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Comparative urinary excretion of ethoxyacetic acid in man and rat after single low doses of ethylene glycol monoethyl ether
Toxicology Letters, 41 (1988) 57-68
57
Elsevier
TXL 01939
Comparative urinary excretion of ethoxyacetic acid in man and rat after single low doses of ethylene glycol monoet hyl et her D. Groeseneken, Laboratory
H. Veulemans, R. Masschelein and E. Van Vlem
of U~~Mpaiio*a~ Hygiene and Toxicology,
M~d~eine~ K. U. Leaven,
Department
of ~~~~~a~iona~ and Insurance
3-3000 Louvailz, Beigium
(Received 21 September 1987) (Revision received 4 December 1987) (Accepted 7 December 1987)
Key words: Ethylene glycol monoethyl
ether; Ethoxyacetic acid; (Urinary excretion; Man; Rat)
SUMMARY Male rats were given a single oral dose of ethylene gfycol monoethyl ether (EGEE), the dose ranging from plausible human exposures (0.5-l mg/kg) to doses reported in the literature (100 mg/kg). Urinary excretion of ethoxyacetic acid (EAA) and its glycine conjugate was followed up to 60 h after dosing and compared to data of experimentally exposed human volunteers. In rats, the mean elimination half-life of free as well as conjugated EAA was 7.2 h for all doses. BAA was excreted partly as a glycine conjugate (on average 27%), the extent of conjugation being independent of the dose. The conjugation with glycine showed a clearly diurnal variation, the lowest extent being found during the night. The relative amount of EGEE recovered in urine as EAA was only 13.4% for the lowest dose, but increased as the administered dose of EGEE was higher, indicating that EGEE was metabolised at least in two parallel pathways of which one pathway becomes saturated at relatively low doses. In man, urinary excretion of EAA for equivalent low doses of EGEE differed from that in the rat by a longer elimination half-life (mean 42 h), by the absence of EAA conjugates and by a higher recovery.
Address for correspondence: D. Groeseneken, Laboratory of Occupational Hygiene and Toxicology, Provisorium I, Minderbroedersstraat 17, B-3000 Leuven, Belgium. Abbreviations: EGEE, ethylene glycol monoethyl ether; BAA, ethoxyacetic acid; EGEE-AC, EGEE acetate ester. 0378-4274/88/~ 03.50 Q 1988 Elsevier Science Publishers B.V. (Biomedical Division)
58
INTRODUCTION
Ethylene glycol ethers are a group of solvents which is widely used in industrial operations and consumer products. They were formerly thought to be rather benign substances exhibiting low to moderate toxicity [ 11. Recent animal studies, however, have demonstrated that some glycol ethers produce bone marrow suppression, teratogenic effects and testicular atrophy [2-51. As a basis for biological monitoring of occupational uptake, the relations between exposure concentration, route of exposure, blood concentration, metabolism and disposition have been studied to some extent in laboratory animals. The metabolic studies revealed that the urinary excretion of the respective alkoxyacetic acids, partly conjugated with glycine, seems to be the major disposition route of the ethylene glycol monoalkyl ethers, accounting for 60-80% of the parent glycol ether [6-lo]. When rats were dosed with ethylene glycol monoethyl ether (EGEE), the urinary excretion of ethoxyacetic acid (EAA) was complete within 48 h and the biological half-life of EGEE was estimated to be 10-12.5 h [lo]. The other 10-15’70 of the administered glycol ether is eliminated through the lungs as CO;! suggesting partial 0-dealkylation of the ether and further metabolism of the ethylene glycol and/or alkoxy moiety [6]. We recently studied in humans the urinary excretion of EAA after respiratory uptake of EGEE or its acetate ester (EGEE-AC) under controlled experimental conditions [ll, 121. The elimination half-life of EAA was estimated to be at least 24 h, suggesting the urinary excretion was far from complete after 48 h: within 42 h only 22% of the inhaled dose was recovered as EAA. On the other hand, studies of the urinary excretion of EAA in subjects occupationally exposed to both EGEE and EGEE-AC suggest that EAA elimination half-life may be as high as 48 h [13]. Moreover, attempts to determine the glycine conjugate of EAA in urine of exposed humans indirectly after acid hydrolysis have yet failed. These findings suggest that the metabolic fate of ethylene glycol ethers in humans may differ qualitatively and quantitatively from that of laboratory animals. On the other hand, there is a large discrepancy between the animal doses currently used (2 100 mg/kg) and the amount of EGEE or EGEE-AC inhaled by the human volunteers in our experiments (< 1 mg/kg). In order to investigate whether the metabolic differences could be attributed to the lower doses in man or to species differences, urinary excretion of EAA was studied in rats after administration of EGEE in doses ranging from plausible occupational uptakes in humans to doses reported in the literature. These data were compared with EAA excretion in experimentally exposed human volunteers. This question is relevant in view of the extrapolation of animal toxicity data to man, since EAA appears to be responsible for EGEE toxicity.
