ETBE uptake, indicating further metabolism of TBA or other routes of metabolism and elimination. The kinetics of ETBE and TBA were linear up to 50 ppm. Based upon blood profile, levels in blood and urine, and kinetic profile we suggest that TBA is a more appropriate biomarker for ETBE than the parent ether itself. The acetone level in blood was higher after ETBE exposures compared to control exposure, and acetone is probably partly formed from ETBE. © 1998 Society of Toxicology. Key Words: biological monitoring; ethyl tert-butyl ether; gasoline; human; inhalation exposure; oxygenated fuel; tert-butyl alcohol; toxicokinetics.
Aliphatic ethers, such as methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether may be used as additives (oxygenates) in gasoline (Iborra et al., 1988). These oxygenates enhance the octane number and are thought to improve combustion efficiency, thereby reducing To whom correspondence should be addressed. Fax: 146-8-730 1967. E-mail: [email protected]. 1
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1096-6080/98 $25.00 Copyright © 1998 by the Society of Toxicology. All rights of reproduction in any form reserved.
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explore the toxicokinetics and acute effects in man before ETBE is introduced in large scale in the environment. The main aim of our study was to determine the uptake, distribution, metabolism, and elimination in male volunteers after short-term exposure to ETBE (0, 5, 25, and 50 ppm). In addition, acute health symptoms were subjectively rated and objectively measured during and after exposure and are presented in an accompanying article (Nihle´n et al., 1998a).
series, Grant Instruments Ltd., UK) and stored in a personal computer as 1-min averages during each 2-h exposure. A high-performance liquid chromatography pump (305 piston pump, Gilson, France) transferred the ETBE liquid to a heated (90°C) glass cylinder, which was a part of the exposure chamber influent air system. ETBE was added to the air by means of aerosol formation followed by complete evaporation. To prevent leakage from the chamber the air pressure in the chamber was about 5 Pa lower than in the surrounding laboratory. Sampling and Chemical Analysis
MATERIAL AND METHODS Subjects Eight Caucasian male volunteers, with a mean age of 29 years (range 21– 41 years) and a mean body weight of 82 kg (range 70 –97 kg) participated in the chamber study. All subjects were healthy as judged by clinical examination. In addition, a standard clinical blood chemistry test (Nihle´n et al., 1998b) was performed in order to exclude subjects with liver and metabolic disorders and other diseases. All subjects were nonsmokers and were not occupationally exposed to ETBE or any other solvent. They had to refrain from alcoholic beverages and drugs 2 days before and throughout each experiment. The study was carried out according to the Declaration of Helsinki, after approval by the Regional Ethical Committee at the Karolinska Institute Stockholm, Sweden, and after written consent by the volunteers. Chemicals Technical ETBE was offered as a gift from Ecofuel S.P.A (Ravenna, Italy). The ETBE liquid was redistilled (Department of Organic Chemistry, University of Stockholm, Stockholm, Sweden) since impurities were found. ETBE was approximately 98.5% pure after the redistillation. The major residues were identified by gas chromatography–mass spectrometry (Miljo¨laboratoriet, Nyko¨ping, Sweden) as approximately 0.4% TBA and 1.1% MTBE. For calibration standard preparations TBA (.99.5% purity, Merck, Darmstadt, Germany) and acetone (.99.5% purity, Merck) were used. Experimental Design The study design was similar to that of our former MTBE study (Nihle´n et al., 1998b). In brief, the exposures were conducted during 2 h of light physical exercise (nominal level 50 W) on a computer-controlled bicycle ergometer (Monark Ergomedic 829 E, Sweden) in an exposure chamber. Individual heart rate and actual work load were recorded once every minute throughout the exposure. The subjects interrupted the bicycle exercise for approximately 5 min after 110 min of exposure to allow an acute effect (ocular) measurement in the exposure chamber. All sampling of blood and exhaled air was performed during exercise. Each subject was exposed on four occasions, at nominal levels of 0, 5, 25, and 50 ppm ETBE, with at least 2 weeks between two successive exposures. The highest exposure level was chosen considering the current Swedish limit for occupational exposure to MTBE (Swedish National Board of Occupational Safety and Health, 1996) since restrictions have yet been assigned to ETBE. Exposure Chamber The exposures occurred in a 20-m3 exposure chamber with controlled climate. The humidity and temperature in the chamber were measured by a humidity and temperature probe (HMP 36, Vaisala, Finland) and the air flow was measured by an air flow meter (MiniAir 6, Schiltknecht Messtechnik AG, Switzerland). In addition, the pressure difference of the surrounding laboratory and the chamber was measured (Modus Instruments Inc., Northborough, MA) and the amount of carbon monoxide in the chamber was indicated (Telaire, Martionics AB, Sweden). These measurements were made every 15 s and the signals were sampled with a data logger (Grant Squirrel meter/logger 1200
The time schedule, used equipment, and procedure of collecting samples of blood, urine, and exhaled air (before, during, and after the exposure) were similar to our previously performed MTBE study (Nihle´n et al., 1998b). Time points of collecting blood and urine are shown in Figs. 1 and 3, respectively. ETBE and TBA were analyzed in all biological samples; acetone was analyzed in blood and urine only. Chamber air. The concentration of ETBE in the chamber air was analyzed in 5-min intervals during the exposures by a gas chromatography (GC-Auto System, Perkin–Elmer, UK). The gas chromatograph was equipped with a gas sample injection loop (0.5 ml), a flame ionization detector, and a nonpolar capillary column (Poraplot U, Chrompack, The Netherlands, 10 m, 0.53 mm inner diameter, 20 mm phase thickness). Chamber air was drawn through a Teflon tubing into the gas sampling loop with the use of a pump (15 liters/min, Model DDA-P101-BN, Gast Corp., Benton Harbor, MI). The temperatures of the gas chromatograph injector, column, and detector were 200, 175 (isothermal), and 250, respectively. Nitrogen was used as carrier gas at a flow rate of 4.7 ml/min. Turbochrom chromatography workstation (Version 4.1, PE Nelson, Norwalk, CT) was used to sample and compute chromatographic data. Calibration standards were prepared in Tedlar bags (5–25 liters, SKC Inc., Eighty Four, PA) filled with known volumes of clean air by means of a calibrated pump (AirChek sampler, Model 224-PCXR8, SKC Inc.) and known amounts of ETBE. At least four different concentration levels were prepared at each exposure occasion. Air was drawn from each Tedlar bag into the gas sampling loop with the use of a pump and analyzed by the gas chromatograph as described above. Exhaled air. To determine the respiratory excretion of ETBE, exhaled air was collected once before the exposure, four times during (every 30 min), and six times after the exposure. Each exhalation period lasted for 5– 6 min and the exhaled air was analyzed during the last 2 min. Exhaled air was collected through a mouthpiece connected with two valves to separated in and outlets. During each exhalation period, the volunteers wore a nose clip to prevent nasal breathing. An electric spirometer (K. L. Engineering, USA) was connected to the inlet valve, and the individual’s pulmonary ventilation and respiratory frequency were recorded as 1-min averages during each exhalation period of 5– 6 min. The outlet valve was connected to a stainless-steel tube and a pump transferred the exhaled air to a mixing chamber (7 liters) from which some exhaled air was drawn into a gas sample injection loop. The concentration of ETBE was directly analyzed by the gas chromatograph as described under Chamber. All material in contact with expired air was heated to 40°C to avoid condensation of water. The sampled air was either directed to the gas chromatograph (as described above) or, for later analysis, pumped (590 liters/min, AirChek sampler, Model 224-PCXR8, SKC Inc.) through two sorbent sample tubes (stainless steel, length 90 mm, diameter 6.4 mm, Perkin–Elmer) containing active charcoal (Carbotrap, 20/40 mesh, Supelco Inc., Bellefonte, PA). Direct gas chromatographic analyses of ETBE were applied during the exposure and when the volunteers exhaled sufficiently high ETBE concentrations, i.e., during the entire exposure day at the 25 and 50 ppm exposure levels and until 1 h after the exposure at the 5 ppm exposure level. TBA could not be analyzed by the direct gas chromatographic method and was therefore not determined during the exposure. Sorbent tubes were used postexposure and TBA could then be analyzed. All sorbent tubes were purged with nitrogen before use (10 min, 350°C, and 56 ml/min) to avoid possible contamination and then stored in a
TOXICOKINETICS OF ETBE IN MALE VOLUNTEERS
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FIG. 1. Concentration of (a) ethyl tert-butyl ether (ETBE), (b) tert-butyl alcohol (TBA), and (c) acetone in capillary blood sampled from eight male volunteers exposed to ETBE (2 h, 50 W, mean values). Vertical lines indicate the 95% confidence intervals (only partially for acetone).
