55, 274 –283 (2000)
Copyright © 2000 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Biotransformation and Kinetics of Excretion of tert-Amyl-methyl Ether
in Humans and Rats after Inhalation Exposure
Alexander Amberg, Elisabeth Rosner, and Wolfgang Dekant 1
Institut für Toxikologie, Universität Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany
Received November 16, 1999; accepted February 4, 2000
tert-Amyl methyl ether (TAME) may be widely used as an additive
to gasoline in the future. The presence of this ether in gasoline reduces
the tail pipe emission of pollutants. Therefore, widespread human
exposure to TAME may occur. To contribute to the characterization
of potential adverse effects of TAME, its biotransformation was
compared in humans and rats after inhalation exposure. Human
volunteers (three males and three females) and rats (five males and
five females) were exposed to 4 (3.8 ⴞ 0.2) and 40 (38.4 ⴞ 1.7) ppm
TAME for 4 h in a dynamic exposure system. Urine samples were
collected for 72 h in 6-h intervals and blood samples were taken at
regular intervals for 48 h in humans. In urine, the TAME metabolites
tert-amyl alcohol (t-amyl alcohol), 2-methyl-2,3-butane diol, 2-hydroxy-2-methylbutyric acid, and 3-hydroxy-3-methylbutyric acid
were quantified. TAME and t-amyl alcohol were determined in blood
samples. After the end of the exposure period, blood concentrations of
TAME were 4.4 ⴞ 1.7 ␮M in humans and 9.6 ⴞ 1.4 ␮M in rats after
40 ppm TAME, and 0.6 ⴞ 0.1 ␮M in humans and 1.4 ⴞ 0.8 ␮M in
rats after 4 ppm. TAME was rapidly cleared from blood in both rats
and humans. The blood concentrations of t-amyl alcohol were 9.2 ⴞ
1.8 ␮M in humans and 8.1 ⴞ 1.5 ␮M in rats after 40 ppm TAME, and
1.0 ⴞ 0.3 ␮M in humans and 1.8 ⴞ 0.2 ␮M in rats after 4 ppm
TAME. t-Amyl alcohol was also rapidly cleared from blood. In urine
of humans, 2-methyl-2,3-butane diol, 2-hydroxy-2-methylbutyric
acid, and 3-hydroxy-3-methylbutyric acid were recovered as major
excretory products in urine. In rats, 2-methyl-2,3-butane diol and its
glucuronide were major TAME metabolites. t-Amyl alcohol and its
glucuronide were minor TAME metabolites in both species. All metabolites of TAME excreted with urine in rats were rapidly eliminated, with elimination half-lives of less than 6 h. Metabolite excretion in humans was slower and elimination half-lives of the different
metabolites were between 6 and 40 h in humans. The obtained data
indicate differences in TAME biotransformation and excretion between rats and humans. In rats, TAME metabolites are rapidly
excreted. In humans, metabolic pathways are different and metabolite excretion is slower. Recovery of TAME metabolites in urine was
higher in humans as compared to rats, suggesting more intensive
biotransformation of TAME in humans.
Key Words: biotransformation; F344 NH rats; humans; inhalation exposure; tert-Amyl methyl ether (TAME).
To whom correspondence should be addressed. Fax: ⫹49-0931-201-3865.
E-mail: [email protected].
1
Increased oxygen content in gasoline is required by the 1990
Amendments to the Clean Air Act in certain areas of the United
States that fail to meet the National Ambient Air Quality
standard for carbon monoxide or ozone. The chemicals
blended with gasoline hydrocarbons to meet the required oxygen content are referred to as oxygenates (Costantini, 1993).
At present, the oxygenates most often used are methyl tertbutyl ether and methanol. However, other ethers are also used
or considered for use. tert-Amyl methyl ether (TAME) is a
well-suited oxygenate due to its relatively low production
costs, lower vapor pressure than other ethers, and its ability to
act as a high-octane gasoline-blending compound (HEI, 1996;
Vainiotalo et al., 1998).
Due to the potential widespread exposure of humans to
oxygenates in fuel (HEI, 1996; Vainiotalo et al., 1999), studies
on the toxicity of oxygenates including TAME are underway.
The major toxic effect seen in rodents after inhalation of high
concentrations of TAME is central nervous system depression.
Subchronic inhalation studies with TAME for 4 weeks (6 h per
day, 5 days per week) showed significantly reduced body
weights and relative increases in adrenal, kidney, testes, brain,
and lung weights in male rats exposed to 4000 ppm TAME, but
no treatment-related histopathologic findings (White et al.,
1995). The toxicity of the initial metabolite formed from
TAME, tert-amyl alcohol (t-amyl alcohol), is also low. In rats
exposed to concentrations of up to 1000 ppm TAME for 90
days by inhalation, increased liver weights in male rats were
the only effect observed (Nolan et al., 1976, 1981).
The biotransformation of TAME has been studied in rats and
humans, and metabolites have been identified (Fig. 1). Free and
glucuronidated 2-methyl-2,3-butane diol and a glucuronide of
t-amyl alcohol were major urinary metabolites excreted in rats;
2-hydroxy-2-methyl-butyric acid and 3-hydroxy-3-methyl-butyric acid were minor excretory products formed from TAME.
In humans, 2-hydroxy-2-methylbutyric acid and 3-hydroxy-3methylbutyric acid were major excretory products in addition
to 2-methyl-2,3-butane diol (Amberg et al., 1999a). These
results suggest further intensive biotransformation of t-amyl
alcohol by metabolic oxidation reactions.
In this work, we studied the uptake of TAME and quantified
excretion of metabolites in humans and rats after exposure to
274
275
TAME BIOTRANSFORMATION
volunteers had to refrain from alcoholic beverages and drugs 2 days before and
throughout each experiment. Subjects did not abuse alcohol and were nonsmokers or occasional smokers. Subjects were healthy as judged by medical
examination and clinical blood chemistry, stated no previous occupational
exposure to TAME, and did not refuel their cars during the 2 days prior to
exposure and during sample collection period. Exposures started at 8 am. The
study was carried out according to the Declaration of Helsinki, after approval
by the Regional Ethical Committee of the University of Wuerzburg, Germany,
and after written informed consent by the volunteers. A time interval of 4
weeks was kept between the two exposures. No significant differences in
temperature in the chamber, number of air exchanges, and relative humidity
were observed between the exposures.
