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Appendices 1-18 to: Report on the Health Effects of Selected Pesticide Coformulants

67     Animal toxicity

67.1     Single dose toxicity

The acute toxicity of ethylene glycol in experimental animals closely mirrors the acute effects seen in humans (see part 3). One exception is the fourth clinical stage of delayed cranial neurological consequences observed in humans; no studies have been found that reported this stage in animals (LaKind et al 1999).

Susceptibility to ethylene glycol intoxication varies with species, sex, and individual (NTP 1993). Cats appear to share the susceptibility of humans; however, because cats are unusually sensitive to ethylene glycol due to their high baseline production of oxalic acid, they are not representative of experimental animal species predictive of human responses to ethylene glycol exposure (LaKind et al 1999).

67.1.1     Inhalation

According to Cavender & Sowinski (1994), the one-hour LC₅₀-value in rats is 10.9 g/m³.

In a study of rats, all animals survived an 8-hour exposure to saturated atmosphere (20 ºC: ca. 200 mg/m³ (calculated)) of ethylene glycol (BASF 1961 – quoted from IUCLID 2000).

67.1.2     Oral intake

The reported oral LD₅₀-values for ethylene glycol in rats ranged from >2.0 to 11.3 g/kg b.w. (IUCLID 2000, LaKind et al. 1999, NTP 1993, BUA 1991, Cavender & Sowinski 1994, A&H 1993).

In other experimental animals, the reported oral LD₅₀-values ranged from 5.89 to 15.4 g/kg b.w. in mice, from 7.0 to 9.3 g/kg b.w. in rabbits, and from 4.0 to 8.2 g/kg b.w. in guinea pigs (IUCLID 2000, LaKind et al. 1999, NTP 1993, BUA 1991, Cavender & Sowinski 1994, A&H 1993).

For the cat, oral LD₅₀-values of 1670 and 4700 mg/kg b.w. have been reported and for the dog, from 4000 to 8200 mg/kg b.w. (IUCLID 2000, A&H 1993).

A minimal lethal dose of 3800 mg/kg b.w. has been reported for rats, of 1000 mg/kg b.w. for cats, and of 6700 and 7300 mg/kg b.w. for dogs (LaKind et al. 1999).

Monkeys (Macaca fascicularis) received ethylene glycol orally at doses of 1000, 2000, or 4000 mg/kg b.w. and were observed 14 days after dosing. At the highest dose level, the two animals were found comatose 22 hours after dosing and died 26 and 28 hours after treatment. No further details are given. (ICI 1979 – quoted from IUCLID 2000).

67.1.3     Dermal contact

Dermal LD₅₀-values for ethylene glycol of 9.53 g/kg b.w. (Cavender & Sowinski 1994, A&H 1993) and of 10.6 g/kg b.w. have been reported for the rabbit (IUCLID 2000, BUA 1991).

67.1.4     Skin irritation

Guillot et al. (1982) have evaluated skin irritation in rabbits by determination of the primary cutaneous irritation index under patch-test and by determination of the cumulative irritation index after repeated exposure (6 weeks, MMII) according to official French methods but with some complements or modifications.

In the primary cutaneous irritation test (the experimental procedure is quoted from BUA 1991), 0.5 ml of neat ethylene glycol was applied to undamaged and scarified skin of 6 rabbits and covered with an occlusive dressing. The duration of exposure was 23 hours and assessments of the effects were made one hour and 48 hours after removal of the dressing. Ethylene glycol showed a primary irritation index of 0.08 (an index of up to 0.5 is considered non-irritant and an index of 0.5-2.0 as slightly irritant).

In the cumulative cutaneous irritation test (the experimental procedure is quoted from IUCLID 2000), three rabbits were treated daily with 2 ml of neat ethylene glycol or with a 10% aqueous solution for 6 weeks.  Ethylene glycol undiluted showed a mean maximum irritation index of 0.47 interpreted as ’well tolerated’ and the 10% solution an index of 0 interpreted as ’very well tolerated (the maximum index that could be scored is not stated; the highest index scored was 1.83 for polypropylene glycol and interpreted as ’relatively well tolerated).

Ethylene glycol was tested for skin irritative properties according to the Draize test in female New Zealand rabbits. Skin response was evaluated 24 and 72 hours after treatment. Ethylene glycol had a low potential for skin irritation (score 0.4, with a score of <2.0 meaning mild or no irritation). (Clark et al. 1979).

67.1.5     Eye irritation

Moderate to severe eye irritation was observed in rats and rabbits exposed continuously for 90 days to ethylene glycol (vapour) at a concentration of 12 mg/m³. Two rats developed corneal opacity after 8 days and appeared to be blind for the remainder of the exposure. Erythema, oedema, and discharge began in rabbits after 3 days of exposure, the oedema being severe enough to result in virtual closure of the eyes. Guinea pigs, dogs, and monkeys exposed similarly showed no effects on the eyes. See also part 4.2.1. (Coon et al. 1970).

Rats, guinea pigs, rabbits, dogs, and monkeys were exposed to a concentration of 57 mg/m³ of ethylene glycol (vapour) 8 hours a day, 5 days per week for 6 weeks; none of the animals showed any signs of ocular irritation. In rabbits exposed similarly to 10 mg/m³, mild conjunctivitis was noted in one eye of each of 2 rabbits during the 4^th and 5^th weeks, which persisted until the end of the exposure; each of these rabbits also developed a small lesion over the irritated eye. These signs were, according to the authors, probably brought on by accidental trauma, which may have been aggravated by the exposure. See also part 4.2.1. (Coon et al. 1970).

Diluted ethylene glycol (10, 20, or 50 % solution in water) caused only slight oedema and erythema under occlusive conditions in the eyes of rabbits. Instillation of neat ethylene glycol produced moderate to severe oedema and erythema. No effects were observed 48 hours post-treatment. According to IUCLID, ethylene glycol is evaluated as being moderately irritating to the eyes. (Star 1980 – quoted from IUCLID 2000 and from BUA 1991).

Toxicity and irritation of ethylene glycol were assessed in rabbit eyes following multiple topical or multiple intraocular (anterior chamber) administrations. The concentration of the test solutions was 0.04, 0.4, 4.0, and 40% (topical administration only) in balanced salt solution. The balanced salt solution and saline were used as negative controls. Only sterile test solutions were used. (McDonald et al. 1972).

