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Carcinogenicity

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Description of key information

DePass et al. (1982) identified a NOAEL of 1000 mg/kg/day (without LOAEL) for male and female Fischer rats exposed for 24 months to dietary doses of 0.04, 0.2 and 1.0 mg/kg.

B6C3F1 mice (60/sex/dose) received MEG in the diet for up to 2 years (NTP, 1993). A NOAEL of 1500 mg/kg/day and a LOAEL of 3000 mg/kg/day for liver histopathology were identified in male B6C3F1 mice.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Assessing the potential oncogenicity and chronic toxicity when fed to rats for two years.
GLP compliance:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Housing: in suspended stainless steel wire front and bottom cages, 3 males or 5 females per cage
- Diet: Ground diet, ad libitum
- Water: provided by an automatic dispensing system with demand-controlled values in each cage
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Ethylene glycol was added to pilot batches consisting of ground Purina Lab Chow and the pilot batches were mixed for 15 minutes in a Hobart vertical mixer. Aliquots of the pilot batches then were mixed for 5 minutes with basic Purina Lab Chow to prepare diets containing the percentage necessary to attain the dosage levels 1000, 200 or 40 mg/kg bw/day. Group mean body weights were predicted for the middle of the diet consumption period. This procedure was followed every 2 weeks until the body weights stabilized at 580 days on study. Group mean body weights were derived from the biweekly group/sex mean weight and the diet consumed was derived from diet consumption measurements recorded for the first week in each biweekly period. Thus, percentages in diets were adjusted every 2 weeks at which times fresh diets were prepared.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
24 months
Frequency of treatment:
daily
Post exposure period:
none
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
771 males, 783 females
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
During the study all rats were observed daily for mortality, physical condition and for signs of clinical or behavioral effects.
All animals were weighed every two weeks for the first year and monthly thereafter.
Food consumption was determined every other week on the first 16 cages of rats from each sex and dosage group until the body weights stabilised at 580 doses.
Sacrifice and pathology:
Interim sacrifices were performed at 6, 12 and 18 months. 10 rats per sex per treatment group were sacrificed at 6 and 12 months, and 20 per sex per group at 18 months. Prior to each sacrifice, clinical status was determined by examination of selected haematology, urinalysis and clinical chemistry parameters. Criteria of effect also included mortality, clinical signs, diet and water consumption, body weight change, organ weights, and the incidence of tumors and other pathologic findings.
The following clinical chemistry parameters were measured at approximately 6-month intervals on the animals that were to be sacrificed: total bilirubin, serum urea nitrogen, blood glucose, alkaline phosphatase, serum glutamic oxoacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), serum albumin, creatinine and calcium.
The following hematologic parameters were also measured at approximately 6-month intervals on the animals selected for sacrifice: red blood cell count, haematocrit, haemoglobin mean cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell and differential counts.
Blood samples for clinical chemistry and haematology were collected by retro-orbital sinus puncture from rats under methoxyflurane anaesthesia.
Urinalysis, performed on the same group of animals as above, included measurements of urine volume, pH, specific gravity, protein, glucose, ketones, bilirubin, occult blood and nitrate. Urine samples were also examined for colour, turbidity, presence of phosphate, calcium oxalate, uric acid, amorphous crystals, BBC's, WBC's, urinary epithelial cells, spermatozoa, bacteria and yeasts.
Post-mortem examination: With the exception of a few animals where autolytic change or cannibalization precluded the evaluation of certain organs, rats that died or were sacrificed when moribund were examined for gross anatomic and histologic alterations. The rats selected for sacrifice were anesthetized with methoxyflurane and killed by severing the brachial vessels to permit exsanguination. A complete necropsy was performed on each rat. Weights of the liver, kidneys, spleen, heart, brain, lung and testes were recorded. Tissues were preserved in 10% neutral buffered formalin and processed for histologic examination.
Statistics:
To evaluate the statistical significance of possible changes in continuous data, the analysis of variance validated by Bartlett's test for homogeneity of variance was used. Individual mean differences were identified by Duncan's multiple range test when indicated by a significant F value. In the case of heterogeneous variances, as indicated by Bartlett's test, an F test and Cochran or Student's t-test were used to identify significant differences. Enumerative data were evaluated statistically by use of the NXR Chi-square test, the 2 x 2 Chi-square test correct for continuity or Fisher's Exact Test, where appropriate. Non-parametric data were evaluated statistically by the multiple sum of ranks test. Mortality and tumor incidence were evaluated by life table techniques. The fiducial limit of 0.05 was employed as the critical level of differences not attributable to chance.
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
The data for the male rats show that the cumulative mortality in the high dose males was consistently higher than that of both control groups from the 9th to the 16th month. By 16 months, mortality was 100% for the high level males. Among female rats, there was no increase in mortality associated with the ingestion of EG.
