Toxicokinetics of Methyl tert-Butyl Ether and Its Metabolites in Humans after Oral Exposure

Alexander Amberg, Elisabeth Rosner and Wolfgang Dekant,1

Institut für Toxikologie, Universität Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany

Received September 26, 2000; accepted February 6, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Methyl tert-butyl ether (MTBE) is widely used as an additive to gasoline, to increase oxygen content and reduce tailpipe emission of pollutants. Widespread human exposure to MTBE may occur due to leakage of gasoline storage tanks and a high stability and mobility of MTBE in ground water. To compare disposition of MTBE after different routes of exposure, its biotransformation was studied in humans after oral administration in water. Human volunteers (3 males and 3 females, identical individuals, exposures were performed 4 weeks apart) were exposed to 5 and 15 mg 13C-MTBE dissolved in 100 ml of water. Urine samples from the volunteers were collected for 96 h after administration in 6-h intervals and blood samples were taken in intervals for 24 h. In urine, MTBE and the MTBE-metabolites tert-butanol (t-butanol), 2-methyl-1,2-propane diol, and 2-hydroxyisobutyrate were quantified, MTBE and t-butanol were determined in blood samples and in exhaled air in a limited study of 3 male volunteers given 15 mg MTBE in 100 ml of water. MTBE blood concentrations were 0.69 ± 0.25 µM after 15 mg MTBE and 0.10 ± 0.03 µM after 5 mg MTBE. MTBE was rapidly cleared from blood with terminal half-lives of 3.7 ± 0.9 h (15 mg MTBE) and 8.1 ± 3.0 h (5 mg MTBE). The blood concentrations of t-butanol were 1.82 ± 0.63 µM after 15 mg MTBE and 0.45 ± 0.13 µM after 5 mg MTBE. Approximately 30% of the MTBE dose was cleared by exhalation as unchanged MTBE and as t-butanol. MTBE exhalation was rapid and maximal MTBE concentrations (100 nmol/l) in exhaled air were achieved within 10–20 min. Clearance of MTBE by exhalation paralleled clearance of MTBE from blood. T-butanol was cleared from blood with half-lives of 8.5 ± 2.4 h (15 mg MTBE) and 8.1 ± 1.6 h (5 mg MTBE). In urine samples, 2-hydroxyisobutyrate was recovered as major excretory product, t-butanol and 2-methyl-1,2-propane diol were minor metabolites. Elimination half-lives for the different urinary metabolites of MTBE were between 7.7 and 17.8 h. Approximately 50% of the administered MTBE was recovered in urine of the volunteers after both exposures, another 30% was recovered in exhaled air as unchanged MTBE and t-butanol. The obtained data indicate that MTBE-biotransformation and excretion after oral exposure is similar to inhalation exposure and suggest the absence of a significant first-pass metabolism of MTBE in the liver after oral administration.

Key Words: methyl tert-butyl ether (MTBE); MTBE elimination half-life; excretion and exhalation clearance.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Methyl tert-butyl ether (MTBE) is presently used as a fuel additive to reduce carbon monoxide and hydrocarbon emissions or to boost the octane number of high-performance gasoline (Stern and Kneiss, 1997Go). Exposure of the general population to MTBE may occur at service stations and inside cars, but also from consumption of drinking water contaminated with MTBE. MTBE was detected (analysis performed between 1989 and 1995) in 5 states of the U.S. where MTBE has been used as a fuel additive, with average levels between 5 and 200 µg/l in 2.7% of public wells and in 5.5% of private wells (Davidson, 1995Go). Contamination of domestic water supplies occurs due to leakage of MTBE from underground gasoline storage tanks. At present, contamination of ground water and some drinking water supplies with MTBE, which seems to be highly mobile and stable in ground and surface water, represent a major possible source of exposure of the general population to MTBE.

