TOXICOLOGICAL SCIENCES 81, 502–511 (2004)
doi:10.1093/toxsci/kfh206
Advance Access publication June 30, 2004
Subchronic Toxicity of Ethylene Glycol in Wistar and F-344 Rats
Related to Metabolism and Clearance of Metabolites
George Cruzan,*,1 Richard A. Corley,† Gordon C. Hard,‡ Jos J. W. M. Mertens,§ Kenneth E. McMartin,¶
William M. Snellings,jj Ralph Gingell,jj and James A. Deyojj
*ToxWorks, Bridgeton, New Jersey; †Pacific Northwest National Laboratory, Richland, Washington; ‡Tairua, 2853, New Zealand; §WIL Research
Laboratories, Inc., Ashland, Ohio; ¶Louisiana State University Health Sciences Center, Shreveport, Louisiana; jjAmerican Chemistry Council,
Ethylene Oxide/Ethylene Glycol CHEMSTAR Panel, Arlington, Virginia
Received April 22, 2004; accepted June 24, 2004
Key Words: ethylene glycol; nephropathy; metabolism; oxalate.
Ethylene glycol (CAS RN 107–21–1) can cause kidney toxicity via
the formation of calcium oxalate crystals in a variety of species,
including humans. Numerous repeated dose studies conducted in
rats have indicated that male rats are more susceptible than female
rats. Furthermore, subchronic and chronic studies using different
dietary exposure regimens have indicated that male Wistar rats may
be more sensitive to renal toxicity than male Fischer-344 (F-344)
rats. This study was conducted to compare the toxicity of ethylene
glycol in the two strains of rats under identical exposure conditions
and to evaluate the potential contribution of toxicokinetic differences to strain sensitivity. Ethylene glycol was mixed in the diet at
concentrations to deliver constant target dosage levels of 0, 50, 150,
500, or 1000 mg/kg/day for 16 weeks to groups of 10 male Wistar and
10 male F-344 rats based on weekly group mean body weights and
feed consumption. Kidneys were examined histologically for calcium oxalate crystals and pathology. Samples of blood, urine, and
kidneys from satellite animals exposed to 0, 150, 500, or 1000 mg/kg/
day for 1 or 16 weeks were analyzed for ethylene glycol, glycolic acid,
and oxalic acid. Treatment of Wistar rats at 1000 mg/kg/day resulted
in the death of two rats; in addition, at 500 and 1000 mg/kg/day,
group mean body weights were decreased compared to control
throughout the 16 weeks. In F-344 rats exposed at 1000 mg/kg/
day and in Wistar rats receiving 500 and 1000 mg/kg/day, there
were lower urine specific gravities, higher urine volumes, and
increased absolute and relative kidney weights. In both strains of
rats treated at 500 and 1000 mg/kg/day, some or all treated animals
had increased calcium oxalate crystals in the kidney tubules and
crystal nephropathy. The effect was more severe in Wistar rats than
in F-344 rats. Accumulation of oxalic acid in the kidneys of
both strains of rats was consistent with the dose-dependent and
strain-dependent toxicity. As the nephrotoxicity progressed over
the 16 weeks, the clearance of ethylene glycol and its metabolites
decreased, exacerbating the toxicity. Benchmark dose analysis indicated a BMDL05 for kidney toxicity in Wistar rats of 71.5 mg/kg/
day; nearly fourfold lower than in F-344 rats (285 mg/kg/day). This
study confirms that the Wistar rat is more sensitive to ethylene
glycol–induced renal toxicity than the F-344 rat and indicates
that metabolism or clearance plays a role in the strain differences.
1
To whom correspondence should be addressed at ToxWorks, 1153
Roadstown Road, Bridgeton, NJ 08302; E-mail: [email protected]
Toxicological Sciences vol. 81 no. 2
#
Ethylene glycol (CAS RN 107–21–1) is an intermediate in the
synthesis of a number of commercial chemical products, including polyethylene terephthalate (PET) resins, unsaturated polyester resins, and polyester fibers and films. It is also a constituent
in antifreeze, deicing fluids, surface coatings, heat transfer fluids
and industrial coolants, hydraulic fluids, surfactants, and
emulsifiers (CERHR, 2003). General population, or consumer,
exposure occurs primarily from the use of ethylene glycol
in automotive antifreeze. There have been a number of acute
human poisonings from accidental or intentional ingestion of
antifreeze, with the kidney being the most sensitive target organ.
Regimens for the treatment of acute ethylene glycol poisoning
are designed to prevent metabolism to the toxic acidic metabolites, to treat acidosis, and to prevent kidney damage (Barceloux
et al., 1999; Brent et al., 1999).
Numerous repeated-dose studies in a variety of species have
evaluated the toxicity of ethylene glycol. Although considerable variability has been observed in sensitivity across species, strains, and sexes, these studies have consistently identified
the kidney as a primary target organ, with rats being more
sensitive than mice and males more sensitive than females
after chronic exposure. Furthermore, there appears to be a
strain difference in sensitivity to ethylene glycol–induced
nephrotoxicity in rats that may affect human health risk
assessments. In the 1970s, the British Industrial Biological
Research Association [BIBRA] fed Wistar rats diets with
constant concentrations of ethylene glycol (0, 500, 1000,
2500, or 10,000 ppm) for 16 weeks (Gaunt et al., 1974). As
typical for studies starting when the animals are young, the
amount of feed consumed per kilogram of body weight
decreased about threefold over the 16 weeks of the study. Therefore, the dosage of ethylene glycol in each group was considerably higher during the first week (1410 mg/kg/day for males
and 1782 mg/kg/day for females in the high concentration
Society of Toxicology 2004; all rights reserved.
ETHYLENE GLYCOL METABOLISM AFFECTS SUBCHRONIC TOXICITY IN RATS
group) than during the 16th week (512 and 910 mg/kg/day for
males and females, respectively). The authors reported calcium
oxalate crystals in the kidneys of high-dose male rats (average
daily dosage of 715 mg/kg/day) resulting in degeneration
of kidney tubules. One rat at an average daily dosage of
180 mg/kg/day had observation of individual nephrons with
degenerative changes and occasional oxalate crystals. The
no observable adverse effect level (NOAEL) was reported as
71 mg/kg/day.
A 2-year chronic toxicity/oncogenicity study was conducted
a few years later in a U.S. laboratory using Fischer-344 (F-344)
rats (DePass et al., 1986). Target dosages in mg/kg/day were
determined before study start, and the concentration of ethylene
glycol in the diet was adjusted periodically to maintain the
appropriate mg/kg/day dosage based on group mean body
weights and feed consumption. At 40 and 200 mg/kg/day,
no toxicity or increased tumor incidence was seen in male
or female rats. At 1000 mg/kg/day, no early mortality or
increased tumor incidence was reported in female rats;
however, nephropathology was seen. In male rats exposed to
1000 mg/kg/day, early mortality was seen beginning at about
12 months. By 15 months, all high-dose males had died. Pathologic evaluation revealed extensive kidney damage as the cause
of death. In this study the NOAEL was 200 mg/kg/day, approximately threefold higher than observed in the 16-week Wistar
rat study.
Mice have been shown to be considerably less sensitive than
rats after chronic dietary exposures to ethylene glycol (NTP,
1993). No increased tumors or toxicity were reported in male
mice exposed to calculated average doses of 1500, 3000, or
6000 mg/kg/day or in female mice exposed to 3000, 6000, or
12,000 mg/kg/day for 2 years.
