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

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

70 Evaluation

Ethylene glycol (EG) is rapidly and almost completely absorbed (inhalation, rats: 75-80% of inhaled EG (vapour or aerosol); oral, rats and mice: 90-100%; dermal, rats: 30%; dermal, mice: 85-100%), distributed, metabolised and cleared (almost completely 12 to 48 hours after dosing) following exposure. It should be noted that dermal uptake generally is greater in rodents than in humans because the skin of rodents is thinner than that of humans.

EG is metabolised by oxidation via glycolaldehyde and glycolic acid to glyoxylic acid, which is converted either to carbon dioxide or to oxalic acid. The kinetic study published by Pottenger et al. (2001) indicated that the metabolic conversion of EG to glycolic acid was approaching saturation at EG blood levels obtained following oral administration (gavage) of 2500 mg/kg b.w., and that glycolic acid was being formed at a maximum rate between 1000 and 2500 mg/kg b.w.

The major excretory end products are carbon dioxide in exhaled air, and glycolate and unchanged EG in the urine. The study published by Pottenger et al. (2001) showed that urinary elimination demonstrated dose-dependency, with the high dose groups (2500 mg/kg b.w.) eliminating almost 70% of the administered dose in urine, compared with about 16% in the low dose groups (10 mg/kg b.w.); the shift in urinary elimination was mainly due to increased urinary glycolic acid and EG, and not to increased elimination of oxalic acid. The study also showed that oxalate was a very minor metabolite in both blood and urine at all dose levels (up to 2500 mg/kg b.w.).

There are numerous case reports in the literature of poisoning in humans due to accidental or intentional ingestion of EG; the minimal lethal oral dose for humans has been estimated to be about 1600 mg/kg b.w. for adults. The clinical symptoms of acute EG poisoning in humans can be divided into three (possibly four) fairly distinct stages (effects on the CNS, cardiopulmonary effects, renal effects, and delayed neurological effects); toxicity in stage 2 and 3 is characterised by severe metabolic acidosis. The severity of these stages and the advance from one stage to the next depends greatly on the amount of EG absorbed.

The acute toxicity of EG in experimental animals closely mirrors the acute effects seen in humans; one exception is the fourth clinical stage as no studies have been found that reported this stage in animals. EG is of low acute toxicity in experimental animals, except the cat, with reported oral LD₅₀-values ranging from >2000 to 15400 mg/kg b.w.; a minimal lethal dose of 3800 mg/kg b.w. has been reported for rats, of around 7000 mg/kg b.w. for dogs and, of 1000 mg/kg b.w. for cats. Comparing the lethal oral dose in humans to the minimum lethal dose in experimental animals, EG appears to be two to five times more acutely toxic to humans and cats, on a body weight basis, than to rats and dogs. EG is classified for acute toxic effects following oral administration according to the EU classification criteria.

The very limited data on acute inhalation and dermal toxicity in experimental animals also indicate a low acute toxicity by these routes with a reported LC₅₀-value (one hour) of 10.9 g/m³ in rats and dermal LD₅₀-values of around 10600 mg/kg b.w. for the rabbit.

EG has not shown a particularly irritating potential to eyes or skin in humans and did not show irritating properties when applied to the skin of rabbits. Prolonged dermal exposure to humans can result in skin maceration.

The data on eye irritation in experimental animals are conflicting but overall, the data indicate an eye irritating potential following instillation of either neat EG or solutions of EG to rabbit eyes. Moderate to severe eye irritation has been observed in rats and rabbits exposed continuously for 90 days to EG (vapour, 12 mg/m³) but not following exposure to a concentration of 57 mg/m³ (vapour, 8 hours a day, 5 days per week for 6 weeks); guinea pigs, dogs, and monkeys exposed similarly showed no effects on the eyes.

Male volunteers complained of irritation of the throat following exposure to 17 to 49 mg/m³ (aerosol, 20-22 hours a day for 30 days); the irritation became common at about 140 mg/m³ when the volunteers re-entered the exposure chamber in which the concentration of EG (during the absence of volunteers) was raised and concentrations greater than about 200 mg/m³ were intolerable due to strong irritation of the upper respiratory tract.

EG is not considered to have a sensitising potential in humans although some case reports are available. No data on sensitisation in experimental animals have been found.