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desiccator. The flow rate through each sorbent tube was determined with a mass flow meter (Aalborg, GFM17, SKC Inc.) and a pump (590 liters/min, AirChek sampler, Model 224-PCXR8, SKC Inc.) since flow rate variations were seen. This is probably due to the heterogeneous mesh size of the charcoal. Sorbent tubes used for calibration standards had all uniform flow rates (430 ml/min), and sample sorbent tubes were adjusted for discrepancies (range 330 to 430 ml/min) during calculations. Calibration standards in air containing ETBE and TBA were drawn from Tedlar bags to sorbent tubes with the use of a pump in the same way as described for exhaled air. The sorbent tubes were analyzed with an automated thermal desorption unit (ATD-400, Perkin–Elmer) connected to a gas chromatograph (GC-Auto System, Perkin–Elmer Auto System) equipped with a capillary column (Poraplot Q, Chrompack 25 m, 0.32 mm inner diameter, 20 mm phase thickness) and a flame ionization detector. Temperatures were desorption oven 300°C; desorption trap (Tenax TA 60/80 mesh, Supelco Inc.) 230 to 300°C; transfer line 150°C; injector 200°C; column 120°C for 5 min, 5°C/min to 165°C, then 165°C for 20 min; and flame ionization detector 250°C. Nitrogen flow rates were desorption 56 ml/min, outlet split 8.3 ml/min, and column 2 ml/min. Turbochrom chromatography workstation was used. Blood and urine. Capillary blood (200 ml) was sampled from the fingertips of the volunteers, before, during, and up to 48 h after exposure. During exposure the subject held out his hand through a closable hole in the chamber wall. After rinsing the hand with water (40°C) capillary blood was taken with two 100-ml heparinized micropipetes (Drummond Scientific Co., Broomall, PA) inside a glove box, which was flushed with clean air. The blood sample was transferred to a head space vial (22.4 ml, Perkin–Elmer), which was immediately capped with a Teflon-lined butyl rubber membrane (Perkin– Elmer). All urine samples were collected in 500-ml glass bottles. The volunteers were instructed to always empty the bladder completely and to note the time on the bottle. Samples were collected prior to exposure and at 0, 2, and 4 h and approximately 7, 11, 20, and 22 h after the exposure. In addition, a spot urine sample was collected 46 h after the exposure. The 7-, 11-, and 20-h samples were collected at home and stored at room temperature until the morning when the subject returned to the institute. ETBE, TBA, and acetone in blood and urine were stable in room temperature at least up to 24 h. The urine samples were treated and analyzed as the blood samples, except that 2 ml urine instead of 0.2 ml blood was added to each head-space vial. Gas chromatographic analyses of ETBE, unconjugated TBA, and acetone were performed as soon as possible after sampling. Calibration standards of ETBE, TBA, and acetone were prepared in Tedlar bags (as described under Chamber Air), and known amounts were injected to precapped head-space vials which contained either 200 ml blood (venous blood) or 2 ml urine. The reason for using a gas-phase standard is that ETBE has a rather low water solubility. However, a comparison between gas-phase and liquid (water) standard curves showed close agreement. No internal standard was used because of the risk of evaporation losses with the sampling technique used. Based upon 20 samples from a Tedlar bag, the variation (relative standard deviation, RSD) within the method was below 5% for all three substances in either blood or urine, which illustrates that the method of automatic head-space injection and flame ionization detection has a good reproducibility. The gas phase of each vial was then analyzed with a gas chromatograph (Perkin–Elmer 8700), equipped with a head-space autoinjector (Perkin–Elmer HS-101), a nonpolar capillary column (CP Sil 8, Chrompack, 50 m, 0.53 mm inner diameter, 2 mm phase thickness), and a flame ionization detector. The samples were thermostated for 25 min at 37°C before gas chromatographic analysis. The temperatures were autoinjector 37°C, needle 100°C, transfer line 100°C, injector 180°C, detector 270°C, and column initial 50°C. The column temperature was increased with 5°C/min to 90°C and thereafter 10°C/min to 150°C. Nitrogen was used as carrier gas at a flow rate of 5.7 ml/min. The Turbochrom chromatography workstation was used. The detection limit for ETBE in blood was approximately 0.15 mM, and in urine it was 0.05 mM. The detection limits for TBA and acetone were 0.5 mM in blood and 0.2 mM in urine.
Toxicokinetic Calculations The toxicokinetic calculations were carried out according to the previously published MTBE study (Nihle´n et al., 1998b). In brief, individual concentrations of ETBE in blood versus sampling times, personal net respiratory uptake, and amount of ETBE exhaled postexposure were fitted to the algebraic solution of a linear pharmacokinetic four-compartment model with zero order uptake (G. Johanson, unpublished data). A four-compartment model was chosen to conform with the previous MTBE study. Optimization was carried out by minimizing the unweighted residual sum of squares using Microsoft Excel (Version 5.0) and the Solver add-in macro. In these calculations the absorbed dose is regarded as the sum of the net respiratory uptake and the amount exhaled during exposure. Previously in studies of this kind, the absorbed dose has been estimated as equal to the difference between the concentration in inhaled and exhaled air multiplied by the pulmonary ventilation. However, by inclusion of the exhalation during exposure, dose-related parameters becomes independent of the length of exposure, and a more accurate estimation of body burden is achieved. Thus, absorbed dose in the present study is referred in two ways: the respiratory uptake (new approach) and the net respiratory uptake (old approach). Decay curves of ETBE in urine (starting at 2– 4 h) were fitted to biexponential functions by nonlinear regression analysis using Solver in Microsoft Excel. The elimination phase of TBA in blood and urine, starting approximately at 6 h, was fitted to a monoexponential function again by nonlinear regression analysis. The blood concentrations at steady state for ETBE were calculated for each subject as the respiratory uptake rate divided by the total clearance. The area under the concentration–time curve (AUC) of ETBE in blood was obtained from the four-compartment model, whereas the AUCs of TBA and acetone was calculated by the trapezoidal rule in Microsoft Excel. The renal clearance of TBA was calculated as the total amount TBA excreted in urine divided by the AUC of TBA in blood. Statistical Analysis Analysis of variance repeated measures model (SuperANOVA Version 1.11, Abacus Concepts Inc., Berkeley, CA), and the Student’s paired t test (Microsoft Excel VERSION 5.0) were used to compare results from different exposure occasions (5, 25, and 50 ppm ETBE) and between the present study and the previous MTBE study. The level of significance was set to 0.05. Unless otherwise stated, the results are presented as mean values 695% confidence intervals.
RESULTS
Exposure Conditions The measured mean levels of ETBE in the chamber air were 4.8, 25, and 50 ppm. The variations of the chamber air concentration between than exposure occasions were less 6% at 5 ppm, 4% at 25 ppm, and 3% at the 50 ppm exposure levels, expressed as RSD. The variation (RSD) within each 2-h exposure session was less than 7, 5, and 4%, respectively. With the exception of a slight temperature difference between the 5 and 25 ppm exposure levels (18.6 and 19°C, respectively, p , 0.05) no differences were seen between the exposure occasions regarding relative humidity (mean 43%, 95% confidence interval 42.3– 43.7%), air exchanges per hour (16 h21, 15.4 –16.6 h21), chamber temperature (18.7°C, 18.5– 18.9°C), actual physical exercise (49 W, 48.5– 49.5 W), and heart rate (95 beats/min, 89 –101 beats/min). The variation, expressed as RSD, were relative humidity, less than 2% be-
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TOXICOKINETICS OF ETBE IN MALE VOLUNTEERS
TABLE 1 Uptake and Excretion of Ethyl tertiary-Butyl Ether (ETBE) and tertiary-Butyl Alcohol (TBA) in Eight Volunteers Exposed to ETBE for 2 h (Mean Values 6 95% Confidence Interval) Exposure level
ETBE Inhaled amount (mmol) Net respiratory uptake (%)a Respiratory uptake (%)b Net respiratory excretion (% of net respiratory uptake) Respiratory excretion (% of respiratory uptake) Urinary excretion (% of respiratory uptake) TBA Net respiratory excretion (% of ETBE net respiratory uptake)c Urinary excretion (% of ETBE respiratory uptake)
Time span (h)
5 ppm
25 ppm
50 ppm
0–2 0–2 0–2
0.58 6 0.05 26 6 3.4 32 6 4.4
2.9 6 0.19 26 6 2.9 34 6 5.3
5.8 6 0.39 26 6 2.9 34 6 3.4
2–24 0–24 0–24
30 6 3.5 45 6 4.0 0.12 6 0.053
31 6 4.2 50 6 5.6 0.061 6 0.021
30 6 4.7 46 6 5.5 0.056 6 0.022*
2–24 0–24
1.4 6 0.8 0.71 6 0.34
3.3 6 1.4 0.66 6 0.24
3.8 6 0.9 0.90 6 0.49
Net respiratory uptake 5 [amount inhaled 2 amount exhaled] during exposure. Respiratory uptake 5 [net respiratory uptake 1 amount exhaled during exposure]. c Net respiratory excretion 5 [(TBA exhaled postexposure 2 TBA net uptake during exposure)/ETBE net uptake], assuming 35–50% net respiratory uptake of TBA. * Significant different from 5 ppm level (p , 0.05, repeated-measures ANOVA). a b
tween exposure occasions and 7% within each 2-h exposure session; air exchanges, 6 and 2%; chamber temperature, 3 and 2%; actual physical exercise, 3 and 9%; and heart rate, 17 and 9%, respectively. Further, no differences in pulmonary ventilation or respiratory frequency were seen between exposure occasions. The pulmonary ventilation was on average 23.7 (22.9 –24.5) liters/min during the exposures and decreased postexposure to 11.4 (10.7–12.1) liters/min. The respiratory frequency was on average 18 (16.8 –19.2) breaths/min during and 15 (13.8 –16.2) breaths/min after the exposure. TABLE 2 Elimination Half-Times (t21) of Ethyl tertiary-Butyl Ether (ETBE) and tertiary-Butyl Alcohol (TBA) in Eight Volunteers Exposed to ETBE for 2 h (Mean Values 6 95% Confidence Interval) Exposure level Half-time ETBE in blood First phase (min) Second phase (min) Third phase (h) Fourth phase (h) ETBE in urine First phase (min) Second phase (h) TBA in blood (h) TBA in urine (h) Note. n.d., not determined.
5 ppm
25 ppm
50 ppm
1.8 6 1.1 20 6 11 2.1 6 0.59 33 6 6.0
1.2 6 0.75 15 6 9.1 1.5 6 0.45 24 6 9.7
2.0 6 1.3 1.9 6 9.3 1.5 6 0.20 27 6 11
6.3 6 4.3 8.8 6 4.3 n.d. n.d.