59
MATERIALS AND METHODS
Animals, doses and urine collections White, male Wistar rats of an inbred strain, weighing 240-320 g were obtained from the breeding laboratory of the K.U. Leuven. Five rats were used per dose. Following acclimatization to the metabolic cages for 1 day prior to the dosing, the rats were treated by oral intubation with a single dose of EGEE (Fluka, Buchs, Switzerland), dissolved in distilled water. The dilutions were chosen to attain doses of approximately 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 50 mg/kg or 100 mg/kg by a gavage of 0.5-0.65 ml of solution, depending on the body weight of the rat. The first two doses were equivalent to the intake of EGEE during the experimental human exposures to respectively 20 mg/m3 and 40 mg/m3 EGEE for 200 min [l 11. During the period of urine collection, the animals had free access to food and drinking water. Blank urine was obtained during the acclimatization period. Further urine collections were performed at 12 h intervals up to 60 h after the dosing. The urine was stored at -20°C until analyzed. Subjects, exposure conditions and urine collections The human data were drawn from our previous EGEE inhalation study [ 111. The experimental conditions will be described briefly. The five subjects were sitting in an armchair and breathed air containing respectively 10 mg/m3, 20 mg/m3 or 40 mg/m3 EGEE through a respiratory mask connected with a syringe injection generation system [14]. During exposure, EGEE concentration in both inhaled and exhaled air was determined at distinct time intervals, while respiratory minute volume (pn - l/min), oxygen consumption (PO, l/min) and heart rate were monitored continuously. The total uptake of EGEE was calculated by trapezoidal integration of the uptake rate (0 - mg/min) over the whole exposure period. The uptake rate was calculated from the concentration difference of EGEE in inhaled (Cr - mg/l) and exhaled (Cn - mg/l) air, and pn: 0 (mg/min)
= (Cr - Cn). PE
(1)
The total uptake is then given by: Uptake (mg) =
c +. Oi+l
Zr
At
2
It was found that over a 200 min exposure period doses of approximately 0.25 mg/kg, 0.5 mg/kg and 1 mg/kg were obtained when the subjects were exposed to the above-described conditions (Table I). Blank urine was taken immediately before the start of the exposure. The further
60
TABLE
1
RESPIRATORY UPTAKE HUMAN VOLUNTEERS
AND
DOSE
OF EGEE
DURING
a 200 min EXPOSURE ___~__
PERIOD
_____.-_-..-_ Body weight
Uptake
Dose
(mg/m’) -_____.__I_
(kg)
(mg)
(mg/kg) ~____
10
70.6
+ 10.0
18.2 + 5.9
0.26
+ 0.07
20
71.0
* 10.1
35.1
+ 3.8
0.50
rt 0.04
40
70.5 ZIG10.1 _____.
69.1
z!z 1.2
0.98 + 0.05 __.__. -.~
Exposure
concentration
OF
.- -~_
._~
urine collections were pooled to collections of 12 h intervals and reanalyzed. The pooling was performed in order to obtain the same time base as in our animal experiments. Analytical procedures and calculations For the human exposure experiments, EGEE concentration in both inhaled and exhaled air was determined by gas chromatography. Air samples were taken by pumping a known amount of air (usually 3 liter) into glass tubes containing 150 mg silica gel in the sampling section @KC, Palo Alto, CA, U.S.A.). The silica gel was desorbed with 1 ml methanol containing 50 mg/l ethylene glycol monobutyl ether as an internal standard. Gas chromatographic analysis was performed after the injection of 1 ~1 of the me~anoli~ solution on a CP-Wax 57 CB, WCOT fused silica column (25 m x 0.33 mm ID; 0.22 ,urn film thickness). Oven temperature was kept at 30°C for 30 s and programmed to 90°C at a rate of 40”Clmin. This temperature was held for 3 min. Thereafter, the oven temperature was raised to 120°C at a rate of lO”C/min. The gas chromatograph (Hewlett Packard 5890 A) was equipped with a flame ionisation detector and a COa-cryosystem; the flow rate of the He-carrier gas was 1 ml/min. The 12 h urinary volume of both rats and humans was determined by weight and urinary density. The urine was assessed for the presence of ethoxyacetic acid (EAA) and its glycine conjugate. EAA was determined as described elsewhere [ 151. The conjugate was determined as the increase of EAA after acid hydrolysis of the urine. The hydrolysis was performed in test tubes (10 ml) equipped with a microcondensor to avoid losses of EAA by evaporation. Preliminary data on conjugate hydrolysis indicated that the hydrolysis was complete within 22 h at pH 1 and 90°C. Conjugated EAA concentrations ranging from 1 to 120 mg/l could be quantitatively hydrolysed by this method. When necessary, the urine was diluted with saline prior to hydrolysis. Excretion rate of free as well as total EAA was expressed as pg EAA/12 h taking into account the respective EAA concentrations and the 12 h urinary volume.