Male volunteers (exposed for 30 days, 20-22 hours a day, aerosol, mean concentrations of 17 to 49 mg/m³) did not experience any serious signs of toxicity and no indications of renal toxicity (alterations in urea nitrogen and creatinine in the blood) were observed. Similarly, no indications of renal toxicity were observed in rats, guinea pigs, rabbits, dogs and monkeys exposed to EG (vapour) either continuously (12 mg/m³ for 90 days), or repeatedly (10 or 57 mg/m³ for 8 hours a day, 5 days per week for 6 weeks). Inflammatory changes were observed in the lungs of exposed animals and to a lesser degree in controls as well as some effects in the liver in a few animals; most of the observed effects were interpreted, by the authors, as being unrelated to the exposure to EG, however, these interpretations cannot be evaluated from the data provided in the publication (Coon et al. 1970).

Repeated oral administration of EG to rats results primarily in toxic effects in the kidneys. Male rats are far more sensitive to the renal effects of EG than are female rats as renal effects occur in male rats at dose levels from about 200-250 mg/kg b.w./day (16-week and 2-year feeding studies) but in female rats at dose levels above 1000 mg/kg b.w./day. Mice appear to be relatively resistant to EG induced kidney damage when compared to rat as incidence and severity of nephropathy were not affected in either sex at dietary levels (2-year feeding study) of up to 6000 mg/kg b.w./day in males and of up to 12000 mg/kg b.w./day in females.

The incidence and severity of the renal effects appear to depend on the dose level as well as on the exposure duration. A NOAEL for renal effects (in male rats) of about 550 mg/kg b.w./day can be considered from a 90-day drinking water study (Robinson et al. 1990) and of about 625 mg/kg b.w./day from a 13-week feeding study (Melnick 1984) whereas 2-year feeding studies have revealed NOAELs of 100 (Blood 1965) and 200 mg/kg b.w./day (DePass et al. 1986a), respectively. This difference in the NOAELs could be a result of different sensitivity in the rat strains used in the two studies (Blood: Sprague-Dawley; DePass et al.: Fischer 344/N), but no data are available to further elucidate this aspect. However, the validity of the study by DePass et al. is considered to be better than that of the study by Blood, e.g., 130 animals of each sex per group compared to 16 animals of each sex per group; more detailed descriptions of results including incidence and severity of renal effects in the various dose groups. The lowest NOAEL (about 70 mg/kg b.w./day) for damage to the kidneys in male Wistar rats has been observed in a 16-week dietary study (Gaunt et al. 1974 – quoted from BUA 1991 and IUCLID 2000); however, BUA and IUCLID do not give any details about the kidney damage (type, incidence, severity) and the study report is not public available. Overall, a NOAEL for renal effects in male rats, the most sensitive species, of 200 mg/kg b.w./day is considered taken into account the reliability of the various studies as discussed above. According to NTP (1993), the study by DePass et al. (1986a) was considered adequate to evaluate the chronic toxicity of EG in F344 rats; therefore, NTP only has conducted a 2-year study in mice.

According to DePass et al. (1986), the greater susceptibility of male rats to EG induced renal toxicity may be the result of more efficient conversion of EG to toxic metabolites including oxalate in male rats, as well as to the more rapid progression of spontaneous nephropathy in the male. High-dose females had significant amounts of urinary oxalate crystals at 12, 18, and 24 months, so conversion of EG to oxalate clearly occurred in females also. The critical factor responsible for the more severe nephrotoxicity in male rats was probably the greater incidence and severity of spontaneous nephropathy in the male.

No data on toxicity to reproduction in humans have been found.

Dietary exposure of male and female F344 rats to EG at dose levels up to 1000 mg/kg b.w./day (the highest dose level in the study) for three generations produced no effects on fertility, fecundity, or reproductive performance. When EG was administered to CD-1 mice in the drinking water for 14 weeks (continuous breeding study), reduced fertility and fecundity, and foetotoxic effects, including malformations were observed at about 1640 mg/kg b.w./day; the NOAEL was about 840 mg/kg b.w./day.

