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These recent data indicate that GA is the primary causativeagent for the high-dose developmental effects of EG in rodents,with the accompanying metabolic acidosis playing a second-ary, exacerbating role at higher dose levels.

The pharmacokinetics of EG metabolism have been studiedin different animal models by several investigators. Most re-cently, Frantz et al. (1996a,b,c) reported on the dose- androute-dependency of EG metabolism in male and nonpregnantfemale rats and mice. In particular, these authors reporteddose-proportional pharmacokinetics for the elimination fromblood of orally (gavage) administered EG in female rats andmice over the dose range investigated (101000 mg EG/kgbw), with a shift to increasing urinary elimination of EG and itsmajor metabolite GA, with increasing dose. However, theirdata on blood levels of GA indicated nonlinearity across doses,with a 1000- to 10,000-fold increase in GA blood levels for a100-fold increase in administered EG dose. This disproportion-ality for GA was also reected by an increased urinary elimi-nation of GA with increasing administered EG doses. Thus,administration of high doses of EG via gavage had the potentialto result in accumulation of GA, the proximate developmentaltoxicant. In contrast, dermal exposures of up to 1000 mg/kgshowed no such shift in metabolism, with the majority of theEG dose being eliminated as CO 2.

No data have been published on the potential effect of pregnancy on EG metabolism. The physiology of pregnancyresults in major changes in physiological parameters thatcould have considerable impact on pharmacokinetics andmetabolism of EG. Examples include overall weight gainand increase in organ size, particularly liver; increase intotal body water and blood volume; increased blood ow to

uterus; decreased overall plasma protein level but increasesin specic plasma proteins; altered hormone blood levels;decreased GI motility and increased GI transit time (Miller,1983). These parameters do not offer a comprehensive listof pregnancy-related physiological changes, but do repre-sent a few that could easily affect the pharmacokinetics andmetabolism of EG. Any pregnancy-related changes in EGmetabolism and pharmacokinetics would be expected tohave impact on the formation and elimination of the prox-imate developmental toxicant, GA, and could be critical tounderstanding the species-specic developmental toxicity of EG. This study investigated the effect of pregnancy (GD 10)

on the pharmacokinetics and metabolism of EG in femaleSprague-Dawley rats, comparing blood concentration-timeproles and urinary excretion of parent and metabolitesacross doses between pregnant and nonpregnant rats. GD 10was chosen based on prior in vivo (Khera, 1991) and whole-embryo culture studies (Carney et al., 1996) showing thisperiod to be highly sensitive to EG toxicity. This study wasconducted in compliance with the requirements of GoodLaboratory Practices (EPA-TSCA, 1989; OECD, 1982).

MATERIALS AND METHODS

Chemicals. Ethylene glycol (EG; 13C 2-labelled) was obtained from IsotecInc. (Miamisburg, OH; purity 96.7% via 1H-NMR). Pentauorobenzoyl chlo-ride and N -(tert-butyldimethylsilyl)- N -methyltriuoroacetamide (MTBSTFA)were obtained from Aldrich Chemical Company (Milwaukee, WI). All othercompounds and solvents were reagent grade or better.

Test animals. Adult female (nonpregnant and time-mated) Sprague-Daw-ley rats were purchased from Hilltop Lab Animals, Scottsdale, Pennsylvania.Rats were shipped on GD 8 and arrived at our laboratory on GD 9. All rats hadan in-dwelling jugular vein cannula implanted at the suppliers facility on GD6 (or the same calendar day for the nonpregnant rats). The rats were allowedto recover for 2 days prior to being shipped. The cannula was exteriorizedunder light methoxyurane anesthesia upon arrival at the laboratory. Similarsurgical procedures in pregnant animals have been used in the past in thislaboratory, and no untoward effects on pregnancy were observed (Carney etal., 1999). Upon receipt the animals were examined by a veterinarian andfound to be in good health. The animals were acclimated to the laboratoryenvironment for 1 day prior to dose administration. The rooms in which theanimals were housed had a 12-h photocycle and are designed to maintainadequate environmental temperature, relative humidity, and airow for the rat.Municipal drinking water and Purina Certied Rodent Chow #5002 (PurinaMills, Inc., St. Louis, MO) were provided ad libitum during the predosing

period, except that on the day prior to dosing, a uniform amount of chow wasfed (approximately 15 gram/rat). Also, food was completely withdrawn ap-proximately 2 h prior to the administration of the test material and was returnedabout 4 h postdosing. These steps were intended to minimize between-animalvariation in absorption of test material, yet limit any deleterious effects of feedrestriction on the pregnant animal. Rats were selected from those with patent jugular vein cannulae on GD 10 and then were randomly assigned to treatmentgroups using a computerized procedure based on animal body weight. Ratswere identied by a uniquely numbered metal eartag.