61
Statistical analysis Data were always expressed as mean f SD. The data were analyzed using Student’s t-tests. Where appropriate, one- or two-way analysis of variance, including dose as a main source of variation, and multiple linear regression analysis were also used. RESULTS
Time course of EAA excretion Before dosing with EGEE, neither EAA nor its glycine conjugate could be de-
0.5
0
12
mg/kg
2L 36 Time ihl
48
60
0 900
^
0
12
24 36 48 Time Ii-d
60
x
24 36 Ttme (hi
48
60
rf 10 mglkg
tf
0
2
12
12
24 36 Time (h)
40
60
12
24 36 48 Time (h)
60
a
c 24 :: w
0
12
24 36 48 Time (h)
60
0
Fig. 1. Urinary excretion rate of EAA in rats after a singte dose of EGEE. The open bars represent the excretion rate of free EAA. The hatched bars indicate the excretion rate of free and conjugated EAA as determined after acid hydrolysis. Statistical analysis by paired t-test: *P
***P
62
tected in the urine of the rats. Maximal excretion rate of EAA was found within 12 h after dosing. Thereafter, a fast exponential decline to almost undetectable levels after 48 h was observed for doses up to 5 mg/kg (Fig. 1). The same time course was observed for the higher doses, but even after 60 h trace amounts of EAA were still detectable. After acid hydrolysis of the urine, EAA concentrations increased (paired t-test: ta2.19; PcO.05 or less) for all collections, indicating that the glycine conjugate was always present. At any time, free and total EAA excretion rate was higher (F> 187; P
HALF-LIFE
OF EAA IN RAT AND MAN, DOSED WITH EGEE
Dose (mg/kg)
b2 01)
rat
~VZ(h)
man
0.25
-
42.4 rt 4.7
0.5
7.36 rt: 1.56 6.78 + 2.52 6.48 lr. 1.31
42.1 + 4.6
1 5
40.9 z+z5.7 -
10
6.60 f
0.90
-
50
7.48 it 1.07 8.47 f 1.13
-
7.20 rt 1.54
42.0 + 4.7
100 Overall
~.._.
63
1 mglkg 60 5 mglkg
TT
TT
Fig. 2. Urinary excretion rate of EAA in man after respiratory uptake of EGEE. The symbols are the same as in Fig. 1.
jugated acid was clearly affected by the time of urine collection (J’ti,, = 9.84; P
= 27.02 + 2.56 dummy (r = 0.406; P
Besides diurnal variation, also individual factors may affect the extent of glycine conjugation (Find. = 2.18; PC 0.01): the lowest and highest individual conjugation averaged respectively 20.4 + 2.0 and 45.6 rt 4.8%. In urine of man exposed to EGEE, virtually no increase in EAA levels was found after acid hydrolysis indicating that it is unlikely that EAA is conjugated with glycine or other substances (e.g. glucuronic acid).
Recovery Since in rats the elimination of EAA and its glycine conjugate was almost complete within 60 h, the total amount of EAA (free and conjugated) was estimated by the area under the curve of the excretion data (Fig. 3). The recovery was only 13.4 ~fr:2.1% for the lowest dose (0.5 mg/kg) and increased (F = 23.7; P~O.001) as the administered dose of EGEE was higher: at a dose of 100 mg/kg, the recovery mounted to 36.8 + 5.4%. In man, the elimination of EAA was far from complete after 48 h. At that time,
64
(I
20
1
40
I
I
60
II
I
80
100
Dose lmg/kgl
Fig. 3. Relative amount of EGEE recovered from urine as EAA within 60 h in rats.
on average 23% of the inhaled EGEE was excreted as EAA in urine (Table III). The 48 h recovery was not affected by the dose (P = 0.98; ns,). Total recovery was estimated to be 3~35~0 by extrapolation using an elimination half-life of 42 h. Urinary excretion of EAA in man and rat at equivalent few doses Two doses of EGEE given to the rats were chosen equal to those inhaled by man (resp. 0.5 mg/kg and 1 mg/kg). To compare directly the urinary excretion of EAA for these doses, the urinary data of both species were normalised for body weight and expressed as pg/kg/l2 h (Fig. 4). Despite a lower metabolic conversion to EAA in rat (- 15%) compared to man (30-35%) in this dose range, the rats excreted EAA at a higher rate than man. The higher excretion rate in rats could be determined, at least partly, by a higher clearance per kg body weight in rats. Renal clearance (expressed in ml/kg/h) was calculated from the elimination rate constant and the volume of distribution, which was assumed to be the total body water (800 mI/kg body weight) in both species. The clearance in rats mounted to 79.5 t- 17.4 ml/kg/h, while in man the clearance was only 13.3 it 1.5 ml/kg/h (unpaired t-test: t = 12.0; P
FROM URINE AS EAA WITHIN 48 h IN MAN Recovery (070) 24.2 + 8.9 21.9 -+ 5.3 22.2 It 7.9
-.-..
.._
RAT
/I
Time (hl
A
0
12
MAN
24 36 Time Lh!
18
Fig. 4. Urinary excretion of EAA per kg body weight in rats (II = 5) and humans (n = 5) for equivalent low doses of EGEE. The open area indicates the excretion rate of free EAA. The hatched area represents EAA excretion rate recorded after acid hydrolysis.