Dead or Moribund Animals: A total of 284 rats were euthanatized when moribund or died during the study. A variety of neoplastic and non-neoplastic lesions were observed in rats from all groups. Those lesions associated with the ingestion of ethylene glycol were found only in the high level male rats and included mineralization of the following organs: cardiac vessels, cardiac muscle, vas deferens, stomach and pulmonary vessels. In addition, other deleterious histologic effects found in these male rats included cellular hyperplasia of the parathyroids, hemosiderosis of the spleen, myocardial fibrosis, portal fibrosis of the liver, bile duct hyperplasia, and hydronephrosis and oxalate nephrosis of the kidneys.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights were actually measured every 2 weeks for the first year of the study, but only monthly values are presented for the sake of brevity. Marked body weight depression, first seen for the high level male rats at 257 doses, recurred in each succeeding weighing period until all the male rats at the high level were dead. No depressions in body weight were observed in any of the other dose groups in either sex for the 2-year period. Because of greatly increased mortality prior to one year of doses, weights for the high level males were not included.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The dosages attained closely approximated the dosage goals. None of the 6 -month diet consumption means of the dosage groups differed significantly from those of the controls. Therefore, diet consumption was not altered by the ingestion of EG.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was measured during 3 intervals of the study, at approx. 365, 547 and 730 doses. At 365 doses, water consumption in the high dose males was significantly higher than that of control group A. This increase in water consumption is probably associated with increased urine volume, renal degeneration and consequent inability of the rate to concentrate urine by reabsorbing water or the impairment of renal tubule reasorption of water. At 547 doses, a slight reduction in water consumption was seen for the males at the low dosage level, but a similar reduction also was present in control group A relative to control group B. Water consumption for the female rats was not affected by treatment.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
EG has shown to have a deleterious hematopoietic effect in both sexes at the high dose level. In the males such effects included: Significant decrease in red blood cells, hematocrit, hemoglobin and mean cell volume at 12 months; significant increase of neutrophils at 12 months. Effects found in the females included: Significant decrease in mean corpuscular hemoglobin concentration at 24 months (730 doses); significant increase in mean cell volume at 24 months. In the intermediate dose level females, marginal increases in red blood cells, hematocrit and hemoglobin were found at 24 months.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following determinations for male and female rats were made: total bilirubin, serum urea nitrogen, blood glucose, alkaline phosphatase, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), serum albumin, creatinine and calcium. At 12 months, SGPT was reduced significantly while serum urea nitrogen and serum creatinine were increased significantly in the high level male rats.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Significant alterations of urine parameter were induced by the ingestion of EG in both sexes at the high dose. Deleterious effects for the males included: Significant increase in urine volume and calcium oxalate crystals at 12 months. At necropsy, calculi analyzed as calcium oxalate were recovered from the kidneys, ureters and urinary bladders of these rats. Significant decrease in urine-specific gravity and pH at 12 months. A decrease in urine-specific gravity was observed for the intermediate level male rats at 24 months of exposure was additional evidence for the renal effect of EG.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
No statistical comparisons were made among the ethylene glycol-treated and control groups sacrificed at 24 months since many of the organs had tumors or other age-related lesions. It is expected that any chronic toxic effects of ethylene glycol on individual organs would have been observed in the animals sacrificed at 6, 12 or 18 months when age-related changes were less pronounced. The effect of EG on the organ weights was observed only in the high dose level in both sexes. In males such effect included: Significant increase in absolute and relative kidney weights at 6 months (180 doses); significant reduction in absolute and relative liver weights at 12 months (365 doses); significant reduction in absolute lung weight at 12 months; significant increase in relative kidney, heart and brain weights at 12 months. In the female rats, such effects included: Significant increase in absolute and relative kidney weights at 6 months; significant increase in absolute kidney weight at 18 months (547 doses). A limited number of transient weight changes seen for liver and spleen were deemed to be of no biological importance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
6-month interim sacrifice: Statistically significant incidences of renal lesions were found in the high level male rats. These lesions included tubular cell hyperplasia, tubular dilation and peritubular nephritis. An increased frequency of focal granulomatous nephritis, while not statistically significant, was most probably related to calcium oxalate crystal deposition in the kidneys.