Regarding multiple topical administration, one drop (approximately 0.05 ml) of the test solution was instilled into the cul-de-sac of the test eye (6 eyes per concentration) at 10-minute intervals for a total of 36 applications in a 6-hour period. All eyes were examined with a biomicroscope at 6 hours after the first application and the eyes were graded (iris, flare, cornea) according to an arbitrary numerical score. Eyes were also scored for palpebral and bulbar conjunctival irritation by the method of Draize at 2, 4, and 6 hours after treatment and daily thereafter, and scored. Based on the Draize scores, 0.4% was the highest concentration to be non-irritating. Irritation at higher concentrations consisted of chemosis, swelling, and conjunctival redness. No test concentration elicited significant toxic findings on biomicroscopic examination. All eyes were normal by 7 days.

Regarding multiple intraocular administration, 0.5 ml of the test solution was instilled once per day for 5 days into the anterior chamber. All eyes were examined with a biomicroscope before test and on days 2, 4, 7, and 14 after the first injection and the eyes were graded (iris, flare, cornea) according to an arbitrary numerical score. Based on the biomicroscopic scores, 0.4% was the highest concentration to be non-toxic.

No evidence of systemic toxicity was, according to the authors, observed following extraocular and intraocular administration, based on behaviour, appearance, and body weight.

Twenty-four hours after the application of 0.5 ml 80% ethylene glycol into the eye of rabbits, no eye irritation could be observed. The lowest non-irritating concentration of ethylene glycol was 20% when applied as 0.1 ml solution 5 times a day for 21 consecutive days. (McDonald et al. 1977 – quoted from IUCLID 2000 and from BUA 1991).

Guillot et al. (1982) have evaluated eye irritation in rabbits by determination of the ocular irritation index according to official French methods but with some complements or modifications. Ethylene glycol (0.1 ml) was instilled into the eyes of six rabbits. It is not stated whether the test substance was washed out or not. Assessments of the irritative effect were made 1, 24, 48, 72, 96, and 168 hours after treatment. Ethylene glycol showed an ocular irritation index of 11.33; a compound was not considered to provoke any significant injury to the eye mucous membrane when no opacity of the cornea occurred and when the ocular irritation index was less than 15. The experimental procedure is quoted from BUA (1991).

Ethylene glycol was tested for eye irritative properties according to the Draize test in female New Zealand rabbits. Eyes were inspected 1, 24, 48, 72 and 96 hours after instillation of 0.1 ml of the fluid (>99% ethylene glycol). A slight irritation was observed one hour after treatment (score: 3.0), but continually diminished with time and could not be noticed after 96 hours. The maximum score possible for a single evaluation was 110. (Clark et al. 1979).

67.1.6     Sensitisation

No data have been found.

67.2     Repeated dose toxicity

67.2.1     Inhalation

67.2.1.1     Rats

Rats (15 Sprague-Dawley and Long-Evans male and female animals per group, no information about distribution between species and sexes) were exposed to ethylene glycol (vapour) continuously at a concentration of 12 ± 2 mg/m³ for 90 days (continuous study), or at concentrations of 10 ± 1 or 57 ± 14 mg/m³ for 8 hours a day, 5 days per week for 6 weeks (repeated study). The control group consisted of 123 animals. (Coon et al. 1970).

In the continuous study, 1 rat died during exposure and 4 rats in the control group. All haematological data were within normal limits. Observations during necropsy revealed normal organs and tissues. Histopathological examination showed inflammatory changes in the lungs of exposed animals and to a lesser degree in controls. Moderate to severe eye irritation was observed and 2 animals developed corneal opacity after 8 days and appeared to be blind for the remainder of the exposure.

In the repeated study, there were no deaths at both exposure levels and all haematological values were within normal limits. At 10 mg/m³, histopathological examination revealed fatty changes and focal necrosis in the liver in 1 of 8 rats. At 57 mg/m³, histopathological examinations revealed non-specific inflammatory changes in the lungs and occasionally the hearts of exposed animals. None of the animals showed any signs of ocular or nasal irritation.

Rats (10 animals) were exposed to ethylene glycol at concentrations of 350 to 400 mg/m³ 8 hours a day, 5 days per week for 16 weeks. One rat died. The histological examination of brain, lung, heart, liver, pancreas, spleen, lymph nodes, kidneys, adrenals, testes, stomach, and caecum did not show any substance-related pathological changes. (Wiley et al. 1936 – quoted from BUA 1991, ACGIH 2001, Cavender & Sowinski 1994 and from IUCLID 2000).

67.2.1.2     Mice

Mice (20 animals) were exposed to ethylene glycol at concentrations of 350 to 400 mg/m³ 8 hours a day, 5 days per week for 16 weeks. Three mice died. The histological examination of brain, lung, heart, liver, pancreas, spleen, lymph nodes, kidneys, adrenals, testes, stomach, and caecum did not show any substance-related pathological changes. (Wiley et al. 1936 – quoted from BUA 1991, ACGIH 2001, Cavender & Sowinski 1994 and from IUCLID 2000).

67.2.1.3     Guinea pigs

Guinea pigs (15 male and female animals per group, no information about distribution between sexes) were exposed to ethylene glycol (vapour) continuously at a concentration of 12 ± 2 mg/m³ for 90 days (continuous study), or at concentrations of 10 ± 1 or 57 ± 14 mg/m³ for 8 hours a day, 5 days per week for 6 weeks (repeated study). The control group consisted of 73 animals. (Coon et al. 1970).

In the continuous study, 3 animals died during exposure. All haematological data were within normal limits. Observations during necropsy revealed normal organs and tissues. Histopathological examination showed inflammatory changes in the lungs of exposed animals and to a lesser degree in controls. Occasional foci of inflammatory cells were seen in kidneys from several animals, but this was not interpreted, by the authors, as being specific chemically induced changes.

In the repeated study, there were no deaths at both exposure levels and all haematological values were within normal limits. At 10 mg/m³, histopathological examination revealed hepatic fatty changes in 2 of 8 animals and focal necrosis in the liver in 1 of 8 animals; focal necrosis of the liver was also seen in 1 of 3 control guinea pigs. At 57 mg/m³, histopathological examinations revealed non-specific inflammatory changes in the lungs and occasionally the hearts of exposed animals. The livers of 1 of 8 animals revealed areas of focal necrosis; this was not considered, by the authors, to be chemically induced. None of the animals showed any signs of ocular or nasal irritation.

67.2.1.4     Rabbits

Male New Zealand albino rabbits (3 animals per group) were exposed to ethylene glycol (vapour) continuously at a concentration of 12 ± 2 mg/m³ for 90 days (continuous study), or at concentrations of 10 ± 1 or 57 ± 14 mg/m³ for 8 hours a day, 5 days per week for 6 weeks (repeated study). The control group consisted of 12 animals. (Coon et al. 1970).