Dose administration. 13C-labelled EG, as an aqueous solution, was ad-ministered to 5 groups of 4 time-mated female Sprague-Dawley rats by gavage,with blunted feeding needles, at the following dose levels: 2500, 1000, 500,150, and 10 mg EG/kg body weight. 13C-labelled EG was also administered, inthe same fashion, to 2 groups of nonpregnant female rats (2500 mg EG/kg, n5; 10 mg EG/kg, n 4). The target dose volume for all animal groups was 5

ml/kg. The experiment was divided into 2 replicates of approximately 1415rats each, due to the number of rats involved. Immediately following dosing,the animals were placed in glass Roth-type metabolism cages for bloodsampling and for the separation and collection of urine.

Specimen Collection

Urine. All urine voided during the study was collected in dry ice-cooledtraps at 12-h intervals and the cage was rinsed with a minimal volume ( 10ml) of deionized water. Analyses were done on the combined urine specimenand cage rinse.

Whole blood. Approximately 0.2 ml blood/rat were collected at 0 (pre-dose), 1, 3, 6, 9, 12, 18, and 24 hours after administration of test material.Following collection of each blood sample, approximately 0.2 ml of heparin-ized saline was slowly injected to ush the cannula and provide a heparin

lock.Terminal sacrice. After collection of the last blood sample, the animals

were euthanized via carbon dioxide inhalation. The uterus of each rat then wasexamined for implantation sites to determine pregnancy status. Blood and urinesamples from any time-mated rats that were found to be nonpregnant duringnecropsy were analyzed and included in the comparative nonpregnant group,as appropriate.

Specimen analysis. Samples of whole blood and urine were analyzed viagas chromatography-mass spectrometry (GC/MS) to quantitate parent 13C 2-EG, and the metabolites 13C 2-GA and

13C 2-OX. Samples were derivatized as

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described below to increase analyte volatility and detector response. The use of a stable isotope-labeled form of EG test material ( 13C 2-), resulted in theformation of 13C 2-labeled metabolites (

13C 2-GA and 13C 2-OX). Analysis of

blood and urine samples via mass spectral detection allowed for the determi-nation of 13C 2-EG and the

13C 2-labelled metabolites independent of the pres-ence of background, and perhaps varying levels of unlabelled EG, GA, andOX. Unlabeled GA and OX served as internal standards in the quantitation of 13C 2-labelled GA and OX. These unlabeled analogs were added to the blood

and urine samples at concentrations of approximately 100 g/ml or 1000g/ml, respectively, which was approximately 50 background levels of these

compounds. Deuterium-labelled EG (D 4-EG) was utilized as an internal stan-dard in the quantitation of 13C 2-EG. Quantitation limits for

13C 2-EG, 13C 2-GA,

and 13C 2-OX in blood were 0.1, 2.1, and 4.9 g/g blood, respectively. Quan-titation limits for 13C 2-EG,

13C 2-GA, and 13C 2-OX in urine were 1.0, 0.94, and

2.0 g/g urine, respectively.Aliquots of each blood sample from the time-course experiment (approxi-

mately 0.1 g) were added to 0.9 ml of an internal standard solution (0.9 ml 1NHCl containing 11.5 g/ml D 4-EG) in a 4-ml glass vial. An additional 0.1 mlof a second internal standard solution (approximately 100 g/ml GA and OX)was also added to each sample. Samples were treated with 0.51 g NaCl andextracted with methyl- t -butyl ether (MTBE) containing 0.5% tri- n-octylphos-phine oxide (2 2 ml). The combined MTBE extracts (containing GA andOX) were evaporated to dryness (nitrogen stream), reconstituted in 0.9 ml

toluene and derivatized with 100 l N -methyl- N -(t -butyldimethylsilyl)triu-oroacetamide (60C 1 h). The derivatized sample was transferred to a 2-mlglass vial for analysis of 13 C 2-GA and

13 C 2-OX. A second aliquot of each bloodsample (approx. 0.1 g) was prepared as above for analysis of parent 13C 2-EG,without NaCl, to minimize chromatographic interferences. The aqueous phaseremaining after MTBE extraction (containing EG) was treated with 300 l 5NNaOH and derivatized with 20 l pentauorobenzoyl chloride and 1 mltoluene (vortex-mixed at 45C 30 min). The toluene layer was transferred toa 2-ml glass vial for analysis of 13C 2-EG.