9.1 6 4.0 8.7 6 2.4 12 6 2.2 8.4 6 1.3
9.3 6 4.7 8.2 6 1.8 12 6 1.2 7.6 6 2.5
Toxicokinetics Results of uptake, excretion, half-times, and clearance are presented in Tables 1–3. To be able to compare the present study with previously performed studies of this kind, results on apparent blood clearance, net respiratory uptake, and excretion based on net respiratory uptake are also given. The respiratory uptake of ETBE was in the range of 32–34%, and the respiratory excretion was in the range of 45–50% of the respiratory ETBE uptake (Table 1). In comparison, the calculated net respiratory uptake and net respiratory excretion of ETBE were lower, 26 and 30 –34% respectively (Table 1), since the amount ETBE cleared by exhalation during exposure was not considered. No significant differences relative in uptake and exhalation were seen between exposure levels. The concentration of ETBE in blood increased during the entire exposure with a tendency to level off but without reaching any plateau (Fig. 1a). Average maximum levels of 1.1, 5.4, and 10 mM were reached at 5, 25, and 50 ppm, respectively. The elimination process of ETBE in blood was separated into four phases (Table 2). The two fastest elimination phases had average half-times of 2 and 18 min, the intermediate phase had an average half-time of 1.7 h, and the slowest phase had an average half-time of 28 h. No statistically significant differences in half-times were seen between exposure levels. The AUC of ETBE in blood (Fig. 2) was linearly related to the ETBE exposure level, suggesting linear kinetics up to 50 ppm. The clearance of ETBE in blood was in the range of 0.6 – 0.9 liters/h/kg (Table 3). A higher clearance was obtained at the 25 and 50 ppm exposures, but any statistically significant differences were not seen compared to the 5 ppm level. The elimi-
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TABLE 3 Toxicokinetic Results of Ethyl tertiary-Butyl Ether (ETBE) and tertiary-Butyl Alcohol (TBA), from Exposure of Eight Volunteers to ETBE (Mean Values 6 95% Confidence Interval) Exposure level
ETBE Apparent total clearance (liters/h/kg)a Total clearance (liters/h/kg)b Metabolic clearance (liters/h/kg)b Exhalatory clearance (liters/h/kg)b Volume of distribution (liters/h/kg) Mean residence time (h) TBA Renal clearance (liters/h/kg)c
5 ppm
25 ppm
50 ppm
0.48 6 0.06 0.61 6 0.09 0.35 6 0.06 0.26 6 0.08 5.8 6 1.3 11 6 4.4
0.61 6 0.12 0.87 6 0.19 0.41 6 0.09 0.45 6 0.13 5.9 6 1.8 7.4 6 2.5
0.62 6 0.15 0.79 6 0.17 0.42 6 0.12 0.36 6 0.08 7.1 6 3.2 11 6 7.1
n.d.
0.00064 6 0.00028
0.00096 6 0.00054
Note. n.d., not determined. a Apparent total clearance calculated from net respiratory uptake. b Clearance calculated from respiratory uptake. c Renal clearance calculated from amount TBA excreted in urine and the AUC of TBA in blood.
nation of ETBE in urine could be separated in two linear phases with half-times of 8 min and 8.5 h (Table 2). The excretion rate of ETBE in urine was rather high (Fig. 3a), and the cumulative excretion of ETBE in urine within 22 h postexposure was less than 0.1% of the respiratory uptake (Table 1). The urinary ETBE excretion was higher at the 5 ppm exposure compared to the 25 and 50 ppm exposures, and a statistically significant difference was seen between the 5 and 50 ppm results (p , 0.05) (Table 1). In contrast to the ETBE profile, TBA in blood increased steadily during the exposure and remained high for several hours postexposure. Average maximum levels of TBA in blood were obtained approximately 30 min postexposure (25 ppm, 6.9 mM; and 50 ppm, 12 mM), and the levels started to decline slowly at about 4 h after the end of exposure (Fig. 1b). No calculations were performed for TBA in blood at the 5 ppm exposure level, since the con-
centration of TBA in blood was close to and sometimes below the gas chromatographic detection limit. The AUC of TBA was linearly related to the ETBE exposure level, suggesting linear kinetics of the metabolite up to 50 ppm (Fig. 2). The average half-times of TBA were 12 h in blood and 8 h in urine (Table 2), which were approximately equal to the previously obtained half-times of TBA after MTBE exposures (Pekari et al., 1996; Nihle´n et al., 1998b). TBA was also detected in exhaled air, and 1.4 –3.8% of ETBE net uptake was excreted as TBA through the lungs (Table 1). The excretion rate of TBA was rather slow (Fig. 3b), and less than 1% of the absorbed ETBE was excreted as TBA in urine within 22 h postexposure (Table 1). A low renal clearance of TBA (less than 1 ml/h/kg) was seen, indicating extensive blood protein binding or tubular reabsorption of the metabolite (Table 3). Acetone was detected in elevated levels in blood after the exposure (Fig. 1c). The average concentration in blood and the AUC of acetone was highest at the 50 ppm exposure, but otherwise poorly related to the exposure level. Acetone was detected in urine as well and the highest cumulative urinary excretion was seen at the 50 ppm exposure level and the lowest was seen after the control exposure (data not shown). The average background levels of acetone as measured in samples collected in the morning prior to ETBE exposure were 42 (95% confidence interval 35–50) mM in blood and 38 (18 –58) mM in urine. A wide interindividual variation in acetone levels (especially in urine) was seen before, during, and after the exposure. DISCUSSION
FIG. 2. The area under the concentration–time curve (AUC) of ethyl tert-butyl ether (ETBE) and tert-butyl alcohol (TBA) in capillary blood versus exposure level of ETBE (mean values and 95% confidence intervals, n 5 8).
The present study shows that ETBE is absorbed in the respiratory tract, is transformed to TBA in the body, and is
TOXICOKINETICS OF ETBE IN MALE VOLUNTEERS
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FIG. 3. Urinary excretion of (a) ethyl tert-butyl ether (ETBE) and (b) tert-butyl alcohol (TBA) versus midpoint time of urine collection after a 2-h exposure to ETBE (mean values, n 5 8). Vertical lines indicate the lower and upper 95% confidence intervals at 5 and 50 ppm, respectively.
partly excreted as ETBE and TBA through urine and exhaled air. Further, the results suggest that ETBE is also biotransformed to acetone. To our knowledge, this is the first time that human toxicokinetic data are presented for ETBE. The chemical used, a commercially available technical grade of ETBE, was contaminated with TBA and MTBE. Even after careful redistillation it was still contaminated with 0.4% TBA and 1.1% MTBE. These contaminations may confound the toxicokinetic analysis and especially TBA should be kept in mind when interpreting the TBA levels in blood, urine, and exhaled air and when comparing ETBE toxicokinetics with that of MTBE. The experimental design of these two studies is similar. Two years elapsed between the two exposure studies, different subjects were exposed, and some of the used analytical equipment was exchanged. In spite of this, the toxicokinetics of ETBE and MTBE in human volunteers were quite similar regarding respiratory uptake and exhalation and concentration– time profiles of blood and urine, as can be seen in Table 4. The small differences seen are consistent with the different blood/ air and olive oil/blood partition coefficients measured in vitro for ETBE and MTBE (Nihle´n et al., 1995; Borghoff et al., 1996). Thus, the lower blood/air partition coefficient of ETBE
compared to MTBE (12 versus 18) suggests a lower respiratory uptake, whereas the higher oil/blood partition coefficient (16 versus 7) implies a longer terminal half-time (Nihle´n et al., 1995). Indeed the respiratory uptake of ETBE was significantly lower (p 5 0.03) than MTBE, whereas a borderline difference (p 5 0.06) was seen regarding the net respiratory uptake. No difference in respiratory excretion was seen between the two ethers. The blood and urinary profiles were quite similar for both ethers apart from somewhat lower ETBE levels in blood. This is illustrated by a 15–30% lower blood AUC for ETBE than for MTBE at the same exposure level. Also, the average blood concentrations at steady state were lower for ETBE and were as follows: mean 1.5 (95% confidence interval, 1.3–1.7) mM at 5 ppm, 5.9 (5.4 – 6.4) mM at 25 ppm, and 13 (12–14) mM at 50 ppm. In comparison, the average MTBE steady-state levels were 2.3 (1.6 –3.0) mM at 5 ppm, 8.8 (7.4 –10) mM at 25 ppm, and 19 (16 –21) mM at 50 ppm. A tendency (p 5 0.07) to a longer terminal half-time in blood (24 –33 h) was seen for ETBE compared to MTBE (17–21 h). Furthermore, a significantly (p 5 0.03) higher volume of distribution was obtained for ETBE, but no difference in blood clearance was seen. In addition, ETBE was excreted significantly slower than MTBE to urine, although the cumulative amount did not differ and
´ ¨ NIHLEN, LOF, AND JOHANSON
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TABLE 4 Comparison of Toxicokinetic Parameters for Ethyl tertiary-Butyl Ether (ETBE), Methyl tertiary-Butyl Ether (MTBE), and the Metabolite tertiary-Butyl Alcohol (TBA) (Average Values 6 95% Confidence Intervals)
Parent ether Net respiratory uptake (%) Respiratory uptake (%) Net respiratory excretion (%) Respiratory excretion (%) Metabolic clearance (liters/h/kg) Exhalatory clearance (liters/h/kg) Volume of distribution (liters/kg) Mean residence time (h) Half-time in blood (terminal, h) Half-time in urine (first phase, h) Half-time in urine (second phase, h) Urinary excretion (%) TBA Net respiratory excretion (%) Half-time in blood (h) Half-time in urine (h) Urinary excretion (%) Renal clearance (liters/h/kg)
ETBE (present study) n 5 24a
MTBE (Nihle´n et al., 1998b) n 5 30a
26 6 1.7 34 6 2.7* 31 6 2.5 47 6 3.1 0.39 6 0.06 0.35 6 0.06 6.4 6 1.4* 9.9 6 3.1 29 6 5.4 0.14 6 0.04* 8.6 6 1.7* 0.08 6 0.02
38 6 4.0 47 6 5.3* 28 6 5.2 39 6 6.3 0.41 6 0.07 0.27 6 0.06 3.9 6 0.6* 6.9 6 1.9 20 6 5.5 0.33 6 0.14* 3.1 6 0.45* 0.08 6 0.01
2.9 6 1.0 12 6 1.5b 7.9 6 1.5b 0.76 6 0.22 0.00080 6 0.00034b
n.d. 10 6 1.4b 8.1 6 2.0b 0.59 6 0.13 0.00067 6 0.00011b
Note. n.d., not determined. a Average of 8 or 10 subjects exposed at three levels (5, 25, and 50 ppm). b Average of 8 or 10 subjects exposed at two levels (25 and 50 ppm). * Statistically significant difference (p , 0.05, Student’s paired t test).
