FUNDAMENTAL AND APPLIED TOXICOLOGY 7,547-565 (1986)

Chronic Toxicity and Oncogenicity Studies of Ethylene Glycol in Rats and Mice’

LINVAL R. DEPASS,*,* ROBERT H. GARMAN,* MURRAY D. WOODSIDE,* W. ELLIS GIDDENS,~?~ ROBERT R. MARONPOT,*,~ AND CARROL S. WEIL*

*Bushy Run Research Center, Mellon Institute-Union Carbide Corporation, Export, Pennsylvania 15632; and TBiomed Research Laboratories, Seattle, Washington 98195

Chronic Toxicity and Oncogenicity Studies of Ethylene Glycol in Rats and Mice. DEPASS,

L. R., GARMAN, R. H., WOODSIDE, M. D., GIDDENS, W. E., MARONPOT, R. R., AND WEIL, C. S.

(1986). Fundam. Appl. Toxicol. 7,547-565. These studies were performed to assess the chronic toxicity and oncogenicity of ethylene glycol (EG) in rats and mice. Groups of 130 Fischer 344 rats and 80 CD-l mice per sex were fed diets yielding approximate dosages of 1.0,0.2, or 0.04 g/kg/day of EG. Two separate control groups in each study received no EG. Mortality rate was increased in high-dose male rats all of which died by 475 days. The following effects were also observed in high dose male rats: reduced body weight gain, increased water intake, increased blood urea nitrogen and creatinine, reduced erythrocyte count, reduced hematocrit and hemo- globin, increased neutrophil count, increased urine volume, reduced specific gravity and pH. Urinary calcium oxalate crystals and increased kidney weight were seen in all high-dose rats. Uric acid crystals were seen in the urine of high-dose female rats at 18 and 24 months. Histopath- ologic changes in high-dose male rats included tubular cell hype@.& tubular dilation, peritu- bular nephritis, parathyroid hyperplasia, and generalized soft tissue mineralization. Fatty change of the liver was seen in high- and intermediate-dose female rats. No clinical signs, or gross or microscopic evidence of toxicity was seen in mice at the dosages used. Water intake and clinical pathologic parameters were not measured in the mouse study. In these studies there was no evidence of an oncogenic effect of EG in rodents. 0 1986 society of Toxicology.

Ethylene glycol (EG) is an important indus- trial chemical which has been used as an anti- freeze and coolant for motor vehicles, in heat exchangers, hydraulic fluids, and as an inter- mediate in the production of glycol esters, ethers and other products. Due to its low vol- atility, there is relatively little potential vapor hazard at ordinary temperatures. However, because of its use in antifreeze mixtures, as a

hydraulic fluid and as a heat exchanger, it may be encountered as a vapor or mist, par- ticularly at elevated temperatures. Oral expo- sure by deliberate or accidental ingestion has also occurred (Balazs et al., 1982; Parry and Wallach, 1974) and is considered a severe problem for veterinarians. Production of EG in the United States in 1980 was estimated to be 4.4 billion pounds (U.S. International Trade Commission, 198 1).

’ Presented in part at the 24th Annual Meeting of the Numerous studies have been performed to

Society of Toxicology in San Diego, March 18-22,1985. determine the potential toxicity of EG and to ’ Present address: Svntex Research, 3401 Hillview elucidate the mechanisms involved (Balazs et

Ave., Palo Alto, Calif. 94304. al., 1982; Parry and Wallach, 1974). The po- 3 Present address: Division of Animal Medicine,

School of Medicine, University of Washington, Seattle, tentia.l chronic toxicity and carcinogenicity

Wash. 98 195. have been assessed in at least four published

4 Present address: National Institute of Environmental studies (Morris et al., 1942; Blood et al., Health Sciences, Research Triangle Park, N.C. 27709. 1962; Blood, 1965; Mason et al., 197 1). Al-

541 0272-0590186 $3.00 Copyri&t 8 1986 by the Society of Toxichw. All rights of qmduction in my form reserved.

548 DE PASS ET AL.

though none of these studies provided evi- dence of carcinogenic activity, there have been more recent reports of mutagenic activ- ity in plants (Bose and Naskar, 1975; Alam et al., 198 1) and mammalian cells (Conan et al., 1979; Brown et al., 1980).

The present studies were performed to more definitively assess the potential oncoge- nicity and chronic toxicity of EG by using much larger numbers of rats than had been used in the previous studies with this species (Morris et al., 1942; Blood, 1965; Mason et al., 197 1). In addition, a second species (the mouse) was used which had not been tested in previous studies.

METHODS

Ethylene glycol. Fifty-five gallons of polyester grade EG were supplied by Union Carbide Corporation, Hahn- ville, Louisiana. The sample was analyzed to be 99.93% EG at the time of shipment, which was approximately 1 year prior to the start ofthe rat study and 2 1 months prior to the start of the mouse study. The diethylene glycol concentration was less than 0.005% by weight. The sam- ple was analyzed again after approximately 38 months of storage at the laboratory. Silylation gas chromatography revealed that the sample contained 99.82% EC and 0.18% diethylene glycol.

Diet preparation. Ethylene glycol was added to ground Purina Laboratory Chow 5008 at concentrations pro- jected to produce dosages of 1.0, 0.2, or 0.04 g/kg/day. Dietary EG concentration was adjusted every 2 weeks for the first 438 or 580 days in the mouse and rat studies, respectively, according to the most recent group mean body weight and diet consumption data. The stability of EG in the test diets was demonstrated using standard gas chromatographic procedures. No significant degradation was observed after 73 days of storage.

Animals and husbandry Young adult Fischer 344 rats and Charles River CrlCOBS CD 1 (ICR)BR mice from Charles River Breeding Laboratories, Wilmington, Mas- sachusetts, were used in these studies. The rats were approximately 38 days old at study initiation. The mice were approximately 42 days of age at the start of the study. Rats were housed in suspended stainless-steel wire front and bottom cages, three males or five females per cage. Mice were housed in similar cages, two per cage for the first year and singly thereafter. Water was provided by an automatic system with demand controlled valves in each cage. Ground diet was supplied ad libitum in opal glass jars for the rats and in stainless-steel feeders for the mice. Animals were identified by toe clips.