Administration of EG via the gastrointestinal route (gavage) at high concentrations has resulted in developmental toxicity, including teratogenicity in rats and mice. Developmental toxicity was observed in CD rats in two gavage studies at dose levels from about 1000 mg/kg b.w./day (gavage) and in Wistar rats from about 860 mg/kg b.w./day whereas one gavage study did not show any developmental toxicity in CD rats at 1250 mg/kg b.w./day. When EG was administered to F344 rats in the diet, no developmental effects were observed at dose levels up to 1000 mg/kg b.w./day (the highest dose level in the study). No serious maternal effects were noted at the dose levels resulting in developmental toxicity. Mice appear to be far more sensitive to the developmental effect exerted by EG with severe developmental toxicity, including malformations, being observed in a gavage study at dose levels from 750 mg/kg b.w./day (the lowest dose level in the study), a dose level where no maternal effects were observed. In another gavage study, lower dose levels were administered and a NOAEL for developmental toxicity of 150 mg/kg b.w./day can be considered from this study. One gavage study in rabbits indicates that this species is fairly resistant to the developmental effects exerted by EG as no developmental effects were observed at dose levels up to 2000 mg/kg b.w./day (the highest dose level in the study).

Following dermal application of EG to CD-1 mice, no developmental effects were observed at dose levels up to about 3550 mg/kg b.w./day (the highest dose level in the study).

Developmental toxicity, including teratogenicity, has been observed in CD-1 mice following whole-body exposures to EG respirable aerosol at concentrations from 1000 mg/m³ (6 hours a day); CD rats exposed similarly exhibited developmental toxicity, but no teratogenicity at the same exposure levels. This is consistent with the results observed in the oral studies discussed above, in which mice appeared to be far more sensitive to the developmental effect exerted by EG than rats. The NOAEL for developmental effects in the whole-body study was 150 mg/m³ for rats and at or below 150 mg/m³ for mice with NOAELs for maternal effects of 1000 and 150 mg/m³, respectively (no concentrations between 150 and 1000 mg/m³). Analysis of EG on the fur of rats and mice during and after the exposure period (2500 mg/m³) showed significant amounts of EG on the fur, which, according to the authors (Tyl et al. 1995a,b), alone could have produced the effects seen in mice if it were ingested by grooming and/or percutaneously absorbed. Therefore, a nose-only study was performed in CD-1 mice in order to evaluate the toxicity of EG aerosol from inhalation exposure alone (500, 1000, and 2500 mg/m³). In this study, the NOAEL for maternal and developmental effects, including teratogenicity, was 500 and 1000 mg/m³, respectively. Assuming (for pregnant mice) an inhalation rate of 25 ml/min (corresponding to 0.036 m³/day), a body weight of 0.035 kg, and 100% absorption of EG by inhalation, the NOAELs correspond to about 500 and 1000 mg/kg b.w./day, respectively.

Most of the mutagenicity and genotoxicity tests available indicate that EG is not a mutagenic or genotoxic substance although some positive results have been reported. In the micronucleus assay in mice, the increased numbers of micronuclei was observed following administration (oral, intraperitoneal injection) of very high doses (2.8 to 13.9 g/kg b.w.) and thus, the result is not considered as being reliable. Overall, EG is considered not to be a mutagenic or genotoxic substance.

No evidence of a carcinogenic effect of EG was observed at dietary concentrations of up to approximately to 2000 mg/kg b.w./day for 2 years in rats or of up to approximately 12000 mg/kg b.w./day for 2 years in mice.

No data on mutagenic and genotoxic effects, or carcinogenic effects of EG in humans have been found.

The toxicity of EG is primarily a result of the effects of its metabolites although the effects on the central nervous system observed shortly after acute ingestion is partly attributed to unmetabolised EG.

The metabolic acidosis is usually attributed to the acidic metabolites of EG and recent studies of cases of human EG poisoning (discussed in Jacobsen & McMartin 1997) have demonstrated that the major determinant of the metabolic acidosis is the degree of glycolic acid accumulation as glycolate accumulation correlates with the increase in anion gap or decrease in arterial bicarbonate concentrations observed in poisoned humans, as well as in animals.