The toxicology of MTBE has been intensively investigated. The acute toxicity of MTBE is low. Both MTBE and t-butanol have been studied in long-term bioassays for tumorigenicity (Fig. 1Go). MTBE and its presumed major metabolite, t-butanol, induce renal tumors in male rats (Bird et al., 1997Go; Burleigh-Flayer et al., 1992Go; Chun et al., 1992Go; Takahashi et al., 1993Go). Renal tumor induction by these compounds may be mediated by the accumulation of {alpha}2u-globulin (Borghoff et al., 1996Go; Prescott-Mathews et al., 1997Go; Scientific Advisory Board on Toxic Air Pollutants 1995Go). An impaired degradation pathway of this protein induced by bound metabolites of t-butanol and MTBE or by t-butanol or MTBE may cause renal toxicity, cell proliferation and, finally, renal tumors (Swenberg et al., 1989Go). MTBE exposure also increased the incidence of liver tumors in female mice and testicular tumors in male rats (Belpoggi et al., 1995Go; Bird et al., 1997Go; Burleigh-Flayer et al., 1992Go; Chun et al., 1992Go). Testicular tumors in male rats were also observed following oral administration of MTBE (Belpoggi et al., 1995Go). MTBE and the presumed major metabolite, t-butanol, are negative in standard genotoxicity studies (Duffy et al., 1992Go).



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FIG. 1. Biotransformation of MTBE and t-butanol (TBA) in rats. Excreted metabolites in urine are underlined.

 
We have previously studied the disposition of MTBE and metabolite excretion in humans after inhalation exposure (Amberg et al., 1999Go). In this study, the biotransformation and kinetics of metabolite excretion were quantified in humans exposed to MTBE in drinking water. The 15-mg oral dose of MTBE administered here was selected to be equivalent to the calculation of received MTBE dose (based on an alveolar ventilation rate of 9 l/min and a retention of 0.4) after a 4-h inhalation exposure to 4 ppm MTBE, to permit a comparison of the relative excretion of metabolites in humans after MTBE exposure by the two most relevant pathways. The obtained data may be used to validate toxicokinetic models for comparison of MTBE disposition after different exposure scenarios. 13C-MTBE was used in this human-exposure study to monitor metabolite excretion in the low-dose range and avoid interference of the large concentrations of endogenously formed 2-hydroxyisobutyrate present in urine with metabolite determination.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
MTBE (99.8+% purity), t-butanol (99.5+% purity) and 2-hydroxyisobutyrate (98+% purity) were obtained from Aldrich Chemical Company (Deisenhofen, Germany). The syntheses of 13C-MTBE and of 2-methyl-1,2-propane diol were described previously (Bernauer et al., 1998Go). All other chemicals were obtained from commercial suppliers in the highest purity available.

Oral exposure of human volunteers to 13C-MTBE.
To study kinetics of excretion and biotransformation of 13C-MTBE, 6 human volunteers (3 male and 3 female, identical individuals with a 4-week interval between exposures, see Table 1Go) were given 5 and 15 mg of MTBE in 100 ml of tap water. Fixed amounts of MTBE were administered to match inhalation exposure, where received doses were not calculated by adjusting to individual parameters. The volunteers consumed the spiked water samples within 30 s. Blood samples were taken in 60-min intervals from h 0 to h 4 and in 120 min intervals from h 4 to h 12 after the exposure. One additional blood sample was taken 24 h after the exposure.


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TABLE 1 Characteristics of Human Volunteers Who Participated in the Oral Exposure Studies
 
Urine samples were taken in 6-h intervals for 96 h after the administration. The volunteers had to refrain from alcoholic beverages and medicinal drugs 2 days before and throughout each experiment. Subjects did not abuse alcohol and were non-smokers or occasional smokers. Subjects were healthy, as judged by medical examination and clinical blood chemistry, and stated having no previous occupational exposure to MTBE. They also did not refuel their cars during the 2 days prior to exposure and during sample collection period. Exposures started at 7 A.M. The study was carried out according to the Declaration of Helsinki, after approval by the Regional Ethical Committee of the University of Wüerzburg, Germany, and after receiving written informed consent from the volunteers. There was a time interval of 4 weeks between the 2 exposures.