In the continuous study, 1 rabbit died during exposure. All haematological data were within normal limits. Observations during necropsy revealed normal organs and tissues. Histopathological examination showed inflammatory changes in the lungs of exposed animals and to a lesser degree in controls. One rabbit had hamartomatosis (a benign tumour-like nodule) in liver bile ducts, but this was not interpreted, by the authors, as being a specific chemically induced change. Moderate to severe eye irritation was observed and erythema, oedema, and discharge began after 3 days of exposure, the oedema being severe enough to result in virtual closure of the eyes. 

In the repeated study, there were no deaths at both exposure levels and all haematological values were within normal limits. At 10 mg/m³, mild conjunctivitis was noted in one eye of each of two rabbits during the 4th and 5th weeks, which persisted to the end of the exposure; each of these rabbits also developed a small lesion over the irritated eye. These signs were, according to the authors, probably brought on by accidental trauma, which may have been aggravated by the exposure. At 57 mg/m³, histopathological examinations revealed non-specific inflammatory changes in the lungs and occasionally the hearts of exposed animals. None of the animals showed any signs of ocular or nasal irritation.

67.2.1.5     Dogs

Male Beagle dogs (2 animals per group) were exposed to ethylene glycol (vapour) continuously at a concentration of 12 ± 2 mg/m³ for 90 days (continuous study), or at concentrations of 10 ± 1 or 57 ± 14 mg/m³ for 8 hours a day, 5 days per week for 6 weeks (repeated study). The control group consisted of 12 animals. (Coon et al. 1970).

In the continuous study, all haematological data were within normal limits. Observations during necropsy revealed normal organs and tissues. Histopathological examination showed inflammatory changes in the lungs of exposed animals and to a lesser degree in controls.

In the repeated study, there were no deaths at both exposure levels and all haematological values were within normal limits. At 10 mg/m³, histopathological examination revealed mild congestion in the spleens of both exposed animals. At 57 mg/m³, histopathological examinations revealed non-specific inflammatory changes in the lungs and occasionally the hearts of exposed animals. None of the animals showed any signs of ocular or nasal irritation.

67.2.1.6     Monkeys

Male squirrel monkeys (2/3 animals per group) were exposed to ethylene glycol (vapour) continuously at a concentration of 12 ± 2 mg/m³ for 90 days (continuous study), or at concentrations of 10 ± 1 or 57 ± 14 mg/m³ for 8 hours a day, 5 days per week for 6 weeks (repeated study). The control group consisted of 8 animals. (Coon et al. 1970).

In the continuous study, all haematological data were within normal limits. Observations during necropsy revealed normal organs and tissues. Histopathological examination showed inflammatory changes in the lungs of exposed animals and to a lesser degree in controls.

In the repeated study, there were no deaths at both exposure levels and all haematological values were within normal limits. At 57 mg/m³, histopathological examinations revealed non-specific inflammatory changes in the lungs and occasionally the hearts of exposed animals. The livers of 2 of 3 animals revealed areas of focal necrosis; this was not considered, by the authors, to be chemically induced. None of the animals showed any signs of ocular or nasal irritation.

Two chimpanzees were exposed by inhalation to air saturated with ethylene glycol (stated to be 256 mg/m³) for 28 days. A biopsy revealed that one animal had oxalate crystals in the kidney. Both animals experienced an unexplained rise in haemoglobin concentration and mean red cell volume, as well as a decreased ability to concentrate urine. (Felts 1969 (abstract) – quoted from LaKind et al. 1999, Cavender & Sowinski 1994).

When monkeys were exposed to an ethylene glycol aerosol (500 mg/m³) for up to 30 weeks, oxalate crystals were found in the kidneys (Harris 1969 (abstract) – quoted from LaKind et al. 1999).

67.2.2     Oral intake

67.2.2.1     Rats

Sprague-Dawley rats (10 males and females per group) received ethylene glycol in their drinking water for 10 or 90 days. In the 10-day study, the concentrations were 0, 0.5, 1.0, 2.0, or 4.0%. Based on a water consumption of 100 ml/kg b.w. per day, these concentrations correspond to 0, 554, 1108, 2216, or 4432 mg/kg b.w./day. In the 90-day study, females received the same concentrations in the drinking water whereas males received 0, 0.25 (227 mg/kg b.w./day), 0.5, 1.0, or 2%. (Robinson et al. 1990 – quoted from BUA 1991 and from IUCLID 2000).

In the 10-day study, high-dose (4%) male rats showed a strong reduction in body weight and in heart, liver, spleen and thymus weights. High-dose female rats had changes in blood parameters (decreased haemoglobin and haematocrit and markedly reduced numbers of erythrocytes and leucocytes) and decreased thymus weights. In male animals at the 2 and 4% dose levels, histopathological examination of the kidneys revealed a dose-dependent increase in the incidence and severity of kidney damage, which involved dilation, degeneration and necrosis of the renal tubules, acute inflammation and intratubular deposits of proteinaceous material and calcium oxalate crystals. In females, the effects on kidneys were limited to tubular dilation and intratubular deposition of proteinaceous material at the highest dose level. A NOAEL of 1% ethylene glycol in the drinking water (1108 mg/kg b.w./day) for 10 days of exposure can be considered.

In the 90-day study, 8 female and 2 male animals from the high-dose group (4% and 2%, respectively) died. Body weight gain was markedly reduced in high-dose (2%) males. In females, the leucocyte count was significantly reduced at 0.5, 2. and 4%. In male animals at the 1 and 2% dose levels and in female animals at the 2 and 4% dose levels, histopathological examination of the kidneys showed a dose-dependent increase in the incidence and severity of kidney damage, which involved dilation, degeneration and inflammation of the renal tubules, and renal pelvis. A NOAEL of 0.5% ethylene glycol in the drinking water (554 mg/kg b.w./day) for 90 days of exposure can be considered based on the renal effects in male animals.

In a 13-week study, ethylene glycol was administered in the feed to Fischer 344/N rats (10 males and 10 females) at dose levels of 0, 0.32, 0.63, 1.25, 2.5, or 5.0% (equivalent to 0, 160, 315, 625, 1250, or 2500 mg/kg b.w./day assuming than an adult rat consumes 50 g feed/kg b.w./day). Four high-dose male rats died. The body weight gain for male rats in the 2.5 and 5% dose groups was depressed by more than 10% when compared to the control group. The relative kidney weight was significantly increased in both male and female rats in the two highest dose groups (2.5 and 5%) and the relative thymus weight was significantly decreased in high-dose (5%) male rats. Serum urea nitrogen and serum creatinine levels were significantly elevated in male animals at the two highest dose levels (2.5 and 5%) and kidney lesions were observed in all male rats at these dose levels. Damaged kidneys contained calcium oxalate crystals mainly located within tubular lumens in the renal cortex, but were also occasionally found in tubules in the medulla. Crystals were also observed in the urinary bladder, the urethral lumen, and in the brain of some high-dose male rats (5%). Severe toxic nephrosis (distension and dilation of renal tubules, necrosis and regeneration of tubule epithelium, thickening of basement membranes, and fibrosis) was diagnosed in the 5% male dose group and moderate toxic nephrosis in the 2.5% male dose group. Toxic lesions in the kidneys of female rats, which were multifocal and tended to be subcapsular, were only observed in the high-dose group (5%); no crystals were observed. Based upon the renal effects observed in male rats, a NOAEL of 1.25% (equivalent to 625 mg/kg b.w./day) can be considered. According to the author, this dose level corresponds to 0.6 to 1.0 g/kg b.w./day. (Melnick 1984).