Thus, there are two repeated-dose studies in two different
strains of rats in which males were more sensitive than females,
and Wistar rats more sensitive than F-344 rats (NOAEL of
200 mg/kg/day in male F-344 rats after 2 years of exposure
vs. NOAEL of 71 mg/kg/day in male Wistar rats after 16
weeks of exposure). This apparent difference in strain sensitivity
could be due to differences in the susceptibility of renal tubule
cells to calcium oxalate crystals or to potential differences in the
toxicokinetics of ethylene glycol leading to a localized build-up
of calcium oxalate crystals. Thus, research is underway to
evaluate the potential toxicokinetic and toxicodynamic contributions to ethylene glycol–induced renal toxicity to assist in
human health risk assessments. As a part of this program, a
16-week subchronic toxicity study was conducted to directly
compare the toxicity of ethylene glycol in male F-344 and Wistar
rats under identical dietary exposure conditions. Subgroups of
animals were also included to determine the levels of ethylene
glycol and its toxic metabolites, glycolic acid and oxalic acid, in
blood, urine, and kidneys after 1 week and 16 weeks of exposure.
A goal of the study was to provide insights into the potential role
of toxicokinetics in the sensitivity differences between
rat strains.
503
METHODS
Study design. Groups of 10 male F-344 and Wistar rats were administered
ethylene glycol via the diet at targeted dosage levels of 0 (control), 50, 150, 500,
or 1000 mg/kg/day for 16 weeks. Dietary concentrations were adjusted weekly
based on group mean body weights and feed consumption for each group of rats.
At the end of 16 weeks of exposure, each animal received a complete necropsy
with kidneys evaluated by histopathology. Toxicokinetic satellite groups consisting of 10 rats/strain/dose level were administered ethylene glycol via the diets
along with the main study animals at 0, 150, 500, and 1000 mg/kg/day. Half of the
satellite animals were euthanized after 1 week of exposure, prior to the onset of
significant renal pathology, and the remainder were euthanized after 16 weeks of
treatment for analysis of ethylene glycol, glycolic acid, and total oxalic acid
(oxalic acid plus calcium oxalate) in blood and kidneys. Urine was collected from
each animal for 24 h prior to each sacrifice and, along with cage wash samples,
analyzed for the same three chemicals.
Animals. Male Wistar (Crl:WI(Glx/BRL/Han)IGSBR and Fischer-344
(CDF(F-344)/CrlBR) rats were obtained from Charles River, Raleigh, NC, at
approximately 26 days of age and acclimated to the testing facility (WIL
Research Laboratories, Inc., Ashland, OH) for 20 days before administration
of ethylene glycol in the diet. The animals were allocated into treatment groups
using a computerized body weight stratified randomization procedure.
Animal husbandry. Animals were housed individually in stainless-steel
mesh caging suspended above cageboard. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal
care program, including the facilities at WIL Research Laboratories, Inc., is
accredited by the Association for Assessment and Accreditation of Laboratory
Animal Care International (AAALAC International).
Mean daily animal room temperature ranged from 71 F to 72 F (21.9 C to
22.4 C) and mean daily relative humidity ranged from 38% to 50% during the
study. Light timers were set to provide a 12-hour light/12-hour dark photoperiod.
Animals were fed NTP2000 diet, lower protein, from Ziegler Bros., ad libitum.
Reverse osmosis–treated municipal water was available ad libitum via an automatic system, except when water consumption was measured from water bottles.
Test materials and chemicals. Polyester grade ethylene glycol (CAS #107–
21–1, 99.99% pure; 0.0089% diethylene glycol) was supplied by the Dow
Chemical Company (Taft, LA) for use in the dietary feeding study. For the
analytical methods used in the toxicokinetic satellite study, ethylene glycol
(Lot No. JR00244CR) and glycolic acid (Lot No. 16802LR) were obtained
from the Aldrich Chemical Company (Milwaukee, WI). Oxalic acid (Lot No.
123H1122) was obtained from Sigma (St. Louis, MO). Deuterated internal standards D2-glycolic acid (Lot No. I1-5086), D4-ethylene glycol (Lot No. P-6136)
were obtained from Cambridge Isotope Laboratories, Inc. (Andover, MA), and
the internal standard, 2-butoxyethanol (Lot No. 07847HN) was obtained from the
Aldrich Chemical Company. Derivatizing reagents, pentafluorobenzoyl chloride
and N-( tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) were
also obtained from the Aldrich Chemical Company. All other compounds and
solvents used in the toxicokinetic analyses were reagent grade or better.
Diet preparation. Doses of 0, 50, 150, 500, and 1000 mg/kg/day were
chosen to cover the range of doses in the studies by Gaunt et al. (1974) and
DePass et al. (1986). Concentrations of ethylene glycol in the diets were calculated weekly for each group of Wistar and F-344 rats based on the most recent
group mean body weights and amounts of feed consumed to achieve the targeted
dosages. For each mixture, the needed amount of ethylene glycol was weighed
and mixed into an appropriate amount of NTP2000 diet (Ziegler Bros, Inc.) using
a Hobart blender. The test diets were prepared weekly and stored under ambient
conditions. The concentrations of ethylene glycol in the diets were confirmed by
gas chromatography during weeks 0, 1, 2, 3, 4, 8, and 15.
Toxicity assessment. Animals were observed for clinical signs of toxicity
daily, and detailed physical examinations were conducted on all animals weekly.
Each animal was weighed weekly. Extensive hematological, urine, and clinical
chemistry analyses had been conducted in the prior subchronic and chronic
504
CRUZAN ET AL.
studies. Therefore, only limited urinalyses were conducted in this study as a
complement to the renal pathology examinations. Urine was collected over
an approximately 24-h period prior to necropsy for all animals (fasted)
using metabolism cages. The following parameters were evaluated: specific
gravity (ATAGO Urine Specific Gravity Refractometer), pH, color, appearance, protein, glucose, bilirubin, urobilinogen, ketones, occult blood,
leukocytes, nitrites (CLINITEK 200 1 Urine Chemistry Analyzer with
reagent strips from Bayer), total volume, color, appearance, and microscopy
of sediment.
Pathology. After 16 weeks of treatment, all survivors were euthanized
and necropsied. Selected tissues (44/animal) were preserved in neutral buffered formalin for potential future evaluations. Since the mode of action of
ethylene glycol is well documented and complete histopathological analyses
were conducted in prior subchronic and chronic studies, the focus of the
pathology examinations in this 16-week study was limited to the kidneys.
The kidneys were weighed and the ratio to the terminal body weight was
calculated for each animal. Sections of each kidney (one longitudinal in a
mid-sagittal position and from the other kidney, a transverse section through
the renal papilla) were imbedded in paraffin, processed, and cut at 5-mm
thickness, mounted on glass microscopic slides, and stained with hematoxylineosin. The slides were examined by normal light (brightfield) for pathologic
lesions, by polarized light for the presence of oxalate crystals (Khan et al.,
1982; Rushton et al., 1981), and by fluorescence microscopy for the presence
of lysosomes (Maunsbach, 1966; Hard and Snowden, 1990). The severity of
compound-induced nephropathy was graded on a scale of 1–5, and crystal
deposition was measured on a scale of 1–4 (see Tables 3 and 4 for description
of grades).
Toxicokinetics. Satellite groups of five rats/strain/dose level were administered ethylene glycol in the diets at 0, 150, 500, and 1000 mg/kg/day for either
1 week or 16 weeks of exposure. For 24 h prior to euthanasia, urine was collected
while the animals were housed separately in metabolism cages. Prior to euthanasia a blood sample was obtained. At euthanasia, the kidneys were removed. All
samples were flash-frozen in liquid nitrogen and shipped on dry ice from WIL
Research Laboratories, Inc., to Battelle Northwest. Samples were stored frozen
(80 C) until analyzed, generally within a few weeks of collection. Previous
studies have shown that samples prepared and stored in this manner remain viable
for analysis of ethylene glycol, glycolic acid and oxalic acid for up to 542 days
(Corley et al., 2002).