Exhalation of MTBE and t-butanol.
The exhalation of unchanged MTBE and of t-butanol was studied in 3 male volunteers exposed to 15 mg 13C-MTBE in 100 ml of tap water. Samples of the exhaled breath from the volunteers were collected in gas sampling bulbs (Supelco, Deisenhofen, Germany) in 10-min intervals. Samples (300 µl) from the collected exhalates were injected into the GC/MS system; separation was performed using a fused silica capillary column (DB1, 1 µm film, 30 m x 0.25 mm ID). Samples were injected in the splitless mode and eluted from the column at 35°C with a flow rate of 1 ml/min using helium as carrier gas. Quantitation of 13C-MTBE and 13C-t-butanol in the air samples was done by selected ion monitoring, using m/z 20, 60, and 74. MTBE and t-butanol concentrations in the samples were determined based on calibration curves obtained by adding known concentrations of MTBE or t-butanol to 2-liter gas sampling bags (Supelco, Deisenhofen, Germany), and the peak obtained for neon (m/z 20) as internal standard. The method was linear in response between 1 pmol (limit of quantitation) and 100 pmol MTBE or t-butanol per ml of air, and calibration samples were run after each analysis series.

Analytical methods for determination of 13C-MTBE and its metabolites in blood and urine.
Methods for determination of 13C-MTBE and its metabolites were adjusted to the content of 13C in the parent compounds or metabolites, with respect to mass spectrometric quantitation. Separation conditions and internal standards were identical as described previously (Amberg et al., 1999Go).

Statistical analysis.
Statistical analyses of the data were performed using Student's t-test in Microsoft Excel spreadsheets. p-Values less than 0.05 were considered significant. To determine possible sex differences, all data sets from male and female human volunteers were compared using the Student's t-test in Microsoft Excel spreadsheets. p-Values of less than 0.05 were considered significant. Half-lives were calculated using exponential regression in Microsoft Excel® spread sheets. The curve-fitting function of the program was used and curves were stripped based on correlation coefficients. r2 values of >0.95 were considered for separation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In these experiments, MTBE, labeled with the stable isotope 13C, was used in order to be able to follow the kinetics of MTBE-metabolite excretion without interference from background. 13C-MTBE is metabolized as 12C-MTBE, but, due to a change in molecular weight by one mass unit, metabolites can be measured by mass spectrometry without interference from the natural background of 12C compounds.

Human volunteers (three males and three females each) were exposed to 13C-MTBE dissolved in local tap water that did not contain detectable concentrations of MTBE. Experimental results on the excretion of MTBE metabolites and half-lives in humans after oral exposure are given in Tables 2, 3, and 4GoGoGo and in Figures 2, 3, and 4GoGoGo.


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TABLE 2 MTBE and t-Butanol Blood Concentrations in Humans 1 Hour after Oral Administration of MTBE in Water
 

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TABLE 3 Cumulative Metabolite Excretion and Half-Lives of Urinary Excretion of MTBE Metabolites in Humans (n = 6) after Oral Exposure to 15 mg MTBE in 100 ml Water and 5 mg MTBE in 100 ml Water MTBE
 

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TABLE 4 Amounts of Metabolites Recovered in Human Urine after Oral Administration of MTBE in Water
 


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FIG. 2. Time course of MTBE (filled circle) and t-butanol (filled square) elimination from blood of 6 human volunteers exposed to 5 mg (A) and 15 mg (B) 13C-MTBE by oral ingestion in water. Due to the use of 13C-labeled ether for the oral studies, there was no detectable background of metabolites.

 


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FIG. 3. Time course of MTBE (filled circle) and t-butanol (filled square) exhalation in 3 male volunteers exposed to 15 mg MTBE by oral ingestion.

 


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FIG. 4. Excretion with urine of 2-hydroxyisobutyrate (filled square), 2-methyl-1,2-propane diol (filled circle) and t-butanol (filled triangle) in 6 human volunteers exposed to 5 mg (panel A) and 15 mg (panel B) MTBE by oral ingestion. Numbers (mean ± SD) given represent total amount of metabolite excreted in the urine samples collected in 6 h intervals. Each sample was analysed in duplicate. Due to the use of 13C-labelled ether for the oral studies, there was no detectable background of metabolites.