Aliquots (0.1 g) of urine samples were added to 0.8 ml 1N HCl, and fortiedwith 5 l or 100 l of internal standard solution (approx. 1000 g/ml GA, OXand D 4-EG) and extracted with MTBE containing 0.5% tri- n-octylphosphineoxide (2 2 ml). Further treatment of the combined MTBE extracts (con-taining GA and OX) was as described above for the blood samples. A secondaliquot (0.1 g) of each urine sample was fortied with 100 l of internal

standard solution (approx. 1000 g/ml GA, OX and D 4-EG), treated with 50l 5N NaOH and derivatized with 50 l pentauorobenzoyl chloride and 2 mltoluene (vortex-mixed at 45C 30 min). The toluene layer was transferred toa 2-ml glass vial for analysis of 13C 2-EG.

GC-MS analyses were performed on a Finnigan TSQ-700, SSQ-710, orHewlett Packard 5989X mass spectrometer (Finnigan MAT, San Jose, CA;Hewlett Packard, Avondale, PA), equipped with a Hewlett Packard 5890 gaschromatograph and a 7673A autosampler. Separations were achieved using aJ&W DB-5 fused silica capillary column (J&W Scientic, Folsom, CA)(GA/OX: 30 m 0.25 mm id 0.25 m lm; EG: 30 m 0.32 mm id1 m lm); helium carrier gas (10 psig) at a ow rate of approximately 0.5ml/min; gas chromatograph oven temperature program for GA/OX: 100C (0.5min initial hold) to 280C at 15/min, then to 300C at 25/min with injectorand capillary transfer line at 200C and 250C, respectively; gas chromato-graph oven temperature program for EG: 100C (0.5 min initial hold) to 280Cat 20/min with injector and capillary transfer line at 250C; 1- l autosamplerinjection (GA/OX: 40 ml split; EG: 0.1 min splitless). The mass spectrometerconditions for GA/OX were electron impact ionization (EI): ion source tem-perature, 150C; ionizing current, 0.4 mA; electron energy, 70 eV. The massspectrometer conditions for EG were negative-ion chemical ionization (NCI):ion source temperature, 150C; ionizing current, 0.4 mA; electron energy, 70eV. Quantitation of the t-butyldimethylsilyl derivatives of GA, 13C 2-GA, OXand 13C 2-OX was achieved by selected ion monitoring (m/z 247, 249, 261 and263 @ 70 msec/ion/scan). Quantitation of the pentauorobenzoyl ester deriv-atives of 13C 2-EG and D 4-EG was achieved by selected ion monitoring (m/z452 and 454 @ 75 msec/ion/scan).

Statistics and data analysis. Descriptive statistics were used (i.e., meanSD). Results were generally expressed as percentage of administered doseand/or as g of parent EG or metabolite. Certain pharmacokinetic parameterswere estimated for blood data, including Cmax and AUC for parent materialand GA (Gibaldi and Perrier, 1982), time to reach maximum blood levels(Tmax), and half-life of elimination ( t1/2). The reported pharmacokinetic (PK)values were estimated using a commercially available computer modelingprogram (PK Solutions v2.02, Summit Research Services, Ashland, OH).

RESULTS

Actual concentrations of EG in the various dose solutionsranged from 95102% of target. Administered dosage of EGranged from 92100% of target across all dose groups. Atdosing, individual animal body weights ranged from 211 to264 g across all dose groups. There were no remarkablechanges in behavior or demeanor recorded during thedaily animal observations. At terminal sacrice, the preg-nancy status of the time-mated rats was determined, and thenumber of implantations/animal ranged from 722 for thepregnant rats.

Blood Concentration-Time Course of EG and Metabolites

Individual blood samples were analyzed for parent EG, GA,and OX concentrations. The mean values for the concentration-time courses of EG, GA, and OX are presented in Table 1, andFigures 1 and 2 depict the concentration-time course prolesfor EG and GA, respectively. The pharmacokinetic parameters,estimated using the blood data, are presented in Table 2. Nopharmacokinetic parameters, including area-under-the-curve(AUC) data, were calculated for the OX concentration-timecurve, as the blood levels of 13C 2-OX were either at or near thequantitation limit of 4.9 g/g blood in all samples analyzed.