was as low as 0.1% of the uptake. The delayed excretion of ETBE is probably a reflection of the slower decay in blood compared to MTBE. The excretion of minor amounts of ETBE and MTBE in the urine is not a surprise, since these ethers are slightly water soluble. In fact, even less water-soluble chemicals e.g., styrene and trimethylbenzenes, are recovered in small amounts in human urine (Ong et al., 1994; Ja¨rnberg et al., 1996). Urinary excretion of small amounts of unmetabolized MTBE has been demonstrated in several volunteer studies (Pekari et al., 1996; Buckley et al., 1997; Nihle´n et al., 1998b). The toxicokinetic calculations indicate that the clearance by exhalation was almost as high as the metabolic clearance of ETBE (Table 3). A theoretical calculation of the exhalatory clearance based on literature values on the blood/air partition coefficient for ETBE (12) (Nihle´n et al., 1995), alveolar ventilation (4 liters/min at rest, 17–22 liters/min at 50 W), and cardiac output (5 liters/min at rest, 7–12 liters/min at 50 W) (Åstrand, 1983) gives respiratory clearance values between 0.3 (rest) and 1.2 liters/h/kg (50 W exercise). As our exposures were performed during 2 hours of light exercise followed by rest, the experimentally obtained expiratory clearance (0.3– 0.5 liters/h/kg, Table 3) is reasonable. The kinetics of the metabolite TBA were similar after the ETBE and MTBE exposures, judging by blood and urine profiles and the toxicokinetic results (Table 4). Somewhat
lower concentrations of TBA in blood were reached after ETBE compared to MTBE exposures; this probably reflects the different blood levels of the parent ether. In the present study there are no signs of metabolic saturation, since the kinetics of ETBE and TBA are linear in the studied exposure interval (0 –50 ppm ETBE) (Fig. 2). Linear kinetics have previously been demonstrated for MTBE and TBA in humans up to 75 ppm (Pekari et al., 1996; Nihle´n et al., 1998b) and in rats up to 300 ppm (Savolainen et al., 1985). About 50% of inhaled ether was recovered as the parent ether and as the metabolite TBA in breath and urine. The low recovery indicates competing pathways, further metabolism of the alcohol, or other excretion routes. After exposing rats for MTBE or ETBE several metabolites (2-methyl-1,2-propanediol, a-hydroxyisobutyric acid, and TBA conjugates) have been found in the urine (Miller et al., 1997; Bernauer et al., 1998). ETBE was contaminated with 0.4% TBA even after careful redistillation. This makes the calculations of the kinetics of TBA more difficult. On average the subjects exhaled 7.6, 39, and 75 mmol TBA as measured 0 – 46 h after the exposure, corresponding to 2.4 –5.2% of the ETBE net uptake. Assuming that the chamber air is contaminated with 0.4% TBA or to 0.027, 0.14, and 0.28 ppm TBA during the 5, 25, and 50 ppm ETBE exposures, respectively, and that the relative uptake of
TOXICOKINETICS OF ETBE IN MALE VOLUNTEERS
TBA is 35–50% (Nihle´n et al., unpublished observation with MTBE), exhaled TBA formed by ETBE biotransformation is in the range of 1.4 –3.8% (Table 1) of the ETBE net uptake. In our previous MTBE study (Nihle´n et al., 1998b) TBA was not detected in exhaled air. Acetone is a metabolite of MTBE, ETBE, and TBA in rodents (Cederbaum et al., 1980; Baker et al., 1982; Bernauer et al., 1998) suggesting that acetone is also a metabolite of ETBE in humans. In the present study, acetone in blood (Fig. 1c) and urine (data not shown) was elevated at 50 ppm ETBE, but no clear dose response was seen when comparing all exposure levels. Acetone is produced endogenously, and the acetone levels may change naturally through the day due to for example physical exercise or food intake (Jones et al., 1993). Thus, tracer techniques would be needed to confirm the formation of acetone from ETBE in humans. Acetone was not measured in our earlier MTBE study. A potentially useful biological exposure marker for ETBE and gasoline-containing ETBE might be TBA, because of the relatively high levels in blood and urine and the rather slow decay after exposure. However, further studies of the metabolism pattern of ETBE are important, since other metabolites might be found in higher quantities in the urine and therefor be better as a biomarkers of ETBE. In this study we did not correct the urine levels for creatinine, since we collected all urine and thus could calculate excretion rates. However, if TBA is used as a biomarker of ETBE creatinine corrections might be more practical. In conclusion, our study has for the first time investigated the kinetics of ETBE in humans. Relative to many other solvents a rather low respiratory uptake of ETBE and a quite high respiratory exhalation were seen. The metabolism and accumulation of ETBE are almost similar to that previously seen for MTBE (Nihle´n et al., 1998b). Thus, from toxicokinetic considerations, such as bioavailability and potential for accumulation, neither ether is preferred over the other. However, further research of other toxicological and biological aspects of ETBE should be performed before ETBE is used in large scale in gasoline and spread in the environment. ACKNOWLEDGMENTS We express our gratitude to Mrs. E. Gullstrand, L. Ernstgård, and A. Dihkan for skillful technical assistance and Dr. A. Toomingas for medical examinations. The authors thank Ecotraffic Research & Development AB, Sweden, for valuable advice and Ecofuel S.P.A, Italy, for offering technical ETBE. The project was financially supported by the National Board for Industrial and Technological Development (NUTEK), Sweden (Grant P3626-1).
REFERENCES Åstrand, I. (1983). Effect of physical exercise on uptake, distribution and elimination of vapors in man. In Modelling of Inhalation Exposure to Vapors: Uptake, Distribution and Elimination (V. Fiserova-Bergerova, Ed.), Vol. II, pp. 107–133. CRC Press, Boca Raton, FL.
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Baker, R. C., Sorensen, S. M., and Deitrich, R. A. (1982). The in vivo metabolism of tertiary butanol by adult rats. Alcohol Clin. Exp. Res. 6, 247–251. Bernauer, U., Amberg, A., and Dekant, W. (1998). Biotransformation of 12Cand 2-13C-methyl-tert-butyl ether, ethyl-tert-butyl ether and tert-butanol in rats: Identification of metabolites in urine by 13C-NMR and GC/MS. In Proceedings, Society of Toxicology, 37th Annual Meeting, Washington State Convention Center, Seattle, WA. Borghoff, S. J., Jayjock, E., and Murphy, J. E. (1996). Methyl tert-butyl ether (MTBE) vs ethyl tert-butyl ether (ETBE): A comparison of blood and tissue partition coefficients in male and female F-344 rats. Fundam. Appl. Toxicol. 30, 34. Brady, J. F., Xiao, F., Ning, S. M., and Yang, C. S. (1990). Metabolism of methyl tertiary-butyl ether by rat hepatic microsomes. Arch. Toxicol. 64, 157–160. Buckley, T. J., Prah, J. D., Ashley, D., Zweidinger, R. A., and Wallace, L. A. (1997). Body burden measurements and models to assess inhalation exposure to methyl tertiary butyl ether (MTBE). J. Air Waste Manage. Assoc. 47, 739 –752. Cain, W. S., Leaderer, B. P., Ginsberg, G. L., Andrews, L. S., Cometto-Mun˜iz, J. E., Gent, J. F., Buck, M., Berglund, L. G., Mohsenin, V., Monahan, E., and Kjaergaard, S. (1996). Acute exposure to low-level methyl tertiary-butyl ether (MTBE): Human reactions and pharmacokinetic response. Inhal. Toxicol. 8, 21– 48. Cederbaum, A. I., and Cohen, G. (1980). Oxidative demethylation of t-butyl alcohol by rat liver microsomes. Biochem. Biophys. Res. Commun. 97, 730 –736. Hartle, R. (1993). Exposure to methyl tert-butyl ether and benzene among service station attendants and operators. Environ. Health Perspect. Suppl. 101, 23–26. HEI (1996). The Potential Health Effects of Oxygenates Added to Gasoline:. A Review of the Current Literature—A Special Report of the Institute’s Oxygenates Evaluation Committee, April 1996., Health Effects Institute, Cambridge, MD. Iborra, M., Izquierdo, J. F., Tejero, J., and Cunill, F. (1988). Getting the lead out with ethyl t-butyl ether. Chemtech. February 1988, 120 –122. Ja¨rnberg, J., Johanson, G., and Lo¨f, A. (1996). Toxicokinetics of trimethylbenzenes in man. Toxicol. Appl. Pharmacol. 140, 281–295. Jones, A. W., Sagarduy, A., Ericsson, E., and Arnqvist, H. J. (1993). Concentrations of acetone in venous blood samples from drunk drivers, type-I diabetic outpatients, and healthy blood donors. J. Anal. Toxicol. 17, 182– 185. Miller, M. J., Ferdinandi, E. S., Klan, M., Andrews, L. S., Douglas, J. F., and Kneiss, J. J. (1997). Pharmacokinetics and disposition of methyl t-butyl ether in Fischer-344 rats. J. Appl. Toxicol. 17, S3–S12. Moolenaar, R. L., Hefflin, B. J., Ashley, D. L., Middaugh, J. P., and Etzel, R. A. (1994). Methyl tertiary butyl ether in human blood after exposure to oxygenated fuel in Fairbanks, Alaska. Arch. Environ. Health 49, 402– 409. Nihle´n, A., Lo¨f, A., and Johanson, G. (1995). Liquid/air partition coefficients of methyl- and ethyl t-butyl ethers, t-amyl methyl ether, and t-butyl alcohol. J. Exp. Anal. Environ. Epidemiol. 5, 573–582. Nihle´n, A., Lo¨f, A., and Johanson, G. (1998a). Controlled ethyl tert-butyl ether (ETBE) exposure of male volunteers. II. Acute effects. Toxicol. Sci. 46, 143–150. Nihle´n, A., Lo¨f, A., and Johanson, G. (1998b). Experimental exposure to methyl tertiary-butyl ether. I. Toxicokinetics in humans. Toxicol. Appl. Pharmacol. 148, 274 –280. Ong, C. N., Shi, C. Y., Chia, S. E., Chua, S. C., Ong, H. Y., Lee, B. L., Ng, T. P., and Teramoto, K. (1994). Biological monitoring of exposure to low concentrations of styrene. Am. J. Ind. Med. 25, 719 –730.
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Pekari, K., Riihima¨ki, V., Vainiotalo, S., Tera¨va¨inen, E., and Aitio, A. (1996). Experimental exposure to methyl-tert-butyl ether (MTBE) and methyl-tertamyl ether (MTAE). In Proceedings, International Symposium on Biological Monitoring in Occupational and Environmental Health, Hanasaari Cultural Centre, Espoo, Finland. Prah, J. D., Goldstein, G. M., Devlin, R., Otto, D., Ashley, D., House, D., Cohen, K. L., and Gerrity, T. (1994). Sensory, symptomatic, inflammatory, and ocular responses to and the metabolism of methyl tertiary butyl ether in a controlled human exposure experiment. Inhal. Toxicol. 6, 521– 538. Savolainen, H., Pfa¨ffli, P., and Elovaara, E. (1985). Biochemical effects of methyl tertiary-butyl ether in extended vapour exposure of rats. Arch. Toxicol. 57, 285–288.
