This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the United Nations Environment Programme, the International Labour Organization, or the World Health Organization.

First draft prepared by R. Gomes, R. Liteplo, and M.E. Meek, Health Canada, Ottawa, Canada

Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organization, and the World Health Organization, and produced within the framework of the Inter-Organization Programme for the Sound Management of Chemicals.

World Health Organization
Geneva, 2002

The International Programme on Chemical Safety (IPCS), established in 1980, is a joint venture of the United Nations Environment Programme (UNEP), the International Labour Organization (ILO), and the World Health Organization (WHO). The overall objectives of the IPCS are to establish the scientific basis for assessment of the risk to human health and the environment from exposure to chemicals, through international peer review processes, as a prerequisite for the promotion of chemical safety, and to provide technical assistance in strengthening national capacities for the sound management of chemicals.

The Inter-Organization Programme for the Sound Management of Chemicals (IOMC) was established in 1995 by UNEP, ILO, the Food and Agriculture Organization of the United Nations, WHO, the United Nations Industrial Development Organization, the United Nations Institute for Training and Research, and the Organisation for Economic Co-operation and Development (Participating Organizations), following recommendations made by the 1992 UN Conference on Environment and Development to strengthen cooperation and increase coordination in the field of chemical safety. The purpose of the IOMC is to promote coordination of the policies and activities pursued by the Participating Organizations, jointly or separately, to achieve the sound management of chemicals in relation to human health and the environment.

WHO Library Cataloguing-in-Publication Data

Ethylene glycol : human health aspects.

(Concise international chemical assessment document ; 45)

1. Ethylene glycol - toxicity 2. Ethylene glycol - adverse effects

3. Risk assessment 4. Environmental exposure

5. No-observed-adverse-effect level I.International Programme on Chemical Safety II.Series

ISBN 92 4 153045 6 (NLM Classification: QD 305.A4)

ISSN 1020-6167

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Concise International Chemical Assessment Documents (CICADs) are the latest in a family of publications from the International Programme on Chemical Safety (IPCS) — a cooperative programme of the World Health Organization (WHO), the International Labour Organization (ILO), and the United Nations Environment Programme (UNEP). CICADs join the Environmental Health Criteria documents (EHCs) as authoritative documents on the risk assessment of chemicals.

International Chemical Safety Cards on the relevant chemical(s) are attached at the end of the CICAD, to provide the reader with concise information on the protection of human health and on emergency action. They are produced in a separate peer-reviewed procedure at IPCS.

CICADs are concise documents that provide summaries of the relevant scientific information concerning the potential effects of chemicals upon human health and/or the environment. They are based on selected national or regional evaluation documents or on existing EHCs. Before acceptance for publication as CICADs by IPCS, these documents undergo extensive peer review by internationally selected experts to ensure their completeness, accuracy in the way in which the original data are represented, and the validity of the conclusions drawn.

The primary objective of CICADs is characterization of hazard and dose–response from exposure to a chemical. CICADs are not a summary of all available data on a particular chemical; rather, they include only that information considered critical for characterization of the risk posed by the chemical. The critical studies are, however, presented in sufficient detail to support the conclusions drawn. For additional information, the reader should consult the identified source documents upon which the CICAD has been based.

Risks to human health and the environment will vary considerably depending upon the type and extent of exposure. Responsible authorities are strongly encouraged to characterize risk on the basis of locally measured or predicted exposure scenarios. To assist the reader, examples of exposure estimation and risk characterization are provided in CICADs, whenever possible. These examples cannot be considered as representing all possible exposure situations, but are provided as guidance only. The reader is referred to EHC 170.¹

While every effort is made to ensure that CICADs represent the current status of knowledge, new information is being developed constantly. Unless otherwise stated, CICADs are based on a search of the scientific literature to the date shown in the executive summary. In the event that a reader becomes aware of new information that would change the conclusions drawn in a CICAD, the reader is requested to contact IPCS to inform it of the new information.

The flow chart on page 2 shows the procedures followed to produce a CICAD. These procedures are designed to take advantage of the expertise that exists around the world — expertise that is required to produce the high-quality evaluations of toxicological, exposure, and other data that are necessary for assessing risks to human health and/or the environment. The IPCS Risk Assessment Steering Group advises the Coordinator, IPCS, on the selection of chemicals for an IPCS risk assessment based on the following criteria:

• there is the probability of exposure; and/or
• there is significant toxicity/ecotoxicity.

Thus, a priority chemical typically

• is of transboundary concern;
• is of concern to a range of countries (developed, developing, and those with economies in transition) for possible risk management;
• is significantly traded internationally;
• has high production volume;
• has dispersive use.

The Steering Group will also advise IPCS on the appropriate form of the document (i.e., EHC or CICAD) and which institution bears the responsibility of the document production, as well as on the type and extent of the international peer review.

The first draft is based on an existing national, regional, or international review. Authors of the first draft are usually, but not necessarily, from the institution that developed the original review. A standard outline has been developed to encourage consistency in form. The first draft undergoes primary review by IPCS to ensure that it meets the specified criteria for CICADs.

The second stage involves international peer review by scientists known for their particular expertise and by scientists selected from an international roster compiled by IPCS through recommendations from IPCS national Contact Points and from IPCS Participating Institutions. Adequate time is allowed for the selected experts to undertake a thorough review. Authors are required to take reviewers’ comments into account and revise their draft, if necessary. The resulting second draft is submitted to a Final Review Board together with the reviewers’ comments. At any stage in the international review process, a consultative group may be necessary to address specific areas of the science.

The CICAD Final Review Board has several important functions:

• to ensure that each CICAD has been subjected to an appropriate and thorough peer review;
• to verify that the peer reviewers’ comments have been addressed appropriately;
• to provide guidance to those responsible for the preparation of CICADs on how to resolve any remaining issues if, in the opinion of the Board, the author has not adequately addressed all comments of the reviewers; and
• to approve CICADs as international assessments.

Board members serve in their personal capacity, not as representatives of any organization, government, or industry. They are selected because of their expertise in human and environmental toxicology or because of their experience in the regulation of chemicals. Boards are chosen according to the range of expertise required for a meeting and the need for balanced geographic representation.

Board members, authors, reviewers, consultants, and advisers who participate in the preparation of a CICAD are required to declare any real or potential conflict of interest in relation to the subjects under discussion at any stage of the process. Representatives of nongovernmental organizations may be invited to observe the proceedings of the Final Review Board. Observers may participate in Board discussions only at the invitation of the Chairperson, and they may not participate in the final decision-making process.

This CICAD on ethylene glycol (human health aspects) was prepared by the Environmental Health Directorate of Health Canada based on documentation prepared as part of the Priority Substances Program under the Canadian Environmental Protection Act (CEPA). The objective of assessments on priority substances under CEPA is to assess potential effects of indirect exposure in the general environment on human health as well as environmental effects, although only aspects related to human health are considered herein. Data identified as of the end of January 2000² were considered in this review. Information on the nature of the peer review and availability of the source documents is presented in Appendix 1. Other reviews that were also consulted include those prepared by the Environmental Criteria Assessment Office of the US Environmental Protection Agency (US EPA, 1987), the Agency for Toxic Substances and Disease Registry of the US Department of Health and Human Services (ATSDR, 1997), and the German Chemical Society (BUA, 1994), as well as reviews prepared under contract by BIBRA International (1996, 1998). Information on the peer review of this CICAD is presented in Appendix 2. This CICAD was approved as an international assessment at a meeting of the Final Review Board, held in Ottawa, Canada, on 29 October – 1 November 2001. Participants at the Final Review Board meeting are presented in Appendix 3. The International Chemical Safety Card (ICSC 0270) on ethylene glycol, produced by the International Programme on Chemical Safety (IPCS, 2000a), has also been reproduced in this document. The effects of ethylene glycol on the environment are not considered herein, since they were addressed in CICAD No. 22 (IPCS, 2000b).

Ethylene glycol (CAS No. 107-21-1) is a colourless, odourless, sweet-tasting, relatively non-volatile liquid. It has a low vapour pressure and is completely miscible in water.