12-month interim sacrifice: Biologically and statistically significant incidences of renal lesions were found in the high level male rats. These lesions included renal calcium oxalate crystalluria with associated chronic nephritis as well as calcium oxalate calculi in urinary bladders and ureters. No other treatment-related changes were found in male or female rats.

18-month interim sacrifice: Treatment of male Fischer 344 rats with 1.0 g/kg/day of EG resulted in the death of all animals prior to the 18-month sacrifice. These deaths were attributable to oxalate nephrosis. Comparison of lesion prevalence between the dosage groups and their controls revealed a few treatment-related increases. These included increased interstitial cell hyperplasia of the testes (0.2 and 0.04 g/kg/day males) and increased hepatocellular fatty metamorphosis (0.04 g/kg/day males). Both lesions are common in the F344 rat and are, therefore, considered to be of no biological importance. A variety of tumor types, none of which was treatment-related, were found in the rats sacrificed at 18 months.

24-month sacrifice: A variety of neoplastic and non-neoplastic lesions were observed in the rats sacrificed at 24 months. When the incidence of given lesions appeared to be different in the ethylene glycol-dosed group than in the control groups, statistical comparisons were made. In comparing tumor incidence, a life table method which included all the animals in the study, rather than only those which were sacrificed at 24 months, was used. Among male rats in the intermediate and low dose, the following lesions were considered to be related to treatment: mineralization of the pulmonary vessels, mineralization of the seminiferous tubules and mineralization of the optic sclerae. Among the female rats at the high dose, the frequency of hemosiderosis of the mesenteric lymph nodes was marginally higher than that of the controls.
Furthermore, a marked increase in the frequency of fatty metamorphosis of the liver concomitant with an increase of mononuclear cell infiltrates was observed in these high level females. Marginal changes found in the intermediate dose females included increased fatty metamorphosis of the liver accompanied by increased mononuclear cell infiltrates as well as an increase in the incidence of myocardial fibrosis. Mineralization of the pulmonary vessels and optic sclerae was of moderate significance. In the low dose females, marginal increases in thymus epithelial cell hyperplasia and hepatic granuloma were found. A moderate increase in mineralization of the optic sclera was observed also. The incidence of mononuclear cell leukemia was statistically higher in the intermediate level male rats that in group group A, but not higher when compared with the pooled controls. This difference deemed to be of toxicological importance for the following reasons: This difference was marginally significant with respect to control group A only not or with respect to the pooled controls. Furthermore, the relatively high incidence of this lesion in all dose groups in the final months of study suggests that this change is primarily age-related. Evaluation of the mononuclear cell leukemia data by the more definitive method of Thomas et al. revealed no treatment-related effects. A marginal increase in the incidence of fibroadenoma of the mammary gland was observed in the low dose females during the final months of the study when compared with separate controls or pooled controls. However, since the incidence of this neoplasm in either the high or intermediate level female rats was approximately one half that of the low level during this period, a dosage relationship was not established by the increased incidence in the low level females. Increases in the incidence of uterine polyp were found in the low level females from months 13 - 18. As was the case for mammary gland fibroadenoma in the females, the high and intermediate level rats were unaffected. Consequently, the increase in incidence of this neoplasm in the low level females is deemed to be not dosage-related and of no toxicological importance. The increased incidence of uterine polyp in the low level females was mitigated when compared with the pooled controls.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg diet
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg diet
System:
other: urogenital
Organ:
kidney
ureter
urethra
Treatment related:
yes
Dose response relationship:
not specified
Conclusions:
Inclusion of ethylene glycol in the diet of Fischer 344 rats for up to 24 months resulted in deleterious effects which were most numerous in the high level male rats but was found to a lesser degree in the high level females as well. The most noteworthy of these effects in the high level male rats was the production of urinary calculi in the kidneys, ureters and urinary bladders concomitant with high levels of calcium oxalate crystals in the urine.

