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.

Concise International Chemical Assessment Document 57

First draft prepared by Drs J. Kielhorn, C. Pohlenz-Michel, S. Schmidt, and I. Mangelsdorf, Fraunhofer Institute for Toxicology and Experimental Medicine, Hanover, Germany

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, 2004

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

Glyoxal.

(Concise international chemical assessment document ; 57)

1.Glyoxal — toxicity 2.Risk assessment 3.Environmental exposure 4. Food contamination
I.International Programme on Chemical Safety II.Series

ISBN 92 4 153057 X          (LC/NLM Classification: QD 305.A6)

ISSN 1020-6167

©World Health Organization 2004

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Technically and linguistically edited by Marla Sheffer, Ottawa, Canada, and printed by Wissenchaftliche Verlagsgesellschaft mbH, Stuttgart, Germany

FOREWORD

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. They may be complemented by information from IPCS Poison Information Monographs (PIM), similarly produced separately from the CICAD process.

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 usually 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.

Procedures

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, it is typical of a priority chemical that

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

The Steering Group will also advise IPCS on the appropriate form of the document (i.e., a standard CICAD or a de novo 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 usually based on an existing national, regional, or international review. When no appropriate source document is available, a CICAD may be produced de novo. 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. When a CICAD is prepared de novo, a consultative group is normally convened.

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 glyoxal was prepared by the Fraunhofer Institute for Toxicology and Experimental Medicine, Hanover, Germany. It is based on reports compiled by the German Advisory Committee on Existing Chemicals of Environmental Relevance (BUA, 1997). A comprehensive literature search of relevant databases was conducted up to February 2003 to identify any relevant references published subsequent to those incorporated in these reports. Information on the preparation and peer review of the source document is presented in Appendix 1. Information on the peer review of this CICAD is presented in Appendix 2. This CICAD was considered and approved as an international assessment at a meeting of the Final Review Board, held in Varna, Bulgaria, on 8-11 September 2003. Participants at the Final Review Board meeting are presented in Appendix 3. The International Chemical Safety Card for glyoxal (ICSC 1162), produced by the International Programme on Chemical Safety (IPCS, 2002), has also been reproduced in this document.

Anhydrous glyoxal (CAS No. 107-22-2) has a melting point of about 15 °C. However, it is generally available as an aqueous solution (typically containing 30-50% glyoxal) in which hydrated oligomers are present. Glyoxal is used as a chemical intermediate in the production of pharmaceuticals and dyestuffs, as a cross-linking agent in the production of a range of different polymers, as a biocide, and as a disinfecting agent. Releases to the environment are primarily emissions to ambient air and water.

The predominant target compartments for glyoxal in the environment are the hydrosphere and soil (at about 46% and 54%, respectively) and, to a lesser extent, air (<1%). Reported concentrations of glyoxal in ambient air in the USA, Europe, and Asia are in the range of about 0.1 µg/m³ up to 10 µg/m³. In European rivers and groundwater, concentrations up to 12 µg/litre are reported. Glyoxal is a by-product of ozone disinfection and has been detected at low µg/litre concentrations in drinking-water.

Due to microbial activity as well as non-enzymatic autoxidation of oil or browning reactions of saccharides, glyoxal is frequently detected in fermented food and beverages. It was found in different brands of beer, wine, and other beverages such as tea at concentrations ranging from about 20 µg/litre (black tea) up to 1556 µg/litre (sherry wine). In addition, it was detected in a range of fermented products such as soybean paste and yoghurt (0.63-4.2 mg/kg), bakery products such as bread (0.07-1.6 mg/kg), different plant materials (3-14 mg/kg), and edible oils (up to 6.5 mg/kg).

Glyoxal released into the environment is rapidly converted by abiotic processes, such as transformation by photochemically produced hydroxyl radicals. Due to the low soil sorption coefficient (K_oc) reported for this compound, it may leach from soil into groundwater. However, it is readily biodegraded and quickly transformed enzymatically by bacteria and fungi. Its low log octanol/water partition coefficient (K_ow) indicates that glyoxal is unlikely to bioaccumulate.