Swedish National Board of Occupational Safety and Health (1996). Occupational Exposure Limit Values, Ordinance, AFS 1996:2. Swedish National Board of Occupational Safety and Health, Solna, Sweden. U.S. EPA (1993). Assessment of Potential Health Risks of Gasoline Oxygenated with Methyl tertiary Butyl Ether (MTBE), EPA/600/R-93/206. Office of Research and Development, U.S. Environmental Protection Agency, Washington, DC. White, M. C., Johnson, C. A., Ashley, D. L., Buchta, T. M., and Pelletier, D. J. (1995a). Exposure to methyl tertiary-butyl ether from oxygenated gasoline in Stamford, Connecticut. Arch. Environ. Health. 50, 183–189. White, R. D., Daughtrey, W. C., and Wells, M. S. (1995b). Health effects of inhaled tertiary amyl methyl ether and ethyl tertiary butyl ether. Toxicol. Lett. 82/83, 719 –724.
46, 1–10 (1998)
TX982516
Controlled Ethyl tert-Butyl Ether (ETBE) Exposure of Male Volunteers I. Toxicokinetics Annsofi Nihle´n,*,†,1 Agneta Lo¨f,* and Gunnar Johanson*,‡ *Department of Occupational Medicine, National Institute for Working Life, SE-171 84 Solna, Sweden; †Institute of Environmental Medicine, Division of Toxicology, Karolinska Institute, Solna, Sweden; and ‡Department of Occupational and Environmental Medicine, University Hospital, Uppsala, Sweden Received December 31, 1997; accepted June 3, 1998
emissions to the atmosphere. Today, MTBE is widely used in gasoline all over the world. One reason for the interest in ETBE is the potential to increase the market for bioethanol, as ETBE can be manufactured from ethanol and isobutene. ETBE has additional advantages compared to MTBE, in terms of lower vapor pressure and higher octane number (Iborra et al., 1988). Examples of occupational exposure to oxygenates are during manufacturing, blending, and transportation of gasoline and in gasoline stations (Hartle, 1993; U.S. EPA, 1993). The general public is exposed to oxygenates mainly during gasoline filling. Furthermore, groundwater could be contaminated with MTBE from leaking underground gasoline storage tanks, and the general public is today exposed to low levels of MTBE from the drinking water in parts of several states in the United States (HEI, 1996). Biotransformation of ETBE appears to be similar to that of MTBE. Thus, O-dealkylation of MTBE and ETBE will result in the same metabolite, tert-butyl alcohol (TBA) (Brady et al., 1990; White et al., 1995b; Miller et al., 1997; Bernauer et al., 1998). After MTBE exposure TBA was detected in blood and urine in animals (Savolainen et al., 1985; Brady et al., 1990; Miller et al., 1997; Bernauer et al., 1998) and in humans (Moolenaar et al., 1994; Prah et al., 1994; White et al., 1995a; Cain et al., 1996; Pekari et al., 1996; Buckley et al., 1997; Nihle´n et al., 1998b). In MTBE-exposed rats, 2-methyl-1,2propanediol, a-hydroxyisobutyric acid, and TBA conjugates were detected in the urine (Miller et al., 1997; Bernauer et al., 1998), which indicates further oxidation of TBA. In addition, other studies show formation of formaldehyde, acetone and carbon dioxide in TBA-treated rats and rat liver microsomes (Cederbaum et al., 1980; Baker et al., 1982). The methyl and ethyl parts of MTBE and ETBE are biotransformed to formaldehyde and acetaldehyde, respectively (White et al., 1995b; HEI, 1996). To our knowledge, no metabolites other than TBA have been identified in biological samples from humans exposed to MTBE, and no studies of the uptake and disposition of ETBE in man have been performed. It is important to further
Controlled Ethyl tert-Butyl Ether (ETBE) Exposure of Male Volunteers. I. Toxicokinetics. Nihle´n, A., Lo¨f, A., and Johanson, G. (1998). Toxicol. Sci. 46, 1–10. Ethyl tert-butyl ether (ETBE) might replace methyl tert-butyl ether (MTBE), a widely used additive in unleaded gasoline. The aim of this study was to evaluate uptake and disposition of ETBE, and eight healthy male volunteers were exposed to ETBE vapor (0, 5, 25, and 50 ppm) during 2 h of light physical exercise. ETBE and the proposed metabolites tert-butyl alcohol (TBA) and acetone were analyzed in exhaled air, blood, and urine. Compared to a previous MTBE study (A. Nihle´n et al., 1998b, Toxicol. Appl. Pharmacol. 148, 274 –280) lower respiratory uptake of ETBE (32– 34%) was seen as well as a slightly higher respiratory exhalation (45–50% of absorbed ETBE). The kinetic profile of ETBE could be described by four phases in blood (average half-times of 2 min, 18 min, 1.7 h, and 28 h) and two phases in urine (8 min and 8.6 h). Postexposure half-times of TBA in blood and urine were on average 12 and 8 h, respectively. The 48-h pulmonary excretion of TBA accounted for 1.4 –3.8% of the absorbed ETBE, on an equimolar basis. Urinary excretion of ETBE and TBA was low, below 1% of the ETBE uptake, indicating further metabolism of TBA or other routes of metabolism and elimination. The kinetics of ETBE and TBA were linear up to 50 ppm. Based upon blood profile, levels in blood and urine, and kinetic profile we suggest that TBA is a more appropriate biomarker for ETBE than the parent ether itself. The acetone level in blood was higher after ETBE exposures compared to control exposure, and acetone is probably partly formed from ETBE. © 1998 Society of Toxicology. Key Words: biological monitoring; ethyl tert-butyl ether; gasoline; human; inhalation exposure; oxygenated fuel; tert-butyl alcohol; toxicokinetics.
Aliphatic ethers, such as methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether may be used as additives (oxygenates) in gasoline (Iborra et al., 1988). These oxygenates enhance the octane number and are thought to improve combustion efficiency, thereby reducing To whom correspondence should be addressed. Fax: 146-8-730 1967. E-mail: [email protected]. 1
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1096-6080/98 $25.00 Copyright © 1998 by the Society of Toxicology. All rights of reproduction in any form reserved.
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explore the toxicokinetics and acute effects in man before ETBE is introduced in large scale in the environment. The main aim of our study was to determine the uptake, distribution, metabolism, and elimination in male volunteers after short-term exposure to ETBE (0, 5, 25, and 50 ppm). In addition, acute health symptoms were subjectively rated and objectively measured during and after exposure and are presented in an accompanying article (Nihle´n et al., 1998a).
series, Grant Instruments Ltd., UK) and stored in a personal computer as 1-min averages during each 2-h exposure. A high-performance liquid chromatography pump (305 piston pump, Gilson, France) transferred the ETBE liquid to a heated (90°C) glass cylinder, which was a part of the exposure chamber influent air system. ETBE was added to the air by means of aerosol formation followed by complete evaporation. To prevent leakage from the chamber the air pressure in the chamber was about 5 Pa lower than in the surrounding laboratory. Sampling and Chemical Analysis
MATERIAL AND METHODS Subjects Eight Caucasian male volunteers, with a mean age of 29 years (range 21– 41 years) and a mean body weight of 82 kg (range 70 –97 kg) participated in the chamber study. All subjects were healthy as judged by clinical examination. In addition, a standard clinical blood chemistry test (Nihle´n et al., 1998b) was performed in order to exclude subjects with liver and metabolic disorders and other diseases. All subjects were nonsmokers and were not occupationally exposed to ETBE or any other solvent. They had to refrain from alcoholic beverages and drugs 2 days before and throughout each experiment. The study was carried out according to the Declaration of Helsinki, after approval by the Regional Ethical Committee at the Karolinska Institute Stockholm, Sweden, and after written consent by the volunteers. Chemicals Technical ETBE was offered as a gift from Ecofuel S.P.A (Ravenna, Italy). The ETBE liquid was redistilled (Department of Organic Chemistry, University of Stockholm, Stockholm, Sweden) since impurities were found. ETBE was approximately 98.5% pure after the redistillation. The major residues were identified by gas chromatography–mass spectrometry (Miljo¨laboratoriet, Nyko¨ping, Sweden) as approximately 0.4% TBA and 1.1% MTBE. For calibration standard preparations TBA (.99.5% purity, Merck, Darmstadt, Germany) and acetone (.99.5% purity, Merck) were used. Experimental Design The study design was similar to that of our former MTBE study (Nihle´n et al., 1998b). In brief, the exposures were conducted during 2 h of light physical exercise (nominal level 50 W) on a computer-controlled bicycle ergometer (Monark Ergomedic 829 E, Sweden) in an exposure chamber. Individual heart rate and actual work load were recorded once every minute throughout the exposure. The subjects interrupted the bicycle exercise for approximately 5 min after 110 min of exposure to allow an acute effect (ocular) measurement in the exposure chamber. All sampling of blood and exhaled air was performed during exercise. Each subject was exposed on four occasions, at nominal levels of 0, 5, 25, and 50 ppm ETBE, with at least 2 weeks between two successive exposures. The highest exposure level was chosen considering the current Swedish limit for occupational exposure to MTBE (Swedish National Board of Occupational Safety and Health, 1996) since restrictions have yet been assigned to ETBE. Exposure Chamber The exposures occurred in a 20-m3 exposure chamber with controlled climate. The humidity and temperature in the chamber were measured by a humidity and temperature probe (HMP 36, Vaisala, Finland) and the air flow was measured by an air flow meter (MiniAir 6, Schiltknecht Messtechnik AG, Switzerland). In addition, the pressure difference of the surrounding laboratory and the chamber was measured (Modus Instruments Inc., Northborough, MA) and the amount of carbon monoxide in the chamber was indicated (Telaire, Martionics AB, Sweden). These measurements were made every 15 s and the signals were sampled with a data logger (Grant Squirrel meter/logger 1200