DISCUSSION
This study was set up to correlate the urinary excretion of EAA in experimental human exposure to EGEE [ 111 to the excretion data from animal studies reported in the literature. Although the human exposures occurred by inhalation, the oral dosing of the rats in this series of experiments was preferred since no data on EGEE retention nor respiratory minute volume of rats were available to calculate precisely the respiratory uptake of EGEE. Since Jonsson et al. [16] observed similar recoveries of EAA and its glycine conjugate in urine of rats from inhaled and ingested EGEE, the metabolism of ethylene glycol ethers was assumed to be independent of the route of administration. The elimination half-life of EAA in rats (mean 7.2 h) would have as a result that about 90% of EAA was excreted within 24 h. This finding agrees with data in the literature. About 88-92% of the radioactivity of a single oral dose of 230 mg/kg [14C]EGEE was recovered in urine during the first 24 h and the biological half-life of EGEE was estimated to be 10-12.5 h in rats [lo]. Compared to rats, the excretion rate of EAA declined at a much slower rate in man. The calculated half-life of
66
elimination (mean 42 h) was greater than the one we reported previously [ll], although the same data were used. This could be mainly attributed to the averaging effect of pooling the urine collections, especially during the first 12 h. Nevertheless, a half-life of 42 h is closer to the one which could be derived from our study of occupationally exposed subjects [ 131. Rats excreted EAA partly as a (glycine) conjugate. The extent of conjugation was independent of the dose, but clearly showed a diurnal variation. To the best of our knowledge, this has never been reported in the literature. This difference could be due to diurnal variation in glycine conjugation or in renal clearance of the conjugate. The latter might be affected by the animal’s activity (Dr. J. Caldwell, personal communication). A study of plasma metabolites with time might elucidate this problem. In man, on the contrary, only free EAA was found in the urine. Humans may have a relative inability to form a number of glycine conjugates and this is sometimes seen in a high excretion of the free acid or in extensive glucuronidation (Dr. J. Caldwell, personal communication). Similar observations were made in the excretion of, e.g., 3,Sdibromoanthranilic acid, a metabolite of bromhexine: rats excreted this metabolite as a mixture of the free acid and its glycine conjugate, while in man only the free acid was found [17]. The dose-dependent recovery of EAA in rats might offer a plausible explanation for the wide range of recoveries reported in the literature, the recoveries ranging from 30 [16] to 80% [9, lo]. Higher recoveries were mainly observed after administration of higher doses. The recovery of other alkoxyacetic acids might also be dose dependent, since Miller et al. [8] reported recoveries of 54.3 + 5.0 and 63.2 f 3.9% for methoxyacetic acid after dosing rats with respectively 1 and 8.7 mmol/kg ethylene glycol monomethyl ether. We found the increase to be statistically significant although it was not stated by the authors. A dose-dependent change in the relative abundancy of a urinary metabolite could indicate that the xenobiotic is metabolized in at least two parallel pathways of which at least one becomes saturated at relatively low doses. If the relative abundancy of the metabolite increases with increasing dose, it is the result of the non-saturated pathway [18]. Therefore the oxidation of EGEE to EAA, presumably by liver alcohol dehydrogenase, appeared not to be saturated, even at a dose of 100 mg/kg. Eventually, above this dose, saturation is likely to occur in this pathway as suggested by in vitro studies with perfused livers [19] and the purified enzyme [20] which reported dose-dependent kinetics with a K, value of 0.6-1.0 mmol/l. The alternative pathway in ethylene glycol ether metabolism (0-dealkylation) might be the saturable pathway. As a consequence, at low doses of EGEE (< 10 mg/kg), oxidation to EAA seemed to be only a minor pathway in EGEE metabolism, but becomes more important when the dose of EGEE increased. In man, the recovery of EAA was higher than in the rat for equivalent low doses of EGEE (0.5 and 1 mg/kg), indicating that the metabolic conversion of EGEE to EAA seemed more important in man that in rat. In man, the 48 h recovery of EAA
67
was found to be independent of dose. However, since the range of EGEE doses was very small compared to the range used in the rat experiments, it is not clear how the recovery will evolve at higher doses in humans. Due to ethical considerations, this problem could not be explored experimentally. When the urinary excretion data of the lower dose range were normalised for body weight in both species, the rats excreted EAA at a higher rate than man for equivalent doses. However, when applying single compartment pharmacokinetics, it can be deduced from the elimination half-life and the renal clearance that the mean blood concentrations of EAA might be at least 3 times higher in man than in rat and that these higher concentrations would be maintained for a considerably longer period. This hypothesis can, however, not be checked since the expected blood concentrations of EAA or EGEE were far below the detection limit of the respective methods. Nevertheless, one should take into account this possibility since it could have consequences for the toxicity of EGEE in man, as the toxic properties of ethylene glycol ethers have been associated with their respective alkoxyacetic acids [21]. ACKNOWLEDGEMENTS
We gratefully acknowledge the excellent technical assistance of Ms. H. Janssens and Ms. C. Van den Bosch. We are also indebted to Dr. J. Caldwell from the St. Mary’s Hospital Medical School, London, for the helpful discussions on part of this work. We also wish to thank Ms. V. De Keyser for preparing the manuscript. This work was supported by the Institute of Hygiene and Epidemiology of the Belgian Ministry of Public Health, contract No. 1l/210-0/1985.