Blood and urine samples were analyzed for ethylene glycol, glycolic acid, and
oxalic acid by gas chromatography/mass spectrometry (GC/MS) using the general extraction and derivatization methods of Pottenger et al.. (2001). 2-Butoxyethanol and deuterated ethylene glycol and glycolic acid were used as internal
standards. Kidneys were first homogenized directly (no diluent) then analyzed by
the method used for analysis of blood. GC/MS analyses of ethylene glycol,
glycolic acid, and oxalic acid were performed on a Hewlett Packard 7683
Mass Selective Detector equipped with a Hewlett Packard 6890 Plus gas chromatograph and 7683 autosampler (Hewlett Packard, Avondale, PA). The limits
of quantitation (LOQ) were: 0.2, 0.6, and 0.1 mg/g for ethylene glycol (EG),
glycolic acid (GA), and total oxalic acid (OX), respectively, in blood; 0.8, 0.5, and
0.6 mg/g for EG, GA, and OX, respectively, in kidneys; and 0.5 and 20.6 mg/g for
GA and OX, respectively, in urine and cage wash samples. For groups with
samples below the LOQ, LOQ/2 was used to calculate the means and standard
deviations.
Because of the high concentrations of ethylene glycol in urine and cage wash
samples, direct analysis of these samples was performed by gas chromatography
with flame ionization detection (GC/FID) on a Hewlett Packard 6890 gas
chromatograph. The limits of quantitation (LOQ) for EG in these samples
was 2.4 mg/g.
Statistics. Body weight, body weight change, feed consumption, urinalysis,
and kidney weight data were subjected to a parametric one-way analysis of
variance (ANOVA) to determine intergroup differences. If the ANOVA revealed
statistical significance ( p 5 0.05), Dunnett’s test was used to compare the test
article-treated groups to the control group.
RESULTS
In-Life
Two Wistar rats treated with ethylene glycol at 1000 mg/kg/
day died (approximately 15 weeks). There were no other deaths
and no clinical signs of toxicity. Wistar rats dosed at 500 and
1000 mg/kg/day had lower mean body weights (Fig. 1) in comparison to the controls throughout the study. After 16 weeks of
treatment, the mean body weight in the 1000 mg/kg/day Wistar
group was approximately 23% lower than in the control group,
while that in the 500 mg/kg/day Wistar group was 9% lower than
the control group. Treatment of Wistar rats at 50 and 150 mg/kg/
day had no effect on body weight. Ethylene glycol had no effect
on body weight at any dosage level in F-344 rats.
Treatment of Wistar rats at 1000 mg/kg/day resulted in
reduced feed consumption, which became more pronounced
after week 8. At 500 mg/kg/day, Wistar rats ate slightly less
feed than the controls. F-344 rats receiving 1000 mg/kg/day
consumed 4% less feed than the F-344 controls (data
not shown).
The calculated compound intakes for both F-344 and Wistar
rats were within 10% of intended targets on each weekly measurement (Fig. 2). For Wistar rats at 1000 mg/kg/day, the
decreasing food consumption after week 8 resulted in increasing
concentrations of ethylene glycol in the feed; even so, for the last
7 weeks, compound intake was 5–10% below the level intended.
In the 1000 mg/kg/day F-344 group and in the 500 and
1000 mg/kg/day Wistar groups, the amounts of water consumed
were significantly increased compared to controls (200%),
which resulted in higher total urine volumes (170–300%),
and lower urine specific gravities (Table 1). These groups
also had higher incidences in the occurrence of white blood
cell (microscopy) and/or leukocyte esterase (dipstick) estimates
(data not shown). Other urine parameters were unaffected by
treatment. There were increased incidences of calcium oxalate
crystals in the urine of the 150, 500, and 1000 mg/kg/day F-344
and Wistar groups (Table 1).
Pathology
Mean absolute and relative (to final body weight) kidney
weights (Table 2) in the 1000 mg/kg/day F-344 and 500 and
1000 mg/kg/day Wistar groups were significantly ( p 5 0.05 or
0.01) higher than the control group at the scheduled necropsy.
With examination by brightfield microscopy (Table 3), all
Wistar rats receiving 500 and 1000 mg/kg/day of ethylene glycol
showed evidence of crystal deposition in the kidney and an
associated compound-induced nephropathy. The crystals were
lodged in the tubule lumens, particularly proximal tubules in
severe cases. The typical cuboidal epithelial lining of tubules
containing crystals was reduced to a flattened and attenuated
appearance. The nephropathy was characterized by foci or linear
tracts of tubule basophilia associated with thickened basement
425
325
400
300
375
275
350
250
325
225
grams
grams
ETHYLENE GLYCOL METABOLISM AFFECTS SUBCHRONIC TOXICITY IN RATS
300
275
505
200
175
250
150
225
125
200
100
175
1
1
2
3 4
5
6
7
8
2
3
4 5
9 10 11 12 13 14 15 16 17
6
7
8
9 10 11 12 13 14 15 16 17
weeks
weeks
Wistar:0
Wistar:50
Wistar:500
Wistar:1000
Wistar:150
F-344:0
F-344:50
F-344:500
F-344:1000
F-344:150
FIG. 1. Body weights of Wistar and F-344 rats exposed to ethylene glycol for 16 weeks: Left panel. Wistar rats. Right panel. F-344 rats. Mean for Wistar rats
at 1000 mg/kg/day was significantly ( p 5 0.05) reduced compared to control from week 1 onward. At 500 mg/kg/day significant weeks 3, 6–12. F-344 rats not
different from control.
membrane (or in advanced cases, interstitial hyalinization),
interstitial inflammation (predominantly mononuclear) and
fibroblastic reaction, occasional single cell degeneration and
mitoses in the affected tubules, usually some tubule dilation,
and in advanced cases, sporadic cellular casts. There was an
associated hyperplasia of the lining of the renal pelvis, and sometimes mild pelvic dilation. Crystals were invariably observed in
the fornix of the renal pelvis, even in the least affected cases, but
in severe cases the papilla tip was disrupted by outpouchings of
crystal deposits. These various microscopic aspects of crystal
nephropathy reflected the range of gross changes seen at autopsy
in these dose groups, namely, rough or pale kidney surface, white
areas in cortex/medulla, multiple pinpoint calculi, and dilated
pelvis. With evaluation under polarized light (Table 4), the crystals appeared as birefringent polycrystalline particles arranged
in rosette or fan-shaped patterns, or individually as nearrectangular plates.
Nephropathy with crystal deposition was most severe in
Wistar rats administered ethylene glycol at 1000 mg/kg/day,
where end-stage (grade 5) change affected 4 of the 10 rats, 2
of which died before the scheduled necropsy. Crystal deposition
in the kidney and associated nephropathy were also observed in
all F-344 rats at the highest dose of 1000 mg/kg/day, but at a
lesser severity. Crystal-induced nephropathy was observed in all
Wistar rats treated at 500 mg/kg/day, but in only 1 of 10 F-344
rats treated at 500 mg/kg/day. Six additional rats in the F-344
group dosed with 500 mg/kg/day had evidence of crystals, but
without involvement of the renal parenchyma. In each case these
were represented by a solitary, small, birefringent crystal lodged
in the fornix of the renal pelvis.
The severity of crystal nephropathy at 1000 mg/kg/day in F344 rats was approximately equivalent to that seen at 500 mg/kg/
day in Wistar rats. No crystal nephropathy was seen in either
Wistar or F-344 rats at 50 or 150 mg/kg/day.