Experimental design and observations. One hundred thirty rats and eighty mice of each sex were assigned ran- domly to each of five treatment groups. Two untreated control groups were used in each study in addition to the three EG-treated groups to provide an estimate of the variation between two groups treated alike. The animals were examined daily for clinical condition, and weighed every 2 weeks for the first year and monthly thereafter. Food consumption was recorded every 2 weeks for 16 cages in each group until the body weights stabilized and dietary concentration of EG was held constant. Rat water consumption was measured at 12,18, and 24 months for 10, 20, and 10 animals/sex/group, respectively. Except for the 24-month sacrifice, these animals were chosen randomly at the start of the study and were not replaced by others when deaths occurred.

Blood samples were collected by retroorbital sinus puncture under methoxyflurane anesthesia. Clinical chemistry and hematology measurements were per- formed only on the rats. Evaluated were 10, 10, 20, and 10 rats/sex/group from the 6-, 12-, 18-, and 24-month sacrifices, respectively. Total bilirubin, serum urea nitro- gen, glucose, alkaline phosphatase, serum glutamic oxa- loacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), serum albumin, cmatinine, and calcium were measured using the Centrifichem centrifu- gal analyzer (Baker Instruments, Pleasantville, N.Y.). Hematologic measurements and calculations included RBC count, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscu- lar hemoglobin concentration, total WBC, and differen- tial counts performed on a Coulter Counter S-Plus (Coulter Electronics, Inc., Hialeah, Fla.).

Urinalysis included measurements of urine volume, pH, specific gravity, protein, glucose, ketones, biirubin, occult blood, and nitrate. Specific gravity was deter- mined with a clinical refractometer (American Optical Co., Buffalo, N.Y.). The other measurements were made with the Ames Multistix Reagent Strip read on the Clini- tek (Ames Division, Miles Laboratories, Elkhart, hid.). Samples were also examined grossly for color and turbid- ity, and microscopically for phosphate, calcium oxalate, uric acid, and amorphous crystals, as well as for blood and epithelial cells, spermatozoa, bacteria, and yeast. The types of crystals were identified by standard morpho- logic criteria (McNeely, 1980).

The duration of each study was 24 months. Ten rats per sex per group were sacrificed at 6 and 12 months and 20 per sex per group were sacrificed at 18 months. Twenty mice per sex per group were sacrificed at 80 weeks of treatment. Complete necropsies were per- formed on all animals except when precluded by autoly- sis or cannibalism. Weights of the liver, kidneys, spleen, heart, brain, lung, and testes were recorded in the rat study from all animals at the interim and terminal sacri- fices. Tissues were preserved in 10% neutral buffered For- malin and processed (paraffin technique) for histologic

examination. Microscopic examination was performed on tissues from ail major organs in the high-dose and control groups plus gross lesions and target tissues from the intermediate- and low-dose groups. The list of tissues evaluated histologically included pituitary, thyroid, para- thyroids, adrenals, heart, spleen, mesenteric lymph node, trachea, lungs, ovaries, oviduct, submandibular salivary gland, esophagus, stomach, duodenum, jejunum, ileum, colon, liver, pancreas, brain, spinal cord (lumbar), uterus, testes, epididymides, prostate, kidneys, urinary bladder, eyes, skin, skeletal muscle, femur, sternum, and gall bladder (in mice). All tissues were examined from animals that died or were sacrificed moribund during the study.

TABLE 1

WATER CONSUMPTION DATA (MEANS + SD)

OF MALE RATS AT 12 MONTHS

Dosage of ethylene glycol Volume Wg/day) (ml/rat/dw)

1.0 41.6 f 6.8“ 0.2 17.7 + 3.8 0.04 15.1 -t_ 3.8 O.OA 16.8 + 3.8 O.OB 20.3 + 2.8

Statistical analyses. For continuous data, the analysis Note. O.OA and O.OB, control groups.

of variance (AOV) validated by Bartlett’s test for homo- ’ Significantly different from controls (p < 0.001).

geneity of variances was used. If AOV was significant, differences from control groups were evaluated by Dun- can’s multiple range test if variances were homogeneous or by appropriate t tests, if variances were heterogeneous. Water Consumption Frequency data were evaluated by x2 or Fisher’s exact tests. Mortality and tumor incidence were evaluated by There was no significant difference be- life-table techniques (Sachs, 1959; Mantel, 1966; Bres- low, 1970). When comparisons were made between indi-

tween the water intake of EG-treated female

vidual dose groups and control groups, the latter were rats and their controls at any time. However, almost always considered as separate groups without at the first measurement (at 12 months), the pooling of the control data. Exceptions are mentioned in high-dose male rats drank approximately the text or tables. twice as much as the controls (Table 1). All

high-dose males had died by the 18-month measurement period. The water consump-

RESULTS tion of male rats that received 0.2 or 0.04 g/ kg/day of EG was not affected by EG treat- ment at any time.

Dosage and Diet Consumption

Body Weights The dosage of EG calculated from the body

weight and diet consumption data using the The weight gain of EG-treated mice was nominal concentrations of EG in the diet similar to that of controls throughout the were always within 10% of the dosage goals. study. As shown in Table 2, the weight gain There were no significant differences between of male rats was normal for the first 6 months the diet consumption of the EG-treated and of the study. However, by the end of the first the control rats or mice at any time. year the high-dose males had gained substan-

The nominal concentrations of EG in the tially less weight than either control group. test diets ranged from 0.01 to 0.05, 0.07 to This difference continued until the death of 0.24, and 0.35 to 1.27% in the low-, mid-, and the last surviving high-dose male in the 15th high-dose groups, respectively, in the mouse month of the study. study. The concentrations used in the rat In contrast to the males, average weight study were substantially higher with values of gain among the high-dose female rats was sta- 0.1, 0.5, and 2.5% for the low-, mid-, and tistically higher than that of the controls at six high-dose groups, respectively, during the last and at 12 months into the study. However, 5 months of the study when the concentra- the relatively small difference from the con- tions were constant. trol means and the absence of a clear dose-