Furthermore, the frequencies of tubular cell hyperplasia, tubular dilation, peritubular nephritis and focal granulomatous nephritis were increased markedly. Other significant findings in these male rats were reduced markedly body weight gain, increased absolute and relative kidney weight, decreased absolute and relative liver weight, various hematopoietic changes and increased water consumption rates. A variety of histological changes was found in the high level male rats with mineralization of the heart, lungs, stomach and vas deferens being the most remarkable. The various deleterious effects in the high dose male rats resulted in a significant shortening of their life-spans: increased mortality became apparent by 8 months and all male rats of this group were dead by 16 months.
While the incidence of calcium oxalate crystals in the urine was significant in the high level female rats, urinary calculi were not formed. Absolute and relative kidney weights were increased in these rats, however. Furthermore, several hepatopoietic changes were observed in these female rats, while fatty metamorphosis of the liver was the most significant histologic finding.

Transient changes in organ weights, erythroid parameters, water consumption rates and urine specific gravity observed for the intermediate and low level rats were considered to be statistical artifacts attributable to chance.
Focal soft tissue mineralization within certain organs was found in rats of both sexes for the intermediate and low levels at the 24-month sacrifice. This change was quite possibly the result of altered calcium metabolism associated with the ingestion of ethylene glycol.
In general, inclusion of EG in the diet of Fischer-344 rats for 2 years resulted in a variety of marked toxic effects at the high dose level, the most pronounced of which was renal toxicity. Under the conditions of this study, EG was not oncogenic.
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
method according to NTP-internal standards
GLP compliance:
yes
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 62 days (males) and 55 days (females)
- Housing: Male mice were housed five per cage for 54 weeks, then individually until study end. Female mice were housed five per cage for 67 weeks , then individually until study end. Cages were rotated within racks and racks were rotated within rooms every 2 weeks.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 19 days
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The dose formulations were prepared by mixing appropriate amounts of ethylene glycol and feed in a blender. Studies to determine the homogeneity and stability of the dosed feed preparations were conducted by the analytical chemistry laboratory. Gas chromatographic methods were used to confirm homogeneity as well as the stability of dose formulations stored protected from light for 2 weeks at 25°C. During the studies, the dose formulations were stored at 5° C before use and at room temperature during use for up to 14 days.
Periodic analyses of the dose formulations of ethylene glycol were conducted at the study laboratory and the analytical chemistry laboratory using gas chromatography. During the 2-year studies, the dose formulations were analyzed at least once every 8 weeks and 98% of the dose formulations were within 10% of the target concentrations. Results of periodic referee analyses performed by the analytical chemistry laboratory were in good agreement with the results obtained by the study laboratory.
Duration of treatment / exposure:
103 weeks
Frequency of treatment:
daily
Dose / conc.:
6 250 ppm (nominal)
Remarks:
males
Dose / conc.:
12 500 ppm (nominal)
Remarks:
males; females
Dose / conc.:
25 000 ppm (nominal)
Remarks:
males; females
Dose / conc.:
50 000 ppm (nominal)
Remarks:
females
No. of animals per sex per dose:
total: 120 mice
Control animals:
yes, concurrent no treatment
Details on study design:
In male mice, potentially progressive renal lesions were seen in the 25000 and 50000 ppm groups and significantly decreased mean weight gain occurred in the 12500 and 50000 ppm groups. Also, ethylene glycol is known to be more toxic to males than females in other rodent species. Therefore, a high dose of 25000 ppm and lower doses of 6250 and 12500 ppm were selected for treatment of male mice in the 2-year feed studies.
Observations and examinations performed and frequency:
All animals were observed twice daily and clinical findings were recorded at each weight check. Individual body weights were obtained weekly through week 13, monthly thereafter, and at the end of the study. After 15 months, 6 male and 9 to 10 female mice from each dose group were evaluated. Organ weights were recorded for the brain, right kidney, and liver of all animals evaluated at 15 months. Blood samples were collected by cardiac puncture.
Sacrifice and pathology:
Necropsy was performed on all animals. During necropsy, all organs and tissues were examined for gross lesions. Tissues for microscopic examination were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned to a thickness of 4 to 6 µm, and stained with hematoxylin and eosin. Complete histopathologic examinations were performed on all control and high-dose mice and all animals that died early in the low- and mid-dose groups. For all other low- and mid-dose mice, organs and tissues examined included all gross lesions, kidney, liver, and thyroid gland in all mice, lung in females, and urinary bladder in males.
Samples of formalin-fixed liver from selected high dose male and female mice were post-fixed in Fowler's solution for 2 days, post-fixed in 1.0% osmium, dehydrated in ethanol, and infiltrated with Epon 812. Resulting blocks were thin-sectioned (approximately 90 nm), mounted on 100-mesh copper-rhodium grids, stained with 2.7% lead citrate and 5.0% uranyl acetate, and examined with a Philips 400 transmission electron microscope.
Microscopic evaluations were completed by the study laboratory pathologist, and the pathology data were entered into the Toxicology Data Management System (TDMS). The slides, paraffin blocks, and residual wet tissues were sent to the NTP Archives for inventory, slide/block match, and wet tissue audit for accuracy of labelin and animal identification and for thoroughness of tissue trimming. The slides, individual animal records and tables were compared for accuracy, slides and tissue counts were verified, and histotechnique was evaluated.
Organ weights were recorded for the brain, right kidney, and liver of all animals evaluated for 15 months.
Blood samples were collected from animals evaluated at 15 months.
Haematology: haematocrit, haemoglobin, erythrocytes, total and differential leukocyte counts.
Clinical chemistry: blood urea nitrogen, creatinine, total bilirubin, alanine aminotransferase, aspartase aminotransferase, lactate dehydrogenase, and sorbitol dehydrogenase.
Complete histopathology was performed on all high dose and control males and females at the 15-month interim evaluations. Complete histopathology was also performed on all control and high dose micat the end of the 2-year study and on animals that died before the end of the 2-year study.
Statistics:
Statistical methods for survival analyses, calculation of incidence, analysis of neoplasm incidences, analysis of non-neoplastic lesion incidences, analysis of continuous variables.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no statistically significant differences in survival between dosed and control groups. Of the high dose males, 65% (35/54) survived to 18 months. Because of several early deaths due to extensive fighting, male mice were housed individually after week 54; female mice were housed individually after week 67.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights of exposed and control male and female mice were similar. No treatment-related clinical findings or gross lesions were noted. Results from all serologic analyses for murine viruses were negative. Feed consumption by exposed male and female mice was similar to that by controls. For male mice, dietary levels of 0, 6250, 12500 and 25000 ppm resulted in average daily EG consumption levels of approx. 1500, 3000 or 6000 mg/kg bw. For females, dietary levels of 0, 12500, 25000 or 50000 ppm resulted in average daily EG consumption levels of approx. 3000, 6000 or 12000 mg/kg bw.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related neoplasms were observed in male or female mice at the 15-month interim evaluations or at the end of the 2-year studies. Several treatment-related or biologically significant nonneoplastic lesions were seen in exposed mice at interim evaluations and in the 2-year study.

Liver: The incidence of hepatocellular hyaline degeneration was increased in exposed male and female mice at the 15-month interim evaluations. At the end of the 2-year study, incidences of centrilobular hepatocyte hyaline degeneration were increased in dosed male and female mice and were considered clearly related to ethylene glycol administration. Severity did not increase with dose. Under light microscopy, this lesion appeared similar to hyaline degeneration seen in the 13-week studies and the 15-month interim evaluations, and consisted of cytoplasmic accumulations of nonbirefringent, eosinophilic, granular to globular material resembling erythrocytes in size, shape, and tinctorial properties. When examined by transmission electron microscopy, irregularly shaped, pleomorphic, nonmembrane bound, intracytoplasmic inclusions were seen in affected hepatocytes. These inclusions were composed of crystalline parallel arrays of alternating electron-dense and electronlucent linear structures (8.5 to 11 nm) with 11 to 13 nm periodicity. Hepatocellular erythrophagocytosis was diagnosed in two high-dose female mice and consisted of subcapsular hepatocytes whose cytoplasm was packed with intact erythrocytes.