Ethylene glycol is used in the manufacture of polyethylene terephthalate, in natural gas processing, and as an antifreeze agent. Monitoring data upon which to base estimates of exposure of the general population to ethylene glycol are extremely limited. In a sample estimate of exposure, intakes in air and soil in the vicinity of a point source were estimated based on modelled data, and that in food was based on reported concentrations in a very limited range of foodstuffs from various countries. Dermal absorption was also estimated for a limited range of products for which data on the proportion of ethylene glycol in the products were identified.

There is convincing evidence that the toxicity of ethylene glycol is mediated principally through metabolites (notably, glycolate and oxalate). Available data also indicate that the likely pathways involved in the metabolism of ethylene glycol are qualitatively similar in humans and other mammalian species; potential quantitative differences have not been well studied.

Ethylene glycol has low acute toxicity in experimental animals following oral, inhalation, or dermal exposure. In both humans and animals, ethylene glycol has induced only minimal dermal irritation. Nasal and/or throat irritation were reported in a small number of subjects inhaling ethylene glycol, while higher concentrations produced severe irritation. In experimental animals, ethylene glycol induces only minimal conjunctival irritation, without permanent corneal damage. Data on the potential of ethylene glycol to induce sensitization have not been identified.

Ethylene glycol has not been carcinogenic in a 2-year bioassay in rats and mice, in primarily limited, early bioassays. In the limited number of identified in vitro and in vivo studies, ethylene glycol has not been genotoxic.

Available data from acute poisoning cases (humans) and repeated-dose toxicity studies (experimental animals) indicate that the kidney is a critical organ for the toxicity of ethylene glycol in both humans and experimental animals. Consistently, metabolic acidosis and degenerative non-neoplastic changes in the kidney (including dilation, degeneration, and deposition of calcium oxalate in the tubules) have been observed at lowest doses in a range of species.

Based on a rather extensive database, ethylene glycol induces developmental effects in rats and mice by all routes of exposure, although at doses greater than those associated with renal effects in male rats. Indeed, ethylene glycol is teratogenic, inducing primarily skeletal variations and external malformations, sometimes at doses less than those that are maternally toxic, with mice being more sensitive than rats. The reproductive toxicity of ethylene glycol has been extensively investigated in adequate studies in mice and rats. In repeated-dose toxicity studies, there has been no evidence of adverse impact on reproductive organs; in specialized studies, including a three-generation study in rats and continuous-breeding protocols in mice, evidence of reproductive effects has been restricted to mice (but not rats or rabbits) exposed to doses considerably greater than those associated with developmental effects in this species or renal effects in rats.

Available data are inadequate to assess potential adverse neurological or immunological effects associated with long-term exposure to ethylene glycol, although neurobehavioural and neurological disorders have been reported in cases of acute ethylene glycol poisoning in humans. In the limited number of investigations identified to date, neurological effects have not been observed at doses below those that have induced renal toxicity. Consistent treatment-related effects on immune system-related parameters have not been observed in available repeated-dose toxicity studies, in which several species have been exposed to ethylene glycol either orally or by inhalation.

A tolerable intake of 0.05 mg/kg body weight per day has been derived for this substance, based on a benchmark dose of 49 mg/kg body weight per day calculated for non-neoplastic renal effects in animals and an uncertainty factor of 1000. However, this tolerable intake is uncertain, owing primarily to lack of information on progression of renal lesions in the most sensitive animal model. Highly uncertain sample estimates of exposure of some age groups in the vicinity of a point source or of adults through absorption from some consumer products approach or exceed the tolerable intake. Additional study to better characterize progression of the renal lesions and to refine exposure estimates is recommended.

Ethylene glycol (CAS No. 107-21-1) belongs to the simplest group of organic chemicals of the chemical family of glycols, which are characterized by two hydroxyl groups at adjacent positions in a hydrocarbon chain (see Figure 1).

Figure 1: Chemical structure of ethylene glycol

Ethylene glycol is a clear, colourless, odourless, relatively non-volatile, viscous liquid. It has a sweet taste and imparts a warming sensation to the tongue when swallowed. Ethylene glycol has a relatively low vapour pressure (7–12 Pa at 20 °C) and a low Henry’s law constant of 5.8 × 10^–6 – 6.0 × 10^–3 Pa·m³/mol. It is completely miscible in water. It is very hygroscopic and will absorb up to 200% of its weight in water at 100% relative humidity. The octanol/water partition coefficient of ethylene glycol is very low (i.e., log K_ow = –1.36). The conversion factors for airborne ethylene glycol at 101.3 kPa and 20 °C are 1 ppm = 2.6 mg/m³ and 1 mg/m³ = 0.39 ppm (Health Canada, 2000). Information on other physical/chemical properties of ethylene glycol is included in the International Chemical Safety Card (ICSC 0270) reproduced in this document.

The common analytical methods used for determining ethylene glycol in biological and environmental samples are presented in Table 1. The primary method is derivatization followed by gas chromatography using either a flame ionization detector or mass spectrometry for quantification. Detection limits for ethylene glycol by these methods range from sub- to low milligrams per kilogram or milligrams per litre (ATSDR, 1997). Ethylene glycol and its metabolites, such as glycolate, hippurate, and oxalate, are determined in blood and urine samples by high-performance liquid chromatography. Water samples may be analysed with preparation, but the determination of ethylene glycol in air requires adsorption onto a surface and subsequent extraction. Ethylene glycol in foods and drugs is usually analysed by chromatography of the sample after hexane is used to extract fat from the sample.

Table 1: Analytical methods for determining ethylene glycol in biological and environmental samples.^a,b

Limited information on production and use from the source country of the national assessment on which this CICAD is based (i.e., Canada) is presented here, principally to set context for the sample risk characterization. Additional information on sources, production, and use is presented in CICAD No. 22: Ethylene glycol: Environmental aspects (IPCS, 2000b).

Ethylene glycol was identified as one of the substances in the edible fungus Tricholoma matsutake (Ahn & Lee, 1986) and has been identified as a metabolite of ethylene, a natural plant growth regulator (Blomstrom & Beyer, 1980).

Based on a review by CIS (1997), total worldwide capacity for ethylene glycol is over 10 000 kilotonnes per year, with major increases expected in the future. World-wide, ethylene glycol is used primarily in the production of polyesters for fibre and polyethylene terephthalate (PETE). It is also used in smaller quantities in a broad range of products, including paints, lacquers and resins, coolants and heat transfer fluids, and medicinals and adhesives (ATSDR, 1993; Lewis, 1993).

Available data indicate that projected annual production capacity for the ethylene glycols (mono-, di-, and triethylene glycols) in Canada increased to 907 kilotonnes in 1999 from 524 kilotonnes in 1992 (CIS, 1997). In 1996, approximately 810 kilotonnes of ethylene glycols (mono-, di-, and tri-) were exported from Canada. Import volumes in 1996 were estimated at 31.3 kilotonnes (CIS, 1997).

In Canada, the majority of ethylene glycol is used in antifreeze formulations (primarily for automotive vehicle engines, but also for aircraft deicing), at 66% (105 kilotonnes) of domestic consumption (CIS, 1997). An estimated 7.7 kilotonnes of ethylene glycol were used in 1996 for aircraft deicing/anti-icing (Environment Canada, 1997). The amount used for the production of the polyester PETE in 1996 was relatively small, at 25 kilotonnes (15.7% of domestic consumption). Six per cent or 9.5 kilotonnes were used in natural gas processing to assist in the removal of water and to prevent ice formation. The remaining 19.5 kilotonnes were used in the production of solvents, including use as a component in latex paint formulations to guard against paint freezing and as an antifreeze liquid injected into hoses used to pump liquid explosives (CIS, 1997). In 1995 and 1996, 1.4 kilotonnes and 2.0 kilotonnes were used in the Canadian paints and coatings sector, respectively (Environment Canada, 1997).

Data on environmental levels other than those directly linked to human exposure have been addressed in the source documents and CICAD No. 22 (IPCS, 2000b). Data on concentrations in the environment primarily from the source country of the national assessment on which this CICAD is based (i.e., Canada) are presented here, as a basis for the sample risk characterization for human health.

Only those media for which relevant data have been identified are addressed below. Information on concentrations in indoor air and drinking-water were not identified.