The design of the chamber and the generation of the chemical/air mixtures
has been described previously (Ertle et al., 1972; Müller et al., 1972, 1974,
1975). The chamber had a total volume of 8 m 3, airflow rate was 28 m 3/h at a
temperature of 22°C, and a relative humidity of 50 – 60 %. After the exposure,
the urine of the volunteers was collected at fixed intervals for 72 h, urine
volumes were determined by the volunteers, and two aliquots (60 ml each)
were rapidly frozen after collection and stored at –20°C until sample preparation. Metabolite concentrations in each urine sample collected were determined in duplicate.
Exposure of rats to TAME. Five male (210 –230 g, 12 weeks of age) and
five female (190 –210 g, 12 weeks of age) F344 NH rats from HarlanWinkelmann (Borchen, Germany) were exposed to targeted concentrations of
4 and 40 ppm TAME in the exposure chamber as described above for human
volunteers. During the exposure, rats were kept separately in Macrolon威 cages
with free access to food and water. After the end of the exposure, the cages
were checked for urine and feces, the animals were transferred to metabolic
cages, and urine was collected on ice for 72 h in 6-h intervals. Blood samples
from the tail vein (100 ␮l) were taken from each rat after the end of the
exposure period to quantify TAME and t-amyl alcohol blood concentrations.
FIG. 1. Biotransformation of TAME in mammals. Metabolites found in
urine are underlined. 1, TAME; 2, t-amyl alcohol; 3, 2-methyl-2,4-butane diol;
4, 3-hydroxy-3-methyl-butyric acid; 5, 2-methyl-2,3-butane diol; 6, glucuronide of 2-methyl-2,3-butane diol; 7, 2-methyl-1,2-butane diol; 8, 2-hydroxy2-methyl-butyric acid; 9, glucuronide of t-amyl alcohol.
TAME by inhalation to obtain data on blood levels, extent of
metabolism in both species, and kinetics of metabolite excretion.
MATERIALS AND METHODS
Chemicals. TAME (97⫹% purity), t-amyl alcohol (99⫹% purity), 2-hydroxy-2-methylbutyric acid, (98⫹% purity), tert-butanol (99.5⫹% purity),
1,2-propane diol (99.5⫹ purity), 2-hydroxyvaleric acid (98⫹% purity), Dglucuronic acid (98⫹ purity), boron trifluoride methanol-solution (14%), and
P 2O 5 (97% purity) were obtained from Sigma-Aldrich Chemical Company
(Deisenhofen, Germany). 2-Methyl-2,3-butane diol was prepared as described
(Amberg et al., 1999a). 3-Hydroxy-3-methylbutyric acid was obtained from
Tokyo Chemical Industry Co. (Tokyo, Japan), hexamethyldisilazane (98%
purity), trimethylchlorosilane solution (1M in tetrahydrofuran), and pyridine
were obtained from Fluka Chemie AG (Deisenhofen, Germany). All other
reagents and solvents were reagent grade or better and obtained from several
commercial suppliers. All GC columns were obtained from J&W Scientific
(Folsom, CA).
Exposure of volunteers to TAME. Three healthy female and three healthy
male volunteers (Table 1) were exposed to targeted concentrations of 4 and 40
ppm TAME for 4 h in a dynamic exposure chamber (Ertle et al., 1972). TAME
concentrations in chamber air were determined at 15-min intervals by GC/MS.
Actual TAME concentrations were 3.8 ⫾ 0.2 and 38.4 ⫾ 1.7 ppm. The
Quantification of TAME concentration in the exposure chamber. Samples (50 ␮l) of the chamber air were taken every 15 min with a gas-tight
syringe. TAME in the atmosphere of the exposure chamber was quantified by
capillary gas chromatography using a Fisons 8000 gas chromatograph coupled
to a Fisons MD 800 mass spectrometer. Separation was performed with a DB-1
fused silica column (30 m, 0.25 mm I.D., film thickness 1 ␮m) at an oven
temperature of 35°C. Injector temperature was 150°C and detector temperature
200°C; split injection with a split ratio of 5:1. During the separation (run time
of 5 min), the intensity of the major fragment ion in the electron impact mass
spectrum of TAME (m/z ⫽ 73) was monitored, with a dwell time of 80 msec.
Quantitation was based on calibration curves obtained with metered TAME
concentrations.
Quantitation of TAME and t-amyl alcohol in blood. Blood samples (10
ml) from the volunteers were taken with heparinized syringes. Volumes for
blood samples from rats were 100 ␮l. Blood samples from humans (0.5 ml)
and rats (0.025 ml) were transferred into GC autosampler vials (2-ml volume
TABLE 1
Characteristics of Human Volunteers Participating in the Study
Volunteer
Gender
Age
Height
(cm)
Body weight
(kg)
% Body fat
A
C
G
D
F
I
Male
Male
Male
Female
Female
Female
28
28
26
40
26
29
175
185
176
166
165
174
75
75
62
56
58
60
17.5
13.5
8.5
26.5
27.5
26.5
Note. Percent body fat was measured according to Donoghue, 1985.
276
AMBERG, ROSNER, AND DEKANT
for human samples and 0.2-ml volume for rat blood samples) immediately after
blood sampling to avoid loss of the volatile analytes during manipulations. The
vials were capped and stored at –20°C for a maximum of 4 weeks.
For TAME and t-amyl alcohol quantitation, 5 ␮l of an aqueous solution of
the internal standard tert-butanol (1000 nmol/ml) was added through the
septum with a microliter syringe, and the vials were then heated to 70°C for
1 h. TAME and t-amyl alcohol-concentrations were quantified by headspace
GC/MS by injecting 200 ␮l of the headspace from the vials using split
injection (split ratio of 10:1). Samples were separated using a DB-1 coated
fused silica column (30 m ⫻ 0.25 mm ID, 1.0-␮m film) at a temperature of
40°C. In addition to monitoring m/z 73 (for TAME), m/z 59 (most intensive
fragment ion in the electron impact mass spectrum of t-amyl alcohol and the
internal standard t-butanol) were monitored during the separation, with dwell
times of 80 msec. Quantitation was performed relative to the content of
t-butanol and referenced to calibration curves with fortified aliquots of blood
samples from controls containing 0 –20 nmol TAME and 0 –20 nmol t. amyl
alcohol/ml blood. The method was linear in the range of concentrations used
and calibration standards were analyzed with every sample series (usually
10 –20 samples) The method permitted the quantitation of 0.1 nmol TAME and
0.2 nmol t-amyl alcohol/ml of blood with a signal-to-noise ratio of 5:1. When
identical samples were repeatedly analyzed, the coefficients of variation were
lower than 10% (n ⫽ 8). TAME and t-amyl alcohol concentrations reported in
blood samples are based on duplicate analysis of samples from every individual.
