* 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; || American Chemistry Council, Ethylene Oxide/Ethylene Glycol CHEMSTAR Panel, Arlington, Virginia
Received April 22, 2004; accepted June 24, 2004
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ABSTRACT |
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Key Words: ethylene glycol; nephropathy; metabolism; oxalate.
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INTRODUCTION |
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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 glycolinduced nephrotoxicity in rats that may affect human health risk assessments. In the 1970 s, 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 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 glycolinduced 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.
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METHODS |
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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 osmosistreated 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 #107211, 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 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 + 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-µm thickness, mounted on glass microscopic slides, and stained with hematoxylin-eosin. 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 15, and crystal deposition was measured on a scale of 14 (see Tables 3 and 4 for description of grades).
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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 µg/g for ethylene glycol (EG), glycolic acid (GA), and total oxalic acid (OX), respectively, in blood; 0.8, 0.5, and 0.6 µg/g for EG, GA, and OX, respectively, in kidneys; and 0.5 and 20.6 µg/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 µg/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 < 0.05), Dunnett's test was used to compare the test article-treated groups to the control group.
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RESULTS |
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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 510% below the level intended.
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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 F-344 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 animals consumed their diets, as the half-life for ethylene glycol is on the order of 1.41.9 h and the levels of metabolites were correspondingly higher in these samples (Pottenger et al., 2001
).
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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 µg/g (background blood levels generally ranged from 1 to 10 µg/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 23-fold higher than levels observed after 1 week of exposure. This difference was less apparent or nonexistent in Wistar rats.
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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
), 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.
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DISCUSSION |
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A distinction needs to be made between the presence of oxalate, a metabolite of ethylene glycol, and calcium oxalateinduced 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 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 renal toxicity in human acute poisoning cases and in repeated-dose 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 23% 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 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 (2325%) as after 1 week (2227%); however, Wistar rats treated at 500 and 1000 mg/kg/day had severely reduced excretion (from 1819% to 35%).
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 1.122.06 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
4.27.4 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 = 1.2 and pka2 = 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 oxalateinduced 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 glycolinduced 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.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at ToxWorks, 1153 Roadstown Road, Bridgeton, NJ 08302; E-mail: toxworks{at}aol.com
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