Comparison of the data collected for pregnant (P) and non-pregnant (NP) groups at both the high (2500 mg EG/kg) andlow (10 mg EG/kg) dose clearly demonstrated that pregnancyat this stage (GD 1011) did not have any signicant impact onthe blood concentration-time proles of EG, GA, or OX. Asshown in Figures 1 and 2, the time-courses for the mean EGand GA blood levels of P and NP rats administered either 2500or 10 mg EG/kg were superimposable. Thus, the pharmacoki-netic parameters summarized in Table 2 did not differ signif-icantly between the P and NP dose groups.

Time course of parent EG. Parent EG was not detectable inany pretreatment blood samples. The Tmax for EG for all dosegroups occurred as expected at 1 h postdosing, the rst sam-

pling time. The blood concentration of EG decreased in a linearfashion following the 1 h Cmax. Blood EG was no longerdetectable for the low dose groups (10 mg EG/kg) by 12 hpostdosing, and for the 150 and 500 mg EG/kg dose groups by24 h postdosing, although parent EG was detected at 24 hpostdosing for the 2500 and 1000 mg EG/kg dose groups.

Comparison of Cmax values across dose levels demon-strated linearity in the dose-response for parent EG for all dosegroups except the 2500 mg EG/kg. The linearity of the dose-

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response for parent EG was also supported by comparison of AUC values for EG, where the AUC increased across doselevels proportionately with dose for all the groups, except thehigh dose. The 1000 and 2500 mg EG/kg doses differed by2.5-fold, while the respective AUC and Cmax values demon-strated about a 4-fold difference. This disproportionate in-crease may indicate that blood clearance of EG was initiallysaturated at the 2500 mg EG/kg dose level. Such initial satu-ration could be due to either saturation of metabolic clearanceof EG from blood (i.e., saturation of formation of GA fromEG) or to saturation of renal elimination of EG from blood.The estimated t 1/2 of elimination of EG from blood was short,less than 2 h for all dose levels, indicating that, overall, parentEG was rapidly cleared from blood. Thus, any initial saturationin blood levels at the high dose did not last long enough to alterits half-life of elimination from blood.

Time course of GA in blood. GA, recognized as an endo-genously present compound (Jolivet et al., 1985; Poore et al.,1997), has been shown to be present in human plasma and

urine at concentrations of approximately 0.1 and 20 g/g,respectively (Hoffman et al., 1989; Tanaka et al., 1980). Anal-ysis of control blood samples from untreated rats, in the ab-sence of added GA internal standard, afforded true concentra-tions of endogenous, background GA of up to 2.1 g/g ratblood (data not shown).

Blood levels of the 13C 2-GA, derived from the test material,increased to a peak at 3 h postdosing, except for the 10 mgEG/kg dose groups, which demonstrated nondetectable levelsof 13C 2-GA over the entire 24-h time course. For all dosegroups, blood levels of GA decreased by 24 h postdosing toundetectable levels. Elimination of GA from blood appearedbiphasic until it reached background levels, with the phasefor each curve dened as follows: 918 h (2500 mg EG/kg),618 h (1000 mg EG/kg), 612 h (500 mg EG/kg), and 39 h(150 mg EG/kg). Estimation of the t1/2 of elimination of GAfrom blood resulted in similar values across dose levels (1.11.9 h), based on these biphasic elimination curves. No phar-macokinetic parameters were estimated for GA data from the10 mg EG/kg dose groups, as those samples were below LOQlevels.

Examination of GA blood levels as a function of dosedemonstrated nonlinear, dose-dependent kinetics. Figure 3 de-picts graphically the Cmax blood levels determined for GA,and compares them with estimated examples of what the Cmax

levels would have been for a linear relationship between Cmaxand administered dose (GA Ex Cmax). At the 10 and 150 mgEG/kg dose levels, GA blood levels appeared to be roughlyproportional to dose, given that GA levels were 2.1 g/g forthe 10 mg EG/kg dose, as compared to 20.6 g/g for the 150mg EG/kg dose (15-fold higher dose). A marked shift in GAblood kinetics was observed as the dose increased from 150 to500 mg EG/kg. Over this 3.3-fold dose interval, the Cmax forGA increased by a factor of 6.4, which was a clear dispropor-

tionate increase. This disproportionality with dose persisted toa somewhat lesser degree between the 500 and 1000 mg EG/kgdose levels, where the 2-fold increase in dose brought about a2.8-fold increase in GA Cmax.