5.1.1 Ambient air

Based on ChemCAN 4.0 modelling and attribution of the reported largest emission to the atmosphere in Canada in 1996 (374 tonnes from ethylene glycol manufacturing plants in Alberta) to one plant, the predicted average airborne concentration in the prairie region of Alberta from this emission would be 1.2 ng/m³. Predicted maximum daily average ground-level concentrations downwind from the plant, responsible for approximately 99% of all releases from manufacturing, were 100, 50, and 25 µg/m³ at distances of 1.8, 4.0, and 6.8 km, respectively, from the property boundary, although annual frequency of occurrence was not presented (Environment Canada, 1997).

Percy (1992) reported concentrations of ethylene glycol in air of 3.2 and 4.1 mg/m³ at the Thunder Bay, Ontario, airport. In Louisiana, USA, during bridge deicing operations, total airborne concentrations were between <0.05 and 10.57 mg/m³; aerosol concentrations were lower (<0.05–0.33 mg/m³) (Abdelghani et al., 1990). Proximity of measurements to source of release and the time period represented by individual measurements were not reported for either of these studies.

5.1.2 Food

The presence of ethylene glycol has been demonstrated in only a small number of foodstuffs. In Italy, ethylene glycol was detected in all 44 samples of wine analysed by gas chromatography–mass spectrometry. The average and maximum concentrations were 2.8 and 6.25 mg/litre, respectively (Gaetano & Matta, 1987). However, the source of ethylene glycol in wines is not known (Gaetano & Matta, 1987; Kaiser & Rieder, 1987). In Japan, ethylene glycol was present in the headspace volatiles of roasted sesame seeds, but quantitative data were not provided (Takei, 1988).

Foods that have been disinfected or preserved with ethylene oxide may contain residual ethylene glycol. In France, Buquet & Manchon (1970) sampled 150 bread loaves preserved with carbonic anhydride and ethylene oxide and packaged in airtight plastic bags. The initial concentrations of ethylene glycol in bread ranged from not detected (detection limit not reported) to 92.2 mg/kg but quickly subsided. In France, Chaigneau & Muraz (1993) sampled 16 spices that had been disinfected using ethylene oxide. Concentrations of ethylene glycol were not reported, and the authors indicated that residual ethylene glycol was rapidly lost.

The potential for ethylene glycol to migrate into beverages contained in PETE bottles and into foods packaged in regenerated cellulose film (RCF) has been demonstrated, resulting from small amounts of unreacted ethylene glycol in such products (Kashtock & Breder, 1980; Castle et al., 1988a; Kim et al., 1990). Kashtock & Breder (1980) measured the migration of ethylene glycol at 32 °C from PETE bottles into 3% acetic acid (intended to simulate carbonated beverages). Time-dependent increases in average concentrations were measured, resulting in a maximum concentration of 104 µg/litre after 6 months’ storage at this elevated temperature.

RCF is widely used as a food packaging material, since its permeability, sealability, and ease of application for twist wrapping are desirable for packing certain foods. In the United Kingdom, Castle et al. (1988a) measured the ethylene glycol content of several foodstuffs wrapped in RCF at random intervals up to the end of their usual maximum shelf-lives. Four samples of boiled sweets contained ethylene glycol at concentrations ranging from 14 to 34 mg/kg. Three of four samples of toffee contained ethylene glycol, with a maximum concentration of 22 mg/kg. Two of four samples of Madeira cake contained ethylene glycol, with a maximum concentration of 22 mg/kg. All four samples of fruit cake contained ethylene glycol, with a maximum concentration of 34 mg/kg. Ethylene glycol was not detected in any of the six samples of meat pie, with a limit of detection of 10 mg/kg.

5.1.3 Consumer products

Several products used in the operation or maintenance of automobiles typically contain ethylene glycol. Concentrations ranging up to 85% may have been present in older automotive brake fluids (US EPA, 1986); however, the ethylene glycol content of current brake fluids is less than 0.1% (ATSDR, 1997). Antifreeze solutions in automobile coolant systems typically have an ethylene glycol content of 50% (Franklin Associates Ltd., 1995). Windshield washer fluids intended for use during winter may contain ethylene glycol at up to 14% by weight (Flick, 1986, 1989). The ethylene glycol content of automobile wax and polish can range up to 3% by weight (US EPA, 1986).

Flick (1986) reported concentrations of ethylene glycol ranging from 1.1% to 1.4% in four types of floor polish intended for use in the home. Concentrations ranging up to 3.5% may be present in floor wax and polishes, according to US EPA (1986).

Ethylene glycol may be present as a slow-evaporating solvent or freeze-thaw stabilizer in latex paints (US EPA, 1986). Chang et al. (1997) estimated that latex paints comprised over 85% of the interior coatings used in the USA in 1992 and reported concentrations of ethylene glycol ranging from 23.3 to 25.8 mg/g (from 2.3% to 2.6% by weight) in four samples of medium-priced paints. Eleven Canadian paint and coatings companies reported that their products may contain up to 5% of ethylene glycol by weight (Environment Canada, 1997).

Flick (1986) also reported that other consumer products that may contain ethylene glycol include tub and tile cleaners (3% by weight) and cement sealer (2.2% by weight). Confirmation of ethylene glycol’s use as a tub and tile cleaner in Canada is currently being sought under CEPA.

Ophthalmic solutions (eyedrops) that have been treated with ethylene oxide as a sterilant may contain ethylene glycol and ethylene chlorohydrin as residues. In the USA, Manius (1979) detected ethylene glycol in 4 of 15 samples of ophthalmic solution, with a range of 10–28 mg/litre (detection limit 6 mg/litre).

The only cosmetic registered for use in Canada that lists ethylene glycol as an ingredient is a solid stick foundation, distributed from Quebec. The concentration of ethylene glycol in this product was not available (C. Denman, personal communication, 1999).

Data on levels of ethylene glycol in environmental media in Canada to serve as a basis for development of estimates of population exposure were identified only for areas near industrial point sources in Alberta. These data are limited to a few predicted concentrations in ambient air at ground level and to measured concentrations in soil. No data were identified concerning the presence or concentrations of ethylene glycol in drinking-water in Canada or elsewhere.

Very meaningful deterministic estimates of average exposure for the general population are precluded due to the limitations of the available data. Worst-case intakes have been estimated for populations near industrial point sources of ethylene glycol, although the limitations of the available data, which serve as the basis for these upper-bound estimates, must be borne in mind in their interpretation. On this basis, intake is estimated to range from 22 to 88 µg/kg body weight per day, as summarized in Table 2.

Data on levels of ethylene glycol in food are limited to results of two earlier studies of migration from RCF and PETE bottles in other countries and to reported concentrations in Italian wines. Worst-case intakes from ingestion of foods assumed to be contaminated with ethylene glycol through contact with food packaging materials have been estimated for the general population. On this basis, intake is estimated to range from less than 2.5 to 41.0 µg/kg body weight per day, as summarized in Table 3. Migration to food from RCF accounts for most of the estimated intakes.

The general population is also exposed periodically to ethylene glycol through the use of several consumer products, including, for example, automobile antifreeze, wax, polish, and windshield washer solution, floor wax and polish, possibly tub and tile cleaner, and latex paint. Estimation of exposure from such products is incomplete, due to the lack of adequate data on the proportions that include ethylene glycol as an ingredient and on the concentrations in the various products available. Although automobile coolant (i.e., antifreeze) and winter windshield washer fluid are expected to contain the highest concentrations of ethylene glycol, human exposure to these products is expected to be infrequent and of short duration for a small number of individuals and negligible for the majority of the general population. Also, while some inhalation exposure is expected during use of the products mentioned above, intakes by inhalation were not estimated, since the physicochemical properties of ethylene glycol limit its rate of evaporation from liquid products and the application of these products does not generally involve formation of aerosols.

Intakes by dermal absorption have been estimated for adults using these consumer products (Health Canada, 2000). Generic estimates of the maximum concentrations of ethylene glycol in these products are assumed, as data from analyses of specific products in Canada are not available for this purpose. Based on the worst-case assumptions of 100% dermal absorption of ethylene glycol from thin films of liquid products containing the maximum expected concentrations of ethylene glycol in standardized scenarios using estimates of frequencies of use and areas of exposed skin, the upper-bounding estimates of daily intakes by adults range from 0.09 to 236 µg/kg body weight per day for the four product types for which generic estimates of ethylene glycol content are available. This information is summarized in Table 4.