TOXICITY OF ETHYLENE GLYCOL IN BATS AND MICE 549

550 DE PASS ET AL.

TABLE 2

MEANS + SD OF BODY WEIGHT GAIN IN MALE AND FEMALE RATS

Dosage of ethylene glycol k/k/day) 6 Months

Weight gain (g)

12 Months 18 Months 24 Months

Male rats 1.0 0.2 0.04

O.OA O.OB

Female rats 1.0 0.2

0.04 O.OA O.OB

284k29 298+48' -e - 288k30 356+31 386+46 383+56 281k32 351+47 386+41 385+50 280+20 353k21 387+34 391+34 281+2-l 349+33 381245 381~~54

118~10" 153k18' 199230 23O-t30 116 + 13 146k 19 198232 231+34

117-tl3 153f22d 202k31 234-t31 114+ 10 145 k 15 193k22 226+41 115+ 9 1462 15 192 f 25 224k32

Note. O.OA and O.OB, control groups. ’ p < 0.0 1 vs control A; p < 0.05 vs control B. ’ p < 0.00 1 vs both control groups. ’ p < 0.00 1 vs control A; p <: 0.0 1 vs control B. d p < 0.0 1 vs both control groups. e Last male died before 18 months.

response relationship suggest that the differ- ence observed was adventitious.

Hematology

Hematology data for female rats revealed no changes of physiological importance.

TABLE 3

TWELVE-MONTH BLEND UREA NITR~CEN (BUN) AND CREATNNE DATA (MEANS + SD) OF MALE RATS

Dosage of ethylene glycol

WWW Creatinine

@g/W

1.0 82+65" 2.2 t 1.8' 0.2 20+ 1 0.4 + 0.2 0.04 20* 1 0.4to.1 O.OA 20* 1 0.4 f 0.2

O.OB 20+ 2 0.6 50.1

Note. O.OA and O.OB, control groups. n Significantly different from both control groups (p

<0.05).

Among the males, statistically significant changes in RBC count (p < 0.0 I), hematocrit (p < 0.05), hemoglobin concentration (p < 0.05), and neutrophil count (p < 0.05) oc- curred in the high-dose group at 12 months. The high-dose means for these parameters were 6.0 X 106/mm3, 32.7’S, 14.0 g/dl, and 3239, respectively, compared with control averages of 7.1 X 106/mm3, 38.6%, 16.6 g/dl, and 2004 (combined control data). No effects were seen at the lower dose levels or in any other hematologic parameter at any dose level at any time.

Serum Chemistry

No changes occurred in female rats, but in high-dose males, urea nitrogen and creati- nine showed fourfold mean increases com- pared to controls after 12 months of treat- ment (Table 3). No changes were observed at the lower dose levels or in any other clinical chemistry measurement at any dose level at any time.

TOXICITY OF ETHYLENE GLYCOL IN RATS AND MICE 551

Urinalysis

No treatment-related changes occurred at 6 months, but at 12 months there were statis- tically significant increases in urine volume (p < 0.001) and reductions in urine specific gravity (p < 0.001) and pH (p < 0.01) in the high-dose male rats. High-dose means were 29.0 ml/rat/day, 1 .016, and 6.0, respectively, for the variables listed compared with 7.0 ml/ rat/day, 1.063, and 7.1 for the controls. The only change Seen in female rats at 12 months was a reduction in mean pH at the highest dose level, 7.0 versus 7.4 in controls (p < 0.05). No treatment-related effects were re- corded at 18 months, but at 24 months, high- dose female rats had significantly lower mean urine volume (p < 0.01) and higher specific gravity (p < 0.05) than controls. The high- dose means were 6.7 ml/rat/day and 1.048, respectively, for the variables listed versus 12.2 ml/rat/day and 1.038 for the controls. The toxicologic significance of the 24-month female rat data is questionable in that the control means were much higher than the means of those parameters at earlier time in- tervals, including 18 months. No EG-related changes were seen in these parameters at lower dosages or in any other parameter ex- cept for a slight reduction (p < 0.05) in urine specific gravity in male rats that received 0.2 g/kg/day of EG. The mean was 1.044 for the EG-treated group versus the control mean of 1.052 at the 24-month interval.

Microscopic examination of rat urine sam- ples for crystals yielded the following results (Table 4). After 12 months of treatment, urine samples from high-dose male rats con- tained no triple phosphate crystals in contrast to the samples from all other dose groups which had many such crystals. Calcium oxa- late crystals were found in urine samples from all but one of the high-dose animals of both sexes. Oxalate crystals were not found in samples from most of the other animals on study. At 18 and 24 months, all but one of the high-dose female specimens contained calcium oxalate crystals. There was also an

increased incidence and amount of uric acid crystals in high-dose females at those times. At 0.2 g/kg/day, there was an increased inci- dence and amount of oxalate crystals at 24 months in animals of both sexes.

Mortality

Mortality curves for male and female rats and mice are presented in Fig. 1. Among the female rats and mice of both sexes, the mor- tality rate was equivalent for all dose groups. However, the high-dose male rats had a statis- tically significant (p < 0.00 1) increase in mor- tality rate from the 9th through the 16th month of the study (Fig. 1). The last high- dose male rat died after 474 days of study.

Organ Weights

Rat kidney and liver weights were affected by EG treatment at various times during the study. At the 6-month sacrifice, male and fe- male rat absolute and relative (to body weight) kidney weights in the high-dose group were significantly higher than those of the controls (Table 5). At the 12-month sacri- fice, the high-dose males were still affected but the females were not. In addition, abso- lute and relative liver weights were reduced in the high-dose males compared to controls. At the l&month sacrifice, there were no effects in the males at 0.2 and 0.04 g/kg/day, but the high-dose female kidney weights were higher than those of their controls. Although the or- gans were weighed at the 24-month sacrifice, no statistical comparisons were made be- cause of the relatively large numbers of tu- mors present. There were no biologically sig- nificant changes in the weights of the other organs at any time.