A similar shift, with a disproportionate increase, was foundfor the AUC values obtained from the GA blood concentration-time courses, particularly between 150 to 500 mg EG/kg. Thisshift is depicted graphically in Figure 3, where the actual AUCvalues for GA are compared with estimated values for a linearrelationship between AUC and administered dose (GA ExAUC). Estimated AUC for GA increased by 7.6- and 2.9-foldfor a 3.3- and a 2-fold increase in administered dose, for the150500 and 500-1000 mg EG/kg dose group intervals, re-spectively. Thus, elimination of GA from blood demonstratedsaturation at dose levels of 500 mg EG/kg and above.

Comparison of GA Cmax values for the 2 top dose levelsshowed a less than dose-proportionate increase, with only a25% increase for a 2.5-fold increase in dose. In fact, the GACmax for 1000 mg EG/kg was basically equivalent to the GACmax for the 2500 mg EG/kg dose level. However, AUCvalues for GA were dose-proportionate for this dose interval of 1000 to 2500 mg EG/kg, indicating that over time, a similarfraction of administered dose of EG was converted to GA forboth 1000 and 2500 mg EG/kg.

Time course of OX in blood. OX, another endogenouslypresent compound (Poore et al., 1997; Ribaya-Mercado andGershoff, 1984), has been found in human urine at concentra-tions of approximately 20 g/g (Tanaka et al., 1980). Analysisof control blood samples from untreated rats, in the absence of added OX internal standard, afforded true concentrations of endogenous, unlabelled OX of up to 4.9 g/g rat blood (datanot shown).

The concentrations of blood 13

C 2-OX, derived from the testmaterial, varied between undetectable and about 2 times thelimit of quantitation (4.9 g OX/g blood) over the 24-h col-lection period. There was no obvious pattern to OX bloodlevels; while the 2500 mg EG/kg dose groups had the mosttime points with detectable OX levels, the highest mean OXvalue was obtained with a sample from the 18-h 1000 mgEG/kg dose group (9.2 5.3). Based on these data, it does notappear that OX accumulates in P or NP female rats followingoral gavage administration of EG. Given what appeared asmostly trace levels of OX, no additional analysis of the OXdata was indicated or feasible. The lack of any obvious dose-response relationship in OX blood levels over such a large doserange (250-fold increase) suggests that OX does not play amajor role in the expression of developmental toxicity of EG inrats.

Urinary Proles and Interval Excretion of EG, GA, and OX

Urinary 13C-labeled-EG, -GA, and -OX levels were quanti-ed over 12-h intervals, and the results are summarized inTable 3, both as total amount excreted ( g) and as fraction of


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TABLE 1Concentration-Time Course for Mean Blood Levels of Ethylene Glycol (EG), Glycolic Acid (GA), and Oxalic Acid (OA)

following Oral Administration to Female S-D Rats

Time

EG GA OX

Mean a SD Mean SD Mean SD

High dose

2500 mg/kg NP0 h NQ NA NQ NA NQ NA1 h 2795 536 252 39.5 NQ NA3 h 1908 221 432 62.1 NQ NA6 h 539 186 364 59.4 NQ NA9 h 180 41.4 234 52.6 NQ NA12 h 49.0 14.6 122 48.4 NQ NA18 h 4.1 4.1 NQ NA NQ NA24 h 1.1 0.9 NQ NA NQ NA

2500 mg/kg P b

0 h NQ c NA NQ NA NQ NA1 h 3528 1599 313 79.2 NQ NA3 h 1693 344 452 125 5.9 0.76 h 482 33.0 359 77.4 NQ NA9 h 155 44.1 253 92.0 5.7 0.612 h 42.1 22.6 126 75.0 6.4 2.218 h 2.0 1.1 4.7 5.6 NQ NA24 h 0.5 0.3 NQ NA 5.5 0.6

1000 mg/kg P0 h NQ NA NQ NA NQ NA1 h 886 90.6 213 44.8 NQ NA3 h 450 51.8 363 89.7 NQ NA6 h 127 30.7 155 16.1 NQ NA9 h 23.6 7.8 40.7 11.7 NQ NA12 h 3.9 1.4 2.0 1.1 NQ NA18 h 0.6 0.2 NQ NA 9.2 5.324 h 0.2 0.1 NQ NA NQ NA

500 mg/kg P0 h NQ NA NQ NA NQ NA1 h 392 46.8 80.7 9.8 NQ NA3 h 188 27.3 131 23.9 NQ NA6 h 40.5 12.0 60.5 6.9 NQ NA9 h 7.1 0.6 2.1 1.5 NQ NA12 h 1.2 0.3 NQ NA NQ NA18 h 0.3 0.4 NQ NA NQ NA24 h NQ NA NQ NA NQ NA

150 mg/kg P0 h NQ NA NQ NA NQ NA1 h 88.9 48.6 15.8 2.8 NQ NA3 h 43.4 32.0 20.6 7.1 7.0 2.96 h 13.3 0.9 2.0 0.7 NQ NA9 h 2.3 0.7 NQ NA NQ NA

12 h 0.5 0.3 NQ NA NQ NA18 h 0.1 0.1 NQ NA NQ NA24 h NQ NA NQ NA NQ NA


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the administered dose. Again, there were no substantial differ-ences between the P and NP dose groups, at comparable doselevels. Therefore, gestation has no impact on urinary elimina-tion of orally administered EG to female rats at this stage of pregnancy (GD 1011).