Wistar rats (15 animals of each sex per group) were administered dietary doses of 0, 0.05, 0.1, 0.25, or 1% (according to IUCLID corresponding to 0, 35/38, 71/85, 180/185, or 715/1128 mg/kg b.w. per day for males and females, respectively) for 16 weeks. In male rats at the two highest dose levels, increased levels of oxalic acid and oxalate crystals were found in the urine and histopathological examination revealed damage to the kidneys. In addition, in males at the highest dose level, increased kidney weight and impairment of renal function was observed. In female animals, the same effects were less severe and only occurred at the highest dose level. Based on the effects observed in male animals, a NOAEL of 71 mg/kg b.w. per day is considered. (Gaunt et al. 1974 – quoted from BUA 1991 and from IUCLID 2000).

Fischer 344 rats (130 males and females per group) were fed diets (0.1, 0.5, or 2.5%) yielding approximate dosages of 40, 200, or 1000 mg/kg b.w. per day of ethylene glycol for 24 months. Two untreated control groups were included. (DePass et al. 1986a).

High-dose (2.5%) male rats had a significant increase in mortality rate from the 9^th through the 16^th month of the study and the last high-dose male rat died after 474 days of the study. Other significant findings in high-dose male rats only included: increased water intake; decreased body weight gain; decreased red blood cell count, haematocrit, and haemoglobin concentration; increased neutrophilic leukocyte count; increased serum levels of creatinine and urea; increased urine volume; increased absolute and relative kidney weights after 6 and 12 months of treatment; and decreased absolute and relative liver weights after 12 months of treatment. In female rats of the highest dosage group, kidney weights were increased after 6 and 18 months of treatment, but not after 12 months. Calcium oxalate crystals were found in urine samples from all but one male of the high-dose animals after 12 months of treatment; at 18 and 24 months, all but one of the high-dose female rats had calcium oxalate crystals.

At the 6-month sacrifice, the incidence of renal lesions (tubular hyperplasia, tubular dilation, peritubular nephritis, and calcium oxalate crystalluria) was significantly increased in the high-dose males (2.5%) and calcium oxalate crystals were present in the urinary bladder in two high-dose male rats; these conditions were absent in the other male dose groups and in females.

At the 12-month sacrifice, all high-dose males (2.5%) had chronic nephritis (multiple severe histopathological changes including tubular dilation and proteinosis, glomerular shrinkage, tubular cell hyperplasia, and chronic interstitial nephritis) with calcium oxalate crystalluria and 50% had oxalate crystals in the urinary bladder; these findings were not present in males at the lower dose levels or in females.

By the time of the 18-month sacrifice, all of the high-dose males (2.5%) had died or were sacrificed moribund. In most of these animals, oxalate nephrosis was the primary cause of death. Kidneys from these animals had tubular obstruction by large crystals with secondary tubular dilation and degeneration. Calculi were sometimes found within the renal pelvic space, urethras, and urinary bladder, often with an associated hydronephrosis. Extrarenal lesions included cellular hyperplasia of the parathyroid glands and a significant increase in the incidence of splenic haemosiderosis.

Among the females sacrificed at 2 years, the incidences of haemosiderosis of the mesenteric lymph node (2.5%) and mild fatty metamorphosis of the liver (0.5 and 2.5%) were significantly increased. No biologically significant lesions were observed in the male rats sacrificed at 2 years.

Based on the effects observed in the kidneys, a NOAEL of 200 mg/kg b.w. per day is considered. For females, a NOAEL of 40 mg/kg b.w. per day can be considered based on the effects observed in the liver of high-dose females.

Sprague-Dawley rats (16 male and female animals per group) received ethylene glycol in their diet at concentrations of 0, 0.1, 0.2, 0.5, 1.0, or 4.0% (equivalent to 0, 50, 100, 250, 500 or 2000 mg/kg b.w./day assuming than an adult rat consumes 50 g feed/kg b.w./day). An increased mortality rate was observed in males of the two highest dose groups (1 and 4%) and in females of the highest dose group (4%). A significant decrease in growth was observed in male rats from week 16 at 4% and after week 70 at 1%, and in female rats after about one year at 4%. A significantly increased water consumption was noted in male rats at 1 and 4% and in female rats at 4%, and in these dose groups protein was found in the urine. The mean terminal kidney, lung and liver weights were lower (whether the reduction is significant is not stated in the publication) in male animals at 0.1% and above compared to controls. Calculi and crystal deposition in the kidneys were observed in male rats from 0.5% (only 1/16 animals at 0.5%) and in female rats at 4% and from 1%, respectively. Morphological changes in the kidneys also included degeneration of the tubular epithelium, manifested mainly as cytoplasmic vacuolisation; however, no details are provided at which dose levels these morphological changes were observed. According to the author, it appears probable that the NOEL is no higher than 0.2% (equivalent to 100 mg/kg b.w./day) ethylene glycol in the diet but may be less. (Blood 1965).

67.2.2.2     Mice

In a 13-week study, ethylene glycol was administered in the feed to B6C3F1 mice (10 males and 10 females) at dose levels of 0, 0.32, 0.63, 1.25, 2.5, or 5.0% (equivalent to 0, 480, 945, 1875, 3750, or 7500 mg/kg b.w./day assuming than an adult mouse consumes 150 g feed/kg b.w./day). There were no deaths, the relative weight gain data did not show any clear dose-related effects, and there were no differences in organ weights. Renal lesions diagnosed as mild toxic nephrosis (tubular dilation, cytoplasmic vacuolisation, and regenerative hyperplasia with piling up of nuclei) were observed in about half of the male high-dose mice (5%) and in one male mouse in the 2.5% dose group. There was no evidence of crystal formation in the affected tubules. A degenerative change (accumulation of an eosinophilic hyaline material in the cytoplasm of hepatocytes adjacent to or close to central veins) was present in the livers of all the male mice in the 2.5 and 5% dose groups. There were no adverse effects observed in female mice at any of the dose levels. Based upon the effects observed in kidneys and livers in male mice, a NOAEL of 1.25% (equivalent to 1875 mg/kg b.w./day) can be considered. (Melnick 1984).