Toxicokinetics
In the satellite toxicokinetic group of animals, trace levels of
glycolic acid and oxalic acid were found occasionally in the
blood, kidneys, and urine of control animals. Both glycolic
acid and oxalic acid are known products of endogenous biosynthesis or are metabolites of dietary constituents, and therefore
they were expected to be present in control samples, albeit at
very low concentrations relative to levels achieved after treatment with ethylene glycol at the high dosage levels used in
this study.
Analysis of blood (Table 5). Although only a single blood
sample was analyzed for each animal, blood levels of ethylene
glycol were generally consistent with first-order, non-saturable
metabolism in both strains of rats, similar to previous observations in Sprague-Dawley rats (Pottenger et al., 2001). Blood
ethylene glycol levels in the 16-week Wistar satellite group
were consistently lower than concurrent F-344 blood and previous 1-week blood levels in Wistar rats. This likely reflected
differences in the timing of the blood collections vs. the time the
506
CRUZAN ET AL.
TABLE 1
Water Consumption and Urine Analysis of Wistar and F-344
Rats Administered Diets Containing Ethylene Glycol after 16
Weeks
A. 1200
mg/kg/day
1000
800
600
EG dose
200
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
Week
Wistar:50
Wistar:150
Wistar:500
Wistar:1000
B. 1400
1200
mg/kg/day
0
50
150
500
1000
400
1000
800
600
400
200
0
1
2
3
4
5
6
7
8
F-344:150
7
12
8
21**
22**
5.8
10.9
6.0
9.5
4.1
8.0
1.070 1.037
.058
.017
5.6
9.8
6.0
6.4
16.3**
23.7**
3.5
6.9
5.5
1.050 1.023** 1.016**
.023
.010
.003
0/10
1/10
5/10
6
7
4.6
5.3
1.8
1.040
.007
5.2
5.3
4.0
1.041
.008
8.1
6.8
4.2
1.038
.015
1/10
0/10
3/10
10
10/10
10
5.6
5.3
3.3
1.050
.027
10/10
4/8
18**
8.4
17.5**
3.5
1.017**
.005
7/10
9 10 11 12 13 14 15 16
*Statistically different from control, p 5 0.05; **p 5 0.01.
Week
F-344:50
Wistar rats
Water consumption
(g/animal/day)
S.D.
Urine volume–24 h (mL)
S.D.
Specific gravity
S.D.
Incidence of CaOx
crystal in urine
F-344 rats
Water consumption
(g/animal/day)
S.D.
Urine volume–24 h (mL)
S.D.
Specific gravity
S.D.
Incidence of CaOx crystal
in urine
F-344:500
F-344:1000
FIG. 2. Compound intake of rats exposed to ethylene glycol for 16 weeks.
A. Wistar rats. B. F-344 rats. In F-344 rats, standard deviations were less
than 10% of mean, except on three occasions. In Wistar rats at 50, 150, and
500 mg/kg/day, standard deviations were less than 10%, except on four
occasions; at 1000 mg/kg/day, standard deviations were more than 10% on
nine occasions, but no more than 15%.
animals consumed their diets, as the half-life for ethylene glycol
is on the order of 1.4–1.9 h and the levels of metabolites were
correspondingly higher in these samples (Pottenger et al., 2001).
Glycolic acid levels in blood remained very low at 150 and
500 mg/kg/day in both strains of rats after 1 week and 16 weeks
of exposure. This was expected because these lower dietary
exposures did not generate sufficient glycolic acid levels to result
in saturation of its metabolism (Pottenger et al., 2001; Carney
et al., 2001; Corley et al., 2002). At 1000 mg/kg/day, blood
levels of glycolic acid were increased in F-344 and Wistar
rats after 1 and 16 weeks of exposure, indicating that this
dose level resulted in the saturation of glycolic acid metabolism.
Oxalic acid levels in blood were generally near background
levels in all groups except for small increases in the F-344 and
Wistar rats exposed for 16 weeks to the 1000 mg/kg/day.
Even at 1000 mg/kg/day, blood levels of oxalic acid rarely
exceeded 20 mg/g (background blood levels generally ranged
from 1 to 10 mg/g in previous studies; Pottenger et al., 2001;
Carney et al., 2001; Corley et al., 2002), primarily because of
solubility limits (Burgess and Drasdo, 1993; Hodgkinson,
1981).
Analysis of kidneys (Table 6). Ethylene glycol and glycolic
acid levels in the kidneys of both F-344 and Wistar rats were
very similar to their corresponding blood levels with levels
increasing as exposure duration increased. In F-344 rats,
ethylene glycol kidney levels after 16 weeks were consistently 2–3-fold higher than levels observed after 1 week of
exposure. This difference was less apparent or nonexistent
in Wistar rats.
For glycolic acid, a nearly fivefold increase in the concentration in the kidneys of Wistar rats was observed after 16 weeks of
exposure to 500 and 1000 mg/kg/day than was observed after
only 1 week of treatment. In F-344 rats, exposure-duration
effects on glycolic acid levels in kidneys were apparent only
at 1000 mg/kg/day, where levels were increased approximately
threefold over 1 week of exposure.
After 1 week of exposure, oxalic acid levels in the kidneys of
F-344 rats at 1000 mg/kg/day were elevated, whereas in Wistar
rats at 500 and 1000 mg/kg/day, oxalic acid levels in the kidneys
were elevated. After 16 weeks of exposure, oxalic acid levels
were elevated in the kidneys of F-344 rats at 500 and 1000 mg/
kg/day and at 150, 500, and 1000 mg/kg/day in Wistar rats. Thus,
oxalic acid levels demonstrated significant dose-responses and
time-responses to ethylene glycol dietary exposures, with significantly higher levels achieved in Wistar rats, consistent with
the observed strain differences in renal toxicity.
Analysis of urine (Table 7). Elimination of ethylene glycol
into the urine was largely a first-order process, consistent with
previous studies (Pottenger et al., 2001; Corley et al., 2002),
507
ETHYLENE GLYCOL METABOLISM AFFECTS SUBCHRONIC TOXICITY IN RATS
TABLE 2
Kidney Weights of Wistar and F-344 Rats Administered Diets Containing Ethylene Glycol for 16 Weeks
EG dose
Wistar rats
wt (g)
wt (g/100g bw)
F-344 rats
wt (g)
wt (g/100g bw)
0
50
150
500
1000
2.56 6 0.22
0.66 6 0.03
2.47 6 0.32
0.63 6 0.05
2.48 6 0.16
0.64 6 0.03
3.11 6 0.53*
0.89 6 0.17**
3.52 6 0.56**
1.20 6 0.17**
2.09 6 0.14
0.71 6 0.03
1.99 6 0.17
0.67 6 0.04
2.09 6 0.11
0.71 6 0.02
2.07 6 0.13
0.71 6 0.02
2.45 6 0.28**
0.88 6 0.07**
*Statistically different from control, p 5 0.05; **p 5 0.01.