The time schedule, used equipment, and procedure of collecting samples of blood, urine, and exhaled air (before, during, and after the exposure) were similar to our previously performed MTBE study (Nihle´n et al., 1998b). Time points of collecting blood and urine are shown in Figs. 1 and 3, respectively. ETBE and TBA were analyzed in all biological samples; acetone was analyzed in blood and urine only. Chamber air. The concentration of ETBE in the chamber air was analyzed in 5-min intervals during the exposures by a gas chromatography (GC-Auto System, Perkin–Elmer, UK). The gas chromatograph was equipped with a gas sample injection loop (0.5 ml), a flame ionization detector, and a nonpolar capillary column (Poraplot U, Chrompack, The Netherlands, 10 m, 0.53 mm inner diameter, 20 mm phase thickness). Chamber air was drawn through a Teflon tubing into the gas sampling loop with the use of a pump (15 liters/min, Model DDA-P101-BN, Gast Corp., Benton Harbor, MI). The temperatures of the gas chromatograph injector, column, and detector were 200, 175 (isothermal), and 250, respectively. Nitrogen was used as carrier gas at a flow rate of 4.7 ml/min. Turbochrom chromatography workstation (Version 4.1, PE Nelson, Norwalk, CT) was used to sample and compute chromatographic data. Calibration standards were prepared in Tedlar bags (5–25 liters, SKC Inc., Eighty Four, PA) filled with known volumes of clean air by means of a calibrated pump (AirChek sampler, Model 224-PCXR8, SKC Inc.) and known amounts of ETBE. At least four different concentration levels were prepared at each exposure occasion. Air was drawn from each Tedlar bag into the gas sampling loop with the use of a pump and analyzed by the gas chromatograph as described above. Exhaled air. To determine the respiratory excretion of ETBE, exhaled air was collected once before the exposure, four times during (every 30 min), and six times after the exposure. Each exhalation period lasted for 5– 6 min and the exhaled air was analyzed during the last 2 min. Exhaled air was collected through a mouthpiece connected with two valves to separated in and outlets. During each exhalation period, the volunteers wore a nose clip to prevent nasal breathing. An electric spirometer (K. L. Engineering, USA) was connected to the inlet valve, and the individual’s pulmonary ventilation and respiratory frequency were recorded as 1-min averages during each exhalation period of 5– 6 min. The outlet valve was connected to a stainless-steel tube and a pump transferred the exhaled air to a mixing chamber (7 liters) from which some exhaled air was drawn into a gas sample injection loop. The concentration of ETBE was directly analyzed by the gas chromatograph as described under Chamber. All material in contact with expired air was heated to 40°C to avoid condensation of water. The sampled air was either directed to the gas chromatograph (as described above) or, for later analysis, pumped (590 liters/min, AirChek sampler, Model 224-PCXR8, SKC Inc.) through two sorbent sample tubes (stainless steel, length 90 mm, diameter 6.4 mm, Perkin–Elmer) containing active charcoal (Carbotrap, 20/40 mesh, Supelco Inc., Bellefonte, PA). Direct gas chromatographic analyses of ETBE were applied during the exposure and when the volunteers exhaled sufficiently high ETBE concentrations, i.e., during the entire exposure day at the 25 and 50 ppm exposure levels and until 1 h after the exposure at the 5 ppm exposure level. TBA could not be analyzed by the direct gas chromatographic method and was therefore not determined during the exposure. Sorbent tubes were used postexposure and TBA could then be analyzed. All sorbent tubes were purged with nitrogen before use (10 min, 350°C, and 56 ml/min) to avoid possible contamination and then stored in a
TOXICOKINETICS OF ETBE IN MALE VOLUNTEERS
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FIG. 1. Concentration of (a) ethyl tert-butyl ether (ETBE), (b) tert-butyl alcohol (TBA), and (c) acetone in capillary blood sampled from eight male volunteers exposed to ETBE (2 h, 50 W, mean values). Vertical lines indicate the 95% confidence intervals (only partially for acetone).
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desiccator. The flow rate through each sorbent tube was determined with a mass flow meter (Aalborg, GFM17, SKC Inc.) and a pump (590 liters/min, AirChek sampler, Model 224-PCXR8, SKC Inc.) since flow rate variations were seen. This is probably due to the heterogeneous mesh size of the charcoal. Sorbent tubes used for calibration standards had all uniform flow rates (430 ml/min), and sample sorbent tubes were adjusted for discrepancies (range 330 to 430 ml/min) during calculations. Calibration standards in air containing ETBE and TBA were drawn from Tedlar bags to sorbent tubes with the use of a pump in the same way as described for exhaled air. The sorbent tubes were analyzed with an automated thermal desorption unit (ATD-400, Perkin–Elmer) connected to a gas chromatograph (GC-Auto System, Perkin–Elmer Auto System) equipped with a capillary column (Poraplot Q, Chrompack 25 m, 0.32 mm inner diameter, 20 mm phase thickness) and a flame ionization detector. Temperatures were desorption oven 300°C; desorption trap (Tenax TA 60/80 mesh, Supelco Inc.) 230 to 300°C; transfer line 150°C; injector 200°C; column 120°C for 5 min, 5°C/min to 165°C, then 165°C for 20 min; and flame ionization detector 250°C. Nitrogen flow rates were desorption 56 ml/min, outlet split 8.3 ml/min, and column 2 ml/min. Turbochrom chromatography workstation was used. Blood and urine. Capillary blood (200 ml) was sampled from the fingertips of the volunteers, before, during, and up to 48 h after exposure. During exposure the subject held out his hand through a closable hole in the chamber wall. After rinsing the hand with water (40°C) capillary blood was taken with two 100-ml heparinized micropipetes (Drummond Scientific Co., Broomall, PA) inside a glove box, which was flushed with clean air. The blood sample was transferred to a head space vial (22.4 ml, Perkin–Elmer), which was immediately capped with a Teflon-lined butyl rubber membrane (Perkin– Elmer). All urine samples were collected in 500-ml glass bottles. The volunteers were instructed to always empty the bladder completely and to note the time on the bottle. Samples were collected prior to exposure and at 0, 2, and 4 h and approximately 7, 11, 20, and 22 h after the exposure. In addition, a spot urine sample was collected 46 h after the exposure. The 7-, 11-, and 20-h samples were collected at home and stored at room temperature until the morning when the subject returned to the institute. ETBE, TBA, and acetone in blood and urine were stable in room temperature at least up to 24 h. The urine samples were treated and analyzed as the blood samples, except that 2 ml urine instead of 0.2 ml blood was added to each head-space vial. Gas chromatographic analyses of ETBE, unconjugated TBA, and acetone were performed as soon as possible after sampling. Calibration standards of ETBE, TBA, and acetone were prepared in Tedlar bags (as described under Chamber Air), and known amounts were injected to precapped head-space vials which contained either 200 ml blood (venous blood) or 2 ml urine. The reason for using a gas-phase standard is that ETBE has a rather low water solubility. However, a comparison between gas-phase and liquid (water) standard curves showed close agreement. No internal standard was used because of the risk of evaporation losses with the sampling technique used. Based upon 20 samples from a Tedlar bag, the variation (relative standard deviation, RSD) within the method was below 5% for all three substances in either blood or urine, which illustrates that the method of automatic head-space injection and flame ionization detection has a good reproducibility. The gas phase of each vial was then analyzed with a gas chromatograph (Perkin–Elmer 8700), equipped with a head-space autoinjector (Perkin–Elmer HS-101), a nonpolar capillary column (CP Sil 8, Chrompack, 50 m, 0.53 mm inner diameter, 2 mm phase thickness), and a flame ionization detector. The samples were thermostated for 25 min at 37°C before gas chromatographic analysis. The temperatures were autoinjector 37°C, needle 100°C, transfer line 100°C, injector 180°C, detector 270°C, and column initial 50°C. The column temperature was increased with 5°C/min to 90°C and thereafter 10°C/min to 150°C. Nitrogen was used as carrier gas at a flow rate of 5.7 ml/min. The Turbochrom chromatography workstation was used. The detection limit for ETBE in blood was approximately 0.15 mM, and in urine it was 0.05 mM. The detection limits for TBA and acetone were 0.5 mM in blood and 0.2 mM in urine.
Toxicokinetic Calculations The toxicokinetic calculations were carried out according to the previously published MTBE study (Nihle´n et al., 1998b). In brief, individual concentrations of ETBE in blood versus sampling times, personal net respiratory uptake, and amount of ETBE exhaled postexposure were fitted to the algebraic solution of a linear pharmacokinetic four-compartment model with zero order uptake (G. Johanson, unpublished data). A four-compartment model was chosen to conform with the previous MTBE study. Optimization was carried out by minimizing the unweighted residual sum of squares using Microsoft Excel (Version 5.0) and the Solver add-in macro. In these calculations the absorbed dose is regarded as the sum of the net respiratory uptake and the amount exhaled during exposure. Previously in studies of this kind, the absorbed dose has been estimated as equal to the difference between the concentration in inhaled and exhaled air multiplied by the pulmonary ventilation. However, by inclusion of the exhalation during exposure, dose-related parameters becomes independent of the length of exposure, and a more accurate estimation of body burden is achieved. Thus, absorbed dose in the present study is referred in two ways: the respiratory uptake (new approach) and the net respiratory uptake (old approach). Decay curves of ETBE in urine (starting at 2– 4 h) were fitted to biexponential functions by nonlinear regression analysis using Solver in Microsoft Excel. The elimination phase of TBA in blood and urine, starting approximately at 6 h, was fitted to a monoexponential function again by nonlinear regression analysis. The blood concentrations at steady state for ETBE were calculated for each subject as the respiratory uptake rate divided by the total clearance. The area under the concentration–time curve (AUC) of ETBE in blood was obtained from the four-compartment model, whereas the AUCs of TBA and acetone was calculated by the trapezoidal rule in Microsoft Excel. The renal clearance of TBA was calculated as the total amount TBA excreted in urine divided by the AUC of TBA in blood. Statistical Analysis Analysis of variance repeated measures model (SuperANOVA Version 1.11, Abacus Concepts Inc., Berkeley, CA), and the Student’s paired t test (Microsoft Excel VERSION 5.0) were used to compare results from different exposure occasions (5, 25, and 50 ppm ETBE) and between the present study and the previous MTBE study. The level of significance was set to 0.05. Unless otherwise stated, the results are presented as mean values 695% confidence intervals.