B6C3F1 mice (60 animals of each sex per group) were fed diets containing ethylene glycol for 103 weeks. Male mice received 0, 6250, 12500, or 25000 ppm (equal to average daily levels of approximately 0, 1500, 3000, or 6000 mg/kg b.w./day) and female mice 0, 12500, 25000, or 50000 ppm (equal to average daily levels of approximately 0, 3000, 6000, or 12000 mg/kg b.w./day). There were no significant differences in survival between dosed and control groups. Mean body weights of exposed and control animals were similar and no treatment-related clinical findings or gross lesions were noted. Hepatocellular hyaline degeneration was seen in mid- and high-dose male and high-dose female mice. Pulmonary arterial medial hyperplasia was observed at an increased incidence in exposed females but not in exposed males. Incidence and severity of nephropathy were not affected by treatment in either sex. Small numbers of oxalate-like crystals, calculi, or both were noted in renal tubules, urethras, and/or urinary bladders in a few high-dose male mice. Based upon the effects observed in livers of female mice, a NOAEL of 12500 ppm (equal to approximately 3000 mg/kg b.w./day) can be considered. (NTP 1993).

CD-1 mice (80 males and females per group) were fed diets (0.1-0.05, 0.7-0.24, or 0.35-1.27%) yielding approximate dosages of 40, 200, or 1000 mg/kg b.w. per day of ethylene glycol for 24 months. Two untreated control groups were included. Renal tubular degeneration occurred slightly more frequently in high-dose females than in controls at the 18-month sacrifice, but was absent in the 15 high-dose females sacrificed at 24 month. A NOAEL of 1000 mg/kg b.w. per day can be considered. (DePass et al. 1986a).

67.2.2.3     Monkeys

Rhesus monkeys (2 males and 1 female) were given ethylene glycol in their diet at 0.2% (males) or 0.5% (female) for 3 years. No calculi or abnormal calcium deposits were demonstrated by X-ray examination and no other effects on organs and tissues were observed at the histopathological examination. (Blood et al. 1962).

67.2.3     Dermal contact

No data have been found.

67.3     Toxicity to reproduction

67.3.1     Inhalation

CD rats and CD-1 mice (25 animals per group) were exposed to a respirable ethylene glycol aerosol (mass median aerodynamic diameter (MMAD) 2.3 µm) on gestational days (GD) 6 to 15, 6 hours a day, by whole-body exposures (target concentrations: 0, 150, 1000, or 2500 mg/m³). Rats were sacrificed on gestation day 21, and mice on gestation day 18. (Tyl et al. 1995a).

The total concentrations of ethylene glycol (aerosol plus vapour) were 79, 89, or 84% of target concentrations, respectively (119 ± 13, 888 ± 149, or 2090 ± 244 mg/m³). The vapour phase was 82% of the total concentration for the 150 mg/m³ group; for the higher aerosol concentrations, the vapour phase was 19-20% of the total concentration.

All rat dams survived to scheduled termination. Food and water consumption, maternal body weights and weight gain, and maternal organ weights (other than liver) were unaffected by exposure. A significant increase in absolute and relative liver weight was observed at 2500 mg/m³. Gestational parameters (pre- and post-implantation loss, live foetuses/litter, sex ratio, and foetal body weight/litter) were unaffected by exposure. There was no significant increase in the incidence of any individual malformation, in the incidence of pooled external, visceral, or skeletal malformations, or in the incidence of total malformations by foetus or by litter. There were no significant increases in the incidence in any foetal external or visceral variations. There was some evidence of treatment-related reductions in ossification of the foetal skeleton, including an increase in the incidence of poorly ossified humerus (forelimb) and zygomatic arch (face) at the highest exposure level (2500 mg/m³) and an increase in the incidence of poorly ossified metatarsals and proximal phalanges of the hindlimb at 1000 mg/m³ but not at 2500 mg/m³. According to the authors, the NOAEL was 1000 mg/m³ for maternal and 150 mg/m³ for developmental toxicity.

All mouse dams survived to scheduled termination. One female at 2500 mg/m³ had a totally resorbed litter at termination; all other pregnant animals had one or more live foetuses at sacrifice. Clinical signs included only wet fur for all ethylene glycol exposed mice. Reduced body weight and body weight gain were observed at 1000 and 2500 mg/m³ both during and after the exposure period. Gravid uterine weight was also reduced at the two highest exposure levels so that body weight corrected for gravid uterine weight was unaffected by treatment. Liver and kidney weights were unaffected by treatment. The following gestational parameters were affected: the number of viable implantations per litter was reduced at 2500 mg/m³, the number of nonviable implantations per litter was increased at 1000 and 2500 mg/m³, the number of early resorptions was increased (not significantly) at 2500 mg/m³; the sex ratio was reduced at 1000 mg/m³ but not at 2500 mg/m³, and the foetal body weights per litter (male, female, and total) were reduced at 1000 and 2500 mg/m³. There was a significant increase in the incidence of a number of external, visceral, and skeletal malformations at 1000 and 2500 mg/m³, as well as in the incidence of pooled external, visceral, and skeletal malformations, and in the incidence of total malformations. Malformations were found in the head (exencephaly), face (cleft palate, foreshortened and abnormal face, and abnormal facial bones), and skeleton (vertebral fusions, and fused, forked, and missing ribs). The incidences of many foetal variations were also increased at the two highest dose levels, but only a few at the lowest dose level (150 mg/m³). According to the authors, the NOAEL was 150 mg/m³ for maternal and at or below 150 mg/m³ for developmental toxicity.

Ethylene glycol was teratogenic to mice by whole-body exposure to aerosol (1000 and 2500 mg/m³), see the study described above. According to the authors, the results were confounded by possible exposure from ingestion after grooming and/or from percutaneous absorption. Therefore, CD-1 mice (30 animals per group) were exposed to ethylene glycol aerosol (MMAD 2.6 ± 1.7 µm) on gestational days 6 to 15, 6 hours a day, by nose-only (target concentrations: 0, 500, 1000, or 2500 mg/m³) or whole-body exposures (target concentrations: 0 or 2100 mg/m³). Control environments were water aerosol (4200 mg/m³ for nose-only and 2700 mg/m³ for whole-body). On gestation day 18, the dams were sacrificed. (Tyl et al. 1995b).