Overall urinary elimination of EG and its metabolites dem-onstrated dose-dependency, with the high dose groups (2500

mg EG/kg) eliminating almost 70% of administered dose inurine, compared with about 16% of administered dose elimi-nated via urine by the low dose groups (10 mg EG/kg).

Quantitation of individual metabolites in urine demonstratedthat the shift in urinary elimination was mainly due to in-creased urinary GA and EG, and not to increased eliminationof OX (Table 3). Parent EG was always detectable in urine.

FIG. 1. Concentration-time prole of unchanged parent ethylene glycol(EG) in the blood of pregnant or nonpregnant female S-D rats following oraladministration of EG. Symbols represent the mean SD from 5 rats. LOQ0.1 g EG/g blood.

FIG. 2. Concentration-time prole of glycolic acid (GA) in the blood of pregnant or nonpregnant female S-D rats following oral administration of EG.Symbols represent the mean SD from 5 rats. GA was not quantiable at anytime points for both of the 10 mg EG/kg dose groups. LOQ 2.1 g GA/gblood.

TABLE 1 Continued

Time

EG GA OX

Mean a SD Mean SD Mean SD

Low dose

10 mg/kg NP0 h NQ NA NQ NA NQ NA1 h 9.3 2.5 NQ NA NQ NA3 h 3.8 0.5 NQ NA NQ NA6 h 0.9 0.3 NQ NA NQ NA9 h 0.2 0.1 NQ NA NQ NA12 h NQ NA NQ NA NQ NA18 h NQ NA NQ NA NQ NA24 h NQ NA NQ NA NQ NA

10 mg/kg P0 h NQ NA NQ NA NQ NA1 h 7.9 0.6 NQ NA NQ NA3 h 3.4 0.5 NQ NA NQ NA6 h 0.7 0.3 NQ NA NQ NA9 h 0.1 0.1 NQ NA NQ NA12 h NQ NA NQ NA NQ NA

18 h NQ NA NQ NA NQ NA24 h NQ NA NQ NA 5.3 0.6

Note. EG, GA, and OX given in g/g blood.a Values represent mean SD from 5 rats.b P, pregnant; NP, nonpregnant.c NQ, not quantiable (LOQ: 0.1 g EG/g blood; 2.1 g GA/g blood; 4.9 g OX/g blood); NA, not applicable.


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The fraction of administered dose represented by urinary EGincreased with increasing dose, reaching a maximum of about42% of administered dose at the 500 mg EG/kg dose group,above which urinary EG remained around 40%. Therefore, the

3 highest dose groups all resulted in elimination of a similarfraction of administered dose as urinary EG. This suggests thatrenal elimination of EG was never saturated, even at thehighest dose level. This supports the hypothesis that the initialsaturation in EG blood levels was due to saturation of meta-bolic conversion of EG to GA and not to saturation of renalelimination of EG.

The increase in urinary EG elimination probably corre-sponded in large part with saturation of metabolism of EG toGA, resulting in an increased amount of EG available for renalelimination. In fact, the rst collection intervals for the 500 and1000 mg EG/kg dose groups showed 23 times as muchurinary EG as GA. Then, once the metabolic conversion was

no longer saturated and the excess EG had cleared (i.e., duringthe 1224 h collection interval), the ratio of urinary GA/EGwas roughly 1, while the overall percentage of the administereddose eliminated in the second interval was considerably de-

creased.Urinary GA was quantiable at all intervals, except the

lowest dose levels for the 1224-h interval. The 150 mg EG/kgand 10 mg EG/kg dose levels resulted in similar percentages of administered dose eliminated as urinary GA, representing onlyabout 1% of the administered dose, clearly a minor fraction.However, the fraction of the administered dose eliminated asurinary GA then increased with increasing dose above 150 mgEG/kg, until it was present in roughly equal proportions withEG, as mentioned above. Comparison across dose levels dem-onstrated a disproportionate increase in urinary GA startingwith the 500 mg EG/kg dose group, where the percentage of administered dose eliminated as urinary GA increased about11-fold compared with 150 mg EG/kg, over a dose range of only 3.3-fold. The fraction of administered dose represented byurinary GA increased for the 1000 mg EG/kg and again for the2500 mg EG/kg dose groups, up to about 20% and 33% of theadministered dose, respectively. The shift in urinary GA levelscorresponded with the shift in blood GA levels discussedearlier, indicating that renal elimination of GA was neversaturated.