Histopathology

Rats. Nonneoplastic histopathologic le- sions are presented in Table 6. This is a sum-

552 DE PASS ET AL.

TABLE 4

RESULTS OF EXAMINATION OF RAT URINARY CRYSTALS AT 12,18, AND 24 MONTHS

Triple phosphate Calcium oxalate Uric acid Dosage

h&/day) 12 18 24 12 18 24 12 18 24

Male rats” 1.0 O(O)b -c -c +I (9) - - O(0) - - 0.2 +3(10) +2(19) 0 (3) 0 (4) 0 (0) +I (6) 0 (0) 0 (0) 0 (0) 0.04 +3 (10) +2 (20) +1(7) O(l) 0 (0) 0 (2) 0 (0) 0 (0) 0 (0) O.OA +3 (IO) +2 (20) +1(b) O(l) 0 (0) 0 (2) 0 (0) 0 (0) 0 (0) O.OB +3 (9) +2 (18) +I (6) 0 (0) 0 (0) 0 (2) 0 (0) 0 (0) 0 (0)

Female rats 1.0 +3 (10) +1(18) +1(b) +2 (10) +3 (20) +3 (9) 0 (0) +1(13) +l (8) 0.2 +3(10) +2 (20) +1 (6) 0 (0) O(l) +1(b) 0 (0) 0 (0) 0 (0) 0.04 +2 (10) +2 (18) +1(9) 0 (0) 0 (0) O(l) 0 (0) 0 (0) 0 (0) O.OA +3 (10) +2(18) +I@) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) O.OB +3 (10) +2(18) +1(8) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

Note. O.OA and O.OB, control groups. a Ten specimens per sex per group were examined at 12 and 24 months, 20 at 18 months. bThe first number in each column is the median amount of crystals present on a scale of 0 to +3; +I = few,

+2 = moderate number, f3 = numerous; numbers in parentheses are the number of animals showing crystals. c There were no high-dose male survivors at the 18 and 24 month periods.

mary of the most important lesions observed in the rats that were sacrificed at 6, 12, 18, and 24 months plus those that died or were sacrificed moribund during the study. For ease of presentation the data are not listed by time of appearance of the lesion. However, the time of appearance will be discussed in the text of this section.

At the 6-month sacrifice, the incidence of the following renal lesions was significantly increased in the high-dose males: tubular hy- perplasia, tubular dilation, peritubular ne- phritis, and calcium oxalate crystalluria. Not statistically significant but interesting be- cause of their rarity were two cases of granu- lomatous nephritis; also, in two high-dose males calcium oxalate crystals were present in the urinary bladder. These conditions were absent in the other male dose groups and in females. Among females there was an in- creased incidence of granulomatous myelitis of the femoral marrow at the highest dose level. Special staining indicated the absence of collagen, reticulin, or calcium oxalate. Finely granular periodic acid-Schiff positive

deposits were observed in many of the le- sions. This condition was not present in EG- treated animals sacrificed later in the study.

At the 1Zmonth sacrifice all high-dose males had chronic nephritis with calcium ox- alate crystalluria and 50% had oxalate crys- tals in the urinary bladder. The term “chronic nephritis” is used to indicate the presence of multiple severe histopathologic changes in- cluding tubular dilation and proteinosis, glo- men&r shrinkage, tubular cell hyperplasia, and chronic interstitial nephritis. These find- ings were not present in males at the lower dose levels or in females.

By the time of the 1 S-month sacrifice, all of the high-dose males had died or were sacri- ficed moribund. In most ofthese animals, oxa- late nephrosis was the primary cause of death. Kidneys from these animals had tubular ob- struction by large birefiingent crystals with secondary tubular dilation and degeneration. Calculi were sometimes found within the re- nal pelvic space, ureters, and urinary bladder, often with an associated (secondary) hydrone- phrosis. Associated extrarenal lesions in-

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554 DEPASSETAL.

TABLE5

MEANS (*SD) OF RAT KIDNEY WEIGHTS AT 6,12, AND 18 MONTHS

Dosage of ethylene glycol

WWW

Absolute kidney weight (g) Relative kidney weight (%)

6 12 18 6 12 18

Male rats 1.0 2.74 kO.34" 3.43 f 1.54 -c 0.70 + 0.06d 0.84 f 0.28' -c 0.2 2.29 kO.27 2.87 k 0.28 3.15 +- 1.20 0.61 kO.04 0.65 + 0.02 0.65 too.02 0.04 2.23 kO.12 2.62 kO.08 2.84 k0.36 0.6OkO.03 0.64 kO.02 0.6OkO.04 O.OA 2.26 kO.20 2.80 k0.21 2.72kO.19 0.61 +0.03 0.62 kO.04 0.59 -to.03 O.OB 2.38 kO.35 2.67 20.10 2.95 kO.67 0.60 20.03 0.62 f 0.03 0.64 zkO.13

Female rats 1.0 1.46 k 0.06” 1.66 20.03 1.93 kO.176 0.73 f 0.03@ 0.72 kO.03 0.70 +0.04 0.2 1.34kO.10 1.66 kO.09 1.79 kO.14 0.68 kO.04 0.72 +0.07 0.66 +0.04 0.04 1.42kO.15 1.60 + 0.09 1.83 20.14 0.67 kO.03 0.7 1 f 0.03 0.66 rto.05 O.OA 1.31 kO.06 1.59 *0.09 1.81 20.11 0.69 kO.03 0.71 +0.04 0.67 f 0.03 O.OB 1.37 kO.08 1.61 kO.12 1.81 kO.11 0.67 kO.02 0.70*0.05 0.65 +- 0.04

Note. O.OA and O.OB, control groups. ’ p -c 0.0 1 vs both control groups. b p -C 0.05 vs both control groups. ’ Last male died before 18 months. dp < 0.001 vs both control groups.

eluded cellular hyperplasia of the parathyroid glands and metastatic calcification within nu- merous tissues, especially the gastric glandular mucosa, vasculature, heart, smooth muscle, and renal tubular and glomerular basement membranes. Among the high-dose males, there was also a significant increase in the inci- dence of splenic hemosiderosis, a commonly occurring lesion in the rat.

Among the females sacrificed at 2 years, the incidences of hemosiderosis of the mesen- teric lymph node (1.0 g/kg/day), and mild fatty metamorphosis of the liver (1 .O and 0.2 g/kg/day) were significantly increased over the control incidence. No biologically sig- nificant lesions were noticed in the male rats sacrificed at 2 years.