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57
Elsevier
TXL 01939
Comparative urinary excretion of ethoxyacetic acid in man and rat after single low doses of ethylene glycol monoet hyl et her D. Groeseneken, Laboratory
H. Veulemans, R. Masschelein and E. Van Vlem
of U~~Mpaiio*a~ Hygiene and Toxicology,
M~d~eine~ K. U. Leaven,
Department
of ~~~~~a~iona~ and Insurance
3-3000 Louvailz, Beigium
(Received 21 September 1987) (Revision received 4 December 1987) (Accepted 7 December 1987)
Key words: Ethylene glycol monoethyl
ether; Ethoxyacetic acid; (Urinary excretion; Man; Rat)
SUMMARY Male rats were given a single oral dose of ethylene gfycol monoethyl ether (EGEE), the dose ranging from plausible human exposures (0.5-l mg/kg) to doses reported in the literature (100 mg/kg). Urinary excretion of ethoxyacetic acid (EAA) and its glycine conjugate was followed up to 60 h after dosing and compared to data of experimentally exposed human volunteers. In rats, the mean elimination half-life of free as well as conjugated EAA was 7.2 h for all doses. BAA was excreted partly as a glycine conjugate (on average 27%), the extent of conjugation being independent of the dose. The conjugation with glycine showed a clearly diurnal variation, the lowest extent being found during the night. The relative amount of EGEE recovered in urine as EAA was only 13.4% for the lowest dose, but increased as the administered dose of EGEE was higher, indicating that EGEE was metabolised at least in two parallel pathways of which one pathway becomes saturated at relatively low doses. In man, urinary excretion of EAA for equivalent low doses of EGEE differed from that in the rat by a longer elimination half-life (mean 42 h), by the absence of EAA conjugates and by a higher recovery.
Address for correspondence: D. Groeseneken, Laboratory of Occupational Hygiene and Toxicology, Provisorium I, Minderbroedersstraat 17, B-3000 Leuven, Belgium. Abbreviations: EGEE, ethylene glycol monoethyl ether; BAA, ethoxyacetic acid; EGEE-AC, EGEE acetate ester. 0378-4274/88/~ 03.50 Q 1988 Elsevier Science Publishers B.V. (Biomedical Division)
58
INTRODUCTION
Ethylene glycol ethers are a group of solvents which is widely used in industrial operations and consumer products. They were formerly thought to be rather benign substances exhibiting low to moderate toxicity [ 11. Recent animal studies, however, have demonstrated that some glycol ethers produce bone marrow suppression, teratogenic effects and testicular atrophy [2-51. As a basis for biological monitoring of occupational uptake, the relations between exposure concentration, route of exposure, blood concentration, metabolism and disposition have been studied to some extent in laboratory animals. The metabolic studies revealed that the urinary excretion of the respective alkoxyacetic acids, partly conjugated with glycine, seems to be the major disposition route of the ethylene glycol monoalkyl ethers, accounting for 60-80% of the parent glycol ether [6-lo]. When rats were dosed with ethylene glycol monoethyl ether (EGEE), the urinary excretion of ethoxyacetic acid (EAA) was complete within 48 h and the biological half-life of EGEE was estimated to be 10-12.5 h [lo]. The other 10-15’70 of the administered glycol ether is eliminated through the lungs as CO;! suggesting partial 0-dealkylation of the ether and further metabolism of the ethylene glycol and/or alkoxy moiety [6]. We recently studied in humans the urinary excretion of EAA after respiratory uptake of EGEE or its acetate ester (EGEE-AC) under controlled experimental conditions [ll, 121. The elimination half-life of EAA was estimated to be at least 24 h, suggesting the urinary excretion was far from complete after 48 h: within 42 h only 22% of the inhaled dose was recovered as EAA. On the other hand, studies of the urinary excretion of EAA in subjects occupationally exposed to both EGEE and EGEE-AC suggest that EAA elimination half-life may be as high as 48 h [13]. Moreover, attempts to determine the glycine conjugate of EAA in urine of exposed humans indirectly after acid hydrolysis have yet failed. These findings suggest that the metabolic fate of ethylene glycol ethers in humans may differ qualitatively and quantitatively from that of laboratory animals. On the other hand, there is a large discrepancy between the animal doses currently used (2 100 mg/kg) and the amount of EGEE or EGEE-AC inhaled by the human volunteers in our experiments (< 1 mg/kg). In order to investigate whether the metabolic differences could be attributed to the lower doses in man or to species differences, urinary excretion of EAA was studied in rats after administration of EGEE in doses ranging from plausible occupational uptakes in humans to doses reported in the literature. These data were compared with EAA excretion in experimentally exposed human volunteers. This question is relevant in view of the extrapolation of animal toxicity data to man, since EAA appears to be responsible for EGEE toxicity.