Table 4: Deterministic estimates of upper-bounding daily intakes for adults by dermal absorption
from consumer products.^a

The highest estimates of daily intake by adults due to dermal absorption of ethylene glycol from consumer products result from the standardized scenarios involving the use of tub and tile cleaner. Although mid-point estimates of event frequencies are assumed (i.e., 156 and 48 events per year), these frequencies are considerably higher than the conservative estimates of event frequencies assumed in the scenarios involving the three other consumer product types in Table 4. Therefore, daily intakes are higher for the tub and tile cleaner, even though higher concentrations of ethylene glycol may be present in the other product types.

It should be noted that these values represent worst-case estimates. Estimated daily intakes by dermal absorption of ethylene glycol from use of these consumer products are several orders of magnitude less when based on less conservative assumptions. These include the assumptions that dermal absorption is proportional to the concentration of ethylene glycol in the products and that steady-state penetration occurs for periods equivalent to the average durations of product uses in the standardized scenarios (Health Canada, 2000). However, available data on permeability through human skin are inadequate as a basis for confident estimation of exposure, and, as a result, these estimates are not presented here. This is due to lack of evidence of adequate viability of the skin in the most comprehensive investigation conducted to date, i.e., that by Sun et al. (1995), which may have been compromised due to use of samples of full thickness and lack of identified data on uptake from product formulations (R. Moody, personal communication, 1999; Health Canada, 2000).

A proportion of the population is also exposed to ethylene glycol as passengers during deicing operations for aircraft. While the pattern of exposure of individuals in the population is expected to be highly variable, depending upon frequency of aeroplane travel during the winter season, identified data are inadequate to quantitatively estimate intake from this source.

Based on the US National Occupational Exposure Survey conducted by the National Institute of Occupational Safety and Health (NIOSH, 1990) during 1981–1983, an estimated 1.5 million workers are potentially exposed to ethylene glycol each year. Contact with the skin and eyes is the most likely route of worker exposure to ethylene glycol.

Workers with greatest potential for exposure to ethylene glycol are those in industries involved in the manufacture or use of products containing high concentrations (e.g., antifreeze, coolants, deicing fluids, brake fluids, solvents), particularly in operations involving heating or spraying of these materials (e.g., aircraft deicing). In these cases, inhalation may be an important route of human exposure (Rowe & Wolf, 1982). Air samples taken from the breathing zones of workers applying deicing fluids (50% ethylene glycol) to bridge surfaces contained the compound at concentrations of <0.05–2.33 mg/m³ as aerosols and <0.05–3.37 mg/m³ as vapours (LDOTD, 1990). Trace quantities of ethylene glycol have been detected in artificial theatrical smoke used in plays, concerts, and amusement parks (NIOSH, 1994).

As a small molecular weight alcohol, ethylene glycol readily passes through biological membranes and will be effectively absorbed from the gastrointestinal tract and via inhalation exposure. It is rapidly distributed in body water (Jacobsen et al., 1988).

Based on studies involving oral administration of single doses of ethylene glycol by gavage, absorption is rapid and nearly complete, with peak plasma concentrations increasing linearly with dose among various species (i.e., rats, mice, monkeys) (Carney, 1994). Following the oral administration of 10–1000 mg ethylene glycol/kg body weight per day to Sprague-Dawley rats (both sexes) and CD-1 mice (females), peak plasma concentrations in each species were observed within 1–4 h, with absorption of 90–100% of the administered dose within 24 h (Frantz et al., 1996a,b). Ethylene glycol is rapidly cleared from the blood following absorption into the systemic circulation, with reported plasma half-lives in rodents, monkeys, and dogs (receiving 1–1000 mg/kg body weight) ranging from 1 to 4 h (McChesney et al., 1971; Hewlett et al, 1989; Frantz et al., 1996b).

Following the inhalation exposure (nose only) of F344 rats (n = 15 per sex) to [¹⁴C]ethylene glycol vapour (32 mg/m³) for 30 min or [¹⁴C]ethylene glycol aerosol (184 mg/m³) for 17 min, approximately 60% of the administered radioactivity was absorbed into the systemic circulation (Marshall & Cheng, 1983). Peak plasma levels were observed within 1 h, with a half-life for plasma clearance of 34–39 h (Marshall & Cheng, 1983).

The results of available studies also indicate that, compared with oral exposure, ethylene glycol was absorbed more slowly and to a lesser extent into the systemic circulation following dermal contact. Unlike the extensive bioavailability of ethylene glycol administered orally, the bioavailability of (unchanged) ethylene glycol within the first 6 h following dermal exposure was only 20–30% and 5% in rats and mice, respectively, administered a dose of 1000 mg/kg body weight (Frantz et al., 1996a,b).

Ethylene glycol is oxidized in experimental animals and in humans in successive steps, first to glycoaldehyde (in a reaction catalysed by alcohol dehydrogenase), then to glycolic acid, glyoxylic acid, and oxalic acid (Figure 2). Glyoxylic acid is metabolized in intermediary metabolism to malate, formate, and glycine. Ethylene glycol, glycolic acid, calcium oxalate, and glycine (and its conjugate, hippurate) are excreted in urine. The metabolites of ethylene glycol that have been typically detected are carbon dioxide, glycolic acid, and oxalic acid.

In an early (limited) investigation conducted in one male subject orally administered 8, 10, or 12 ml of ethylene glycol, the serum half-life for this compound was 4.5 h, with 25% of the administered dose (not specified) eliminated in the urine as the parent compound and 2.3% eliminated as oxalic acid (Reif, 1950). Other reported values for the half-life of ethylene glycol in serum following acute ingestion have ranged from 2.5 h (in children) to 8.4 h (in adults) (ATSDR, 1997). In one study of four individuals presenting with acute ethylene glycol poisoning, the half-life for the elimination of glycolic acid from the blood (without haemodialysis) was approximately 10 h (Moreau et al., 1998).

Quantitative information on the absorption of inhaled ethylene glycol by humans was not identified. In a clinical study reported by Wills et al. (1974), in which male volunteers were exposed continuously (via inhalation) to mean daily (20–22 h/day) concentrations of 17–49 mg/m³ (range 0.8–66.8 mg/m³) aerosol for 30 days, ethylene glycol was considered to be poorly absorbed from the respiratory tract, based on comparison of haematological and clinical chemistry parameters and the similar levels of ethylene glycol measured in the blood and urine of exposed (n = 20) and unexposed subjects; the level of ethylene glycol metabolites was not determined.

Reliable quantitative information on the absorption of ethylene glycol following dermal exposure in vivo in humans or in vitro in human skin has not been identified. However, this compound is likely to be absorbed through the skin, based upon its physicochemical properties (Fiserova-Bergerova et al., 1990) and the results of in vitro permeability studies conducted with samples of human skin (Loden, 1986; Driver et al., 1993; Sun et al., 1995).³

Ethylene glycol has low acute toxicity via oral, inhalation, or dermal exposure. LD₅₀s for the oral administration of ethylene glycol in rats range from 4000 to 10 020 mg/kg body weight, while reported values in guinea-pigs and mice are 6610 mg/kg body weight and 5500–8350 mg/kg body weight, respectively. The minimum lethal oral dose in rats is 3.8 g/kg body weight (Clark et al., 1979). Oral LD₅₀s of 5500 and 1650 mg ethylene glycol/kg body weight have also been reported in dogs and cats, respectively. A dermal LD₅₀ of 10 600 mg/kg body weight has been reported for rabbits. In rats and mice, the lethal concentration following 2-h inhalation exposure has been reported to be >200 mg/m³.

Signs of acute ingestion of ethylene glycol are dose dependent and include central nervous system depression, paralysis, ataxia, respiratory arrest, tachycardia, tachypnoea, coma, and death (BUA, 1994). Based on numerous case-studies of accidental acute ingestion of ethylene glycol or ethylene glycol-containing antifreeze in domestic animals (i.e., dogs and cats), metabolic acidosis has been consistently observed; morphologically, congestion and haemorrhage in the lungs, haemorrhage in the stomach, degeneration in the renal tubules, focal necrosis in the liver, and calcium oxalate in the kidneys and brain have been reported (DFG, 1991; BUA, 1994; Health Canada, 2000). Histopathological lesions observed in the kidney included mild tubular nephrosis, necrosis, sloughing of cells, vacuolation, and deposition of oxalate crystals in the cortex and medulla. Electron microscopic myocardial changes have also been noted in rats following acute oral administration (by gavage) of ethylene glycol (7.2 g/kg body weight), including mitochondrial swelling, myofibrillar oedema and necrosis, and enlargement of the smooth endoplasmic reticulum (Bielnik & Szram, 1992; Bielnik et al., 1992).