The frequencies of primary neoplasms are presented in Table 7. The only tumor type for which there was a statistically significant (p < 0.05) difference was fibroadenoma of the female mammary gland at the 0.04 g/kg/day dose level. Because of the absence of an effect at the two higher dose levels, it is most proba-

ble that the difference was the result of chance and unrelated to ethylene glycol treatment.

Mice. Although the mouse study was de- signed primarily as an oncogenicity study, nonneoplastic lesions were diagnosed and re- corded. These lesions are not presented here for the sake of brevity and because there were no treatment-related increases. Renal tubular degeneration occurred slightly more fre- quently in high-dose females than in controls at the 1 g-month sacrifice (5/20 vs l/40) but was absent in the 15 high-dose females sacri- ficed at 24 months.

Primary neoplasms are presented in Table 8. The only tumor type for which there was any evidence of a possible increased inci- dence was lymphosarcoma in females. Statis- tical analysis revealed no significant differ- ence among groups when the unadjusted or time-adjusted proportions of animals with lymphosarcoma were compared. When the data were analyzed to detect a dose-related trend, the unadjusted trend test and Mantel- Cox trend test yielded p values of approxi-

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phos

is

Mon

onuc

lear

ce

ll in

hhra

tes

Hep

atic

gra

nulo

mas

/his

tiocy

tosi

s Lu

ngs

Min

eral

izatio

n,

inte

rstit

ial

Mes

ente

ric l

ymph

nod

e H

emos

ider

osis

Pa

rath

yroi

ds

Cel

lula

r hy

perp

lasi

a St

omac

h M

iner

aliza

tion

Vasc

ular

M

iner

aliza

tion

70/l

lob

l/l10

51/l

10”

O/l

10

10/l

16

1011

29

6/l

16

7112

9 10

/116

” l/l2

9 2/

l 16

O

/l29

72/l

16”

3112

9 95

/l 16

’ O

/l29

16/l

166

O/1

29

5/l

16

1071

129

2189

21

89

lo/l2

9 41

129

O/1

29

O/l2

9 O

/129

O

/129

O

/129

10

3/12

9

65/1

09b

l/113

l/8

9

4519

06

3/10

1 31

84

7211

07’

l/l07

l/89

5817

8 b

2175

21

71

l/l28

O/1

28

lo/l2

8 21

128

O/l2

8 O

/l28

l/128

O

/l28

O/l2

8 10

6112

8

l/127

l/l13

l/l26

o/9

1

l/127

O

/127

1 l/1

28

4112

8 O

/l28

O/1

28

4/ 1

28

O/l2

8 O

/128

10

6112

8

l/127

l/l15

2112

8

o/90

8112

6 25

185’

61

89

7112

6 51

127

8147

31

20

4123

41

5 1

4155

2112

8 l/l2

5 91

128

9112

5 O

/l28

O/l2

5 O

/l28

O/1

25

3112

8 21

125

2112

8 O

/l25

O/1

28

O/1

25

1041

128

9611

25

7112

9 lo

/129

O

/129

O

/l29

O/1

29

O/l2

9 O

/l29

9411

29

4112

8 81

128

O/l2

8 O

/l28

2112

8 O

/128

O

/128

10

0/12

8

O/1

28

7112

8 O

/l28

O/1

28

2112

8 O

/l28

O/1

28

9911

28

35/1

2gd

3511

28’

10/1

28’

2711

25

1611

29

2911

25

2011

29

3112

5 10

/129

’

17/1

28

1711

28

1711

28

3011

28

2112

8 O

/l28

1211

28

O/8

0 61

84

6/11

8 51

121

Not

e. A

and

B, c

ontro

l gr

oups

. ,I

Num

erat

or

equa

ls nu

mbe

r of

rats

with

sp

ecifi

ed f

indi

ng.

Den

omin

ator

eq

uals

num

ber

of ra

ts f

or w

hich

sp

ecifi

ed o

rgan

was

exa

min

ed.

b p <

0.0

01; ‘

p <

0.05

; dp

< 0.

0 1.

Pro

babi

lity

valu

es w

ere

obta

ined

us

ing

the

com

bine

d co

ntro

l da

ta.

Org

an/fi

ndin

g

TABL

E 7

FREQ

UENC

V OF

PRI

MAR

Y NE

OPLA

SMS

OBS

ERVE

D IN

F34

4 RA

TS

Ethy

lene

giy

col

in d

iet

(g/k

g/da

y)

Mal

es

Fem

ales

1.0

0.2

0.04

0.

0 (A

) 0.

0 (B

) 1.

0 0.

2 0.

04

0.0

(A)

0.0

(B)

Abdo

min

al

cavi

ty (s

ee a

lso p

erito

neum

) Pa

raga

nglio

mas

Le

iom

yosa

rcom

a C

arci

nom

a,

poor

ly d

iffer

entia

ted

Sarc

oma,

poo

rly d

iffer

entia

ted

Adip

ose

tissu

e Li

pom

a Ad

rena

ls C

ortic

al a

deno

ma

Cor

tical

car

cinom

a Ph

eoch

rom

ocyt

oma

Pheo

chro

moc

ytom

a,

mal

igna

nt

Neu

robl

asto

ma

Gan

ghon

euro

ma

Brai

n Gra

nula

r ce

ll tu

mor

G

liom

a/As

trocy

tom

a C

ervi

x C

ervi

cal

poly

p C

litora

l gl

and

Aden

oma

Car

cinom

a H

eart Schw

anno

ma/

neur

ilem

oma/

ne

urof

ibro

ma

Inte

stin

es

Duo

denu

m

Leio

myo

ma

Jeju

num

Le

iom

yom

a

2122

"

O/11

6

O/l1

6 O

/l16

O/l1

6 O

/l16

O/l0

4

O/l

10

- - - -

414

719

l/109

l/8

7

2311

09

1218

7

o/10

9 l/8

7 O

/l09

l/87

o/10

9 O/

87

O/l2

8 31

129

2111

0 l/8

9

O/l

l/l

6129

4112

2

2411

22

l/122

o/

122

o/12

2

O/l2

8

O/l2

8

6128

3112

4

18J1

24

O/l2

4 O

/l24

l/l24

l/l27

O/l2

7

O/l l/l

8/27

1211

26

O/12

6 31

126

l/l26

l/l27

O

/l27

l/72

l/2

l/2

3112

8

o/12

1

l/l24

O/1

O

/l

O/3

6185

l/8

5

8185

Of85

O/12

3 O

/l23

l/69

2/2

012

l/85

O/80

O/83

O/l

O/l

ll/ll

1218

9 O/

89

3189

O/89

O/12

6 O

/l26

O/72

4J4

O/4 l/89

O/87

O/86

l/l

O/l

4124

9112

6 l/l

26

3112

6

l/l26

O/12

6 31

126

O/69

l/l

O/l

l/l27

O/l2

2

o/12

0

- - 8130

Fz

7112

7 ;r!