The main routes of occupational exposure to glyoxal during use as a disinfectant are via inhalation of aerosol and dermal absorption. The general population is exposed mainly through ingestion of glyoxal-containing food, but could be exposed through polluted air in urban regions and through traces of glyoxal in drinking-water.

Glyoxal is endogenously produced during normal cellular metabolism by a multitude of enzyme-independent pathways. Glyoxal is also a product of the metabolism and microsomal oxidation of other compounds, such as glycolaldehyde, ethylene glycol, and beta-hydroxy-substituted N-nitrosamines. The concentration of glyoxal in human blood plasma has been reported to be 0.1-1 µmol/litre, with higher levels reported for patients with diabetes or renal failure. In biological materials, less than 10% of the glyoxal present is in unbound forms in aqueous solution (free glyoxal and hydrates), as most of the reactive carbonyl groups are reversibly bound to cysteinyl, lysyl, and arginyl residues of proteins.

Glyoxal, which attacks amino groups of proteins, nucleotides, and lipids, is considered an important intermediate in the formation of advanced glycation end-products (AGEs). AGE modification alters protein function and inactivates enzymes, resulting in disturbance of cellular metabolism, impaired proteolysis, and inhibition of cell proliferation and protein synthesis. The deleterious effects of the highly reactive glyoxal are counteracted by a ubiquitous glutathione (GSH)-dependent glyoxalase system, which converts glyoxal to the less reactive glycolate.

The acute toxicity of glyoxal in experimental animals is low to moderate, depending on the actual concentration of glyoxal in the tested product. In rats, for 40% glyoxal, the LC₅₀ for a single 4-h inhalation of aerosol is 2440 mg/m³, the oral LD₅₀ value ranges from 3000 to 9000 mg/kg body weight (with higher sensitivity in females), and dermal LD₅₀ values are >2000 mg/kg body weight. After inhalation exposure, local irritations of the eyes and respiratory organs as well as hyperaemia and foamy secretion in the lungs predominate. After oral exposure to glyoxal, macroscopic observations include irritations of the gastrointestinal tract and congestion in the gastrointestinal tract, lung, kidney, and adrenal glands. In the prominent target organs, pancreas and kidney, the toxic action of glyoxal leads to severe degenerative changes resembling those induced during diabetes.

Studies into short-term (29-day) inhalation exposure of rats to glyoxal showed a no-observed-effect level (NOEL) of 0.6 mg/m³ (nominal concentration was 0.4 mg/m³) for local effects in the larynx and a NOEL of >8.9 mg/m³ (nominal concentration was 10 mg/m³) for systemic effects (examination of body weight, haematological and biochemical parameters, urine analysis, macroscopic and histological examination). A 28-day study in which glyoxal was administered to rats in drinking-water resulted in a no-observed-adverse-effect level (NOAEL) of 100 mg glyoxal/kg body weight per day. The 90-day feeding of glyoxal to rats resulted in a NOAEL of 125 mg/kg body weight per day (dosage corresponding to 100% glyoxal). Effects stated at higher dosages in these two latter studies were reduced water and food intake (first study only) and retardation of body weight gain (both studies). In a study examining more sensitive end-points (serum clinical biochemistry), the lowest tested dosage of 107 mg/kg body weight per day (99% glyoxal) corresponded to the lowest-observed-adverse-effect level (LOAEL) for a 90-day exposure of rats via drinking-water. A 90-day feeding study in dogs failed to reveal any substance-related changes at the top dose of 115 mg/kg body weight per day (dose corresponding to 100% glyoxal).

In animal studies, 30% and 40% aqueous glyoxal caused slight to definite skin irritations, depending on the application time. Glyoxal is irritating to mucous membranes and acts as a skin sensitizing agent in humans and experimental animals.

Fetotoxic effects occurred only with doses of glyoxal that induced maternal toxicity. In developmental toxicity studies with rats, a NOEL for embryotoxicity was >300 mg glyoxal dihydrate/kg body weight per day (corresponding to >185 mg glyoxal/kg body weight per day), whereas a lowest-observed-effect level (LOEL) (decreased body weight gain) for maternal toxicity was 200 mg glyoxal dihydrate/kg body weight per day (corresponding to 123 mg glyoxal/kg body weight per day). Developmental toxicity range-finding studies in rabbits yielded a NOEL of 200 mg glyoxal dihydrate/kg body weight per day (corresponding to 123 mg glyoxal/ kg body weight per day) for both maternal toxicity and embryotoxicity.