In either exposure regimen, body weights, body weight gain (absolute or corrected for the weight of the gravid uterus), and liver weights (absolute or relative) were unaffected by treatment. Microscopic examination of maternal kidneys indicated no treatment-related incidence or severity of renal lesions. There were no treatment-related differences among groups in the number of corpora lutea per dam, the number of total or viable implantations per litter, or on foetal sex ratio. There was no significant increase in the incidence of any individual external or visceral malformation or variations, or of all external or visceral malformations or in total variations.

In the nose-only experiment, maternal clinical signs associated with the animals struggling while in restraint were observed. Maternal kidney weights were increased at concentrations of 1000 (absolute) and 2500 (absolute and relative) mg/m³ and there was a trend toward reduced gravid uterine weight at these concentrations as well. The percentage of live foetuses was reduced slightly (but not significantly) at the two highest concentrations. At the highest concentration (2500 mg/m³), the foetal body weights per litter were significantly reduced and the incidences of one skeletal malformation (fused ribs) and 18 skeletal variations were increased. According to the authors, exposure of CD-1 mice to a respirable ethylene glycol aerosol during organogenesis by nose-only inhalation resulted in minimal maternal toxicity at 1000 and 2500 mg/m³ and developmental toxicity at 2500 mg/m³; the NOAEL was 500 mg/m³ for maternal and 1000 mg/m³ for developmental toxicity.

In the whole-body dose group (2100 mg/m³), the gravid uterine weight, the percentage of live foetuses (due to an increase in late resorptions), and the foetal body weights per litter were significantly reduced. There was an increase in the incidence of a number of skeletal malformations, including fused thoracic arches, extra thoracic arches, fused lumbar arches and centra, fused ribs, and extra ribs between existing ribs, as well as in the incidence of pooled skeletal malformations and a total of 63 skeletal variations exhibited significantly increased incidences.

67.3.2     Oral intake

67.3.2.1     Rats

To assess the possible effects of ethylene glycol on reproductive performance, a three-generation reproduction study was performed in Fischer 344 rats. Ethylene glycol was administered in the diet at approximate doses of 40, 200, or 1000 mg/kg per day (weekly calculated doses ranged from 40-50, 200-300, or 1000-1300 mg/kg/day for males and from 40-60, 200-300, or 900-1200 for females). Two untreated diet control groups were included. At approximately 100 days of age, 10 males were mated to 20 females in each dose group. Necropsies and microscopic examinations of several organs and tissues were performed (5 animals of each sex from each dose level) on the F₂ parents and on the F₃ weanlings. There was no treatment-related effect on body weight gain or diet consumption, nor was there any mortality among parental rats. No treatment-related effect was observed for any of the reproductive indices (fertility index, gestation index, gestation survival index, survival indices, and days from first mating to litter) for all three generations, or on neonatal body weight at days 4, 14, or 21 postpartum. The histopathological examinations revealed no treatment-related findings in the F₂ parents and in the F₃ weanlings, including kidney damage. The NOAEL for reproductive toxicity was 1000 mg/kg b.w./day. (DePass et al. 1986b).

Fischer 344 rats were fed ethylene glycol in their diet from gestation day 6 to 15 so that the animals received doses of 0, 40, 200, or 1000 mg/kg b.w./day. Animals were sacrificed on gestation day 21 and the foetuses were examined. The only effects found were a non-significant increase in pre-implantation losses at the highest dose level and a delay in ossification of the foetal skeleton. The incidence of malformations was not significantly different to the negative controls. No maternal effects were observed at any of the dose levels; however, only body weight of the dams was examined. (Maronpot et al. 1983 – quoted from BUA 1991 and from IUCLID 2000).

In order to determine a NOAEL for developmental toxicity of ethylene glycol administered orally, CD rats received 0, 150, 500, 1000, or 2500 mg/kg b.w. per day by gavage on gestation days 6 to 15. At the highest dose level, water consumption was increased during treatment and body weights were reduced throughout gestation; liver and kidney weights were increased at sacrifice (gestation day 21). Relative liver weights were also increased at 1000 mg/kg/day. Effects observed in foetuses at 2500 mg/kg/day included hydrocephaly; gastroschisis; umbilical hernia; fused, duplicated, or missing arches, centra, and ribs; poor ossification in thoracic and lumbar regions; and reduced body weights. At 1000 mg/kg/day, reduced body weights; duplicated or missing ribs, centra, and arches; and poor ossification were observed. According to the authors, the NOAEL for developmental toxicity was 500 mg/kg b.w./day. (Neeper-Bradley et al. 1995 – abstract quoted from TOXLINE 1995-1998).

Timed-pregnant CD rats (at least 20 animals per group) were dosed by gavage with ethylene glycol in distilled water on gestational days 6 through 15 at doses of 0, 1250, 2500, or 5000 mg/kg b.w. No maternal deaths or distinctive clinical signs were noted, except for piloerection which was seen in all treated groups but not in controls. Maternal body weight gain during treatment was significantly reduced in all dose groups. Gravid uterine weight was reduced at the mid and high doses, and corrected maternal gestational weight gain showed a significant decreasing trend. Absolute liver weight was significantly decreased at the high dose and relative kidney weight was increased in the mid- and high-dose groups. Dose-related increases in post-implantation loss per litter were observed with the high dose significantly above controls. The number of live foetuses per litter and foetal body weight per litter was significantly reduced at the mid and high doses. The percentage of malformed live foetuses per litter and/or the percentage of litters with malformed foetuses were significantly elevated in all dose groups and more than 95% of litters were affected at the high dose. A wide variety of malformations (external, visceral and skeletal) were observed with the most common being craniofacial and neural tube closure defects and axial skeletal dysplasia. (Price et al. 1985).

Price et al. (1988 – quoted from BUA 1991 and from IUCLID 2000) also have administered ethylene glycol by gavage to CD rats from gestation day 6 to 20 at doses of 0, 250, 1250, or 2250 mg/kg b.w./day. The offspring were reared by untreated dams and examined in respect to postnatal growth and survival rate, bodily development, the onset of sexual maturity, locomotive activity, and performance in a complex test of learning ability. At the two highest dose levels, there was a significant increase in the duration of gestation; microscopic examination revealed an increase in kidney damage. At the highest dose level, maternal body weight gain was reduced. In offspring from dams exposed at the highest dose level, foetal mortality was increased, there was a reduction in live litter size and in the weight of the neonates, and there was a significant increase in the number of malformations (particularly in the form of hydrocephaly and abnormalities of the axial skeleton). Prenatal exposure to ethylene glycol had no adverse effect on postnatal learning behaviour.