TABLE 3
Group Incidence and Severity of Compound-Induced Crystal
Nephropathy Based on Brightfield Examination
TABLE 4
Group Incidence and Severity of Crystal Deposition in Kidney
Based on Polarized Light Examination
Rats with severity gradea
Dose group
(mg/kg/day)
Wistar
0
50
150
500
1000
F-344
0
50
150
500
1000
1
2
3
Rats with severity gradea
Rats in group
0
4
5
10
10
10
10
10
10
10
10
0
0
0
0
0
1
0
0
0
0
4
1
0
0
0
3
0
0
0
0
1
5
0
0
0
1
4
10
10
10
10
10
10
10
10
9
0
0
0
0
1
0
0
0
0
0
2
0
0
0
0
6
0
0
0
0
2
0
0
0
0
0
Dose group
(mg/kg/day)
Wistar
0
50
150
500
1000
F-344
0
50
150
500
1000
Rats in group
0
1
2
3
4
10
10
10
10
10
10
10
10
0
0
0
0
0
0
0
0
0
0
4
1
0
0
0
5
0
0
0
0
1
9
10
10
10
10
10
10
10
10
3
0
0
0
0
7
0
0
0
0
0
0
0
0
0
0
8
0
0
0
0
2
a
Severity grades as follows: 0–no basophilic foci of crystal nephropathy in
cortex or OSOM; 1–solitary focus only; 2–very low scatter of foci or tracts
through cortex/OSOM; 3–frequent scatter of foci or tracts through cortex/
OSOM but majority of cortex normal; 4–majority of cortex/OSOM affected;
5–virtually all of parenchyma affected (end-stage); OSOM 5 outer stripe of outer
medulla.
a
Severity grades as follows: 0–no crystals in kidney parenchyma or pelvis; 1–
only very small, solitary crystals in pelvic fornix or in urothelial lining of papilla;
2–scattered to relatively frequent crystals in pelvis, mainly in the fornix and/or
papilla, but virtually none in cortex; 3–low to mild scatter of crystals in cortex,
and usually frequent in pelvis and/or papilla; 4–frequent crystals in cortex,
medulla, papilla and pelvis.
except for Wistar rats exposed for 16 weeks to 500 and
1000 mg/kg/day, where the clearance of ethylene glycol and
its metabolites were significantly reduced by the extensive
toxicity. The amounts of glycolic acid excreted in the urine
also demonstrated non-linearities, with significantly higher
amounts excreted by both strains of rats after 1 week of exposure
to ethylene glycol at 1000 mg/kg/day. However, after 16 weeks of
exposure, renal toxicity observed in F-344 rats at 1000 mg/kg/day
and in Wistar rats at 500 and 1000 mg/kg/day appeared to reduce
the amounts of glycolic acid excreted in the urine. Oxalic acid
levels in urine, although variable, were clearly elevated at the top
two dose levels in F-344 rats and at all dose levels in Wistar rats,
except at 16 weeks, where renal toxicity impaired excretion.
As shown in Figure 3, the percentage of administered doses
accounted for by ethylene glycol, glycolic acid, and oxalic acid
in urine was remarkably consistent in every group of animals
except the Wistar rats exposed for 16 weeks to ethylene glycol at
500 and 1000 mg/kg/day. At these higher dose levels in Wistar
rats, the severe renal toxicity likely impaired the ability to
excrete ethylene glycol and its metabolites, resulting in significant reductions in the percentage of the dose eliminated in the
urine. This reduced clearance may have further exacerbated the
renal toxicity.
Benchmark Dose Analysis
Benchmark dose analysis of the incidence of crystal nephropathy by brightfield microscopic examination in Wistar rats
using the multistage model (the USEPA BMDS program, version 1.3.1) resulted in a BMD05 of 161 mg/kg/day and a
BMDL05 of 71.5 mg/kg/day. The same analysis of the F-344
data resulted in a BMD05 of 417 mg/kg/day and a BMDL05 of
285 mg/kg/day.
508
CRUZAN ET AL.
TABLE 5
Concentrations (Mean 6 SD)a of Ethylene Glycol (EG), Glycolic
Acid (GA), and Oxalic Acid (OX) in the Blood of Male F-344 and
Wistar Rats Administered Ethylene Glycol via the Diet
Strain week
F-344
1
1
1
1
16
16
16
16
Wistar
1
1
1
1
16
16
16
16
Dose
(mg/kg)
EG
(mg/g)
GA
(mg/g)
OX
(mg/g)
0
150
500
1000
0
150
500
1000
nd
5.9 6 1.5
22.0 6 5.2
57.4 6 12.5
nd
8.5 6 2.4
30.5 6 8.5
133.0 6 23.0
nd
nd
2.7 6 0.4
40.0 6 10.5
0.6 6 0.2
1.6 6 0.3
5.6 6 1.9
118.8 6 7.
0.9 6 0.3
0.2 6 0.2
0.4 6 0.2
0.1 6 0.1
7.2 6 0.1
6.2 6 2.5
5.5 6 1.7
10.2 6 2.0
0
150
500
1000
0
150
500
1000
nd
11.8 6 2.4
30.9 6 16.1
78.8 6 39.8
nd
1.7 6 1.3
15.0 6 8.5
45.1 6 40.3
nd
1.7 6 1.1
6.7 6 5.9
48.8 6 31.9
1.4 6 1.3
0.6 6 0.3
12.7 6 15.4
84.3 6 58.5
nd
0.9 6 1.2
1.1 6 0.4
1.4 6 0.7
6.5 6 1.8
3.1 6 0.2
5.2 6 1.4
18.0 6 4.4
a
TABLE 6
Concentrations (Mean 6 SD)a of Ethylene Glycol (EG), Glycolic
Acid (GA), and Oxalic Acid (OX) in the Kidneys of Male F-344
and Wistar Rats Administered Ethylene Glycol via the Diet
Strain week
F-344
1
1
1
1
16
16
16
16
Wistar
1
1
1
1
16
16
16
16
Dose
(mg/kg)
EG
(mg/g)
GA
(mg/g)
OX
(mg/g)
0
150
500
1000
0
150
500
1000
2.1 6 2.0
5.9 6 2.2
16.6 6 4.0
44.6 6 12.4
nd
9.8 6 3.1
34.6 6 9.7
159.0 6 19.5
2.0 6 0.3
2.6 6 0.4
4.0 6 0.6
46.4 6 11.0
1.1 6 0.5
2.4 6 0.6
6.0 6 1.9
140.4 6 15.3
8.2 6 1.8
2.8 6 1.7
1.7 6 0.7
30.8 6 13.0
nd
13.6 6 6.7
32.9 6 12.0
20,616 6 19,857
0
150
500
1000
0
150
500
1000
nd
3.7 6 1.0
20.1 6 14.0
72.3 6 36.7
2.6 6 1.9
6.3 6 2.9
18.4 6 12.0
58.1 6 51.2
0.9 6 0.3
1.4 6 0.3
7.4 6 5.8
53.3 6 36.4
0.6 6 0.5
1.3 6 0.4
35.4 6 29.3
232.5 6 29.6
2.9 6 1.4
1.8 6 0.5
15.4 6 5.8
1,972 6 3,615
5.4 6 3.8
32.6 6 32.4
33,108 6 46,787
100,812 6 31,899
a
The limits of quantitation (LOQ) were 0.2, 0.6, and 0.1 mg/g for EG, GA,
and OX, respectively. For groups with samples below the limits of
quantitation, the LOQ/2 was used to calculate the means and standard
deviations; nd: not detected.
The limits of quantitation (LOQ) were: 0.8, 0.5, and 0.6 mg/g for EG, GA, and
OX, respectively. For groups with samples below the limits of quantitation, the
LOQ/2 was used to calculate the means and standard deviations; nd: not detected.
DISCUSSION
tubule lumen and without kidney pathology. Investigators
believe that calcium oxalate crystals in the renal tubule lumen
result in renal cell injury leading to cell death. As more of the
kidney becomes impaired, normal kidney water regulation is
compromised, as evidenced by increased urine volume and
decreased urine specific gravity, leading to increased water consumption (Table 3). Ethylene glycol itself may also act as an
osmotic diuretic in renal tubules, contributing to the increased
volume and decreased specific gravity of urine.