Quantitation of TAME and TAME metabolites in urine. TAME and free
t-amyl alcohol in urine samples were quantified by headspace GC/MS using
0.5 ml of human urine and 0.2 ml of rat urine. TAME and t-amyl alcohol in the
urine samples was quantified as described above for blood samples. Two
different methods were used to quantify t-amyl alcohol and 2-methyl-2,3butane diol glucuronides. The first method involves direct analysis of the
glucuronides by GC/MS-determination of trimethylsilyl derivatives. To quantify glucuronide excretion, 50 ␮l of a 1000-nmol/ml solution of the internal
standard glucuronic acid were added to 100 ␮l of human or rat urine, and the
mixtures were lyophilized. The obtained residues were treated for 30 min with
1 ml of a mixture of hexamethyldisilazane, trimethylchlorosilane solution, and
pyridine (2:1:9, v:v:v) at 80°C in a closed reaction vial. From the obtained
solution, 2 ␮l were injected into the GC/MS. Separation was performed using
a DB-1 coated fused silica column (30 m ⫻ 0.25 mm ID, 1-␮m film). Injector
temperature was 310°C, and transfer line temperature was 310°C. Samples
were injected using split injection (split ratio of 10:1); oven temperature was
100°C and increased to 310°C with a rate of 10°C/min. Samples were monitored using m/z 204 and 217. Quantitation was performed relative to the
content of glucuronic acid and referenced to calibration curves with fortified
aliquots of urine samples from controls containing 0 –1000 nmol/ml of the
glucuronides isolated from urine of TAME-treated rats by prep HPLC (Amberg et al., 2000). This method was not very sensitive and could be used only
with samples containing high concentrations of the glucuronides. In addition,
the injected mixture resulted in a rapid deterioration of the performance of the
mass spectrometer. Therefore, all samples were analyzed by a simpler and
more sensitive method using acid hydrolysis of the glucuronides. Enzymatic
hydrolysis was not very effective with reference compounds. Glucuronidase
did not completely cleave the glucuronides within 24 h. Under the conditions
of the acid hydrolysis, the alcohols formed by the acid hydrolysis were further
converted by an acid-catalyzed dehydration of t-amyl alcohol to give 2-methyl2-butene and of 2-methyl-2,3-butane diol to form 3-methyl-2-butanone. The
efficiency of the acid hydrolysis and the dehydration was checked by NMR
using urine samples from rats treated with 13C-TAME (Amberg et al., 1999a)
monitoring the disappearance of the glucuronide signals. To quantify content
of free alcohols and glucuronides, t-butanol (25 ␮l from a 1000-nmol/ml
solution in water; t-butanol is cleaved to 2-methylpropene under acidic conditions) as internal standard and 60 ␮l of 10 M sulfuric acid were added to 200
␮l of urine in a closed vial. After 1 h at 90°C, 500 ␮l of the gas phase from
the vial was analyzed by GC/MS. Separation was performed using a DB-1
coated fused silica column (30 m ⫻ 0.25 mm ID, 1-␮m film). Samples were
injected using splitless injection; oven temperature was 40° C. Samples were
monitored using m/z 56, 70, and 86 by selected ion monitoring. Quantitation
was performed relative to the formed 2-methylpropene and referenced to
calibration curves with fortified aliquots of urine samples from controls containing 0 –1000 nmol/ml of t-amyl alcohol and 2-methyl-2,3-butane diol. The
method was linear in the range of concentrations used and calibration standards
were analyzed with every sample series (usually 20 –30 samples). The method
permitted the quantitation of 0.1 nmol of t-amyl alcohol and 0.5 nmol 2methyl-2,3-butane diol glucuronide with a signal-to-noise ratio of 3:1. When
identical samples were repeatedly analyzed, the coefficients of variation were
lower than 10% (n ⫽ 8). This method determined the content of free t-amyl
alcohol and free 2-methyl-2,3-butane diol and their glucuronides in the samples. Concentrations of the two glucuronides were obtained by subtraction of
the content of the free alcohols determined as described below.
To quantify 2-methyl 2,3-butane diol, 25 ␮l of a solution of the internal
standard 1,2-propane diol (1000 nmol/ml in water) was added to 0.1 ml of
human or rat urine. Urine samples were then diluted with 0.9 ml methanol, and
2-methyl-2,3-butane diol content was quantified by GC/MS by injecting 1 ␮l
of the obtained mixtures. Separation was achieved using a fused silica column
coated with DB-FFAP (30 m ⫻ 0.32 mm, film thickness 0.25 ␮m) with helium
as carrier gas (2 ml/min). Samples were separated using a linear temperature
program from 50°C to 230°C with a heating rate of 10°C/min. Injector and
transfer line temperatures were 230°C. The concentrations of 2-methyl-2,3butane diol were determined by monitoring m/z 59 and m/z 45 during the gas
chromatographic separation, with dwell times of 80 msec. Split injection (split
ratio of 10:1) was used. Quantitation was performed relative to the content of
1,2-propane diol and referenced to calibration curves with fortified aliquots of
urine samples from controls containing 0 –1000 nmol/ml 2-methyl 2,3-butane
diol. The method was linear in the range of concentrations used and calibration
standards were analyzed with every sample series (usually 20 –30 samples)
The method permitted the quantitation of 1 nmol 2-methyl 2,3-butane diol/ml
of urine with a signal-to-noise ratio of 5:1. When identical samples were
repeatedly analyzed, the coefficients of variation were lower than 15% (n ⫽ 8).