Studies were not identified concerning the potential of ethylene glycol to induce sensitization in experimental animals. Ethylene glycol induces mild dermal irritation in rabbits and guinea-pigs (Clark et al., 1979; Guillot et al., 1982; Anderson et al., 1986). Single or short-term ocular exposure to ethylene glycol (liquid or vapour) produces minimal conjunctival irritation without permanent corneal damage in rabbits (McDonald et al., 1972; Clark et al., 1979; Guillot et al., 1982; Grant & Schuman, 1993).

The results of short-term studies (in agreement with longer-term studies) confirm that the kidney is the principal target organ following oral exposure to ethylene glycol. In a 10-day study in which a wide range of end-points was examined in Sprague-Dawley rats administered drinking-water containing 0.5–4.0% ethylene glycol (650–5300 mg/kg body weight per day in males; 800–7300 mg/kg body weight per day in females), significant alterations in serum chemistry parameters were observed at all doses in males and at ³ 1500 mg/kg body weight per day in females (Robinson et al., 1990). The incidence and severity of histopathological lesions in the kidney were significantly increased in males at >2600 mg/kg body weight per day and in females at 7300 mg/kg body weight per day.

In a 4-week study in Wistar rats administered (by gavage) 2000 mg ethylene glycol/kg body weight per day, effects in the kidney (including discoloration, tubulopathy, hyperplasia, and crystalline deposits), changes in urinary parameters, and increased relative kidney weights (10–14%) were observed in both sexes (Schladt et al., 1998).

In studies in which groups (n = 5–10 per sex) of B6C3F₁ mice were administered (by gavage) 50, 100, or 250 mg ethylene glycol/kg body weight per day (in water) for 4 days, no clear treatment-related effects on survival, relative organ weights, haematology, or histopathology in major organs (including the liver, kidney, lung, and bone marrow) were observed. However, exposure produced depression of progenitor cells at all doses, hypocellularity at >100 mg ethylene glycol/kg body weight per day, and decreased erythropoiesis at 250 mg/kg body weight per day in the bone marrow (Hong et al., 1988). As effects on haematological parameters in blood were not observed, the biological significance of these effects is unclear.

In a study for which reporting was limited, male macaque monkeys exposed to 0.25–10% (1–152 g/kg body weight) ethylene glycol in drinking-water for 6–157 days had dose-related renal effects at >17 g ethylene glycol/kg body weight (Roberts & Seibold, 1969). No significant renal histopathology was observed among females receiving up to 152 g ethylene glycol/kg body weight.

In a 90-day study in which Sprague-Dawley rats ingested drinking-water containing 0.25–2.0% ethylene glycol (205–3130 mg/kg body weight per day in males; 600–5750 mg/kg body weight per day in females), alterations in haematological parameters in females (P < 0.05) were observed at lowest doses (Robinson et al., 1990). There were increased relative kidney weights in males at >950 mg/kg body weight per day, decreased body weights in males at 3130 mg/kg body weight per day, and dose-related histopathological changes (tubular dilation and degeneration, intratubular crystals) in the kidney in males (>950 mg/kg body weight per day) and females (>3100 mg/kg body weight per day).

In a well conducted study in which Fischer 344 rats (both sexes) were administered 165, 325, 640, 1300, or 2600 mg ethylene glycol/kg body weight per day in the diet for 13 weeks, significant effects were observed at 1300 mg/kg body weight per day and above, including reduced growth in males, increased kidney weight in both sexes, renal histopathology (dilation, necrosis, fibrosis, and crystal deposition in renal tubules) in males, and alterations in serum clinical chemistry parameters in males (Melnick, 1984). At 2600 mg/kg body weight per day, in males, mortality was increased and relative thymus weights were decreased, and in females, microscopic changes in the kidney (infiltration of inflammatory cells, increased vacuolation, and enlarged nuclei in renal tubules) were increased.

In an unpublished study by Gaunt et al. (1974), in which a wide range of end-points was examined in Wistar rats (n = 25 per sex per dose) administered ethylene glycol in the diet (males: 35, 71, 180, or 715 mg/kg body weight per day; females: 38, 85, 185, or 1128 mg/kg body weight per day) for up to 16 weeks, statistically significant specific microscopic changes in the kidney (i.e., dilation, degeneration, protein casts, deposition of calcium oxalate crystals in nephrons) were observed at the highest dose. Among male rats, histopathological changes were observed in the two highest dose groups (see Table 5).⁴ With one exception, severe tubular damage was observed in all male rats having oxalate crystals in the kidney. An increased incidence of kidney damage, although not statistically significant, was observed among females receiving the highest dose, 1128 mg ethylene glycol/kg body weight per day. The occurrence of inflammation of the Harderian gland in the exposed males, "pneumonial changes" in the lungs of males and females, and salivary adenitis was not considered related to exposure to ethylene glycol. [No-observed-adverse-effect level (NOAEL) = 71 mg/kg body weight per day (males); lowest-observed-adverse-effect level (LOAEL) = 180 mg/kg body weight per day (males)]

^a From Gaunt et al. (1974); P.G. Brantom, personal communication (2000).

Histopathological analyses of the kidneys in small (n = 5) groups of animals terminated at interim sacrifice at 2 or 6 weeks revealed no statistically significant increase in the incidence of specific histological changes, although the overall incidence of animals with tubular damage was significantly elevated in the high-dose group after 6 weeks’ exposure (Gaunt et al., 1974).

In B6C3F₁ mice receiving 400–6700 mg ethylene glycol/kg body weight per day in the diet for 13 weeks, treatment-related effects were limited to hyaline degeneration in the liver and minimal to mild tubule dilation, cytoplasmic vacuolation, and regenerative hyperplasia in the kidney in males at >3300 mg/kg body weight per day. There was no evidence of deposition of oxalate crystals in renal tubules. No effects on body or organ weights, clinical chemistry, haematology and urinary parameters, gross pathology, or histopathology in a wide range of organs were observed in females (Melnick, 1984; NTP, 1993).

Identified data on effects following inhalation are restricted to a few early, limited, short- and medium-term studies; in none of these was intake of ethylene glycol resulting from ingestion or via dermal absorption (i.e., see Tyl et al., 1995a,b) assessed. No treatment-related effects on survival, behaviour, physical appearance, locomotor activity, haematology, clinical chemistry parameters, or histopathology in selected organs (including lung, kidney, and liver) were observed in rats, guinea-pigs, rabbits, dogs, or monkeys exposed (whole body) to 10 or 57 mg ethylene glycol vapour/m³ for 8 h/day, 5 days/week, for 6 weeks (Coon et al., 1970). In a review by Browning (1965), it was reported that exposure of rats to 500 mg/m³ for 28 h over 5 days resulted in slight narcosis.

No clear treatment-related effects on histopathology in lung, liver, or kidney, on haematology, or on clinical chemistry parameters were observed in rats (n = 15), guinea-pigs (n = 15), rabbits (n = 3), dogs (n = 2), or monkeys (n = 3) exposed continuously to 12 mg ethylene glycol vapour/m³ for 90 days (Coon et al., 1970). Although mortality was observed among rabbits (n = 1), guinea-pigs (n = 3), and rats (n = 1) exposed in this study, none of the deceased animals was reported to exhibit "any specific signs of toxicity" (Coon et al., 1970). Moderate to severe irritation of the eyes was reported for the continuously exposed rabbits (i.e., erythema, oedema, discharge) and rats (i.e., corneal opacity and apparent blindness in 2 of 15 animals); however, these effects were not observed in a separate study involving exposure of these species to 57 mg ethylene glycol/m³, 8 h/day, 5 days/week, for 6 weeks (Coon et al., 1970).