O/12

7 g

IO/1

27

i O

/l27

’

0112

7 Of

127

O/66

w

o/2

l/l27

l/l21

o/12

2

Leio

myo

sarc

oma

Ileum

C

arcin

oid

Aden

ocar

cinom

a C

ecum

H

eman

giop

eric

ytom

a C

olon

Ad

enoc

arcin

oma

Kidn

eys

Embr

yona

l ne

phro

ma

Tera

toid

ne

phm

ma

Tubu

lar

aden

oma

Aden

ocar

cinom

a Tr

ansit

iona

l ce

ll ca

rcin

oma

Squa

mou

s ce

ll ca

rcin

oma

Lipo

sarc

oma

Lacr

imal

gl

and,

ext

raor

bita

l Ly

mph

oepi

thel

iom

a Ad

enom

a Li

ver

Neo

plas

tic n

odul

es

Hep

atoc

ellu

lar

aden

oma

Hep

atoc

ellu

lar

carc

inom

a Lu

ngs

Pulm

onar

y ad

enom

a Al

veol

ar

cell

carc

inom

a Ad

enoc

arcin

oma/

carc

inom

a C

hord

oma,

m

etas

tatic

Ly

mph

nod

es, b

ronc

hial

Ly

mph

angi

oma

Lym

ph n

odes

, cer

vica

l Sa

rcom

a, p

oorly

diff

eren

tiate

d Ly

mph

osar

com

a Ly

mph

nod

es, m

esen

teric

Ly

mph

angi

oma

Hem

angi

oma,

ca

vern

ous

Mam

mar

y gl

and

Aden

oma

Fibr

oma

Fibr

oade

nom

a

Of96

O

/l06

l/85

o/12

1 O

/l24

O/96

O/

106

l/85

l/121

O/

124

O/28

O/

78

o/7

1 O

/l 1

l/74

O/l0

3 O

/l03

l/81

O/l

15

o/12

1

O/l

16

O/l2

9 O

/l29

l/l28

Of12

8

O/l1

6 O

/l29

O/12

9 O

/l28

l/l28

O

/l 16

l/l

29

O/l2

9 O

/l28

O/l2

8

O/l

16

l/l29

O

/l29

O/l2

8 l/l

28

Of11

6 O

/l29

O/12

9 O

/l28

2112

8 O

/l16

l/129

O/

129

O/12

8 O

/l28

- - o/11

0 21

129

o/11

0 31

129

O/I

10

l/129

o/10

9

o/10

9 l/1

09

o/10

9

O/68

O/86

o/27

o/

21

l/27

O/4

O/

4 11

2 11

2

o/11

3 o/

113

o/11

3 o/

113

O/l2

9 91

128

5112

8 21

129

O/l2

8 21

128

O/12

9 3/

128

2112

8

l/89

l/l27

O/

127

O/89

l/1

27

Of12

1

3/89

O/

127

2/12

1 O/

89

l/l21

l/l

27

o/95

l/l

9

O/98

Of

19

l/69

O/62

O/

69

O/62

l/6

9 41

62

O/10

2

o/11

3

O/5

5 l/5

5 3/

55

O/l2

8 O

/l25

O/l2

9 O

/l28

l/l28

O/l2

8 l/l

25

O/l2

9 O

/l28

O/l2

8

o/2

012

l/128

O/

125

O/l2

9 O/

128

l/128

l/128

O

/l25

O/l2

9 O

/l28

O/l2

8

l/128

o/

90

l/91

l/l27

O

/l25

l/l28

O/

90

o/91

o/

121

l/l25

O

/l28

l/90

l/91

l/l27

O

/l25

O/12

3 l/8

0 O/

84

O/l1

8 o/

121

l/l03

O

/l16

Of7

7 O/

87

l/l07

O

/l13

O/l1

6 o/

77

O/81

21

101

l/113

O/l2

3 l/8

0 O/

84

O/l1

8 O

/l21

l/l16

O/62

2/

101

l/77

l/84

2110

0 O/

89

O/62

o/

101

Of7

7 O/

84

2110

0 O/

89

Of62

13

/101

13

/v 21

184

12/1

00

9189

O/l2

4 l/8

3 O/

86

O/l2

0 O

/l22

Org

an/fi

ndin

g 1.

0 0.

2

TABL

E l-C

ontin

ued Et

hyle

ne g

lyco

l in

diet

(g/

kg/d

ay)

Mal

es

Fem

ales

0.04

0.

0 (A

) 0.

0 (B

) 1.

0 0.

2 0.