Concentrations of 2-hydroxy-2-methylbutyric acid and 3-hydroxy-3-methylbutyric acid in urine were quantified by GC/MS after transformation to the
corresponding methyl esters. Urine samples (0.1 ml for humans and rats) were
mixed with 2-hydroxyvaleric acid (internal standard, 25 ␮l of a 1000-nmol/ml
solution in water). Samples were then taken to dryness using anhydrous P 2O 5
in an evacuated desiccator. The obtained residues were treated with 500 ␮l of
BF 3/methanol (14%) at 60°C for 30 min. Samples were then diluted with 250
␮l water and extracted with 1 ml chloroform. The chloroform layers were dried
over sodium sulfate and 2 ␮l of the obtained solutions was analyzed by GC/MS
(splitless injection). Samples were separated on a DB-WAX column (30 m ⫻
0.25 mm, 0.25 ␮m film thickness) using a linear temperature program from
50°C to 230°C with a heating rate of 10°C/min. The intensities of m/z 55 and
59 were monitored during the separation with dwell times of 80 msec.
Quantitation was performed relative to the content of 2-hydroxyvaleric acid
and referenced to calibration curves with fortified aliquots of urine samples
from controls containing 0 –1000 nmol/ml 2-hydroxy-2-methylbutyric acid and
3-hydroxy-3-methylbutyric acid. The method was linear in the range of concentrations used and calibration standards were analyzed with every sample
series (usually 20 –30 samples) The method permitted the quantitation of 3
nmol 2-hydroxy-2-methylbutyric acid and 3-hydroxy-3-methylbutyric /ml of
urine with a signal-to-noise ratio of 3:1. When identical samples were repeatedly analyzed, the coefficients of variation were lower than 10% (n ⫽ 8).
GC/MS analysis. GC/MS analyses were performed on a Fisons MD 800
mass spectrometer coupled to a Fisons 8000 GC and equipped with an AS 800
autosampler and an electron impact source (Fisons Instruments, Mainz, Germany). Samples were also analyzed with electron impact ionization using a
Hewlett-Packard 5970 mass spectrometer coupled to a 5890 GC or a HewlettPackard 5973 mass spectrometer coupled to a 6890 GC. Both instruments were
equipped with a CTC Combi-PAL autoinjector with capability for headspace
injection.
277
TAME BIOTRANSFORMATION
TABLE 2
TAME and t-Amyl Alcohol Blood Concentrations in Humans Exposed to TAME for Four Hours
38.4 ⫾ 1.7 ppm
TAME
Exposure
concentrations
Max. conc.
(␮M)
A
3.4 ⫾ 0.0
C
5.6 ⫾ 0.0
G
7.2 ⫾ 0.1
D
2.9 ⫾ 0.0
F
4.2 ⫾ 0.2
I
3.1 ⫾ 0.2
⌽
4.4 ⫾ 1.7
3.8 ⫾ 0.2 ppm
t-Amyl alcohol
Half-lives* (h)
1.1
3.2
1.7
4.0
0.9
2.2
1.2
4.1
1.0
4.0
1.0
3.6
1.2 ⫾ 0.3
3.5 ⫾ 0.7
TAME
Max. conc.
(␮M)
Half-life (h)
Max. conc.
(␮M)
8.4 ⫾ 1.0
7.3
0.58 ⫾ 0.02
7.5 ⫾ 0.4
7.4
0.57 ⫾ 0.03
8.2 ⫾ 0.0
7.0
0.83 ⫾ 0.11
7.9 ⫾ 0.1
6.9
0.51 ⫾ 0.06
10.9 ⫾ 0.4
6.6
0.63 ⫾ 0.08
12.0 ⫾ 0.2
6.2
0.66 ⫾ 0.02
9.2 ⫾ 1.8
6.9 ⫾ 0.4
0.63 ⫾ 0.11
t-Amyl alcohol
Half-lives* (h)
1.3
3.4
1.5
6.2
1.2
3.9
1.3
5.8
1.6
2.8
1.3
5.3
1.4 ⫾ 0.2
4.6 ⫾ 1.4
Max. conc.
(␮M)
Half-life (h)
0.73 ⫾ 0.10
4.8
0.70 ⫾ 0.02
5.6
1.33 ⫾ 0.42
2.5
0.90 ⫾ 0.08
8.6
1.18 ⫾ 0.13
4.3
1.27 ⫾ 0.13
5.5
1.02 ⫾ 0.28
5.2 ⫾ 2.0
Note. Elimination of TAME from blood occurred in two phases.
* Half-lives for both phases were calculated.
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 the male and female animals and male and female human volunteers were
compared using 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 R spreadsheets. The curve-fitting
function of the program was used and curves were stripped based on correlation coefficients. r 2-Values of ⬎ 0.95 were considered for separation.
RESULTS
Biotransformation of TAME in Humans
During all experiments, the deviations between the targeted
concentrations and the actual concentrations of TAME in the
chamber were less than 10% of the targeted values. Average
concentrations of TAME in the chamber were 3.8 ⫾ 0.2 ppm
and 38.4 ⫾ 1.7 ppm (mean ⫾ SD of 16 determinations in
15-min intervals over 4 h). Experimental results on the excretion of TAME metabolites and half-lives in humans are given
in Tables 2– 4 and in Figures 2 and 3. TAME and t-amyl
alcohol were not detected in blood samples from the volunteers
taken before the exposure. The maximal concentrations of
TAME and t-amyl alcohol in blood were determined directly
after the end of the exposure period. TAME concentrations
rapidly decreased to reach the limit of detection 12 h (40 and
4 ppm) after the end of the exposure period. Elimination of
TAME from blood was rapid and could be separated into two
phases with half-lives of 1.2 h and 3.5 h. Blood samples taken
from the volunteers after exposure to 4 and 40 ppm TAME
showed detectable concentrations of t-amyl alcohol for the
time period between the end of the exposure and the 36-h blood
sampling after 40 ppm TAME and between the end of the
exposure and the 6-h blood sampling point after 4 ppm TAME
(data not shown). Clearance of t-amyl alcohol from blood
followed first-order kinetics and was slower than that of
TAME.
In urine samples of the volunteers collected before TAME
exposure and in samples collected from control subjects, low
concentrations of 2-methyl-2,3-butane diol were present. In
addition, high and variable concentrations of 2-hydroxy-2methylbutyric acid and 3-hydroxy-3-methylbutyric acid were
observed. In the urine samples from TAME-exposed individuals, the concentrations of 2-methyl-2,3-butane diol were significantly increased in all urine samples collected until 72 h
after the end of the exposure period after both 4 and 40 ppm
TAME. Elimination of 2-methyl-2,3-butane diol was slow and
not complete within the period of observation. Statistically
significant increases in the concentrations of 2-hydroxy-2methylbutyric acid were observed only in urine samples taken
between 0 and 30 h after the end of the 40-ppm exposure.