Humans appear to handle ethylene glycol in a qualitatively
similar manner to animals. After ingestion of intentional high
acute oral doses, ethylene glycol has been observed to be converted to glycolic and oxalic acid, leading to metabolic acidosis
and eventually kidney damage. Treatment of acute ethylene
glycol poisoning in animals and humans has therefore consisted
of administration of ethanol or 4-methylpyrazole to inhibit the
metabolism of ethylene glycol by alcohol dehydrogenase and
gastric lavage and hemodialysis to hasten the elimination of
ethylene glycol and its metabolites (Baud et al., 1987 and
1988; Bowen et al., 1978; Brent et al., 1999; Cheng et al.,
1987; Eder et al., 1998; Gordon and Hunter, 1982; Harry et al.,
1994; Hewlett and McMartin, 1986; Jacobsen et al., 1984, 1988;
Karlson-Stiber and Persson, 1992; Malmlund et al., 1991; Siew,
1979; Simpson, 1985; Spillane et al., 1991; Walder and Tyler,
1994). The mode of action may explain the delays observed in
The postulated mode of action by which ethylene glycol
causes renal toxicity in rats is through the metabolism of ethylene glycol to oxalic acid and the precipitation of oxalic acid with
calcium. Oxalic acid does not appear to be formed from ethylene
glycol in the kidneys of rats, indicating that it must be transported
from the liver (Liao and Richardson, 1972). Oxalic acid has a
very low solubility in aqueous systems; thus blood levels of
oxalic acid typically don’t rise above 10–20 mg/L (0.1–0.2 mM),
regardless of dose (Burgess and Drasdo, 1993; Hodgkinson,
1981; Pottenger et al., 2001). As oxalate ions are concentrated
prior to clearance in urine, they can precipitate with calcium
ions, and this leads to the growth of insoluble calcium oxalate
crystals in the renal tubule epithelium.
A distinction needs to be made between the presence of oxalate, a metabolite of ethylene glycol, and calcium oxalate–
induced toxicity. In the current study, calcium oxalate crystals were seen in the urine of Wistar rats dosed at 150, 500,
and 1000 mg/kg/day, but crystals were seen in the kidney only at
500 and 1000 mg/kg/day, whereas in F-344 rats, crystals were
seen in the urine at 500 and 1000 mg/kg/day, but were observed
in the kidney parenchyma of only 1 of 10 rats at 500 mg/kg/day
and in all 10 at 1000 mg/kg/day. Thus calcium oxalate crystals
can be present in the urine without being detected in the renal
ETHYLENE GLYCOL METABOLISM AFFECTS SUBCHRONIC TOXICITY IN RATS
TABLE 7
Total Amounts (Mean 6 SD)a of Ethylene Glycol (EG), Glycolic
Acid (GA), and Oxalic Acid (OX) Eliminated in the Urine and
Cage Wash of Male F-344 and Wistar Rats Collected Over 24 h
after 1 and 16 Weeks of Dietary Exposure to Ethylene Glycol
Strain week
F-344
1
1
1
1
16
16
16
16
Wistar
1
1
1
1
16
16
16
16
Dose
(mg/kg)
EG
(mg/g)
GA
(mg/g)
OX
(mg/g)
0
150
500
1000
0
150
500
1000
nd
5,129 6 601
14,508 6 847
32,033 6 1,604
15 6 8
10,340 6 1,626
35,427 6 8,872
59,877 6 3,642
85 6 10
357 6 50
1,210 6 198
8,554 6 2,622
nd
186 6 74
870 6 296
5,621 6 1,590
251 6 65
272 6 109
1,161 6 586
764 6 487
301 6 183
361 6 153
1,364 6 533
1,205 6 931
0
150
500
1000
0
150
500
1000
nd
5,553 6 895
18,247 6 5,985
24,810 6 14,749
93 6 50
8,233 6 1,193
8,285 6 7,090
4,854 6 3,164
98 6 58
475 6 102
2,530 6 1,199
9,710 6 5,516
nd
226 6 147
341 6 349
2,850 6 971
132 6 64
631 6 252
2,344 6 1,884
5,614 6 2,897
nd
578 6 427
64 6 35
84 6 64
a
The limits of quantitation (LOQ) were: 2.4, 0.5 and 20.6 mg/g for EG, GA, and
OX, respectively. For groups with samples below the limits of quantitation, the
LOQ/2 was used to calculate the means and standard deviations; nd: not detected.
F344 1 wk
Wistar 1 wk
F344 16 wk
Wistar 16 wk
% Dose in Urine
30%
20%
10%
0%
150
500
1000
Dose (mg/kg/d)
FIG. 3. Percentages of the administered doses eliminated in 24-h urine
collections from male F-344 and Wistar rats after 1 week and 16 weeks of
dietary exposure to 150, 500, or 1000 mg/kg/day. Results expressed as the
means 6 SD of the total amounts of ethylene glycol, glycolic acid, and oxalic
acid eliminated in the 24-h urine and cage wash samples from four to five
animals/group.
509
renal toxicity in human acute poisoning cases and in repeateddose animal studies. Kidney damage occurs after metabolic
conversion of ethylene glycol in the liver, the slow transport
of oxalic acid to the kidneys (due to low blood solubility), and the
precipitation of the resulting oxalic acid with calcium. In human
poisoning cases, the damage to the kidneys can be either reduced
or prevented if treatment is instituted soon after consumption
(Brent et al., 1999).
The presence of calcium oxalate crystals in the kidney tubule
prevents normal kidney function at that site and leads to cell
death. Eventually, the kidney may be so compromised that it has
a reduced capacity to excrete oxalates, resulting in greater kidney burden of oxalic acid and calcium oxalate. Because of the
use of strong acid in extraction procedures, calcium oxalate is
converted to oxalic acid in all chemical analyses. Thus, particularly in kidney tissues, the oxalic acid detected was likely to
consist primarily of calcium oxalate. After 16 weeks of ethylene
glycol dietary exposures, oxalic acid accounted for 2–3% of
the kidney weights of Wistar rats in the 500 mg/kg/day group and
of F-344 rats in the 1000 mg/kg/day group. In Wistar rats in the
1000 mg/kg/day group, oxalic acid accounted for as much as
10% of the kidney weight or as much as 18%, if it was all in
the form of calcium oxalate. If the damage is sufficient as it was
with the some of Wistar rats dosed at 1000 mg/kg/day, death
may occur.
In F-344 rats, exposure to 1000 mg/kg/day for 12 months
resulted in oxalate crystal nephropathy leading to the death of
all male rats before the end of the 24-month study. However,
no oxalate crystal nephropathy was seen in rats dosed at
200 mg/kg/day for 24 months (DePass et al., 1986). On the
other hand, the unpublished study in Wistar rats (Gaunt et al.,
1974) was reported to have produced nephropathy in all male
rats at 715 mg/kg/day, while 1 of 15 developed renal lesions with
oxalate crystals at 180 mg/kg/day. These two studies used different dosing procedures (constant concentration in feed
[decreasing dose] vs. constant dose [increasing concentration]),
different diets, different criteria for pathology diagnoses, and
different strains of rat. The present study was therefore necessary
to clarify the results from the previous studies by using current
techniques and identical dietary exposure regimens to provide a
direct species comparison. Based on the results of this 16-week
study, Wistar rats are about twice as sensitive to ethylene glycol
as F-344 rats, if one compares the incidence and severity
of nephrotoxic effects (i.e., incidences and severity in
Wistar rats after 16 weeks of dosing with ethylene glycol at
500 mg/kg/day is about the same as seen in F-344 rats at
1000 mg/kg/day). Benchmark dose analysis of the incidences
of crystal nephropathy indicated that the BMDL05 for
F-344 rats is about fourfold higher than that of Wistar rats.