59
MATERIALS AND METHODS
Animals, doses and urine collections White, male Wistar rats of an inbred strain, weighing 240-320 g were obtained from the breeding laboratory of the K.U. Leuven. Five rats were used per dose. Following acclimatization to the metabolic cages for 1 day prior to the dosing, the rats were treated by oral intubation with a single dose of EGEE (Fluka, Buchs, Switzerland), dissolved in distilled water. The dilutions were chosen to attain doses of approximately 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 50 mg/kg or 100 mg/kg by a gavage of 0.5-0.65 ml of solution, depending on the body weight of the rat. The first two doses were equivalent to the intake of EGEE during the experimental human exposures to respectively 20 mg/m3 and 40 mg/m3 EGEE for 200 min [l 11. During the period of urine collection, the animals had free access to food and drinking water. Blank urine was obtained during the acclimatization period. Further urine collections were performed at 12 h intervals up to 60 h after the dosing. The urine was stored at -20°C until analyzed. Subjects, exposure conditions and urine collections The human data were drawn from our previous EGEE inhalation study [ 111. The experimental conditions will be described briefly. The five subjects were sitting in an armchair and breathed air containing respectively 10 mg/m3, 20 mg/m3 or 40 mg/m3 EGEE through a respiratory mask connected with a syringe injection generation system [14]. During exposure, EGEE concentration in both inhaled and exhaled air was determined at distinct time intervals, while respiratory minute volume (pn - l/min), oxygen consumption (PO, l/min) and heart rate were monitored continuously. The total uptake of EGEE was calculated by trapezoidal integration of the uptake rate (0 - mg/min) over the whole exposure period. The uptake rate was calculated from the concentration difference of EGEE in inhaled (Cr - mg/l) and exhaled (Cn - mg/l) air, and pn: 0 (mg/min)
= (Cr - Cn). PE
(1)
The total uptake is then given by: Uptake (mg) =
c +. Oi+l
Zr
At
2
It was found that over a 200 min exposure period doses of approximately 0.25 mg/kg, 0.5 mg/kg and 1 mg/kg were obtained when the subjects were exposed to the above-described conditions (Table I). Blank urine was taken immediately before the start of the exposure. The further
60
TABLE
1
RESPIRATORY UPTAKE HUMAN VOLUNTEERS
AND
DOSE
OF EGEE
DURING
a 200 min EXPOSURE ___~__
PERIOD
_____.-_-..-_ Body weight
Uptake
Dose
(mg/m’) -_____.__I_
(kg)
(mg)
(mg/kg) ~____
10
70.6
+ 10.0
18.2 + 5.9
0.26
+ 0.07
20
71.0
* 10.1
35.1
+ 3.8
0.50
rt 0.04
40
70.5 ZIG10.1 _____.
69.1
z!z 1.2
0.98 + 0.05 __.__. -.~
Exposure
concentration
OF
.- -~_
._~
urine collections were pooled to collections of 12 h intervals and reanalyzed. The pooling was performed in order to obtain the same time base as in our animal experiments. Analytical procedures and calculations For the human exposure experiments, EGEE concentration in both inhaled and exhaled air was determined by gas chromatography. Air samples were taken by pumping a known amount of air (usually 3 liter) into glass tubes containing 150 mg silica gel in the sampling section @KC, Palo Alto, CA, U.S.A.). The silica gel was desorbed with 1 ml methanol containing 50 mg/l ethylene glycol monobutyl ether as an internal standard. Gas chromatographic analysis was performed after the injection of 1 ~1 of the me~anoli~ solution on a CP-Wax 57 CB, WCOT fused silica column (25 m x 0.33 mm ID; 0.22 ,urn film thickness). Oven temperature was kept at 30°C for 30 s and programmed to 90°C at a rate of 40”Clmin. This temperature was held for 3 min. Thereafter, the oven temperature was raised to 120°C at a rate of lO”C/min. The gas chromatograph (Hewlett Packard 5890 A) was equipped with a flame ionisation detector and a COa-cryosystem; the flow rate of the He-carrier gas was 1 ml/min. The 12 h urinary volume of both rats and humans was determined by weight and urinary density. The urine was assessed for the presence of ethoxyacetic acid (EAA) and its glycine conjugate. EAA was determined as described elsewhere [ 151. The conjugate was determined as the increase of EAA after acid hydrolysis of the urine. The hydrolysis was performed in test tubes (10 ml) equipped with a microcondensor to avoid losses of EAA by evaporation. Preliminary data on conjugate hydrolysis indicated that the hydrolysis was complete within 22 h at pH 1 and 90°C. Conjugated EAA concentrations ranging from 1 to 120 mg/l could be quantitatively hydrolysed by this method. When necessary, the urine was diluted with saline prior to hydrolysis. Excretion rate of free as well as total EAA was expressed as pg EAA/12 h taking into account the respective EAA concentrations and the 12 h urinary volume.