Significantly increased concentrations of 3-hydroxy-3-methylbutyric acid were seen only in urine samples taken between 0
and 12 h after the end of exposure. After exposure to 4 ppm
TAME, none of the urine samples contained significantly increased concentrations of 2-hydroxy-2-methylbutyric acid and
3-hydroxy-3-methylbutyric acid due to the high and variable
background.
Due to the absence of background, the concentrations of
278
AMBERG, ROSNER, AND DEKANT
TABLE 3
Cumulative Metabolite Excretion and Half-Lives of Urinary Excretion of TAME Metabolites in Humans (n ⴝ 6)
after Exposure to 38.4 ⴞ 1.7 and 3.8 ⴞ 0.2 ppm TAME
38.4 ⫾ 1.7 ppm
Exposure concentrations
TAME
t-Amyl alcohol
t-Amyl alcoholglucuronide
2-Methyl-2,3-butane
diol
2-Methyl-2,3-butane
diol glucuronide
2-Hydroxy-2methylbutyric acid
3-Hydroxy-3methylbutyric acid
3.8 ⫾ 0.2 ppm
Total excretion
(␮mol)
Background
(␮mol)
Half-life (h)
1.5 ⫾ 1.1
(⌺ 12 h)
5.7 ⫾ 2.0
(⌺ 30 h)
13.6 ⫾ 2.4
(⌺ 72 h)
261.3 ⫾ 76.3**
(⌺ 72 h)
ND
ND
4.3 ⫾ 1.4
ND
6.0 ⫾ 1.6
ND
7.0 ⫾ 0.9
7.9 ⫾ 3.3
31.5 ⫾ 6.1
ND
—
95.6 ⫾ 53.7
12.3 ⫾ 3.0
200.0 ⫾ 68.9
9.8 ⫾ 3.2
291.8 ⫾ 80.1**
(⌺ 36 h)
330.9 ⫾ 106.1*
(⌺ 24 h)
Total excretion
(␮mol)
Background
(␮mol)
Half-life (h)
0.12 ⫾ 0.04
(⌺ 6 h)
0.51 ⫾ 0.25
(⌺ 12 h)
1.8 ⫾ 0.5
(⌺ 36 h)
26.8 ⫾ 2.4
** (⌺ 72 h)
ND
ND
8.1 ⫾ 1.5
ND
Not determined
ND
8.9 ⫾ 1.7
4.7 ⫾ 1.1
39.8 ⫾ 10.3
ND
—
56.0 ⫾ 31.4
—
192.6 ⫾ 46.9
—
85.7 ⫾ 30.2
(⌺ 36 h)
219.3 ⫾ 64.1
(⌺ 18 h)
Note. Metabolite amounts recovered are calculated by using only urine samples with metabolite concentrations significantly above background excretion (Figs.
2 and 3). Background levels given were adjusted for sample collection periods. Background levels were determined in all volunteers before the exposure and
in control subjects (n ⫽ 6). Numbers represent mean ⫾ SD. ND, not detected.
* Statistically significant difference from background levels (p ⬍ 0.05).
** Statistically significant difference from background levels (p ⬍ 0.01).
TAME and t-amyl alcohol could be quantified with higher
precision. TAME was detectable in all urine samples from the
volunteers collected between 0 and 12 h after the end of the
exposure after 40 ppm TAME and between 0 and 6 h after the
end of exposure after 4 ppm TAME. t-Amyl alcohol and its
glucuronide were also detected in all urine samples collected
between 0 and 36 h after the end of the 40-ppm exposure in low
concentrations. Excretion of these compounds with urine was
rapid and occurred with half-lives of less than 10 h.
Based on the recovered amounts of 2-methyl-2,3-butane diol
and 2-hydroxy-2-methylbutyric acid, these compounds represent the major excretory metabolites formed from TAME (Tables 3 and 4) in humans. In addition, 3-hydroxy-3-methylbutyric acid was a TAME metabolite present in higher
concentrations in urine, whereas free and conjugated t-amyl
alcohol and unchanged TAME were only minor excretory
products. Large variations in the extent of TAME biotransformation (Table 4) between the individuals and in the rates of
TABLE 4
Received Doses of TAME in Humans and Amount of Metabolites Recovered in Urine
38.4 ⫾ 1.7 ppm
3.8 ⫾ 0.2 ppm
Exposure concentrations
⌺ of excreted metabolites
(␮mol)
Metabolite excretion
(% of received dose)
⌺ of excreted a metabolites
(␮mol)
Metabolite excretion
(% of received dose)
A
C
G
D
F
I
⌽
593
394
500
513
675
927
600 ⫾ 186
57
38
48
50
65
90
58 ⫾ 18
29
77
41
44
51
84
54 ⫾ 21
29
75
40
43
50
82
53 ⫾ 21
Note. Received doses were calculated as 1033 ␮mol (38.4 ⫾ 1.7 ppm) and 102 ␮mol (3.8 ⫾ 0.2 ppm) based on an alveolar ventilation rate of 9 l/min and
a retention of 0.3. Urine samples were collected in 6-h intervals for 72 h. Numbers are mean of two determinations of each metabolite/collected urine sample.
The results of repeated measurement of the same sample showed deviations ⬍ 10 % in the analyte measured. Numbers were corrected for metabolite excretion
in unexposed individuals.
a
Without 3-hydroxy-3-methyl-butyric acid.
TAME BIOTRANSFORMATION
FIG. 2. Excretion with urine of 3-hydroxy-3-methylbutyric acid (square),
2-hydroxy-2-methylbutyric acid (octagon) (Panel A), and 2-methyl-2,3-butane
diol (octagon), t-amyl alcohol glucuronide (square), t-amyl alcohol (triangle),
and unchanged TAME (diamond) (Panel B) in six human volunteers exposed
to 38.4 ⫾ 1.7 ppm TAME for 4 h in a dynamic exposure chamber. Numbers
(mean ⫾ SD) given represent total amount of metabolite excreted in the urine
samples collected in 6-h intervals. Each sample was analyzed in duplicate.
Statistically significant differences as compared to background in controls
(**p ⬍ 0.01; * p ⬍ 0.05).
279
blood as compared to human blood. The concentrations of
t-amyl alcohol were not significantly different between rats and
humans after 4 and 40 ppm TAME.