The toxicokinetic data generated in this study support the
mode of action and differing susceptibility of Wistar and
F-344 rats. After 1 week of exposure at 150 mg/kg/day, there
was essentially no difference between Wistar and F-344 rats in
urinary excretion of ethylene glycol, glycolic acid, or oxalic
510
CRUZAN ET AL.
acid. At 500 and 1000 mg/kg/day, F-344 rats excreted more
ethylene glycol and glycolic acid, but less oxalic acid than
Wistar rats. The effect of kidney pathology on ethylene glycol
removal was evident in Wistar rats, but not F-344 rats after
16 weeks of treatment. F-344 rats excreted the same proportion
of the administered dosage after 16 weeks (23–25%) as after
1 week (22–27%); however, Wistar rats treated at 500 and
1000 mg/kg/day had severely reduced excretion (from 18–19%
to 3–5%).
The reduced clearance of ethylene glycol and its metabolites
ultimately led to significantly greater target tissue levels of
oxalic acid (presumably in the form of calcium oxalate) in
male Wistar rats than in male F-344 rats. Based on results
from an ongoing series of in vitro metabolism studies, there
do not appear to be significant differences between strains of
rats in the relative rates of metabolism when glycolic acid is used
as a substrate (manuscript in preparation). Furthermore, there
do not appear to be significant strain differences in susceptibility to calcium oxalate crystals in ongoing in vitro toxicity
studies (manuscript in preparation). It is thus likely that the
differential susceptibility to ethylene glycol toxicity may be
due to differences in renal clearance of oxalic acid (or calcium
oxalate).
Oxalic acid does not appear to bind to plasma proteins and is
freely filtered by the glomerulus. The ratio of oxalic acid renal
clearance (CLOX) to inulin clearance (CLIn), a measure of glomerular filtration rate, is generally 1.12–2.06 in a variety of
species (Boer et al., 1985; Cattell et al., 1962; Knight et al..,
1979a, b; McIntosh and Belling, 1975; Osswald and Hautmann,
1979; Prenan et al., 1982), suggesting that there is net active
secretion of oxalate into the tubule for excretion. Oxalic acid can
either be excreted in urine within its solubility limits (solubility
of calcium oxalate in deionized water is 4.2–7.4 mg/l; Burgess
and Drasdo, 1993; Hodgkinson, 1981) or precipitate with calcium to begin the process of forming crystals (which may also
be excreted in the urine). With such a low pka (pka1 5 1.2 and
pka2 5 3.8; Greger, 1981), no ion-trapping is expected as oxalic
acid is in its ionized state at all physiological pH’s. At present,
these data do not indicate whether major differences between
male Wistar rats and other rat strains is likely associated with
differences in the active transport of oxalic acid into the tubule
or in glomerular filtration.
High acute oral doses of ethylene glycol can cause calcium
oxalate–induced kidney toxicity in humans who may have accidentally or intentionally consumed antifreeze, but chronic exposures to ethylene glycol in humans are apparently quite low, and
nephropathy has not been observed in workers as a consequence
of ethylene glycol exposure. In an occupation with perhaps the
greatest potential for exposure to ethylene glycol, no demonstrable kidney damage was observed in 33 adult male Canadian
airport deicing workers (Gerin et al., 1997). In addition, no clear
evidence of kidney toxicity was observed in a group of 10 male
Finnish auto mechanics, although urinary ethylene glycol levels
were significantly elevated relative to age-matched male office
workers (Laitinen et al., 1995). Thus, under typical use conditions where exposures are low, metabolism and clearance of
ethylene glycol and its metabolites are not saturated and kidney
toxicity is unlikely.
In summary, this study has confirmed the considerable strain
variability in sensitivity to ethylene glycol–induced renal toxicity and that this sensitivity may be related to differences in the
ability to clear ethylene glycol and its metabolites, most notably
oxalic acid (or calcium oxalate), in the urine. To explain the
mechanism for the strain differences, additional studies are in
progress to examine (1) the inherent sensitivity of Wistar rat,
F-344 rat, and human proximal tubule epithelial cells; (2) the
conversion of ethylene glycol to glycolic and oxalic acid; and
(3) the renal clearance of oxalate as a function of strain and
ethylene glycol exposure. These additional studies, along
with the results from this 16-week study and an ongoing chronic
toxicity/toxicokinetic study, should provide a more definitive
and quantitative basis for extrapolating results from either strain
of rat in human health risk assessments.
ACKNOWLEDGMENTS
The study was sponsored by the American Chemistry Council CHEMSTAR
Ethylene Glycol/Ethylene Oxide Panel. Members of the Panel include: BASF
Corporation, The Dow Chemical Company, Eastman Chemical Company,
Equistar Chemicals, L. P., Huntsman Corporation, and Shell Chemical, L.P.;
Mr. William Gulledge is the Panel Manager. Dr. Cruzan has provided toxicology
consulting to Equistar. Dr. McMartin has provided consulting to the panel and has
received grants from the panel for in vitro mode of action studies. The biophase of
the study was conducted under the direction of Dr. Mertens at WIL Research
Laboratories, Inc; the technical expertise from all those involved is greatly
appreciated. Metabolic analyses were conducted under the direction of
Dr. Corley at PNNL; thanks to Jolen J. Soelberg and Karl K. Weitz for analytical
support. Thanks to Dr. Chris Kirman, The Sapphire Group, for benchmark dose
analysis. The authors of this article are either employed by, or serve as consultants
to, companies that manufacture ethylene glycol or the ACC Chemstar Ethylene
Glycol/Ethylene Oxide Panel, or performed this work under contract to the
ACC CHEMSTAR Panel.
REFERENCES
Barceloux, D. G., Krenzelok, E. P., Olson, K., Watson, W. (1999). American
Academy of Clinical Toxicology practical guidelines on the treatment of
ethylene glycol poisoning. J. Toxicol. Clin. Toxicol. 37, 537–560.
Baud, F. J., Bismuth, C., Garnier, R., Galliot, M., Astier, A., Maistre, G., and
Soffer, M. (1987). 4-Methylpyrazole may be an alternative to ethanol therapy
for ethylene glycol intoxication in man. Clin. Toxicol. 24, 463–483.
Baud, F. J., Galliot, M., Astier, A., Vu Bien, D., Garnier, R., Likforman, J., and
Bismuth, C. (1988). Treatment of ethylene glycol poisoning with intravenous
4-methylpyrazole. Med. Intel. 319, 97–100.
Boer, P., Prenen, J. A. C., Koomans, H. A., and Dorhout Mees, E. J. (1985).
Fractional oxalate clearance in subjects with normal and impaired renal
function. Nephron 41, 78–81.
Bowen, D. A. L., Minty, P. S. B., and Sengupta, A. (1978). Two fatal cases of
ethylene glycol poisoning. Med. Sci. Law 18, 102–107.
ETHYLENE GLYCOL METABOLISM AFFECTS SUBCHRONIC TOXICITY IN RATS
511
Brent, J., McMartin, K., Phillips, S., Burkhart, K.K., Donovan, J. W., Wells, M.,
and Kulig, K. (1999). Fomepizole for the treatment of ethylene glycol poisoning. N. Engl. J. Med. 340, 832–838.
Burgess, J., and Drasdo, D. N. (1993). Solubilities of calcium salts of dicarboxylic
acids in methanol-water mixtures; transfer chemical potentials of dicarboxylate anions. Polyhedron 12, 2905–2911.
Jacobsen, D., Ovrebo, S., Ostborg, J., and Sejersted, O. M. (1984). Glycolate
causes the acidosis in ethylene glycol poisoning and is effectively removed by
haemodialysis. Acta Med. Scand. 216, 409–416.
Jacobsen, D., Hewlett, T. P., Webb, R., Brown, S. T., Ordinario, A. T., and
McMartin, K. E. (1988). Ethylene glycol intoxication: evaluation of kinetics
and crystalluria. Am. J. Med. 84, 145–152.