The mechanism of the renal toxicity is not yet known. Calcium oxalate precipitation within the renal tubules has long been accepted as an important pathogenic factor for the development of renal toxicity; however, there is no evidence directly linking oxalate precipitation with development of renal tubular necrosis and renal damage can occur at exposure levels where no or few oxalate crystals are detected. The renal toxicity has also been suggested to occur from a metabolite-induced cytotoxicity, such as from glycolaldehyde or glyoxylate, which are both highly toxic in vitro, or via the metabolic acidosis resulting from accumulation of glycolic acid. As a result of the decrease in the pH in body fluids, an elevated anion gap develops and the serum osmolal gap across cells increases, resulting in renal oedema that compromises intrarenal blood flow and promotes renal failure. These suggestions are in concordance with the data published by Pottenger et al. (2001) showing that oxalate was a very minor metabolite in rats in both blood and urine at oral (gavage) dose levels up to 2500 mg/kg b.w. and the data discussed in Jacobsen & McMartin (1997) showing that glycolate accumulation correlates with the increase in anion gap or decrease in arterial bicarbonate concentrations observed in poisoned humans, as well as in animals.

Although the mechanism(s) behind the developmental effects is not yet known, a link between maternal metabolic acidosis and developmental toxicity has been suggested with glycolic acid being the predominant toxic metabolite.

70.1.1 Conclusion

The critical effects following exposure to EG are the effects in the kidneys, which are observed in both humans and experimental animals; the developmental effects observed in experimental animals; and the irritative effects observed in humans and experimental animals following inhalation of EG.

In female rats, effects on the liver (mild fatty metamorphosis) has been observed with a NOAEL of about 200 mg/kg b.w./day; however, the liver effects observed are not considered to be as serious as the renal lesions observed in male rats although a NOAEL of about 200 mg/kg b.w./day for renal effects in male rats has been considered as well.

The mechanism(s) behind the nephrotoxic and developmental effects are not known but are probably due to the metabolic acidosis resulting from an accumulation of the EG metabolite glycolic acid. Glycolic acid is a major metabolite in both humans and experimental animals whereas oxalate appears to be a minor metabolite.

Based on the results reported in the available oral studies, male rats are far more sensitive to the renal effects than are female rats and mice (both sexes). No data are available in order to evaluate the sensitivity of humans to the renal effects as no long term studies in humans are available. Following acute ingestion of EG, the same type of renal effects are observed in humans as in experimental animals. Therefore, humans are considered to be as sensitive as male rats to the nephrotoxic effects of EG; a NOAEL of 200 mg/kg b.w./day has been considered for renal effects in male rats from a 2-year dietary study as discussed above. The exposure levels in the available inhalation studies (volunteers: 17-49 mg/m³ for 30 days; experimental animals: 12 mg/m³ continuously for 90 days or 10-57 mg/m³ for 8 hours a day, 5 days per week for 6 weeks) are considered to be far too low to result in any renal effects.

Mice appear to be far more sensitive to the developmental toxicity exerted by EG than are rats and rabbits. No data on reproductive toxicity in humans are available. Therefore, humans are considered to be as sensitive as mice to the developmental effects of EG. In a nose-only inhalation study, the NOAEC for developmental effects, including teratogenicity, in mice was 1000 mg/m³ while the NOAEC was at or below 150 mg/m³ in a whole-body inhalation study (no concentrations between 150 and 1000 mg/m³). Assuming (for pregnant mice) an inhalation rate of 25 ml/day (corresponding to 0.036 m³/day), a body weight of 0.035 kg, and 100% absorption of EG by inhalation, these NOAECs correspond to about 1000 and 150 mg/kg b.w./day, respectively. A NOAEL for developmental toxicity in mice following gavage of 150 mg/kg b.w./day can be considered as discussed above.

Male volunteers complained of irritation of the throat following exposure to mean concentrations of 17 to 49 mg/m³ (aerosol, 20-22 hours a day for 30 days). Moderate to severe eye irritation has been observed in rats and rabbits exposed continuously for 90 days to EG (vapour, 12 mg/m³) but not following exposure to a concentration of 57 mg/m³ (vapour, 8 hours a day, 5 days per week for 6 weeks); furthermore, the data indicate an eye irritating potential following instillation of either neat EG or solutions of EG to rabbit eyes. Based on these data, a LOAEC for irritative effects of 17 mg/m³ is considered. Assuming (for adults) an inhalation rate of 20 m³/day, a body weight of 70 kg, and 100% absorption of EG by inhalation, this LOAEC corresponds to about 5 mg/kg b.w./day, which is far below the NOAELs considered for renal (200 mg/kg b.w./day) and developmental toxicity (150 mg/kg b.w./day).