61
Statistical analysis Data were always expressed as mean f SD. The data were analyzed using Student’s t-tests. Where appropriate, one- or two-way analysis of variance, including dose as a main source of variation, and multiple linear regression analysis were also used. RESULTS
Time course of EAA excretion Before dosing with EGEE, neither EAA nor its glycine conjugate could be de-
0.5
0
12
mg/kg
2L 36 Time ihl
48
60
0 900
^
0
12
24 36 48 Time Ii-d
60
x
24 36 Ttme (hi
48
60
rf 10 mglkg
tf
0
2
12
12
24 36 Time (h)
40
60
12
24 36 48 Time (h)
60
a
c 24 :: w
0
12
24 36 48 Time (h)
60
0
Fig. 1. Urinary excretion rate of EAA in rats after a singte dose of EGEE. The open bars represent the excretion rate of free EAA. The hatched bars indicate the excretion rate of free and conjugated EAA as determined after acid hydrolysis. Statistical analysis by paired t-test: *P
***P
62
tected in the urine of the rats. Maximal excretion rate of EAA was found within 12 h after dosing. Thereafter, a fast exponential decline to almost undetectable levels after 48 h was observed for doses up to 5 mg/kg (Fig. 1). The same time course was observed for the higher doses, but even after 60 h trace amounts of EAA were still detectable. After acid hydrolysis of the urine, EAA concentrations increased (paired t-test: ta2.19; PcO.05 or less) for all collections, indicating that the glycine conjugate was always present. At any time, free and total EAA excretion rate was higher (F> 187; P
HALF-LIFE
OF EAA IN RAT AND MAN, DOSED WITH EGEE
Dose (mg/kg)
b2 01)
rat
~VZ(h)
man
0.25
-
42.4 rt 4.7
0.5
7.36 rt: 1.56 6.78 + 2.52 6.48 lr. 1.31
42.1 + 4.6
1 5
40.9 z+z5.7 -
10
6.60 f
0.90
-
50
7.48 it 1.07 8.47 f 1.13
-
7.20 rt 1.54
42.0 + 4.7
100 Overall
~.._.
63
1 mglkg 60 5 mglkg
TT
TT
Fig. 2. Urinary excretion rate of EAA in man after respiratory uptake of EGEE. The symbols are the same as in Fig. 1.
jugated acid was clearly affected by the time of urine collection (J’ti,, = 9.84; P
= 27.02 + 2.56 dummy (r = 0.406; P
Besides diurnal variation, also individual factors may affect the extent of glycine conjugation (Find. = 2.18; PC 0.01): the lowest and highest individual conjugation averaged respectively 20.4 + 2.0 and 45.6 rt 4.8%. In urine of man exposed to EGEE, virtually no increase in EAA levels was found after acid hydrolysis indicating that it is unlikely that EAA is conjugated with glycine or other substances (e.g. glucuronic acid).
Recovery Since in rats the elimination of EAA and its glycine conjugate was almost complete within 60 h, the total amount of EAA (free and conjugated) was estimated by the area under the curve of the excretion data (Fig. 3). The recovery was only 13.4 ~fr:2.1% for the lowest dose (0.5 mg/kg) and increased (F = 23.7; P~O.001) as the administered dose of EGEE was higher: at a dose of 100 mg/kg, the recovery mounted to 36.8 + 5.4%. In man, the elimination of EAA was far from complete after 48 h. At that time,
64
(I
20
1
40
I
I
60
II
I
80
100
Dose lmg/kgl
Fig. 3. Relative amount of EGEE recovered from urine as EAA within 60 h in rats.
on average 23% of the inhaled EGEE was excreted as EAA in urine (Table III). The 48 h recovery was not affected by the dose (P = 0.98; ns,). Total recovery was estimated to be 3~35~0 by extrapolation using an elimination half-life of 42 h. Urinary excretion of EAA in man and rat at equivalent few doses Two doses of EGEE given to the rats were chosen equal to those inhaled by man (resp. 0.5 mg/kg and 1 mg/kg). To compare directly the urinary excretion of EAA for these doses, the urinary data of both species were normalised for body weight and expressed as pg/kg/l2 h (Fig. 4). Despite a lower metabolic conversion to EAA in rat (- 15%) compared to man (30-35%) in this dose range, the rats excreted EAA at a higher rate than man. The higher excretion rate in rats could be determined, at least partly, by a higher clearance per kg body weight in rats. Renal clearance (expressed in ml/kg/h) was calculated from the elimination rate constant and the volume of distribution, which was assumed to be the total body water (800 mI/kg body weight) in both species. The clearance in rats mounted to 79.5 t- 17.4 ml/kg/h, while in man the clearance was only 13.3 it 1.5 ml/kg/h (unpaired t-test: t = 12.0; P
FROM URINE AS EAA WITHIN 48 h IN MAN Recovery (070) 24.2 + 8.9 21.9 -+ 5.3 22.2 It 7.9
-.-..
.._
RAT
/I
Time (hl
A
0
12
MAN
24 36 Time Lh!
18
Fig. 4. Urinary excretion of EAA per kg body weight in rats (II = 5) and humans (n = 5) for equivalent low doses of EGEE. The open area indicates the excretion rate of free EAA. The hatched area represents EAA excretion rate recorded after acid hydrolysis.