Carney, E. W., Liberacki, A. B., Tornesi, B., and Markham, D. A. (2001).
Ethylene Glycol: Effect of Dose-Rate on Developmental Toxicity. R&D
Report K-002558–017 of the Dow Chemical Company, Midland, MI.
Karlson-Stiber, C., and Persson, H. (1992). Ethylene glycol poisoning: Experiences from an epidemic in Sweden. Clin. Toxicol. 30, 565–574.
Cattell, W. R., Spencer, A. G., Taylor, G. W., and Watts, R. W. E. (1962). The
mechanism of the renal excretion of oxalate in the dog. Clin. Sci. 22, 43–51.
CERHR (NTP Center for Evaluation of Risks to Human Reproduction)
(2003). NTP-CERHR Expert Panel Report on the Reproductive and
Developmental Toxicity of Ethylene Glycol. NIEHS, Research Triangle
Park, NC, pp. 1–120.
Cheng, J. T., Beysolow, T. D., Kaul, B., Weisman, R., and Feinfeld, D. A. (1987).
Clearance of ethylene glycol by kidneys and hemodialysis. Clin. Toxicol.
25, 95–108.
Corley, R. A., Weitz, K. K., Luders, T. M., Studnisky, K. G., Blessing, J. C.,
Gies, R. A., and Carney, E. C. (2002). Pharmacokinetics of Ethylene Glycol in
Pregnant SD Rats Following Bolus Oral Gavage or Continuous Subcutaneous
Infusion. Final Report. Battelle Northwest Project 29812 for the Ethylene
Glycol Panel, American Chemistry Council.
DePass, L. R., Garman, R. H., Woodside, M. D., Giddens, W. E., Maronpot, R. R.,
and Weil, C. S. (1986). Chronic toxicity and oncogenicity studies of ethylene
glycol in rats and mice. Fundam. Appl. Toxicol. 7, 547–565.
Eder, A .F., McGrath, C. M., Dowdy, Y. G., Tomaszewski, J. E., Rosenberg, F. M.,
Wilson, R. B., Wolf, B. A., and Shaw, L. M. (1998). Ethylene glycol poisoning:
toxicokinetic and analytical factors affecting laboratory diagnosis.
Clin. Chem. 44, 168–177.
Gaunt, I. F., Hardey, J., Gangolli, S. D., Butterworth, K. R.., and Lloyd, A. G.
(1974). Short-Term Toxicity of Monoethylene Glycol in the Rat. Carshalton,
Surrey, UK: BIBRA International; Research Report 4/1974.
Gerin, M., Patrice, S., Begin, D., Goldberg, M. S., Vyskocil, A., Adib, G.,
Drolet, D., and Viau, C. (1997). A study of ethylene glycol exposure and
kidney function of aircraft de-icing workers. Int. Arch. Occup. Environ. Health
69, 255–265.
Gordon, H. L., and Hunter, J. M. (1982). Ethylene glycol poisoning. Anesthesia
37, 332–338.
Greger, R. (1981). Renal transport of oxalate. In Renal Transport of Organic
Substances (Greger, R., Lang, F., and Silbernagel, S., eds.). Springer-Verlag,
Berlin. pp. 224–233.
Hard, G. C., and Snowden, R. T. (1991). Hyaline droplet accumulation in rodent
kidney proximal tubules; an association with histiocytic sarcoma. Toxicol.
Pathol. 19, 88–97.
Harry, P., Turcant, A., Bouachour, G., Houze, P., Alquier, P., and Allain, P.
(1994). Efficacy of 4-methylpyrazole in ethylene glycol poisoning: clinical
and toxicokinetic aspects. Hum. Exp. Toxicol. 13, 61–64.
Hewlett, T. P., and McMartin, K. E. (1986). Ethylene glycol poisoning. The value
of glycolic acid determinations for diagnosis and treatment. Clin. Toxicol.
24, 389–402.
Hodgkinson, A. (1981). Sampling errors in the determination of urine calcium
and oxalate: Solubility of calcium oxalate in HCL-urine mixtures. Clin. Chim.
Acta 109, 239–244.
Khan, S. R., Finlayson, B., Hackett, R. L. (1982). Experimental calcium
oxalate nephrolithiasis in the rat. Role of the renal papilla. Am. J. Pathol.
107, 59–69.
Knight, T. F., Senekjian, H. O., and Weinman, E. J. (1979a). Effect of
para-aminohippurate on renal transport of oxalate. Kidney Int. 15, 38–42.
Knight, T. F., Senekjian, H. O., Taylor, K., Steplock, D. A., and Weinman, E. J.
(1979b). Renal transport of oxalate: Effects of diuretics, uric acid, and calcium.
Kidney Int. 16, 572–576.
Laitinen, J., Liesivuori J., and Savolainen, H. (1995). Exposure to glycols and
their renal effects in motor servicing workers. Occup. Med. 45, 259–262.
Liao, L. L., and Richardson, K. E. (1972). The metabolism of oxalate precursors
in isolated perfused rat livers. Arch. Biochem. Biophys. 153, 438–448.
Malmlund, H. O., Anders, B., Karlman, G., Magnusson, A., and Ullman, B.
(1991). Considerations for the treatment of ethylene glycol poisoning
based on analysis of two cases. Clin. Toxicol. 29, 231–240.
Maunsbach, A. B. (1966). Observation on the segmentation of the proximal
tubule in rat kidney. Comparison of results from phase contrast, fluorescence,
and electron microscopy. J. Ultrastruct. Res. 16, 239–258.
McIntosh, G. H., and Belling, G. B. (1975). An isotopic study of oxalate excretion
in sheep. Aust. J. Exp. Biol. Med. Sci. 53, 479–487.
NTP. (1993). Toxicology and carcinogenesis studies of ethylene glycol (CAS
No. 107–21–1) in B6C3F1 Mice (feed studies). National Institutes of Health,
National Toxicology Program; 1993.
Osswald, H. and Hautmann, R. (1979). Renal elimination kinetics and plasma
half-life of oxalate in man. Urol. Int. 34, 440–450.
Pottenger, L. H., Carney, E. W., and Bartels, M. J. (2001). Dose-dependent
nonlinear pharmacokinetics of ethylene glycol metabolites in pregnant
(GD10) and nonpregnant Sprague-Dawley rats following oral administration
of ethylene glycol. Toxicol. Sci. 62, 10–19.
Prenan, J. A. C., Boer, P., Dorhout, E. J., Endeman, H. J., Spoor, S. M., and
Oei, H. Y. (1982). Renal clearance of [14C]oxalate: Comparison of constantinfusion with single injection techniques. Clin. Sci. 63, 47–51.
Rushton, H. G., Spector, M., Rodgers, A. L., Hughson, M., Magura, C. E. (1981).
Developmental aspects of calcium oxalate tubular deposits and calculi induced
in rat kidneys. Invest. Urol. 19, 52–57.
Siew, S. (1979). Investigation of crystallosis of the kidney by means of polarization, scanning, transmission and high-voltage electron microscopy.
Isr. J. Med. Sci. 15, 698–710.
Simpson, E. (1985). Some aspects of calcium metabolism in a fatal case of
ethylene glycol poisoning. Ann. Clin. Biochem. 22, 90–93.
Spillane, L., Roberts, J. R., and Meyer, A. E. (1991). Multiple cranial
nerve deficits after ethylene glycol poisoning. Ann. Emerg. Med. 20,
208–210.
Walder, A. D., and Tyler, C. K. G. (1994). Ethylene glycol antifreeze poisoning.
Anaesthesia 49, 964–967.