In a carcinogenicity bioassay reported by DePass et al. (1986a), no tumours were observed in Fischer 344 rats (n = 130 per sex per dose) receiving 40, 200, or 1000 mg ethylene glycol/kg body weight per day in the diet for up to 2 years, based on microscopic examination of an extensive range of organs. At >200 mg/kg body weight per day, calcium oxalate crystals were observed in the urine of both sexes. At 1000 mg/kg body weight per day, females had a transient increase in kidney weight and mild fatty changes in the liver,⁵ while males had 100% mortality by 15 months (attributed to calcium oxalate nephrosis), reduced growth, organ weight changes (liver and kidney), microscopic lesions in the kidney (including dilation, proteinosis, glomerular shrinkage, hyperplasia, nephritis), and alterations in haematological, clinical chemistry, and urinary parameters. Among male rats, the incidence of tubular dilation, hydronephrosis, oxalate nephrosis, and calcium oxalate crystalluria was significantly elevated in the highest dose group (Table 6).

^a 0 (A) and 0 (B) are control groups.

Data on the incidence of lesions at interim and final sacrifice additional to those presented in the publication of DePass et al. (1986a) indicate that the terminology for intermediate- and end-stage histological renal lesions in this bioassay was inconsistent (Table 6). Indeed, based on this additional information, the reported incidence of early-stage lesions (tubular hyperplasia, tubular dilation, and calcium oxalate crystalluria) in DePass et al. (1986a) is based on the numbers of animals with these lesions in the small groups killed at interim sacrifice divided by the total number of animals on test, although these lesions were not scored at late stages in the study. Moreover, after 18 months on study, all male rats in the high-dose group either had died or were sacrificed when moribund.

Renal histopathology (calcification or oxalate-containing calculi), reduced growth, and mortality were observed in male and female rats exposed to >250 mg ethylene glycol/kg body weight per day in the diet for 2 years, with effects consistently observed at lower doses in males than in females (Blood, 1965). No tumours were observed in the limited number of tissues examined (including liver, kidney, and lung).

Similarly, calcium oxalate-related renal pathology and slight liver damage but no increase in tumours were presented in an early (limited) chronic bioassay in which only survival, growth, and histopathology in selected organs were examined in small groups of male (n = 6 per group) and female (n = 4 per group) albino rats (strain not specified) fed diets containing 1% or 2% ethylene glycol (500 or 1000 mg/kg body weight per day) for 2 years (Morris et al., 1942).

In an NTP (1993) bioassay, no tumours were observed in B6C3F₁ mice (n = 60 per sex per dose) administered ethylene glycol in the diet (males: 1500, 3000, or 6000 mg/kg body weight per day; females: 3000, 6000, or 12 000 mg/kg body weight per day) for 103 weeks. Females had a treatment-related increase in arterial medial hyperplasia in the lungs at all levels of exposure and hyaline degeneration in the liver at 12 000 mg/kg body weight per day. At 3000 and 6000 mg/kg body weight per day, male mice had dose-related hepatocellular hyaline degeneration and transient kidney damage (nephropathy). No clear evidence of treatment-related effects on survival, body weight, or tissue histopathology was observed in CD-1 mice (n = 80 per sex per dose) given 40, 200, or 1000 mg ethylene glycol/kg body weight per day in the diet for 2 years, based on microscopic examination of an extensive range of organs at 80 weeks and 2 years (DePass et al., 1986a). However, as indicated above in relation to the bioassay in rats reported by the same authors, histological reporting in this study was inadequate.

In a limited early investigation reported by Blood et al. (1962), male (n = 2) and female (n = 1) rhesus monkeys receiving 80 or 200 mg ethylene glycol/kg body weight per day in the diet, respectively, for 3 years had no overt signs of toxicity and no abnormal calcium deposits or histopathological changes in the major tissues examined (including the urogenital system, liver, and bone marrow).

In two studies conducted by subcutaneous injection, no tumours were observed in Fischer 344 rats (Mason et al., 1971) or NMRI mice (Dunkelberg, 1987) following repeated administration of up to 1000 mg ethylene glycol/kg body weight for 52 or 106 weeks.

In in vitro mutagenicity studies in bacterial cells, results have been consistently negative (Clark et al., 1979; Pfeiffer & Dunkelberg, 1980; Zeiger et al., 1987; JETOC, 1996), with and without S9 activation. Results have also been negative for mutagenicity in mouse lymphoma L51784Y cells (with and without activation) (McGregor et al., 1991). Results have been negative for chromosomal aberrations and sister chromatid exchange in cultured Chinese hamster ovary cells (with and without activation) (NTP, 1993) and for DNA damage in rat hepatocytes (Storer et al., 1996) and Escherichia coli (McCarroll et al., 1981; von der Hude et al., 1988).

In in vivo genotoxicity studies, results have been negative for dominant lethal mutations in F344 rats following administration in F₂ males (from a multigeneration study) of up to 1000 mg ethylene glycol/kg body weight per day for 155 days (DePass et al., 1986b). Results have also been negative for chromosomal aberrations in bone marrow cells of male Swiss mice exposed (by intraperitoneal injection) to 638 mg ethylene glycol/kg body weight per day for 2 days (Conan et al., 1979). There was only a slight increase in the incidence of micronuclei in the erythrocytes of Swiss mice administered >1250 mg ethylene glycol/kg body weight by gavage (or by intraperitoneal injection) (Conan et al., 1979). However, it should be noted that the magnitude of the effect was small, was not dose related, and was based on pooled data for treated groups.

In a three-generation reproduction study in which F344 rats (both sexes) received 40, 200, or 1000 mg ethylene glycol/kg body weight per day in the diet, there were no treatment-related parental effects (based on survival, body weight, food consumption, appearance, behaviour, and histopathology in major organs) or effects on fertility index, gestation index, gestation survival index, pup weight, appearance, behaviour, or histopathology in major organs (DePass et al., 1986b).

In F344 rats administered 40, 200, or 1000 mg ethylene glycol/kg body weight per day in the diet during days 6–15 of gestation, statistically significant increases of poorly ossified and unossified vertebral centra were observed in the offspring of dams exposed to 1000 mg ethylene glycol/kg body weight per day (Maronpot et al., 1983). Exposure to ethylene glycol had no effect upon pregnancy rate or number of corpora lutea, litters, live and dead fetuses, total implantations, pre-implantation loss, or resorptions; there was also no evidence of maternal toxicity.

In studies in which pregnant CD rats were admin-istered (by gavage) 150, 500, 1000, or 2500 mg ethylene glycol/kg body weight per day on days 6–15 of gestation, significant dose-related developmental effects were observed at 1000 mg/kg body weight per day and above, including reduced fetal body weight per litter, reduced skeletal ossification, and malformations in the skeleton (missing arches, missing and extra ribs) (Neeper-Bradley et al., 1995). Exposure had no effect upon the number of corpora lutea, live and dead fetuses, or resorption sites. Maternal toxicity was observed at 2500 mg/kg body weight per day, based on an increase (10%, P < 0.001) in relative kidney weight. [NOAEL (offspring) = 500 mg/kg body weight per day; LOAEL (offspring) = 1000 mg/kg body weight per day; NOAEL (maternal) = 1000 mg/kg body weight per day]

Other oral studies conducted in rats (Price et al., 1985; NTP, 1988; Marr et al., 1992) do not contribute additionally to the weight of evidence or dose–response for the reproductive toxicity of ethylene glycol, due to conduct of the studies at very high doses at which there was clear evidence of maternal toxicity (>1250 mg/kg body weight per day by stomach tube).

In a continuous-breeding study in which male and female CD-1 mice received 410, 840, 1640, or 2800 mg ethylene glycol/kg body weight per day in drinking-water, F₁ female pup weight was decreased at >840 mg/kg body weight per day (Lamb et al., 1985; NTP, 1986; Morrissey et al., 1989). Slight reductions in the number of F₁ litters per fertile pair (8%, P < 0.01) and number of live F₁ pups per litter (6%, P < 0.05), as well as unusual facial features and skeletal changes in the skull, sternebrae, ribs, and vertebrae, were observed at 1640 mg/kg body weight per day; however, incidence rates were not reported. There were no clear treatment-related effects on parental survival, body weight gain, or water intake, and no overt signs of toxicity were observed (Lamb et al., 1985; NTP, 1986; Morrissey et al., 1989).