DISCUSSION
This study was set up to correlate the urinary excretion of EAA in experimental human exposure to EGEE [ 111 to the excretion data from animal studies reported in the literature. Although the human exposures occurred by inhalation, the oral dosing of the rats in this series of experiments was preferred since no data on EGEE retention nor respiratory minute volume of rats were available to calculate precisely the respiratory uptake of EGEE. Since Jonsson et al. [16] observed similar recoveries of EAA and its glycine conjugate in urine of rats from inhaled and ingested EGEE, the metabolism of ethylene glycol ethers was assumed to be independent of the route of administration. The elimination half-life of EAA in rats (mean 7.2 h) would have as a result that about 90% of EAA was excreted within 24 h. This finding agrees with data in the literature. About 88-92% of the radioactivity of a single oral dose of 230 mg/kg [14C]EGEE was recovered in urine during the first 24 h and the biological half-life of EGEE was estimated to be 10-12.5 h in rats [lo]. Compared to rats, the excretion rate of EAA declined at a much slower rate in man. The calculated half-life of
66
elimination (mean 42 h) was greater than the one we reported previously [ll], although the same data were used. This could be mainly attributed to the averaging effect of pooling the urine collections, especially during the first 12 h. Nevertheless, a half-life of 42 h is closer to the one which could be derived from our study of occupationally exposed subjects [ 131. Rats excreted EAA partly as a (glycine) conjugate. The extent of conjugation was independent of the dose, but clearly showed a diurnal variation. To the best of our knowledge, this has never been reported in the literature. This difference could be due to diurnal variation in glycine conjugation or in renal clearance of the conjugate. The latter might be affected by the animal’s activity (Dr. J. Caldwell, personal communication). A study of plasma metabolites with time might elucidate this problem. In man, on the contrary, only free EAA was found in the urine. Humans may have a relative inability to form a number of glycine conjugates and this is sometimes seen in a high excretion of the free acid or in extensive glucuronidation (Dr. J. Caldwell, personal communication). Similar observations were made in the excretion of, e.g., 3,Sdibromoanthranilic acid, a metabolite of bromhexine: rats excreted this metabolite as a mixture of the free acid and its glycine conjugate, while in man only the free acid was found [17]. The dose-dependent recovery of EAA in rats might offer a plausible explanation for the wide range of recoveries reported in the literature, the recoveries ranging from 30 [16] to 80% [9, lo]. Higher recoveries were mainly observed after administration of higher doses. The recovery of other alkoxyacetic acids might also be dose dependent, since Miller et al. [8] reported recoveries of 54.3 + 5.0 and 63.2 f 3.9% for methoxyacetic acid after dosing rats with respectively 1 and 8.7 mmol/kg ethylene glycol monomethyl ether. We found the increase to be statistically significant although it was not stated by the authors. A dose-dependent change in the relative abundancy of a urinary metabolite could indicate that the xenobiotic is metabolized in at least two parallel pathways of which at least one becomes saturated at relatively low doses. If the relative abundancy of the metabolite increases with increasing dose, it is the result of the non-saturated pathway [18]. Therefore the oxidation of EGEE to EAA, presumably by liver alcohol dehydrogenase, appeared not to be saturated, even at a dose of 100 mg/kg. Eventually, above this dose, saturation is likely to occur in this pathway as suggested by in vitro studies with perfused livers [19] and the purified enzyme [20] which reported dose-dependent kinetics with a K, value of 0.6-1.0 mmol/l. The alternative pathway in ethylene glycol ether metabolism (0-dealkylation) might be the saturable pathway. As a consequence, at low doses of EGEE (< 10 mg/kg), oxidation to EAA seemed to be only a minor pathway in EGEE metabolism, but becomes more important when the dose of EGEE increased. In man, the recovery of EAA was higher than in the rat for equivalent low doses of EGEE (0.5 and 1 mg/kg), indicating that the metabolic conversion of EGEE to EAA seemed more important in man that in rat. In man, the 48 h recovery of EAA
67
was found to be independent of dose. However, since the range of EGEE doses was very small compared to the range used in the rat experiments, it is not clear how the recovery will evolve at higher doses in humans. Due to ethical considerations, this problem could not be explored experimentally. When the urinary excretion data of the lower dose range were normalised for body weight in both species, the rats excreted EAA at a higher rate than man for equivalent doses. However, when applying single compartment pharmacokinetics, it can be deduced from the elimination half-life and the renal clearance that the mean blood concentrations of EAA might be at least 3 times higher in man than in rat and that these higher concentrations would be maintained for a considerably longer period. This hypothesis can, however, not be checked since the expected blood concentrations of EAA or EGEE were far below the detection limit of the respective methods. Nevertheless, one should take into account this possibility since it could have consequences for the toxicity of EGEE in man, as the toxic properties of ethylene glycol ethers have been associated with their respective alkoxyacetic acids [21]. ACKNOWLEDGEMENTS
We gratefully acknowledge the excellent technical assistance of Ms. H. Janssens and Ms. C. Van den Bosch. We are also indebted to Dr. J. Caldwell from the St. Mary’s Hospital Medical School, London, for the helpful discussions on part of this work. We also wish to thank Ms. V. De Keyser for preparing the manuscript. This work was supported by the Institute of Hygiene and Epidemiology of the Belgian Ministry of Public Health, contract No. 1l/210-0/1985.
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