In urine samples of rats collected before TAME exposure,
and in samples collected from control rats, low concentrations
of 2-hydroxy-2-methylbutyric acid, 3-hydroxy-3-methylbutyric acid, 2-methyl-2,3-butane diol, and t-amyl alcohol were
present. In the urine samples from exposed rats, the concentrations of 2-hydroxy-2-methylbutyric acid were significantly
increased (as compared to controls) in only a few urine samples
collected within 24 h after the end of 40-ppm TAME inhalation
(Fig. 4). Significantly increased concentrations of 2-hydroxy2-methylbutyric acid were not observed in any of the urine
samples collected from the 4-ppm exposure of rats. Statistically significant increases above background excretion rates in
the concentration of 3-hydroxy-3-methylbutyric acid were not
observed in any of the urine samples collected from TAMEexposed rats. Due to much lower background levels, the concentrations of 2-methyl-2,3-butane diol were significantly
higher than controls in all samples collected between 0 and
42 h after both 4 and 40 ppm TAME. Excretion of the glucuronide of 2-methyl-2,3-butane diol was detectable in all urine
samples collected up to 48 h after the end of the exposure after
40 ppm, but not in urine samples after 4-ppm exposure. Excretion of the glucuronide of t-amyl alcohol was detectable in
all urine samples collected up to 24 h after the end of exposure
after 40 ppm and 4 ppm. The other metabolites quantified were
rapidly excreted, and their concentration in urine samples was
below the limit of detection after 24 h. The presence of these
minor metabolites was not detected in urine samples collected
from rats after exposure to 4 ppm TAME.
Based on the amounts of 2-methyl-2,3-butane diol and its
glucuronide, these compounds and t-amyl alcohol glucuronide
represent the major urinary metabolites of TAME (Table 6) in
excretion and the urinary concentrations of 2-hydroxy-2-methylbutyric acid and 3-hydroxy-3-methylbutyric acid were observed. However, no statistically significant differences in the
amounts of these acids, of free and conjugated 2-methyl-2,3butane diol or any of the other metabolites excreted, or in the
rates of excretion of metabolites were seen between the male
and female subjects in the study. The determined half-lives of
elimination with urine were not significantly different after the
4 and 40 ppm TAME exposures (Tables 3 and 4).
Biotransformation of TAME in Rats
Rats were exposed to the same TAME concentrations as
used in the human studies. The experimental results on metabolite concentrations and excretion are compiled in Tables 5–7
and in Figures 4 and 5. The concentrations of TAME in blood
of rats determined after the end of the 4-h exposure period were
twice as high as those seen in humans after identical exposure
concentrations. TAME was more rapidly cleared from rat
FIG. 3. Excretion with urine of 2-methyl-2,3-butane diol (octagon), tamyl alcohol glucuronide (square), and t-amyl alcohol (triangle) in six human
volunteers exposed to 3.8 ⫾ 0.2 ppm TAME for 4 h in a dynamic exposure
chamber. Numbers (mean ⫾ SD) given represent total amount of metabolite
excreted in the urine samples collected in 6-h intervals. Each sample was
analyzed in duplicate. Statistically significant differences as compared to
background in controls (** p ⬍ 0.01; * p ⬍ 0.05).
280
AMBERG, ROSNER, AND DEKANT
TABLE 5
Blood Concentrations at the End of Exposure and Half-Lives of Elimination from Blood after Exposure of Rats (n ⴝ 10)
to 38.4 ⴞ 1.7 and 3.8 ⴞ 0.2 ppm TAME for Four Hours
38.4 ⫾ 1.7 ppm
3.8 ⫾ 0.2 ppm
Concentration (␮M)
Half-life (h)
Concentration (␮M)
Half-life (h)
9.6 ⫾ 1.4
8.1 ⫾ 1.5
0.6 ⫾ 0.1
Not determined
1.4 ⫾ 0.8
1.8 ⫾ 0.2
1.1 ⫾ 0.5
Not determined
TAME
t-Amyl alcohol
Note. Numbers represent mean ⫾ SD.
rat urine. Assuming identical retention of TAME after inhalation exposure, humans excreted a significantly higher proportion (p ⬍ 0.03) of the retained TAME as metabolites in urine
as compared to rats (Tables 5 and 7).
DISCUSSION
In this work, the biotransformation of TAME in rats and in
humans was compared after inhalation exposure. After 4-h
inhalation exposures, lower blood levels of TAME were obtained in humans as compared to rats immediately after the end
of exposure to 4 and 40 ppm TAME. The higher blood levels
observed in rats are likely due to higher alveolar ventilation
rates, resulting in increased delivery and uptake of TAME in
rats as compared to humans. In general, the time course of
elimination of TAME and all metabolites quantified in this
study shows that rats excrete TAME and its metabolites more
rapidly than humans.
Half-lives of elimination of TAME from blood were also
similar to those seen with other ethers intended for use as
oxygenates determined in other studies. Elimination of methyl
tert-butyl ether and ethyl tert-butyl ether also occurs with
half-lives of less than 1 h in rats and (Miller et al., 1997)
between 2 and 4 h in humans (Nihlen et al., 1998; Prah et al.,
1994). As seen with these ethers, no sex differences in the
apparent half-lives of elimination of these compounds were
seen.