Urinary OX represented a constant fraction of administereddose across all dose levels. This resulted in a larger totalamount of urinary OX with increasing dose, despite the con-tinued endogenous/background blood concentrations of OX, asdiscussed above.

DISCUSSION

The data presented here clearly demonstrate that the phar-macokinetic parameters for EG and its metabolites reportedhere were not affected by GD 1011 stage pregnancy inSprague-Dawley rats. This signies that the rich database onEG pharmacokinetics and metabolism, collected in NP rats,

FIG. 3. Disproportionate dose-response relationship between orally ad-ministered dose of ethylene glycol (EG) and resulting blood Cmax and AUCvalues quantied for its metabolite, glycolic acid (GA), in pregnant S-D rats.Experimental data are found in Table 2. Values for a linear response wereestimated based on the 150 mg EG/kg dose measured values for GA, and areshown as dotted lines (- - - -) for comparison.

TABLE 2Pharmacokinetic Parameters Estimated for EG and GA following Oral Administration of EG to Female Rats

Parameter a Units

2500-NP b 2500-P 1000-P 500-P 150-P 10-P c 10-NP c

EG GA EG GA EG GA EG GA EG GA EG EG

Tmax h 1 3 1 3 1 3 1 3 1 3 1 1

Cmax g/g 2795 432 3528 452 886 363 392 131 88.9 20.6 7.9 9.3AUC g-h/g 11,368 3807 11,638 4031 2928 1829 1208 641 292 84 23 27t1/2 h 1.9 1.1 1.7 1.5 1.8 1.6 1.7 1 1.7 1.4 1.4 1.5Corr. (x:y) 0.9913 0.9511 0.9915 0.9717 0.9693 0.8552 0.9714 0.8739 0.9836 0.9032 0.9828 0.9998

a PK parameters estimated using a noncompartmental analysis with PK Solutions (v2.0.2).b NP Nonpregnant; P pregnant.c PK parameters estimated only on EG values for 10 mg/kg dose groups, as GA and OX data were mostly background levels.


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1000 to 2500 mg EG/kg, with about a 4-fold increase in Cmaxvs. a 2.5-fold one in administered dose. This suggests that theapparent initial saturation of EG elimination from blood, seenat the 2500 mg EG/kg dose, was most probably due to satura-tion of the metabolic conversion of EG to GA, rather than tosaturation of renal elimination of EG. The conversion of EG toGA by alcohol/aldehyde dehydrogenase (ADH/ALDH) is oneof 2 known rate-limiting steps in the EG metabolic pathway.The other major rate-limiting step is the further oxidationof GA.

Overall urinary elimination (percentage of the administereddose) was not affected by pregnancy at GD 1011, and P andNP rats demonstrated similar urinary elimination kinetics forEG and its metabolites for the 10 and 2500 mg EG/kg doses,respectively. However, overall urinary elimination did demon-strate dose-dependency, with the high dose groups (2500 mgEG/kg) eliminating almost 70% of the administered dose inurine, compared with about 16% of the administered doseeliminated via urine by the low dose groups (10 mg EG/kg).This is in agreement with previously published data demon-strating a shift in disposition of 14C-EG-derived radioactivityfrom exhalation of 14CO 2 at low doses to urinary elimination of radioactivity at high doses following intravenous and oraladministration of 14C-EG to female rats (Frantz et al., 1996b;Marshall, 1982).

The shift in urinary GA levels corresponded with the shift inblood GA levels discussed earlier, indicating that renal elimi-nation of GA was not saturated. In fact, on a wt/wt basis,urinary elimination of GA increased about 3.5- and 4-fold overa 2- and 2.5-fold increase in administered dose, between 500and 1000 mg EG/kg, and between 1000 and 2500 mg EG/kg,respectively. This suggests that the saturation in elimination of

GA from blood was due to saturation of downstream metabo-lism of GA, and not to saturation of renal elimination of GA.Data from Frantz et al. (1996b), demonstrating a shift in thedisposition of 14C-labeled EG, from formation of 14CO 2 at alow dose level (presumably representing downstream metabo-lism of the GA), to increased urinary radioactivity at a highdose level, supports the conclusion of no saturation of renalelimination of GA. Marshall (1982) showed that GA in urineaccounted for approximately 2% of a 20 or 200 mg EG/kgbolus dose of EG, but approximately 20% of a 1000 or 2000mg EG/kg dose.