Glyoxal is directly genotoxic in vitro in bacterial and mammalian cells, inducing, for example, DNA adducts, mutations, chromosomal aberrations, DNA repair, sister chromatid exchanges, and DNA single strand breaks. In vivo, a genotoxic activity of glyoxal was established at the site of application in the pyloric mucosa of rats by demonstration of unscheduled DNA synthesis and DNA single strand breaks. After oral application, DNA strand breaks were further observed in rat liver. There are no carcinogenesis bioassays with inhalation exposure to glyoxal. Glyoxal showed tumour-promoting activity in a two-stage glandular stomach carcinogenesis model in male Wistar rats, whereas it was inactive in a short-term liver foci assay. In an assay for tumour-initiating activity of glyoxal in skin and in cell transformation assays, glyoxal yielded negative test results.

Taking the 29-day inhalation study in rats exposed to glyoxal, which showed a NOEL of 0.6 mg/m³ for local effects in the larynx, and using uncertainty factors of 10 for interspecies differences and 10 for interindividual differences, a tolerable concentration of 6 µg/m³ for local effects in the larynx for short-term exposure was estimated.

In a sample risk assessment for the general population, an exposure scenario has been compiled as a hypothesized worst case. Using the daily intake of, maximally, 10 mg glyoxal via food, an estimated intake of 0.16 mg glyoxal/kg body weight per day can be calculated. This is similar to the tolerable intake of about 0.2 mg/kg body weight per day for lifetime oral exposure to glyoxal.

In a second sample risk assessment, for a nurse or hospital cleaner or consumer using disinfectant, a typical brand of disinfectant (7.5 g in 100 g = 7.5% glyoxal) is used at a dilution of 1% for disinfection and cleaning of surfaces (i.e., 0.075% glyoxal). Using a rounded-up 0.1% glyoxal solution and a calculation derived from a model gives an uptake of about 4 µg/kg body weight per day, assuming a body weight of 64 kg. This is much (50 times) less than the tolerable intake of about 0.2 mg/kg body weight per day for lifetime oral exposure. However, using a worst-case exposure to 4% glyoxal and the same assumptions as above would give an uptake of about 0.15 mg/kg body weight, which is approximately the same as the tolerable intake of about 0.2 mg/kg body weight per day for lifetime oral exposure.

In the final sample risk assessment, a farmer using a spray application of biocidal products containing glyoxal to disinfect a stable was used as an example. The model calculation using the given assumptions predicts a short-term exposure concentration of 24 µg glyoxal/m³ for a 6-min exposure and 32 µg glyoxal/m³ for a 15-min exposure. This can be compared with the estimated tolerable concentration of 6 µg/m³ for local effects in the larynx for a short-term exposure. There is a perceived risk of local laryngeal effects and irritation to the skin from this spray application of glyoxal.

Exposure to glyoxal has been shown to inhibit the activities of aerobic as well as anaerobic bacteria, green algae (96-h EC₅₀ value of about 149 mg/litre for Pseudokirchneriella subcapitata [formerly Selenastrum capricornutum]), and an invertebrate species. In four fish species tested, the lowest reported 96-h LC₅₀ value was 215 mg/litre (Pimephales promelas).

A sample risk characterization for the aquatic environment was performed by calculating the ratio between a local predicted environmental concentration (PEC), based on recently measured data, and a corresponding predicted no-effect concentration (PNEC). A PNEC of 149 µg/litre for surface water was estimated from the lowest EC₅₀ value of 149 mg/litre by applying an uncertainty factor of 1000. Using the highest recently measured concentration of glyoxal in surface water (1.9 µg/litre), a PEC/PNEC quotient of 0.013 was obtained. As this is less than 1, no further information, testing, or risk reduction measures are required.

A no-observed-effect concentration (NOEC) of 68 mg/litre was determined for the inhibition of the proliferation of rhizome fragments of Helianthus tuberosus, with a corresponding EC₃₀ value of 136 mg/litre, in the only study available. As no additional data characterizing the toxic effects exhibited by glyoxal upon terrestrial microorganisms or invertebrates are available, it was not possible to perform a reliable quantitative risk characterization.