In urine of humans and rats, the known metabolites of
TAME excreted with urine and unchanged TAME were quantified. A minor part of the TAME dose is excreted with urine
in humans. Elimination of the parent ether with urine was also
TABLE 6
Cumulative Metabolite Excretion and Half-Lives of Urinary Excretion of TAME metabolites in Rats (n ⴝ 10)
after Exposure to 38.4 ⴞ 1.7 and 3.8 ⴞ 0.2 ppm TAME
38.4 ⫾ 1.7 ppm
TAME
t-Amyl alcohol
t-Amyl alcohol-glucuronide
2-Methyl-2,3-butane diol
2-Methyl-2,3-butane diol glucuronide
2-Hydroxy-2-methylbutyric acid
3-Hydroxy-3-methylbutyric acid
3.8 ⫾ 0.2 ppm
Total excretion
(␮mol)
Background
(␮mol)
Half-life (h)
ND
0.011 ⫾ 0.007*
(⌺ 18 h)
1.0 ⫾ 0.9
(⌺ 48 h)
4.7 ⫾ 1.1**
(⌺ 48 h)
1.7 ⫾ 1.2
(⌺ 48 h)
0.64 ⫾ 0.12**
(⌺ 64 h)
2.1 ⫾ 0.9
(⌺ 24 h)
ND
0.005 ⫾ 0.002
—
Not determined
ND
4.1 ⫾ 0.9
0.1 ⫾ 0.1
4.6 ⫾ 0.9
ND
4.5 ⫾ 1.4
0.26 ⫾ 0.04
4.8 ⫾ 0.6
1.6 ⫾ 0.8
—
Total excretion
(␮mol)
Background
(␮mol)
Half-life (h)
ND
0.005 ⫾ 0.002
(⌺ 18 h)
0.082 ⫾ 0.047
(⌺ 24 h)
0.71 ⫾ 0.19**
(⌺ 48 h)
ND
ND
0.005 ⫾ 0.002
—
—
ND
6.0 ⫾ 1.6
0.06 ⫾ 0.02
4.7 ⫾ 0.8
ND
—
0.28 ⫾ 0.08
(⌺ 24 h)
1.7 ⫾ 0.8
(⌺ 24 h)
0.23 ⫾ 0.06
—
1.6 ⫾ 1.1
—
Note. Metabolite amounts recovered are calculated by using only urine samples with metabolite concentrations significantly above background excretion (Figs.
4 and 5). Background levels given were adjusted for sample collection periods. Background levels were determined before the exposure and in control rats (n ⫽
10). TAME excretion with urine was observed; however, the concentrations were close to the limit of detection and therefore not quantified. Numbers represent
mean ⫾ SD. ND, not detected.
*Statistically significant difference from background levels (p ⬍ 0.05).
**Statistically significant difference from background levels (p ⬍ 0.01).
281
TAME BIOTRANSFORMATION
TABLE 7
Received Doses of TAME in Rats and Amount of Metabolites Recovered in Urine
TAME concentration in chamber
(ppm)
Dose received
(␮mol)
⌺ of excreted metabolites a
(␮mol)
Metabolite excretion a
(% of received dose)
38.4 ⫾ 1.7
3.8 ⫾ 0.2
19.5
1.9
8.1 ⫾ 2.6
0.78 ⫾ 0.23
42 ⫾ 13
40 ⫾ 12
Note. Received doses were calculated based on an alveolar ventilation rate of 0.169 l/min and a retention of 0.3 in rat. Retention was assumed to be identical
to that of ethyl tert-butyl ether (Nihlen et al., 1998).
a
Numbers were corrected for metabolite excretion in unexposed individuals.
seen with MTBE and ETBE (Amberg et al., 1999b, 2000;
Nihlen et al., 1998; Prah et al., 1994). With these compounds,
urinary elimination of the ether also represents a minor pathway for the clearance of the ethers. In rat urine, the TAME
concentrations in the urine samples were already below the
limit of detection in the first available samples (6 h after the
end of exposure).
The study confirms the structure of TAME metabolites
formed in the rat and demonstrates that identical metabolites of
TAME are formed in humans and excreted with urine. Moreover, the obtained results confirm and extend the semiquantitative findings from our previous study on differences in the
metabolic pattern of TAME between humans and rats (Amberg
et al., 1999a). In rats, TAME is mainly excreted as 2-methyl2,3-butane diol and its glucuronide. Further oxidation of t-amyl
alcohol to other products is of minor importance due to a more
rapid elimination and glucuronide formation. In humans,
2-methyl-2,3-butane diol is eliminated more slowly as compared to rats. In addition, t-amyl alcohol seems to be more
efficiently oxidized to 2-hydroxy-2-methylbutyric acid and
3-hydroxy-3-methylbutyric acid in humans. This reaction occurs only to a minor extent in rats. These differences are likely
due to substrate specificities of the enzymes involved in the
formation of these compounds from TAME and t-amyl alcohol
(cytochrome P450 and glucuronyl transferase).
All TAME metabolites quantified in this study were eliminated in rats with apparent half-lives of elimination of less than
6 h; in humans, the elimination with urine of the metabolites
formed from TAME was considerably slower.
The extent of TAME biotransformation in humans is significantly higher as compared to rats when the amounts of metabolites and the relative concentrations recovered in urine are
compared to the doses received (Tables 5 and 7). Between 40%
(rats) and almost 60% (humans) of the calculated doses of
TAME received by inhalation were recovered as metabolites in
urine. The rest of the TAME taken up by inhalation is likely
exhaled. Exhalation of unchanged TAME was not determined
in this study. However, due to the volatility of TAME and
based on studies with the structurally similar compounds
methyl tert-butyl ether and ethyl tert-butyl ether, it has to be
assumed that the unaccounted part of the received TAME-dose
is exhaled unchanged either by rats or by humans after the
termination of the inhalation period. Exhalation of unchanged
parent ether was a major pathway of elimination of both methyl
tert-butyl ether and ethyl tert-butyl ether in humans and in rats.
Extent of biotransformation of TAME in both rats and humans
seems not to be saturated in the concentration range studied, as
the percentage of dose recovered as metabolites was identical
FIG. 4. Excretion with urine of 2-methyl-2,3-butane diol (octagon),
2-methyl-2,3-butane diol glucuronide (diamond) (panel A), t-amyl alcohol
glucuronide (square), 2-hydroxy-2-methylbutyric acid (octagon), and t-amyl
alcohol (triangle) (panel B) in 10 rats exposed to 38.7 ⫾ 3.2 ppm TAME for
4 h in a dynamic exposure chamber. Numbers (mean ⫾ SD) given represent
total amount of metabolite excreted in the urine samples collected in 6-h
intervals. Each sample was analyzed in duplicate. Statistically significant
differences as compared to background in controls (** p ⬍ 0.01; * p ⬍ 0.05).
282
AMBERG, ROSNER, AND DEKANT
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
manufactures. 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. Part of this work was also
supported by the Biomed Program of the European Union, Contract No.
BMH4-CT96-0184.
REFERENCES
FIG. 5. Excretion with urine of 2-methyl-2,3-butane diol (octagon) and
t-amyl alcohol glucuronide (square) in 10 rats exposed to 3.8 ⫾ 0.2 ppm
TAME for 4 h in a dynamic exposure chamber. Numbers given represent total
amount of metabolite excreted in the urine samples collected in 6-h intervals.