Urinary system: The incidence of nephropathy was increased in male mice at the 15-month interim evaluations; however, there were no treatment-related changes in the incidence or severity of nephropathy in male or female mice at the end of the 2-year study. In several high-dose males, small numbers of pale yellow to clear crystals morphologically compatible with oxalate were seen in the renal cortical tubules (8 mice), urethral lumens (12 mice), and/or renal pelvis (1 mouse). These crystals were birefringent when examined under polarized light. Renal tubule dilatation was often seen in association with renal tubule crystals. Calculi composed of, oxalate-like material were detected grossly or microscopically in four high dose males. Except for one male with both urethral crystals and gross calculus and another male with pelvic crystals and microscopic calculus, there were no simultaneous occurrences of crystals and calculi. The incidence of urethral suppurative inflammation was increased in high-dose males and that of urinary bladder chronic inflammation was increased in all exposed males. Except for two deaths at weeks 35 and 46, all male mice with renal tubule crystals died by week 31. Urethral and/or urinary bladder inflammation was also present in most of these animals. The causes of death or reasons for moribund kill in these males were severe gross and microscopic urogenital and skin lesions including ulcers of the penis and prepuce; alopecia, ulcers, or inflammation of skin in the posterior body; and seminal vesicle and prostate gland inflammation. These lesions were probably related to fight wound trauma and ascending secondary infections. Infections and trauma may have resulted in dehydration, urine stasis, urinary pH alterations, and other physiological changes which could have provided microenvironments favorable for oxalate precipitation in the renal tubules. Therefore, it is uncertain if the presence of this oxalate-like material was a direct toxic result of ethylene glycol administration. All but three males with urethral or pelvic crystals or urinary bladder calculi survived to study end or were killed in a moribund condition at week 84 or later. In these mice, gross and microscopic urogenital and skin lesions possibly related to fight wound trauma were absent or of low severity.

Lung: At the 15-month interim evaluations, two control and three high-dose females had medial hyperplasia of small pulmonary arteries and/or arterioles. At the end of the 2-year study, exposed females had an increased incidence of medial hyperplasia of the small pulmonary arteries and/or arterioles, but severity did not increase with dose. Affected vessels were distributed randomly and had minimal to mild circumferential thickening of mural smooth muscle; it was unclear if this thickening was due to smooth muscle hypertrophy, to hyperplasia, or to both. The vessels were sometimes surrounded by lymphocytic infiltrates. The increased incidence of medial hyperplasia was considered to be clearly related to ethylene glycol administration. Alveolar/bronchiolar adenomas (0/50, 4/50, 4/51, 1/50) and combined adenomas and carcinomas (1/50, 6/50, 6/51, 1/50) were marginally increased in low- and mid dose females. The historical control incidence of combined alveolaribronchiolar adenomas and carcinomas in female mice from recent NTP dosed feed studies is 70/870. No increased incidences of alveolar epithelial hyperplasia were noted in exposed female mice. Since the incidences of these neoplasms were within the historical control range and these increases were not dose-related, these neoplasms were not considered directly related to ethylene glycol administration.

Thyroid gland: The incidence, but not the severity, of follicular cell hyperplasia increased in exposed females (8149, 16/50, 22/51, 14/50). Follicular cell adenomas occurred in one control, one low dose, one mid dose, and three high-dose females. Because the incidences of follicular cell adenomas and carcinomas were within the historical control range, the increased incidences of hyperplasia were not dose-related, and the severity of hyperplasia did not increase in exposed females, the thyroid follicular cell proliferative changes were not considered to be related to ethylene glycol administration.