Following the oral administration (by gavage) in pregnant CD rats of 2500 mg ethylene glycol/kg body weight per day on days 6–15 of gestation, extensive skeletal malformations and variations in the skull, vertebrae, ribs, sternebrae, and centra and significant external (meningoencephalocele, exencephaly, omphalocele, cleft palate, and cleft lip) and visceral (dilated cerebral ventricles) malformations were observed in fetuses from treated dams (Carney et al., 1999). Maternal effects included reduced food consumption and body weight gain and increased liver and kidney weights. Most of the skeletal effects induced by ethylene glycol were similar to those observed among fetuses from dams receiving an equivalent "teratogenic dose" of either glycolic acid (650 mg/kg body weight per day; presence of metabolic acidosis) or sodium glycolate (833 mg/kg body weight per day; absence of metabolic acidosis), while significant external or visceral malformations were not observed among animals exposed to glycolate or sodium glycolate. A reduction in metabolic acidosis (demonstrated with the use of sodium glycolate) ameliorated, but did not completely eliminate, skeletal effects.

Following administration (by gavage) in pregnant CD-1 mice of 750, 1500, or 3000 mg ethylene glycol/kg body weight per day during days 6–15 of gestation, there was a dose-related reduction in average fetal body weights per litter (9–27%) and marked dose-related increases in the incidence of malformed live fetuses per litter (0.25% in the controls and 10–57% in treated groups) and of skeletal malformations in the ribs, arches, centra, and sternebrae (4% in controls and 63–96% in treated groups) at all dose levels (Price et al., 1985). Exposure had no effect on the number of implantations, resorption sites, or live and dead fetuses. At 1500 mg/kg body weight per day and above, there was a significant decrease in maternal body weight gain (32%, P < 0.01) and absolute liver weight (9%, P < 0.01).

In studies in which pregnant CD-1 mice were administered (by gavage) 0, 50, 150, 500, or 1500 mg ethylene glycol/kg body weight per day on days 6–15 of gestation (Neeper-Bradley et al., 1995), exposure to the highest dose produced a statistically significant increase in the incidence of 25 of the 27 skeletal malformations/variations examined. At 1500 mg/kg body weight per day, an increased incidence of skeletal malformations and variations and a reduction in fetal body weight per litter were observed. The administration of 500 mg ethylene glycol/kg body weight produced a statistically significant increase in the incidence of the occurrence of an extra 14th rib. Exposure produced no effects on the number of corpora lutea or viable implantation sites, pre-implantation loss or sex ratio, or maternal toxicity. [No-observed-effect level (NOEL) (offspring) = 150 mg/kg body weight per day; NOAEL (offspring) = 500 mg/kg body weight per day; LOAEL (offspring) = 1500 mg/kg body weight per day; NOAEL (maternal) = 1500 mg/kg body weight per day]

In a study in which sperm counts and motility, histopathology and organ weights in the testes and epididymis, percentage of pregnant females, and number of live and dead implantation sites were examined in CD-1 mice exposed (both sexes) by gavage to 250–2500 mg ethylene glycol/kg body weight per day for up to 19 days, a significant reduction in live implantations per female was observed (Harris et al., 1992). No signs of parental toxicity were observed, based on examination of survival, body weight, clinical signs, and histopathology in selected organs.

Other oral studies conducted in mice (Nagano et al., 1973, 1984; Morrissey et al., 1989; Harris et al., 1992) do not contribute additionally to the weight of evidence of developmental/reproductive toxicity of ethylene glycol, due to the limited range of end-points examined, limited reporting of results, the absence of data on maternal toxicity, or conduct of the studies at higher doses than used in similar studies addressed herein.

A single study has been identified in which the developmental and reproductive toxicities of ethylene glycol have been examined in rabbits following oral exposure. Despite the presence of severe maternal toxicity (mortality and degenerative changes in the kidney) at the highest dose level, there was no evidence of developmental or reproductive effects in fetuses derived from New Zealand white rabbits administered (by gavage) 100–2000 mg ethylene glycol/kg body weight per day during days 6–19 of gestation (Tyl et al., 1993). Parameters examined included the number of corpora lutea, pre- and post-implantation loss, number of fetuses, fetal body weight per litter, litter sex ratio, and external, visceral, or skeletal variations or malformations.

Identified inhalation studies conducted in CD rats and CD-1 mice, in which pregnant animals were exposed (whole body) to up to 2090 mg ethylene glycol/m³ for 6 h/day on days 6–15 of gestation, provide some evidence of treatment-related skeletal variations (reduced ossification, extra ribs, dilated lateral ventricles, malaligned centra) and malformations of the head (exencephaly), face (cleft palate, abnormal face and facial bones), and skeleton (vertebral fusion, and fused, forked, missing, and extra ribs) (Tyl et al., 1995a,b). However, in each of these studies, there was likely considerable intake due to ingestion after grooming and/or percutaneous absorption; it was estimated by the authors that rats and mice received at least 620 mg/kg body weight per day and 910–1400 mg/kg body weight per day, respectively, in this manner.

In a "nose-only" inhalation study, exposure of pregnant CD-1 mice to 360, 779, or 2505 mg ethylene glycol/m³ for 6 h/day on days 6–15 of gestation yielded increases in some skeletal variations (e.g., decreased ossification in centra and sternebrae, unossified phalanges of the forelimb, extra ribs, extra ossification site in the skull)⁶ and a statistically significant 8-fold increase in the number of litters with animals exhibiting 2–12 fused ribs at the highest concentration (i.e., 2505 mg/m³) (Tyl et al., 1995b). Ethylene glycol had no effect upon the incidence of external or visceral malformations or reproductive parameters (including number of corpora lutea, total and viable implantations per litter, pre- and post-implantation loss, and sex ratio). Only minimal maternal toxicity was observed at 2505 mg/m³, based on a slight increase (7%, P < 0.05) in relative kidney weight, without evidence of cellular injury.

In the single dermal study identified, in which pregnant CD-1 mice were exposed (by occluded cutaneous application) to aqueous solutions of 0, 12.5, 50, or 100% ethylene glycol (estimated doses of 0, 400, 1700, or 3500 mg/kg body weight per day) during days 6–15 of gestation, reported effects were limited to maternal toxicity (based on minimal-grade renal lesions and increased corrected gestational body weight change) and a significant increase in the incidence of poorly ossified skull bone and unossified intermediate phalanges of the hindlimb at 3500 mg/kg body weight per day (Tyl et al., 1995c). Dermal exposure to ethylene glycol had no effect upon the number of corpora lutea, implantation and resorption sites, or live and dead fetuses.

Although data are limited, results of identified toxicity studies conducted (via oral, inhalation, or dermal routes) in rodents, rabbits, and monkeys do not indicate that neurological or immunological effects are critical end-points for ethylene glycol. Neurological effects have not been observed at doses below those that have induced renal toxicity (Penumarthy & Oehme, 1975; Clark et al., 1979; Grauer et al., 1987), and consistent treatment-related effects on immune system-related parameters have not been observed in available repeated-dose toxicity studies in which several species have been exposed to ethylene glycol either orally or by inhalation.

The effects observed in laboratory animals and humans are due primarily to the actions of one or more of its metabolites, rather than to the parent compound per se (see Figure 2). In studies in which inhibitors of alcohol dehydrogenase (the enzyme catalysing the first rate-limiting step in the metabolism of ethylene glycol) have been administered in both animals and humans, toxicity has been minimized. In laboratory animals, the concurrent ingestion or infusion of ethanol, pyrazole, or fomepizole prevents the renal toxicity and mortality observed following exposure to ethylene glycol (Grauer et al., 1987; US EPA, 1987). In humans, therapy for acute ethylene glycol poisoning includes administration of ethanol or 4-methylpyrazole to inhibit ethylene glycol metabolism by competition for alcohol dehydrogenase activity, sodium bicarbonate to reduce metabolic acidosis, and dialysis to remove the toxin (Jacobsen & McMartin, 1986; Grant & Schuman, 1993; Brent et al., 1999).

Based upon the available information, toxicological effects resulting from exposure to ethylene glycol may involve one or a combination of the following: development of an increased osmolal gap,⁷ metabolic acidosis,⁸ the formation of calcium oxalate crystals⁹ and their deposition in various tissues, or possible direct toxic effects produced by one or more of its metabolites.