 
13C-MTBE was not detected in blood samples from the volunteers taken before the oral exposure. The maximal concentrations of MTBE in blood were determined in the first blood samples taken 1 h after administration (Table 2Go). Elimination of MTBE from blood could be separated into 3 phases (Table 2Go, Fig. 2Go) and MTBE concentrations decreased to reach the limit of detection at 12 h after exposure (Fig. 2Go). Maximal concentrations of t-butanol were also determined at the first sampling point, 1 h after oral exposure. t-Butanol concentrations in blood decreased more slowly than MTBE concentrations, and t-butanol was still present in blood in small, but detectable concentrations 24 h after dosing. Elimination of t-butanol from blood occurred according to first-order kinetics.

We quantified the exhalation of 13C-MTBE and 13C-t-butanol in 3 male individuals exposed to 15 mg of 13C-MTBE. Due to the close spacing of air samples taken in order to obtain a representative curve for exhalation, time constraints in sample handling limited the number of individuals that could be studied. Excretion of MTBE by exhalation in the 3 volunteers paralleled the determined blood concentrations and also occurred with 3 half-lives (0.25 ± 0.07 h; 0.64 ± 0.15 h; 1.74 ± 0.23 h) similar to those observed for MTBE clearance from blood (Fig. 3Go, Table 2Go). The exhalation of t-butanol was slower, occurring with only one half-life (6.71 ± 2.17 h), and also paralleled the clearance of t-butanol from blood (Table 2Go). The amount of exhaled MTBE after oral ingestion was calculated as 26 ± 5% of dose, t-butanol exhalation accounted for 6 ± 1% of dose. In the breath samples taken, only very low concentrations of 13C-acetone were present (<0.1% of dose) suggesting that acetone is not a major metabolite of MTBE formed in humans after oral MTBE exposure.

In urine of humans exposed to 13C-MTBE by oral ingestion, identical metabolites as observed after inhalation exposure to MTBE were present. After oral MTBE exposure, 2-hydroxyisobutyrate was the major MTBE metabolite excreted with urine; t-butanol and 2-methyl-1,2-propane diol and unchanged MTBE were minor products excreted with urine (Table 3Go). Kinetics of excretion of the metabolites after oral MTBE exposure was not different from excretion kinetics determined after inhalation of MTBE (Amberg et al., 1999Go). Both t-butanol and 2-methyl-1,2-propane diol were eliminated rapidly and reached the limit of detection between 48 and 66 h after oral MTBE administration (Fig. 4Go). In contrast, the elimination of 2-hydroxyisobutyrate occurred slowly and 13C-2-hydroxyisobutyrate was still present in low concentrations in the urine samples collected up to 96 h after administration of 15 mg MTBE (Fig. 4Go). In the urine sample from the volunteers, ~50% of the administered dose of MTBE was recovered in the form of metabolites (Table 4Go). The determined half-lives of elimination for the metabolites excreted with urine also were not different after the 5 and 15 mg MTBE exposures (Table 3Go). Together with the amount of metabolites recovered in urine (Table 4Go), the summed exhalation of MTBE and t-butanol accounted for over 80% of the administered doses of MTBE.

Statistically significant differences (p < 0.05) between male and female volunteers were not observed when comparing maximal MTBE and t-butanol blood levels, elimination of MTBE and t-butanol from blood, and percentage of MTBE dose recovered as metabolites in urine.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The use of 13C-labeled MTBE for the oral exposure study permitted a more exact determination of MTBE disposition after administration of low doses, as compared to our previous work using inhalation exposures (Amberg et al., 1999Go). 13C-labelled MTBE could not be used in the inhalation exposures in the dynamic exposure chamber due to relative large amounts of MTBE required and the cost associated with synthesis of large amounts of 13C-MTBE.