Harderian gland: In male mice, two harderian gland adenomas occurred in the mid dose group, and two adenomas and one carcinoma occurred in the high-dose group. Because the incidences of harderian gland adenomas and carcinomas were only marginally increased and were within the historical control range, these neoplasms were not considered to be rclated to ethylene glycol administration.

All organs: In female mice, the incidence of combined lymphomas had a statistically significant decrease (17150, 13/50, 9/51, 7/50). The relationship of this decrease to ethylene glycol administration is uncertain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 500 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
gross pathology
Key result
Critical effects observed:
no

Selected non-neoplastic lesions in male and female mice in the 2-year feeding study:

 Male mice  0 ppm  6250 ppm  12500 ppm  25000 ppm
 15 -month interim evaluation        
 Liver: hyaline degeneration / overall rates (a)  0/6  3/6  2/6  6/6
 Kidney: nephropathy / overall rates  2/6  2/6  5/6

 6/6*

 2-year study        
 Liver: hyaline degeneration / overall rates  0/54  0/53  24/53**  36/54**
 Female mice  0 ppm  12500 ppm  25000 ppm  50000 ppm
 15-month interim evaluation        
 Liver: hyaline degeneration / overall rates  0/10  0/10  3/9  10/10
 Lung: arterial medial hyperplasia / overall rates  2/10  0/10  0/9  3/10
 2-year study        
 Liver: hyaline degeneration / overall rates  0/50  0/50  1/15  26/50**
 Lung: arterial medial hyperplasia / overall rates  3/50  10/50*  10/51*  23/50**

 *  significantly different (p<0.05) from the control group by logistic regression test
 **  p<0.01
 (a)  number of affected animals / number of animals necropsied or number of animals with tissues examined microscopically
Conclusions:
Under the conditions of this study, there was no evidence of carcinogenic activity of ethylene glycol in male B6C3F1 mice receiving 6250, 12500 or 25000 ppm or in female B6C3F1 mice receiving 12500, 25000 or 50000 ppm. Administration of ethylene glycol resulted in hepatocellular hyaline degeneration in male mice fed diets containing 50000 ppm. An increased incidence of medial hyperplasia of small pulmonary arteries and arterioles occurred in female mice fed diets containing 12500, 25000 or 50000 ppm ethylene glycol.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

BRRC (1982) DePass et al. (1982) investigated male and female Fischer rats exposed for 24 months to dietary doses of 0.04, 0.2 and 1.0 mg/kg bw and day. In the top dose, clear renal toxicity was observed. No oncogenic effects were noted.

CD-1 mice (80/sex/dose) received MEG in approximate dietary doses of 0, 40, 200 or 1000 mg/kg bw/day for up to 2 years (BRRC, 1982; dePass et al., 1986). Clinical signs, body weight, food consumption, and comprehensive histopathology were investigated. No clear treatment-related effects were observed and a NOAEL of 1000 mg/kg/day (without LOAEL) was identified.

In a further mouse study, B6C3F1 mice (60/sex/dose) received MEG in the diet for up to 2 years (NTP, 1993). Estimated average doses were 0, 1500, 3000 and 6000 mg/kg/day in males and 0, 3000, 6000 and 12000 mg/kg bw/day in females. Hematology, clinical chemistry, organ weights (limited), and comprehensive histopathology were investigated. Increased incidences of hepatocellular hyaline degeneration in males at > 3000 mg/kg/day and females at 12000 mg/kg/day and medial hyperplasia of the pulmonary arterioles in females at > 3000 mg/kg/day were noted; the biological significance of the pulmonary lesion was considered as unclear (NTP, 1993). Small numbers of oxalate-like crystals and/or calculi were found in renal tubules, urethrea and urinary bladder in a few males at 6000 mg/kg. A NOAEL of 1500 mg/kg/day and a LOAEL of 3000 mg/kg/day for liver histopathology were identified in male B6C3F1 mice.

In a further mouse study, male and female animals received oral doses of 0.04, 0.2 and 1 g/kg bw over a study period of 24 months (RRRC, 1984). In the top dose there was an equivocal incidence of an accelerated the appearance of lymphosarcomas in female animals. There was no evidence of an increase in any other tumor type. No clear NOAEL was identified due to a large and confounding incidence of age-related pathology.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified as carcinogen under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218.