RESULTS
Exposure Conditions The measured mean levels of ETBE in the chamber air were 4.8, 25, and 50 ppm. The variations of the chamber air concentration between than exposure occasions were less 6% at 5 ppm, 4% at 25 ppm, and 3% at the 50 ppm exposure levels, expressed as RSD. The variation (RSD) within each 2-h exposure session was less than 7, 5, and 4%, respectively. With the exception of a slight temperature difference between the 5 and 25 ppm exposure levels (18.6 and 19°C, respectively, p , 0.05) no differences were seen between the exposure occasions regarding relative humidity (mean 43%, 95% confidence interval 42.3– 43.7%), air exchanges per hour (16 h21, 15.4 –16.6 h21), chamber temperature (18.7°C, 18.5– 18.9°C), actual physical exercise (49 W, 48.5– 49.5 W), and heart rate (95 beats/min, 89 –101 beats/min). The variation, expressed as RSD, were relative humidity, less than 2% be-
5
TOXICOKINETICS OF ETBE IN MALE VOLUNTEERS
TABLE 1 Uptake and Excretion of Ethyl tertiary-Butyl Ether (ETBE) and tertiary-Butyl Alcohol (TBA) in Eight Volunteers Exposed to ETBE for 2 h (Mean Values 6 95% Confidence Interval) Exposure level
ETBE Inhaled amount (mmol) Net respiratory uptake (%)a Respiratory uptake (%)b Net respiratory excretion (% of net respiratory uptake) Respiratory excretion (% of respiratory uptake) Urinary excretion (% of respiratory uptake) TBA Net respiratory excretion (% of ETBE net respiratory uptake)c Urinary excretion (% of ETBE respiratory uptake)
Time span (h)
5 ppm
25 ppm
50 ppm
0–2 0–2 0–2
0.58 6 0.05 26 6 3.4 32 6 4.4
2.9 6 0.19 26 6 2.9 34 6 5.3
5.8 6 0.39 26 6 2.9 34 6 3.4
2–24 0–24 0–24
30 6 3.5 45 6 4.0 0.12 6 0.053
31 6 4.2 50 6 5.6 0.061 6 0.021
30 6 4.7 46 6 5.5 0.056 6 0.022*
2–24 0–24
1.4 6 0.8 0.71 6 0.34
3.3 6 1.4 0.66 6 0.24
3.8 6 0.9 0.90 6 0.49
Net respiratory uptake 5 [amount inhaled 2 amount exhaled] during exposure. Respiratory uptake 5 [net respiratory uptake 1 amount exhaled during exposure]. c Net respiratory excretion 5 [(TBA exhaled postexposure 2 TBA net uptake during exposure)/ETBE net uptake], assuming 35–50% net respiratory uptake of TBA. * Significant different from 5 ppm level (p , 0.05, repeated-measures ANOVA). a b
tween exposure occasions and 7% within each 2-h exposure session; air exchanges, 6 and 2%; chamber temperature, 3 and 2%; actual physical exercise, 3 and 9%; and heart rate, 17 and 9%, respectively. Further, no differences in pulmonary ventilation or respiratory frequency were seen between exposure occasions. The pulmonary ventilation was on average 23.7 (22.9 –24.5) liters/min during the exposures and decreased postexposure to 11.4 (10.7–12.1) liters/min. The respiratory frequency was on average 18 (16.8 –19.2) breaths/min during and 15 (13.8 –16.2) breaths/min after the exposure. TABLE 2 Elimination Half-Times (t21) of Ethyl tertiary-Butyl Ether (ETBE) and tertiary-Butyl Alcohol (TBA) in Eight Volunteers Exposed to ETBE for 2 h (Mean Values 6 95% Confidence Interval) Exposure level Half-time ETBE in blood First phase (min) Second phase (min) Third phase (h) Fourth phase (h) ETBE in urine First phase (min) Second phase (h) TBA in blood (h) TBA in urine (h) Note. n.d., not determined.
5 ppm
25 ppm
50 ppm
1.8 6 1.1 20 6 11 2.1 6 0.59 33 6 6.0
1.2 6 0.75 15 6 9.1 1.5 6 0.45 24 6 9.7
2.0 6 1.3 1.9 6 9.3 1.5 6 0.20 27 6 11
6.3 6 4.3 8.8 6 4.3 n.d. n.d.
9.1 6 4.0 8.7 6 2.4 12 6 2.2 8.4 6 1.3
9.3 6 4.7 8.2 6 1.8 12 6 1.2 7.6 6 2.5
Toxicokinetics Results of uptake, excretion, half-times, and clearance are presented in Tables 1–3. To be able to compare the present study with previously performed studies of this kind, results on apparent blood clearance, net respiratory uptake, and excretion based on net respiratory uptake are also given. The respiratory uptake of ETBE was in the range of 32–34%, and the respiratory excretion was in the range of 45–50% of the respiratory ETBE uptake (Table 1). In comparison, the calculated net respiratory uptake and net respiratory excretion of ETBE were lower, 26 and 30 –34% respectively (Table 1), since the amount ETBE cleared by exhalation during exposure was not considered. No significant differences relative in uptake and exhalation were seen between exposure levels. The concentration of ETBE in blood increased during the entire exposure with a tendency to level off but without reaching any plateau (Fig. 1a). Average maximum levels of 1.1, 5.4, and 10 mM were reached at 5, 25, and 50 ppm, respectively. The elimination process of ETBE in blood was separated into four phases (Table 2). The two fastest elimination phases had average half-times of 2 and 18 min, the intermediate phase had an average half-time of 1.7 h, and the slowest phase had an average half-time of 28 h. No statistically significant differences in half-times were seen between exposure levels. The AUC of ETBE in blood (Fig. 2) was linearly related to the ETBE exposure level, suggesting linear kinetics up to 50 ppm. The clearance of ETBE in blood was in the range of 0.6 – 0.9 liters/h/kg (Table 3). A higher clearance was obtained at the 25 and 50 ppm exposures, but any statistically significant differences were not seen compared to the 5 ppm level. The elimi-
´ ¨ NIHLEN, LOF, AND JOHANSON
6
TABLE 3 Toxicokinetic Results of Ethyl tertiary-Butyl Ether (ETBE) and tertiary-Butyl Alcohol (TBA), from Exposure of Eight Volunteers to ETBE (Mean Values 6 95% Confidence Interval) Exposure level
ETBE Apparent total clearance (liters/h/kg)a Total clearance (liters/h/kg)b Metabolic clearance (liters/h/kg)b Exhalatory clearance (liters/h/kg)b Volume of distribution (liters/h/kg) Mean residence time (h) TBA Renal clearance (liters/h/kg)c
5 ppm
25 ppm
50 ppm
0.48 6 0.06 0.61 6 0.09 0.35 6 0.06 0.26 6 0.08 5.8 6 1.3 11 6 4.4
0.61 6 0.12 0.87 6 0.19 0.41 6 0.09 0.45 6 0.13 5.9 6 1.8 7.4 6 2.5
0.62 6 0.15 0.79 6 0.17 0.42 6 0.12 0.36 6 0.08 7.1 6 3.2 11 6 7.1
n.d.
0.00064 6 0.00028
0.00096 6 0.00054
Note. n.d., not determined. a Apparent total clearance calculated from net respiratory uptake. b Clearance calculated from respiratory uptake. c Renal clearance calculated from amount TBA excreted in urine and the AUC of TBA in blood.
nation of ETBE in urine could be separated in two linear phases with half-times of 8 min and 8.5 h (Table 2). The excretion rate of ETBE in urine was rather high (Fig. 3a), and the cumulative excretion of ETBE in urine within 22 h postexposure was less than 0.1% of the respiratory uptake (Table 1). The urinary ETBE excretion was higher at the 5 ppm exposure compared to the 25 and 50 ppm exposures, and a statistically significant difference was seen between the 5 and 50 ppm results (p , 0.05) (Table 1). In contrast to the ETBE profile, TBA in blood increased steadily during the exposure and remained high for several hours postexposure. Average maximum levels of TBA in blood were obtained approximately 30 min postexposure (25 ppm, 6.9 mM; and 50 ppm, 12 mM), and the levels started to decline slowly at about 4 h after the end of exposure (Fig. 1b). No calculations were performed for TBA in blood at the 5 ppm exposure level, since the con-
centration of TBA in blood was close to and sometimes below the gas chromatographic detection limit. The AUC of TBA was linearly related to the ETBE exposure level, suggesting linear kinetics of the metabolite up to 50 ppm (Fig. 2). The average half-times of TBA were 12 h in blood and 8 h in urine (Table 2), which were approximately equal to the previously obtained half-times of TBA after MTBE exposures (Pekari et al., 1996; Nihle´n et al., 1998b). TBA was also detected in exhaled air, and 1.4 –3.8% of ETBE net uptake was excreted as TBA through the lungs (Table 1). The excretion rate of TBA was rather slow (Fig. 3b), and less than 1% of the absorbed ETBE was excreted as TBA in urine within 22 h postexposure (Table 1). A low renal clearance of TBA (less than 1 ml/h/kg) was seen, indicating extensive blood protein binding or tubular reabsorption of the metabolite (Table 3). Acetone was detected in elevated levels in blood after the exposure (Fig. 1c). The average concentration in blood and the AUC of acetone was highest at the 50 ppm exposure, but otherwise poorly related to the exposure level. Acetone was detected in urine as well and the highest cumulative urinary excretion was seen at the 50 ppm exposure level and the lowest was seen after the control exposure (data not shown). The average background levels of acetone as measured in samples collected in the morning prior to ETBE exposure were 42 (95% confidence interval 35–50) mM in blood and 38 (18 –58) mM in urine. A wide interindividual variation in acetone levels (especially in urine) was seen before, during, and after the exposure. DISCUSSION
FIG. 2. The area under the concentration–time curve (AUC) of ethyl tert-butyl ether (ETBE) and tert-butyl alcohol (TBA) in capillary blood versus exposure level of ETBE (mean values and 95% confidence intervals, n 5 8).