OX blood levels remained consistently at or below quanti-tation levels in blood, even with increasing doses of EG. These

data are consistent with a slower conversion of GA to OX thanmetabolism of OX to downstream products such as CO 2. Incontrast, the fraction of dose eliminated via the urine as OXremained constant across doses, resulting in increasingamounts of urinary OX eliminated with increased dose levelsof EG. Renal formation of OX from EG and/or GA is onepossible explanation of the differences between blood andurinary OX levels. If intrarenally formed OX were then elim-inated via urine without any reabsorption, there would not be

any reection of its formation in systemic blood OX levels.There are no data on intrarenal formation and elimination of OX from EG. In any case, OX was a very minor metabolite inboth blood and urine at all dose levels, suggesting that it is nota major factor in EG developmental toxicity. This conclusion isfurther supported by data from a preliminary study in whichlevels of OX were measured in the exocoelomic uid of GD 10rat conceptuses following gavage exposure to EG. Exocoelo-mic uid OX averaged approximately 16.4 g/g (0.13 mM)after a 2500 mg EG/kg dose, and was nondetectable (LOD5 g/g) following a 500 mg EG/kg dose (Carney et al., 1998).A rat whole embryo culture study with OX indicated onlyminor growth inhibitory effects at a concentration of 126

g/ml (1 mM) oxalate (Klug and Jaeckh, 1999), suggestingthat embryonic OX levels in vivo are too low to cause signif-icant toxicity to the embryo.

Consistent with GA being the proximate developmentaltoxicant, the location of the shift in GA metabolism along thedose response spectrum corresponds quite well with the dose

response for developmental toxicity in the rat, as previouslydiscussed (NOEL 500 mg EG/kg; LOEL 1000 mg EG/kg;Carney et al., 1996, 1999; Neeper-Bradley et al., 1995). Themean Cmax GA blood levels from the 2500 and 1000 mgEG/kg dose groups were 452 and 363 g GA/g blood, respec-tively, indicating that very high maternal blood levels of GAare required to cause developmental toxicity. These valuescorrespond with about 5.9 and 4.8 mM GA, respectively,falling between the concentrations resulting in abnormal em-bryos (12.5 mM GA) and normal embryos (2.5 mM) fromwhole embryo culture studies in vitro (Carney et al., 1996;Klug and Jackh, 1999). Recent evidence also has shown thatlevels of GA in rat exocoelomic uid are almost 2-fold higherthan in maternal rat blood, such that the critical level of GAexposure to the embryo may be closer to 1012 mM followingEG doses at or above 1000 mg/kg (Carney et al ., 1998). To putthese GA levels into perspective, clinical studies of human EGintoxications associated with intentional ingestion showedpeak GA blood levels of 729 mM (Jacobsen et al., 1984).However, blood GA levels associated with normal handlingand use of EG would not be expected to even approach thesevalues, based on the slower dose-rate for dermal and inhalationexposures and the consequently low likelihood of saturatingEG metabolizing enzymes.

In summary, the salient ndings of this study were: (1) the

shift in GA kinetics previously seen in NP rats also occurs inP (GD 10 11) rats; (2) the onset of this shift occurs at a gavagedose level of 150500 mg EG/kg; (3) the correspondence of the shift in GA kinetics with the NOEL and LOEL for devel-opmental toxicity adds further evidence supporting GA as theproximate developmental toxicant; and (4) developmental tox-icity appears to require maternal blood GA levels in the mil-limolar range. Achievement of such high GA levels seemsplausible only for high dose, oral bolus exposures to EG, but is


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extremely unlikely for typical human occupational and ambientexposures.

ACKNOWLEDGMENTS

Expert technical assistance in conducting this study was provided by A.Liberacki, A. Wardynski, K. Gibson, C. Thornton, J. Whalen, J. Ormand, B.Kropscott, J. Hammond, and F. Lee. Expert analytical support was provided byD. McNett, D. Markham, and K. Engle. We are also grateful for the veterinaryexpertise of J. Lacher. Financial support was provided by The MonoethyleneGlycol Sector Group of CEFIC, and the Ethylene Glycol Panel of TheAmerican Chemistry Council.

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