Ethylene glycol was administered to pregnant Wistar rats from gestation day 6 to 15 orally by a stomach tube at dose levels of 253, 638, 858, 1073, or 1595 mg/kg b.w. The foetal body weight and crown-rump length were significantly reduced from 858 mg/kg and 1.8 to 43.6% of foetuses among these groups presented gastroschisis, exencephaly, meningoencephalocele, harelip, and rib malformation; malformation frequencies showed a dose-response relationship. (Longzhan et al. 1989).

67.3.2.2     Mice

Examination of testicular weight in mice, which were administered ethylene glycol by gavage at doses of 0, 500, 1000, 2000, or 4000 mg/kg b.w. per day, 5 days a week for 5 weeks, gave no indication of any testicular damage (Nagano et al. 1984 – quoted from BUA 1991).

In a continuous breeding study, CD-1 mice (20 animals of each sex in the dose groups; 40 animals of each sex in the control group) were given ethylene glycol in their drinking water at concentrations of 0, 0.25, 0.5, or 1% for 14 weeks. As a rough estimate, the dose (on a mg/kg body weight basis) was calculated using the average daily water consumption multiplied by the concentration of the chemical in that dose group and divided by the body weight. Between days 98 to 105, the average doses were 0, 410, 840, and 1640 mg/kg b.w., respectively. (Lamb et al. 1985).

No treatment-related effects were observed on body weight or water consumption, or in clinical signs of toxicity. At the highest dose level (1%), significant decreases in the number of litters per fertile pair, the mean number of live pups per litter, and the mean live pup weight were observed as compared to control F₀ mice. In the F₁ generation, the number of live pups per litter and the live pup weight were lower in the dosed group (1%), but differences were not significant. Facial anomalies were noted in a number of offspring of high-dose mice (1%) and an examination for skeletal defects demonstrated a pattern including reduction in the size of bones in the skull, fused ribs, and abnormally shaped sternebrae and vertebrae; examination by light microscopy of bones from treated mice did not reveal histological alterations. At least six pups from three different litters had cleft lip when observed grossly at birth. The NOAEL for reproductive effects was 0.5% (corresponding to an average dose of 840 mg/kg b.w./day).

In order to determine a NOAEL for developmental toxicity of ethylene glycol administered orally, CD-1 mice received 0, 50, 150, 500, or 1500 mg/kg b.w. per day by gavage on gestation days 6 to 15. There were no apparent treatment-related effects in dams. Effects observed in foetuses at 1500 mg/kg/day included reduced body weights; fused ribs and arches; poor ossification in thoracic and lumbar centra; and increased occurrence of an extra 14^th rib. At 500 mg/kg/day, slight reductions in foetal body weight and increased incidences of extra ribs were observed. According to the authors, the NOAEL for developmental toxicity was 150 mg/kg b.w./day. (Neeper-Bradley et al. 1995 – abstract quoted from TOXLINE 1995-1998).

Timed-pregnant CD-1 mice (at least 20 animals per group) were dosed by gavage with ethylene glycol in distilled water on gestational days 6 through 15 at doses of 0, 750, 1500, or 3000 mg/kg b.w. No maternal deaths or distinctive clinical signs were noted, except for piloerection which was seen in all treated groups but not in controls. Maternal body weight gain during treatment was significantly reduced in mid- and high-dose groups. Gravid uterine weight was reduced at the mid and high doses, and corrected maternal gestational weight gain showed a significant decreasing trend. Absolute liver weight was significantly decreased at the mid and high dose. Dose-related increases in postimplantation loss per litter were observed. The number of live foetuses per litter was significantly reduced at the high dose and foetal body weight per litter was significantly reduced at the mid and high doses. The percentage of malformed live foetuses per litter and/or the percentage of litters with malformed foetuses were significantly elevated in all dose groups and more than 95% of litters were affected at the high dose. A wide variety of malformations (external, visceral and skeletal) were observed with the most common being craniofacial and neural tube closure defects and axial skeletal dysplasia. (Price et al. 1985).

67.3.2.3     Rabbits

New Zealand White (NZW) rabbits (23-24 inseminated animals per group) were administered ethylene glycol by gavage on gestational day 6 through 19 at doses of 0, 100, 500, 1000, or 2000 mg/kg/day. Dams were sacrificed at gestation day 30.

Profound maternal toxicity (42% mortality, three early deliveries, and one spontaneous abortion) was observed at the highest dose (2000 mg/kg b.w./day). At necropsy, there were no significant effects on gravid uterine weight, and liver or kidney weights. Kidney damage observed at 2000 mg/kg b.w./day was limited to the cortical renal tubules and included intraluminal oxalate crystals, epithelial necrosis and tubular dilatation and renal tubular degeneration. There were no effects on pre- or postimplantation loss, the number of foetuses per litter, foetal body weight per litter, or sex ratio (percent male foetuses per litter), and no evidence of teratogenicity based on evaluation of external, visceral including craniofacial, skeletal or total malformations, or variations at any dose level. The NOAEL for maternal toxicity was 1000 mg/kg b.w./day and the NOAEL for developmental toxicity was 2000 mg/kg b.w./day. (NTP 1991, Tyl et al. 1993 – abstract quoted from Toxline 1990-1994).

67.3.3     Dermal contact

CD-1 mice (30 animals per group) were exposed to ethylene glycol on gestation days 6 to 15, 6 hours per day by occluded cutaneous application at 0, 12.5, 50, or 100% ethylene glycol (0.1 ml/animal, equivalent to approximately 0, 404, 1677, or 3549 mg/kg b.w./day). There were no treatment-related maternal effects, and no differences in pre- or postimplantation loss or in foetal body weights/litter, and no increased incidences of any foetal malformations. Two skeletal variations were observed at the highest exposure level. According to the authors, the NOAEL for maternal and developmental toxicity was the highest exposure level (approximately 3549 mg/kg b.w./day). (Tyl et al. 1995 – abstract quoted from TOXLINE 1995-1998).

67.4     Mutagenic and genotoxic effects

67.4.1     In vitro studies

Ethylene glycol was negative when tested in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 at concentrations from 1 to 10000 µg/plate with and without metabolic activation (Various studies quoted in IUCLID 2000, BUA 1991, and ATSDR 1997).

A negative result has been reported for ethylene glycol in the SOS chromotest in Escherichia coli PQ37 when tested at concentrations of up to 60000 µg/plate with and without metabolic activation (von der Hude et al. 1988).

When tested in the DNA damage and repair assay in Escherichia coli (WP2, WP2uvrA, WP67, CM611, WP100, W3100polA+, p3478polA-) at concentrations up to 60000 µg/plate with and without metabolic activation, negative results were obtained (McCarroll et al. 1991 – quoted from IUCLID 2000, BUA 1991 and from ATSDR 1997).