04

0.0

(A)

0.0

(B)

Aden

ocar

cinom

a N

asal

cav

ity

Aden

oma,

po

lypo

id

Cho

ndro

ma,

na

sal t

urbi

nate

Sa

rcom

a, p

oorly

diff

eren

tiate

d O

ral

cavi

ty

Squa

mou

s pa

pillo

ma,

to

ngue

Sq

uam

ous

papi

llom

a,

phar

ynx

Ova

ries

Gra

nulo

sa

cell

tum

or

Panc

reas

D

uct

aden

oma,

pap

illary

Ad

enom

a,

exoc

rine

Isle

t ce

ll ad

enom

a Is

let

cell

carc

inom

a Pa

rath

yroi

ds

Aden

oma

Perit

oneu

m

Mes

othe

liom

a Pl

eura

M

esot

helio

ma

Pitu

itary

Ad

enom

a C

arcin

oma

Prep

utia

l gl

and

Aden

oma

Aden

ocar

cinom

a/ca

rcin

oma

Saliv

ary

glan

d, p

arot

id

Aden

oma

Aden

ocar

cinom

a Sa

rcom

a, p

oorly

diff

eren

tiate

d

o/10

4 O

/104

o/

104

O/9

0

O/8

5 O

/85

111

O/l

O/6

5 O

/98

O/6

5 l/9

8

o/71

o/

7 1

l/72

Of7

2 o/

79

o/79

l/l

- -

l/l07

O/1

07

5110

7

O/8

9 l/8

9 31

89

o/12

1 o/

121

6/12

1

o/10

1 l/8

4

212

o/11

3

515

- l/l

-

19/1

03

l/103

15

188

O/8

8 15

1127

O

/127

415

516

5/6

l/5

0th

116

O/8

1

O/8

1 O

/87

O/8

7

o/7

1 l/7

1 -

o/74

-

O/6

9 -

O/1

26

219

1

O/1

23

O/1

23

3112

3

l/l

15

313

l/126

l/l2

6

o/11

3

l/83

O/8

3

l/78

-

2211

27

36/1

25

3718

9 31

127

2112

5 l/8

9

313

O/3

l/95

o/95

4/10

1

O/9

6

l/77

O/7

6

2184

-

o/73

O/9

2

l/86

O/8

6

O/7

6

4319

3 l/9

3

O/8

4

3110

0 l/8

9

l/l

l/69

-

Of6

8

O/1

24

O/1

23

l/l23

O/1

23

l/120

l/l23

Of1

23

o/10

4

4611

25

4511

25

2112

5 l/1

25

l/96

o/10

1

Sem

inal

ves

icle

s M

esot

helio

ma

Skel

etal

C

emen

tom

a,

man

dibl

e C

hord

oma,

m

alig

nant

, ve

rtebr

a C

hond

rosa

rcom

a,

leg

Skin

Ke

rato

acan

thom

a IS

quam

ous

papi

llom

a Ke

rato

tic

basa

l cel

l epi

thel

iom

a Se

bace

ous g

land

ade

nom

a Fi

brom

a Ba

sal c

ell c

arcin

oma

Seba

ceou

s gla

nd c

arcin

oma

Squa

mou

s ce

ll ca

rcin

oma

Spin

dle

cell

sarc

oma

Mal

igna

nt

fibro

us h

istio

cyto

ma

Sple

en

His

tiocy

tom

a Ly

mph

osar

com

a R

etic

ulum

ce

ll sa

rcom

a Ly

mph

oid

leuk

emia

M

yelo

geno

us

leuk

emia

M

onon

ucle

ar

cell

leuk

emia

St

omac

h Sq

uam

ous

papi

llom

a sl

lbcu

tis

Lipo

ma

Fibr

oma

Fibr

ous

hist

iocy

tom

a An

gios

arco

ma

Spin

dle

cell

sarc

oma

Test

es

Inte

rstit

ial

cell

aden

oma

Thym

us

Thym

oma

Lym

phos

arco

ma

Thyr

oids

Pa

pilla

ry

aden

oma

Fohi

cula

r ad

enom

a

O/l0

3

O/8

8 O

/88

O/8

8 O

/88

O/8

8

O/8

8 O

/88

O/8

8

O/1

08

O/l0

8

O/l0

8 O

/108

o/10

7

O/l

O/l

O/l

O/l

O/l 3110

5

o/9

1

- l/l

o/10

0 o/

100

l/l00

l/l00

l/l00

o/10

0 l/l0

0 o/

100

O/1

29

l/l29

l/l29

1711

29

O/l0

7

l/l8

14/1

8 O

/l8

l/l8

O/l8

9611

30

O/6

9 O

f69

l/l08

O/8

5

- - Of8

0 l/8

0 O

/80

O/8

0 O

/80

O/8

0 l/8

0 l/8

0

O/1

28

l/l28

O/l2

8 91

128

O/8

9

o/13

10

/13

o/13

o/

13

l/13

9411

29

O/6

2 l/6

2

O/8

8

l/94

l/l - l/i20

o/12

0 O

/l20

O/l2

0 o/

120

o/12

0 O

/l20

O/l2

0

O/1

28

l/l28

O/l2

8 lo

/128

l/l26

218

518

O/8

O

/8

O/8

9811

28

l/60

O/6

0

l/l24

O/9

8

- - O/l1

8 21

118

O/l1

8 l/1

18

O/l1

8

l/l18

O/l1

8 O

/118

l/128

l/1

28

O/l2

8 14

1128

O/l2

8

3117

1 l

/l7

l/l7

o/17

o/

17

1001

28

O/6

8 O

/68

l/l26

- O/l2

3

O/l2

3

O/l2

3

O/1

23

o/12

7 l/l2

7 O

/127

9112

7

- - O/6

8 O

/68

l/l25

l/l25

l/l

l/78

O/7

8

l/78

l/78

O/l2

4 O

/l24

O/1

24

9112

4

l/4

314

O/6

0 O

/60

O/8

6 O

/86

- O/8

5

l/85

O/8

5

O/8

5

l/l27

O/l2

7 O

/l27

1011

27

O/3

31

3

O/6

6 O

/66

O/8

8 O

/88

-

O/l1

8

O/l1

8

l/l18

O/1

18

O/l2

6 O

/l26

l/l26

1311

26

O/l II1

o/54

l/5

4

Of1

27

O/l2

7

- l/l28

O/l2

8 O

/l28

lo/1

28

2/66

O

/66

O/l2

3 O

/l23

Org

an/fi

ndin

g 1.

0 0.

2

TABL

E l--

Con

tinue

d Ethy

lene

gly

col

in d

iet

(g/k

g/da

y)

Mal

es

Fem

ales

0.04

0.

0 (A

) 0.

0 (B

) 1.

0 0.

2 0.