A mode of action involving the formation and deposition of calcium oxalate crystals as requisite steps in the induction of renal effects in animals and humans is consistent with available metabolic and histopathological data. For example, species differences in sensitivity to renal effects are consistent with the limited available data on the comparative proportions of ethylene glycol excreted as oxalic acid, these being greater in rats than in mice (7–8% versus not detected at 24 h; Frantz et al., 1996a,b). Based on limited information, values for monkeys are intermediate, i.e., between those for rats and mice (0.3% at 48 h; McChesney et al., 1971), and those for humans are within the range of values reported for rats (Reif, 1950).

Indeed, calcium oxalate crystals are considered to be important etiological agents in the development of the renal failure in humans acutely poisoned by the ingestion of ethylene glycol (Jacobsen & McMartin, 1986; Wiley, 1999). In addition, in almost all cases where examined, extensive renal damage in experimental animals has been observed only where such crystals are present (Gaunt et al., 1974; Melnick, 1984). Also, in all cases where examined, ethylene glycol-associated renal damage has been observed only at doses greater than those at which there were increases in urinary excretion of oxalate or calcium oxalate crystals (Gaunt et al., 1974; DePass et al., 1986a). However, the possible role of less frequently observed hippuric acid crystals and direct cytotoxicity of other metabolites such as glycoaldehyde, glycolic acid, and glyoxylic acid cannot be precluded (Parry & Wallach, 1974; Marshall, 1982).

Sex-related variations in sensitivity to the renal effects of ethylene glycol may be a function of both toxicokinetic and toxicodynamic differences. Although there have been variations in the proportion of metabolites excreted as oxalic acid in male versus female rats in repeated-dose studies, proportions have been similar following single administration. In Sprague-Dawley rats administered (orally via gavage) a single dose of 1000 mg [¹⁴C]ethylene glycol/kg body weight, similar amounts of the administered radioactivity (i.e., 7–8%) were eliminated in the urine as [¹⁴C]oxalic acid in the males and females (Frantz et al., 1996a,b). Similar amounts of radioactivity have been measured in the kidneys of male and female rats administered single doses of [¹⁴C]ethylene glycol (Frantz et al., 1996a,b). In two studies in which Sprague-Dawley rats were provided drinking-water containing 0.5% ethylene glycol for 28 days, the elimination of oxalic acid (expressed as either mg/litre or µmol/litre per 24 h) in the urine was approximately 2.6- and 4.3-fold higher in males than in females (Lee et al., 1992, 1996).¹⁰ In a study in which male and female Wistar rats received similar doses (i.e., from 35 to 180 mg/kg body weight per day) of ethylene glycol from the diet over a 14- to 16-week period, slightly lower (i.e., 1.3- to 2.8-fold) levels of oxalic acid were excreted in the urine of females than in that of males (Gaunt et al., 1974).

In male Sprague-Dawley rats administered drinking-water containing 0.5% ethylene glycol for 28 days, the occurrence of kidney stones (as well as the excretion of oxalic acid in the urine) was reduced in castrated males, compared with controls (Lee et al., 1992, 1996). The effects of castration upon kidney stone formation (and excretion of oxalic acid) were reversed by the administration of exogenous testosterone to the castrated animals (Lee et al., 1996). Based upon the results of a study using an ethylene glycol/vitamin D induced urolithiasis model in oophorectomized Wistar female rats, Iguchi et al. (1999) suggested that the sex-related differences in the occurrence of kidney stones in rats administered ethylene glycol may be attributed to the female sex hormone-induced suppression of urinary oxalate excretion and suppression of osteopontin¹¹ expression in the kidney.

Based on the limited identified data, including those on acute doses that induce renal toxicity in humans, the comparative proportions of total metabolites excreted as the putatively toxic entity (i.e., oxalic acid) in humans and rats (Reif, 1950; Frantz et al., 1996a,b), and the specific activity of relevant enzymes in hepatic extracts of rats versus humans, it is expected that the sensitivity of humans to renal effects is similar to or greater than that of rats. Available data indicate that humans may be more sensitive than rodents to the acute toxicity of ethylene glycol, with available information on reported minimum lethal doses being consistent with an approximately 10-fold greater sensitivity in humans compared with rodents. The specific activity of alcohol dehydrogenase (the first rate-limiting step in the metabolism of ethylene glycol, considered essential in producing the toxicological effects associated with exposure to this substance) has been slightly higher in hepatic extracts obtained from humans, compared with rats (Zorzano & Herrera, 1990).

Less is known about the potential mode of induction of developmental effects, including the role of putatively toxic metabolites, although research conducted to date has focused on glycolic acid (Carney, 1994; Carney et al., 1999).

Evidence implicating a role for glycolic acid as the principal teratogenic agent has been derived from in vivo studies in which developmental effects have been observed in rats administered glycolic acid at doses lower than those for ethylene glycol inducing similar effects (Munley & Hurrt, 1996; Carney et al., 1999).

In studies in rats, a reduction in the metabolic acidosis typically associated with oral exposure to ethylene glycol ameliorated, but did not completely eliminate, teratogenic effects (Khera, 1991; Carney et al., 1999). Most variations and malformations induced by 2500 mg ethylene glycol/kg body weight administered on days 6–15 of gestation were similar to those observed among fetuses from dams receiving an equivalent "teratogenic dose" of either glycolic acid (presence of metabolic acidosis) or sodium glycolate (absence of metabolic acidosis). However, increased incidence of several external malformations (e.g., meningoencephalocele, exencephaly, omphalocele, cleft lip, cleft palate) among the offspring of ethylene glycol-exposed pregnant rats could not be explained on this basis (Carney et al., 1999).

There are numerous case reports concerning the accidental or deliberate ingestion of this compound in humans; mortality in these cases was associated with renal failure due to marked renal pathology (characterized by calcium oxalate deposition and degeneration in the tubules) (HSDB, 2001). However, available data from these studies are generally inadequate as a basis for quantification of intake associated with observed effects and can provide only crude information as a basis for comparison of the sensitivity of experimental animals with that of humans. Published values for the minimum lethal oral dose in humans have ranged from approximately 0.4 g/kg body weight (RTECS, 1999) to 1.3 g/kg body weight (ATSDR, 1997).¹² Systemic signs of toxicity following ingestion generally progress in three stages, commencing with effects on the central nervous system (intoxication, lethargy, seizures, and coma) and metabolic disturbances (acidosis, hyperkalaemia, hypocalcaemia), progressing to effects on the heart and lungs (tachycardia, hypertension, degenerative changes), and ending with renal toxicity (deposition of calcium oxalate, haematuria, necrosis, renal failure). In addition to the immediate central nervous system effects, neurological effects (including facial paralysis, slurred speech, loss of motor skills, and impaired vision due to "bilateral optic atrophy") have been observed up to several weeks following ingestion, suggestive of cranial nerve damage. Ethylene glycol may also produce a local irritant effect on the gut and cause pain and bleeding secondary to gastric erosion. The type and severity of toxicological effects observed following ingestion vary with the amount of ethylene glycol consumed, the interval between ingestion and treatment, and whether there has been concurrent ingestion of ethanol (Health Canada, 2000).

Information concerning adverse effects following inhalation of ethylene glycol is limited to observational data in a single case report (Hodgman et al., 1997) and the results of one laboratory study in which a range of end-points (physical examinations, psychological testing, and analysis of haematological, serum clinical chemistry, and urinary parameters) was examined in 20 male volunteers exposed (whole body) to ethylene glycol aerosol (Wills et al., 1974). In the latter study, no significant adverse effects were observed among individuals exposed (to up to 67 mg/m³) continuously for periods up to 30 days, although some individuals experienced throat irritation, headache, and back pain. Following a gradual increase in the exposure concentration, nasal and/or throat irritation were noted in all test subjects at 140 mg/m³ and above, while concentrations above 200 mg ethylene glycol/m³ produced severe irritation and could not be tolerated (Wills et al., 1974).

Ethylene glycol is a mild ocular irritant in humans. In dermal patch tests, results have been consistently negative in healthy volunteers (Meneghini et al., 1971; Hindson & Ratcliffe, 1975; Seidenari et al., 1990), while dermal irritation has been noted in individuals with dermal sensitivity, including eczema patients (Hannuksela et al.,