Statistically significant differences as compared to background in controls (**
p ⬍ 0.01; * p ⬍ 0.05)
after both exposure concentrations. The interindividual differences in the extent of TAME biotransformation in humans are
likely due to interindividual differences in the cytochrome
P450 profile (Hong et al., 1999). More than 10-fold differences
in the capacity of human liver microsomes to oxidize TAME to
t-amyl alcohol were observed (Hong et al., 1999). Further
studies identifying the cytochrome P450 enzymes involved in
t-amyl alcohol biotransformation should address this problem.
The differences in biotransformation and kinetics of metabolite excretion suggest that humans may respond differently
than rats to potential toxic effects of TAME based on biotransformation. However, as only limited data on toxic effects of
TAME after long-term inhalation exposure in rats are available, conclusions must be taken with caution. The potential of
TAME to induce acute and chronic toxicity in rats is low, and
the role of biotransformation in the observed changes in relative organ weights is unknown. As the formation of reactive
metabolites during TAME biotransformation is not suggested
based on the structures of metabolites and their mechanisms of
formation, covalent binding to macromolecules has to be regarded as unlikely; thresholds are likely for TAME toxicity.
Moreover, the major TAME metabolites found in humans are
also formed endogenously (Liebich and Forst, 1984). TAME
exposures in low concentrations as expected from the environment are not likely to result in a significant increase in the
human body burden of these compounds, making toxic effects
of TAME in humans under realistic exposure conditions unlikely.
In conclusion, the biotransformation of TAME after inhalation is quantitatively different, but qualitatively similar in rats
and humans. Sex differences in biotransformation were not
seen in either species. 2-Methyl-2,3-butane diol concentrations
in urine or blood may represent useful biomarkers of exposure
to low levels of TAME expected from environmental exposures.
Amberg, A., Bernauer, U., and Dekant, W. (1999a). Biotransformation of 12Cand 13C-tert.-amyl methyl ether and tert-amyl alcohol in rodents. Chem.
Res. Toxicol. 12, 958 –964.
Amberg, A., Rosner, E., and Dekant, W. (1999b). Biotransformation and
kinetics of excretion of methyl tert-butyl ether in rats and humans. Toxicol.
Sci. 51, 1– 8.
Amberg, A., Rosner, E., and Dekant, W. (2000). Biotransformation and kinetics of excretion of ethyl tert-butyl ether in humans and rats after inhalation
exposure. Toxicol. Sci. 53, 194 –201.
Costantini, M. G. (1993). Health effects of oxygenated fuels. Environ. Health
Perspect. 101, 151–160.
Donoghue, W. C. (1985). How to measure your % body-fat. Creative Health
Products, Plymouth, Michigan.
Ertle, T., Henschler, D., Müller, G., and Spassowski, M. (1972). Metabolism
of trichloroethylene in man. I. The significance of trichloroethanol in longterm exposure conditions. Arch. Toxicol. 29, 171–188.
Health Effects Institute (HEI) (1996). The potential health effects of oxygenates added to gasoline. Health Effects Institute, Cambridge, MA.
Hong, J. Y., Wang, Y. Y., Bondoc, F. Y., Lee, M., Yang, C. S., Hu, W. Y., and
Pan, J. M. (1999). Metabolism of methyl tert-butyl ether and other gasoline
ethers by human liver microsomes and heterologously expressed human
cytochromes P450: identification of CYP2A6 as a major catalyst. Toxicol.
Appl. Pharmacol. 160, 43– 48.
Liebich, H. M., and Forst, C. (1984). Hydroxycarboxylic and oxocarboxylic
acids in urine: products from branched-chain amino acid degradation and
from ketogenesis. J. Chromatogr. 309, 225–242.
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.
Müller, G., Spassowski, M., and Henschler, D. (1972). Trichloroethylene
exposure and trichloroethylene metabolites in urine and blood. Arch. Toxicol. 29, 335–340.
Müller, G., Spassowski, M., and Henschler, D. (1974). Metabolism of trichloroethylene in man. II. Pharmacokinetics of metabolites. Arch. Toxicol. 32,
283–295.
Müller, G., Spassowski, M., and Henschler, D. (1975). Metabolism of trichloroethylene in man. III. Interaction of trichloroethylene and ethanol. Arch.
Toxicol. 33, 173–189.
Nihlen, A., Löf, A., and Johanson, G. (1998). Experimental exposure to methyl
tertiary-butyl ether. I. Toxicokinetics in humans. Toxicol. Appl. Pharmacol.
148, 274 –280.
Nolan, R. J., Langvardt, P. W., and Blogg, C. D. (1976). Tertiary amyl alcohol:
results of short term inhalation and pharmacokinetic study. Report, Toxicology Research Laboratory, Health and Environmental Research, Dow
Chemical USA, Midland, Michigan.
Nolan, R. J., Langvardt, P. W., and Blogg, C. D. (1981). Tertiary amyl alcohol:
subchronic toxicity and pharmacokinetics in CD-1 mice, Fisher 344 rats and
TAME BIOTRANSFORMATION
male beagle dogs. Report, Toxicology Research Laboratory, Health and
Environmental Research, Dow Chemical USA, Midland, Michigan.
Prah, J. D., Goldstein, G. M., Devlin, R., Otto, D., Ashley, D., Case, M.,
House, D., Willingham, F., Harder, S., Carter, J., Cazares, L., Dailey, L.,
Soupkup, J., Garlington, A., Cohen, K., and Gerrity, T. (1994). Sensory,
symptomatic, inflammatory, and ocular responses to and metabolism of
methyl tertiary butyl ether (MTBE) in a controlled human exposure experiment. Inhal. Toxicol. 6, 521–538.
Vainiotalo, S., Pekari, K., and Aitio, A. (1998). Exposure to methyl tert-butyl
283
ether and tert-amyl methyl ether from gasoline during tank lorry loading and
its measurement using biological monitoring. Int. Arch. Occup. Environ.
Health 71, 391–396.
Vainiotalo, S., Peltonen, Y., Ruonakangas, A., and Pfaffli, P. (1999). Customer
exposure to MTBE, TAME, C6 alkyl methyl ethers, and benzene during
gasoline refueling. Environ. Health Perspect. 107, 133–140.
White, R. D., Daughtrey, W. C., and Wells, M. S. (1995). Health effects of
inhaled tertiary amyl methyl ether and ethyl tertiary butyl ether. Toxicol.
Lett. 82/83, 719 –724.