The results from the oral administration studies show that disposition and elimination of MTBE does not show major route of administration-dependent differences. Elimination half-lives, peak blood levels of MTBE (1.9 ± 0.4 µM after a 4-h inhalation exposure to 4 ppm, and 1.8 ± 0.3 µM in the oral study extrapolated from the one-h blood sampling) and t-butanol (1.7 µM after inhalation of 4 ppm MTBE for 4 h, background corrected, and 1.8 µM after ingestion of 15 mg MTBE) were similar. Oral administration of MTBE also resulted in measurable blood levels of MTBE and t-butanol. The concentrations of these compounds in blood of all volunteers were dependent on the administered doses. At first glance, MTBE blood concentrations after oral administration seem to be lower when compared to equivalent inhalation exposures to 4 ppm MTBE for 4 h. A lower blood concentration of MTBE after oral administration of identical doses as received by inhalation exposure could be expected due to hepatic first-pass biotransformation. However, the differences in measured maximal blood concentrations between inhalation and oral MTBE exposures in humans are most likely due to differences in blood sampling design between the oral and the inhalation exposures. The first blood samples in the inhalation studies were taken at the end of the inhalation period; after oral administration, the first blood samples were taken one h after administration. Elimination of MTBE from blood could be separated into 3 phases (Table 2Go). The difference in the kinetics of elimination of MTBE from blood (2 phases after inhalation and 3 phases after oral administration) is also most likely due to sampling design (spacing of blood samples). After oral administration, the study design included more closely spaced blood sampling (every h for 4 h after administration) for a closer description of elimination kinetics.

Since blood concentration parallels the concentration in exhaled air, extrapolation from the measured 1-h blood levels using MTBE half-lives determined in blood suggest that maximal blood levels after inhalation of 4 ppm for 4 h and oral administration of 15 mg MTBE are both in the range of 2 µM.

To obtain information of MTBE exhalation and complete mass balance, exhalation of MTBE after oral administration was quantified in a limited study in volunteers exposed to 15 mg MTBE. Absence of a first-pass effect after oral administration of MTBE is also indicated by the extent of recovery of the applied dose as MTBE and t-butanol in exhaled air, which accounted for approximately 30% of MTBE-dose. The results on the elimination of MTBE and t-butanol by exhalation indicate a recovery of >80% of the administered dose. As expected, the half-lives determined for MTBE exhalation are similar to those of MTBE clearance from blood. The data on MTBE exhalation, collected after oral administration, suggest that MTBE is rapidly absorbed from the upper gastrointestinal tract and that the maximum rate of exhalation and highest blood levels are achieved within 10 to 20 min after administration.

In urine of orally exposed humans, identical metabolite amounts were present as after inhalation exposure to MTBE. Kinetics of excretion of the metabolites after oral MTBE-exposure was not significantly different to excretion kinetics determined after inhalation of MTBE. In the urine sample from the volunteers after oral administration, ~50% of the administered dose of MTBE was recovered in the form of metabolites. The same percentage of the received dose was metabolized in humans exposed to 4 ppm MTBE for 4 h, showing that there are no significant differences in extent or pathways of MTBE-biotransformation after oral or inhalation exposures to low doses. Possible adverse effects of MTBE in humans are thus expected to be independent of route of exposure.


    ACKNOWLEDGMENTS
 
Research described in this article was conducted under contract from the Health Effects Institute (HEI, Research Agreement No. 96–3), an organization jointly funded by the United States Environmental Protection Agency (EPA) (Assistance Agreement X-816285) and certain motor vehicle and engine manufacturers. The contents of this article do not necessarily reflect the views of HEI, or its sponsors, nor do they necessarily reflect the views and policies of EPA or motor vehicle and engine manufacturers.


    NOTES
 
1 To whom correspondence should be addressed at the Department of Toxicology, University of Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany. Fax: +49(931) 201 3446. E-mail: dekant{at}toxi.uni-wuerzburg.de. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Amberg, A., Rosner, E., and Dekant, W. (1999). Biotransformation and kinetics of excretion of methyl tert-butyl ether in rats and humans. Toxicol. Sci. 51, 1–8.[Abstract]

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