Ethylene glycol was negative when tested in the gene conversion assay in Saccharomyces cerevisiae with and without metabolic activation. According to IUCLID, genetic changes induced by 11% ethylene glycol appeared to be due to elevated osmotic pressure; no further details are given. (BIBRA 1988 – quoted from IUCLID 2000).

A negative result has also been obtained for aneuploidy induction in the fungus Neurospora crassa when tested with and without metabolic activation (Griffiths 1979, 1981 – quoted from ATSDR 1997).

A negative result has been reported for gene mutation in the HGPRT assay in Chinese Hamster Ovary cells with and without metabolic activation (Ballantyne 1985, Slesinski et al. 1986 – both quoted from IUCLID 2000 and from BUA 1991). In another HGPRT assay in Chinese Hamster Ovary cells, there was no dose-related increase in the incidence of mutagenic events either in the presence or absence of metabolic activations; according to IUCLID, the result of the assay was ambiguous (Union Carbide Corporation 1981 – quoted from IUCLID 2000).

Ethylene glycol has been reported to yield a negative result when tested for point mutations in the mouse lymphoma assay in L5178Y with and without metabolic activation (Brown et al. 1980 – quoted from IUCLID 2000 and from BUA 1991, McGregor et al. 1991 – quoted from IUCLID 2000 and from NTP 1993).

Negative results were obtained for sister chromatid exchanges when ethylene glycol was tested in Chinese Hamster Ovary cells with and without metabolic activation (Ballantyne 1985, Slesinski et al. 1986 – both quoted from IUCLID 2000 and from BUA 1991, Galloway et al. 1985,1987 – quoted from NTP 1993) or without metabolic activation at concentrations for 2 to 20% (v/v) (Union Carbide Corporation 1981 – quoted from IUCLID 2000).

A negative result was also obtained for chromosome aberrations when ethylene glycol was tested in Chinese Hamster Ovary cells with and without metabolic activation (Galloway et al. 1985,1987 – quoted from NTP 1993, Ballantyne 1985, Slesinski et al. 1986 – both quoted from IUCLID 2000 and from BUA 1991). When tested in another assay at concentrations from 10 to 100 mg/ml, ethylene glycol did not produce an increase in the incidence of chromosome aberrations; according to IUCLID, the result of the assay was ambiguous (Union Carbide Corporation 1985 – quoted from IUCLID 2000).

Negative results were also obtained for unscheduled DNA synthesis when ethylene glycol was tested in rat hepatocytes with and without metabolic activation (Slesinski et al. 1986, Union Carbide Corporation 1981 – quoted from IUCLID 2000 and from BUA 1991).

67.4.2     In vivo studies

Ethylene glycol has been tested for mutagenic effects in the micronucleus assay in Swiss mice by oral administration of 2.5, 3.125, 6.25, or 12.5 ml/kg b.w. (corresponding to 2.8, 3.5, 7.0, or 13.9 g/kg b.w.; 4, 4, 4, or 2 animals in the dose groups, respectively) and by intraperitoneal injection of 1.25, 2.5, or 6.25 ml/kg b.w. (corresponding to 1.4, 2,8, or 7.0 g/kg b.w.; 5, 5, or 1 animal in the dose groups, respectively). A control group of 19 animals was included. Animals in the high-dose oral group (13.9 g/kg b.w.) showed toxic effects. At all, but the lowest oral dose level (2.8 g/kg b.w.), there was an increase in the numbers of micronuclei in polychromatic erythrocytes when compared to the control. According to IUCLID, a weak positive but not dose dependent result was obtained. (Conan et al. 1979).

When tested in mice for chromosome aberrations following an intraperitoneal injection of 2.5 ml/kg b.w., a negative result was obtained (Conan et al. 1979 – quoted from BUA 1991).

In a combined 3-generation reproduction and dominant lethal study, ethylene glycol was administered to Fischer 344 rats (20 females and 10 males) in their diet at approximate dose levels of 40, 200, or 1000 mg/kg b.w. per day. Two control groups received the same diet without ethylene glycol. Males from each dosage group of the F₂ generation, which had received an ethylene glycol containing diet for 155 days, were bred with 15 untreated females at weekly intervals for 3 weeks. On day 12 of gestation, the females were sacrificed, and uteri and ovaries were examined for the numbers of living and dead foetuses. No significant changes were observed in any of the test groups. (DePass et al. 1986b).

67.5     Carcinogenic effects

67.5.1.1     Rats

Fischer 344 rats (130 males and females per group) were fed diets (0.1, 0.5, or 2.5%) yielding approximate dosages of 40, 200, or 1000 mg/kg b.w. per day of ethylene glycol for 24 months. Two untreated control groups were included. The only tumour type for which there was a significant difference was fibroadenoma of the mammary gland in females at the lowest dose level (0.1%). According to the authors, this finding was most probably unrelated to ethylene glycol treatment because of the absence of an effect at the two higher dose levels. Non-neoplastic findings are described in 4.2.2.1. (DePass et al. 1986a).

In Sprague-Dawley rats (16 male and female animals per group), which received ethylene glycol in their diet at concentrations of 0, 0.1, 0.2, 0.5, 1.0, or 4.0% (equivalent to 0, 50, 100, 250, 500 or 2000 mg/kg b.w./day) for 2 years, mammary tumours developed in many female rats towards the end of the experiment. According to the author, the distribution of the tumours in both males and females was such that it was impossible to correlate incidence with treatment. Few tumours were identified in organs within the body and none was associated with the occurrence of calculi. (Blood 1965).

67.5.1.2     Mice

In B6C3F1 mice (60 animals of each sex per group), which were fed diets containing ethylene glycol for 103 weeks (male mice: approximately 0, 1500, 3000, or 6000 mg/kg b.w./day; female mice: approximately 0, 3000, 6000, or 12000 mg/kg b.w./day), no treatment-related neoplasms were observed at the 15-month interim evaluations or at the end of the 2-year studies. Non-neoplastic findings are described in 4.2.2.2. (NTP 1993).

CD-1 mice (80 males and females per group) were fed diets (0.1-0.05, 0.7-0.24, or 0.35-1.27%) yielding approximate dosages of 40, 200, or 1000 mg/kg b.w. per day of ethylene glycol for 24 months. Two untreated control groups were included. The only tumour type for which there was any evidence of a possible increased incidence was lymphosarcoma in females. The time-adjusted incidence was significantly increased according to one of three trend tests. Comparisons among groups for differences in tumour proportions indicated that the observed results could have occurred by chance alone. Non-neoplastic findings are described in 4.2.2.2. (DePass et al. 1986).

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