04

0.0

(A)

0.0

(B)

Follic

ular

ad

enoc

arcin

oma

Para

follic

ular

ce

ll ad

enom

a Pa

rafo

llicul

ar

cell

carc

inom

a U

rinar

y bl

adde

r Pa

pilla

ry

carc

inom

a Le

iom

yosa

rcom

a Sa

rcom

a, p

oorly

diff

eren

tiate

d M

esot

helio

ma

Ute

rus

Ute

rine

poly

p Le

iom

yom

a Po

lypo

id

aden

oma

Endo

met

rial

carc

inom

a En

dom

etria

l st

rom

al s

arco

ma

Leio

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.

TOXICITY OF ETHYLENE GLYCOL IN RATS AND MICE 561

mately 0.2. However, The Gehan-Breslow trend test yielded a p value of 0.047. The lat- ter test gives greater weight to tumors de- tected early in the study than does the Man- tel-Cox test.

The lymphosarcomas in the highdose fe- males did occur somewhat earlier than those in the control groups. The median times to death with tumor of the high-dose, control A, and control B groups were 546,694, and 567 days, respectively. When the controls were combined the median was 687 days.

Since lymphosarcomas were detected only at death or sacrifice, a dose-related increase in mortality rate unrelated to the presence of lymphosarcoma (if present) could have ac- counted for an apparent reduction in the time to tumor observation. However, there was no evidence of an increase in the mortal- ity rate of ethylene glycol-treated female mice (Fig. 1). In addition, lymphosarcoma, when present, was always considered to be the cause of death, except for one high-dose animal.

DISCUSSION

The present studies were designed to evalu- ate the chronic toxicity and potential oncoge- nicity of ethylene glycol administered in the diet of rats and mice. Male rats were affected far more severely than female rats or mice. The early demise of the high-dose male rats was not expected from the results of 7-day and 90-&y studies (unpublished), which were performed to set dose levels for the pres- ent studies. Our preliminary data showed the presence of mild renal toxicity in male rats after 90 days of treatment with a dose similar to the highest dose used in these studies. In selecting dose levels, we were aware of the sex difference in sensitivity from our data as well as other previously published data. It was our expectation that the highest dose would pro- duce substantial toxicity in male rats, hope- fully without causing excessive mortality, while also causing a lesser degree of toxicity

in female rats and mice of both sexes. At the time these studies were designed, the use of different dose levels for males and females in the same study was not considered scientih- tally sound, although this procedure is now being used by the National Toxicology Pro- gram (Melnick, 1984).

At the highest dose level (1 .O g/kg/day), there was substantial renal toxicity in male rats due, at least in part, to the accumulation of calcium oxalate crystals and calculi in the kidneys. Deposition of oxalate in the kidneys of EG-treated rats and other changes indica- tive of renal toxicity have previously been re- ported (Morris et al., 1942; Blood, 1965; Mel- nick, 1984). The greater susceptibility of males to EG-induced renal toxicity was also reported in the studies cited, and may be the result of more efficient conversion of EG to toxic metabolites including oxalate in male rats (Richardson, 1965), as well as to the more rapid progression of spontaneous ne- phropathy in the male (Elema and Arends, 1975; Bolton et al., 1976). Since the high- dose female rats had significant amounts of urinary oxalate crystals at 12, 18, and 24 months, conversion of EG to oxalate clearly occurred in females. High-dose females also had increased kidney weights at 6 and 18 months. These results suggest that although renal effects of EG were observed in females, the critical factor responsible for the more se- vere nephrotoxicity and lethality in male rats was probably the greater incidence and sever- ity of spontaneous nephropathy in the male.

The deposition of oxalate calculi and the associated chronic nephritis accounted for the increased mortality rate seen in the high- dose male rats. Ultimately these animals lost the ability to concentrate the urine as indi- cated by the polyuria and secondary polydip- sia seen at 12 months. The high-dose males also had generalized soft tissue minerahza- tion which may be explained in part by in- creased loss of calcium as oxalate followed by compensatory parathyroid hyperplasia (ob- served), hypersecretion of parathyroid hor- mone, and increased resorption of calcium

Org

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LL M

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(A)

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TOXICITY OF ETHYLENE GLYCOL IN RATS AND MICE 563

564 DE PASS ET AL.

from bone (Rajagopal et al., 1977). Phos- phate retention, resulting from the oxalate nephropathy, would also be expected to have contributed to the development of hypocal- cemia, with these same compensatory ho- meostatic changes.

The observed reductions in erythroid pa- rameters in high-dose males at 12 months have been previously reported (Patemi et al., 1956). These reductions may have been the result of decreased erythropoietin production by the damaged kidneys.

With regard to primary neoplasms, there was only one tumor type for which there was any evidence of a treatment-related increase in either study. The time-adjusted incidence of lymphosarcoma in female mice was sig- nificantly increased (p < 0.05) according to one of three trend tests. However, compari- sons among groups for differences in tumor proportions indicated that the observed re- sults could easily have occurred by chance alone (p > 0.05).

Because of the difficulty in accurately diag- nosing lymphoreticular tumors, all lesions suggestive of marked hyperplasia or neopla- sia in the mouse study were reviewed by a panel of three experienced pathologists. The results presented here represent a consensus of the original pathologists (J. Boyce and E. Giddens) plus those who later reviewed the slides.

Since there was only one finding of possible equivocal significance for one tumor type among the many tissues that were examined in both studies, it is fair to conclude that this result could have occurred by chance alone and that there was no definitive indication of an oncogenic effect of EG. The difference in time to detection of lymphosarcomata proba- bly reflects the variability in latency in the population studied as suggested by the difference between the two control groups.

The lack of oncogenicity in these large, well-controlled bioassays is consistent with the results of Morris et al. (1942), Blood ( 1965) and Mason et al. (197 1) in the rat and

Blood et al. (1962) in the monkey. Thus, no carcinogenic activity of EG has been demon- strated in any of the three species (rat, mouse, or monkey) studied.

ACKNOWLEDGMENTS

The authors acknowledge the efforts of Dr. J. Boyce for performing part of the histopathology for the mourn study and of Dr. J. M. Ward, Dr. R. A. Squire, and Dr. C. Frith in reviewing the lymphoreticular lesions. We also thank Dr. B. Ballantyne, Dr. W. M. Snellings, and Dr. C. M. Troup for reviewing the manuscript and Sandy Turner for typing it. These studies were funded by the Union Carbide Corporation.

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