The present study shows that ETBE is absorbed in the respiratory tract, is transformed to TBA in the body, and is
TOXICOKINETICS OF ETBE IN MALE VOLUNTEERS
7
FIG. 3. Urinary excretion of (a) ethyl tert-butyl ether (ETBE) and (b) tert-butyl alcohol (TBA) versus midpoint time of urine collection after a 2-h exposure to ETBE (mean values, n 5 8). Vertical lines indicate the lower and upper 95% confidence intervals at 5 and 50 ppm, respectively.
partly excreted as ETBE and TBA through urine and exhaled air. Further, the results suggest that ETBE is also biotransformed to acetone. To our knowledge, this is the first time that human toxicokinetic data are presented for ETBE. The chemical used, a commercially available technical grade of ETBE, was contaminated with TBA and MTBE. Even after careful redistillation it was still contaminated with 0.4% TBA and 1.1% MTBE. These contaminations may confound the toxicokinetic analysis and especially TBA should be kept in mind when interpreting the TBA levels in blood, urine, and exhaled air and when comparing ETBE toxicokinetics with that of MTBE. The experimental design of these two studies is similar. Two years elapsed between the two exposure studies, different subjects were exposed, and some of the used analytical equipment was exchanged. In spite of this, the toxicokinetics of ETBE and MTBE in human volunteers were quite similar regarding respiratory uptake and exhalation and concentration– time profiles of blood and urine, as can be seen in Table 4. The small differences seen are consistent with the different blood/ air and olive oil/blood partition coefficients measured in vitro for ETBE and MTBE (Nihle´n et al., 1995; Borghoff et al., 1996). Thus, the lower blood/air partition coefficient of ETBE
compared to MTBE (12 versus 18) suggests a lower respiratory uptake, whereas the higher oil/blood partition coefficient (16 versus 7) implies a longer terminal half-time (Nihle´n et al., 1995). Indeed the respiratory uptake of ETBE was significantly lower (p 5 0.03) than MTBE, whereas a borderline difference (p 5 0.06) was seen regarding the net respiratory uptake. No difference in respiratory excretion was seen between the two ethers. The blood and urinary profiles were quite similar for both ethers apart from somewhat lower ETBE levels in blood. This is illustrated by a 15–30% lower blood AUC for ETBE than for MTBE at the same exposure level. Also, the average blood concentrations at steady state were lower for ETBE and were as follows: mean 1.5 (95% confidence interval, 1.3–1.7) mM at 5 ppm, 5.9 (5.4 – 6.4) mM at 25 ppm, and 13 (12–14) mM at 50 ppm. In comparison, the average MTBE steady-state levels were 2.3 (1.6 –3.0) mM at 5 ppm, 8.8 (7.4 –10) mM at 25 ppm, and 19 (16 –21) mM at 50 ppm. A tendency (p 5 0.07) to a longer terminal half-time in blood (24 –33 h) was seen for ETBE compared to MTBE (17–21 h). Furthermore, a significantly (p 5 0.03) higher volume of distribution was obtained for ETBE, but no difference in blood clearance was seen. In addition, ETBE was excreted significantly slower than MTBE to urine, although the cumulative amount did not differ and
´ ¨ NIHLEN, LOF, AND JOHANSON
8
TABLE 4 Comparison of Toxicokinetic Parameters for Ethyl tertiary-Butyl Ether (ETBE), Methyl tertiary-Butyl Ether (MTBE), and the Metabolite tertiary-Butyl Alcohol (TBA) (Average Values 6 95% Confidence Intervals)
Parent ether Net respiratory uptake (%) Respiratory uptake (%) Net respiratory excretion (%) Respiratory excretion (%) Metabolic clearance (liters/h/kg) Exhalatory clearance (liters/h/kg) Volume of distribution (liters/kg) Mean residence time (h) Half-time in blood (terminal, h) Half-time in urine (first phase, h) Half-time in urine (second phase, h) Urinary excretion (%) TBA Net respiratory excretion (%) Half-time in blood (h) Half-time in urine (h) Urinary excretion (%) Renal clearance (liters/h/kg)
ETBE (present study) n 5 24a
MTBE (Nihle´n et al., 1998b) n 5 30a
26 6 1.7 34 6 2.7* 31 6 2.5 47 6 3.1 0.39 6 0.06 0.35 6 0.06 6.4 6 1.4* 9.9 6 3.1 29 6 5.4 0.14 6 0.04* 8.6 6 1.7* 0.08 6 0.02
38 6 4.0 47 6 5.3* 28 6 5.2 39 6 6.3 0.41 6 0.07 0.27 6 0.06 3.9 6 0.6* 6.9 6 1.9 20 6 5.5 0.33 6 0.14* 3.1 6 0.45* 0.08 6 0.01
2.9 6 1.0 12 6 1.5b 7.9 6 1.5b 0.76 6 0.22 0.00080 6 0.00034b
n.d. 10 6 1.4b 8.1 6 2.0b 0.59 6 0.13 0.00067 6 0.00011b
Note. n.d., not determined. a Average of 8 or 10 subjects exposed at three levels (5, 25, and 50 ppm). b Average of 8 or 10 subjects exposed at two levels (25 and 50 ppm). * Statistically significant difference (p , 0.05, Student’s paired t test).
was as low as 0.1% of the uptake. The delayed excretion of ETBE is probably a reflection of the slower decay in blood compared to MTBE. The excretion of minor amounts of ETBE and MTBE in the urine is not a surprise, since these ethers are slightly water soluble. In fact, even less water-soluble chemicals e.g., styrene and trimethylbenzenes, are recovered in small amounts in human urine (Ong et al., 1994; Ja¨rnberg et al., 1996). Urinary excretion of small amounts of unmetabolized MTBE has been demonstrated in several volunteer studies (Pekari et al., 1996; Buckley et al., 1997; Nihle´n et al., 1998b). The toxicokinetic calculations indicate that the clearance by exhalation was almost as high as the metabolic clearance of ETBE (Table 3). A theoretical calculation of the exhalatory clearance based on literature values on the blood/air partition coefficient for ETBE (12) (Nihle´n et al., 1995), alveolar ventilation (4 liters/min at rest, 17–22 liters/min at 50 W), and cardiac output (5 liters/min at rest, 7–12 liters/min at 50 W) (Åstrand, 1983) gives respiratory clearance values between 0.3 (rest) and 1.2 liters/h/kg (50 W exercise). As our exposures were performed during 2 hours of light exercise followed by rest, the experimentally obtained expiratory clearance (0.3– 0.5 liters/h/kg, Table 3) is reasonable. The kinetics of the metabolite TBA were similar after the ETBE and MTBE exposures, judging by blood and urine profiles and the toxicokinetic results (Table 4). Somewhat
lower concentrations of TBA in blood were reached after ETBE compared to MTBE exposures; this probably reflects the different blood levels of the parent ether. In the present study there are no signs of metabolic saturation, since the kinetics of ETBE and TBA are linear in the studied exposure interval (0 –50 ppm ETBE) (Fig. 2). Linear kinetics have previously been demonstrated for MTBE and TBA in humans up to 75 ppm (Pekari et al., 1996; Nihle´n et al., 1998b) and in rats up to 300 ppm (Savolainen et al., 1985). About 50% of inhaled ether was recovered as the parent ether and as the metabolite TBA in breath and urine. The low recovery indicates competing pathways, further metabolism of the alcohol, or other excretion routes. After exposing rats for MTBE or ETBE several metabolites (2-methyl-1,2-propanediol, a-hydroxyisobutyric acid, and TBA conjugates) have been found in the urine (Miller et al., 1997; Bernauer et al., 1998). ETBE was contaminated with 0.4% TBA even after careful redistillation. This makes the calculations of the kinetics of TBA more difficult. On average the subjects exhaled 7.6, 39, and 75 mmol TBA as measured 0 – 46 h after the exposure, corresponding to 2.4 –5.2% of the ETBE net uptake. Assuming that the chamber air is contaminated with 0.4% TBA or to 0.027, 0.14, and 0.28 ppm TBA during the 5, 25, and 50 ppm ETBE exposures, respectively, and that the relative uptake of
TOXICOKINETICS OF ETBE IN MALE VOLUNTEERS
TBA is 35–50% (Nihle´n et al., unpublished observation with MTBE), exhaled TBA formed by ETBE biotransformation is in the range of 1.4 –3.8% (Table 1) of the ETBE net uptake. In our previous MTBE study (Nihle´n et al., 1998b) TBA was not detected in exhaled air. Acetone is a metabolite of MTBE, ETBE, and TBA in rodents (Cederbaum et al., 1980; Baker et al., 1982; Bernauer et al., 1998) suggesting that acetone is also a metabolite of ETBE in humans. In the present study, acetone in blood (Fig. 1c) and urine (data not shown) was elevated at 50 ppm ETBE, but no clear dose response was seen when comparing all exposure levels. Acetone is produced endogenously, and the acetone levels may change naturally through the day due to for example physical exercise or food intake (Jones et al., 1993). Thus, tracer techniques would be needed to confirm the formation of acetone from ETBE in humans. Acetone was not measured in our earlier MTBE study. A potentially useful biological exposure marker for ETBE and gasoline-containing ETBE might be TBA, because of the relatively high levels in blood and urine and the rather slow decay after exposure. However, further studies of the metabolism pattern of ETBE are important, since other metabolites might be found in higher quantities in the urine and therefor be better as a biomarkers of ETBE. In this study we did not correct the urine levels for creatinine, since we collected all urine and thus could calculate excretion rates. However, if TBA is used as a biomarker of ETBE creatinine corrections might be more practical. In conclusion, our study has for the first time investigated the kinetics of ETBE in humans. Relative to many other solvents a rather low respiratory uptake of ETBE and a quite high respiratory exhalation were seen. The metabolism and accumulation of ETBE are almost similar to that previously seen for MTBE (Nihle´n et al., 1998b). Thus, from toxicokinetic considerations, such as bioavailability and potential for accumulation, neither ether is preferred over the other. However, further research of other toxicological and biological aspects of ETBE should be performed before ETBE is used in large scale in gasoline and spread in the environment. ACKNOWLEDGMENTS We express our gratitude to Mrs. E. Gullstrand, L. Ernstgård, and A. Dihkan for skillful technical assistance and Dr. A. Toomingas for medical examinations. The authors thank Ecotraffic Research & Development AB, Sweden, for valuable advice and Ecofuel S.P.A, Italy, for offering technical ETBE. The project was financially supported by the National Board for Industrial and Technological Development (NUTEK), Sweden (Grant P3626-1).
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