Glyoxal (CAS No. 107-22-2; C₂H₂O₂) is also known as ethanedial, diformyl, ethanedione, biformal, and oxal. At room temperature, anhydrous glyoxal is a liquid, with a melting point of about 15 °C. It crystallizes in its monomeric form to yield yellow, irregular to prism-like crystals. However, it is generally employed as an aqueous solution (typically containing 30-50% glyoxal), in which hydrated oligomers are present due to nucleophilic addition (Chastrette et al., 1983; Hoechst AG, 1984a).

Glyoxal can undertake rotational isomerization between the planar cis and trans conformations, with trans-glyoxal being the more stable isomer (Bulat & Toro-Labbé, 2002):

trans-glyoxal           cis-glyoxal

Some of the most important hydrated derivatives of glyoxal formed by nucleophilic addition in aqueous solution are shown below (Whipple, 1970; Chastrette et al., 1983). These include the monomer ethane-1,1,2,2-tetraol (I), the dimer 2-dihydroxymethyl-(1,3)dioxolane-4,5-trans-diol (II), and the trimer bis(dioxolane) (i.e., 2,2'-bi-1,3-dioxolanyl-4,4',5,5'-tetraol) (III) — both cis and trans configurations. However, the proportion of the different structures varies with concentration and pH.

Some studies (developmental toxicity) have used glyoxal trimeric dihydrate (CAS No. 4405-13-4).

The environmentally relevant physicochemical properties of glyoxal and of the commercially employed 40% aqueous solution of this compound are summarized in Table 1. Additional physical and chemical properties are presented in the International Chemical Safety Card reproduced in this document.

The conversion factors² for glyoxal in air (at 20 °C and 101.3 kPa) are as follows:

1 ppm in air = 2.41 mg/m³

1 mg/m³ = 0.414 ppm

Accepted methods for the detection and quantification of glyoxal in different matrices are given below. Additional and more detailed information is available in BUA (1997) and references cited therein.

Determination of glyoxal in air usually involves concentration of the alpha-dicarbonyl onto a solid sorbent coated with an appropriate derivatization agent followed by solvent desorption prior to high-performance liquid chromatographic (HPLC) detection. Zhou & Mopper (1990) reported a detection limit of about 0.05 µg/m³ for a 100-litre air sample using 2,4-dinitrophenylhydrazine (DNPH)-coated C18 cartridges, elution with carbon tetrachloride, and subsequent HPLC detection. As an alternative approach, Ho & Yu (2002) employed pentafluorobenzyl hydroxylamine (PFBHA)-coated sorbent sampling followed by thermal desorption and gas chromatography/mass spectrometry (GC/MS) detection of formed oximes and reported a minimum detection limit of 0.24 µg/m³ for a sample volume of 4.8 litres.

Table 1: Physicochemical properties of glyoxal and its commercially employed aqueous solution (40%).

^a Using KowWin v.1.66.

Edelkraut & Brockmann (1990) detected and quantified glyoxal in water samples by using the typical 2,4-DNPH derivatization followed by HPLC with diode array detection at 360 nm. They reported a detection limit of 295 ng/litre. Glaze et al. (1989) employed aqueous-phase PFBHA derivatization, yielding the corresponding pentafluorobenzyl oxime, followed by n-hexane extraction and detection by GC/electron capture detection (ECD) or GC/MS. A minimum detection limit of 5.1 µg/litre was obtained using GC/ECD, whereas GC/MS detection gave a minimum detection limit of 7.7 µg/litre. Method 556.1 of the US Environmental Protection Agency (US EPA, 1999) suggests a similar procedure (aqueous-phase PFBHA derivatization followed by hexane extraction and fast GC/ECD detection), leading to method detection limits in the range of 0.13-0.39 µg/litre. Steinberg & Kaplan (1984) used both HPLC and GC/MS as well as direct insertion probe/MS to detect and quantify glyoxal after 2,4-DNPH derivatization followed by dichloromethane extraction in fog and mist samples. As a viable alternative, derivatization using o-phenylenediamine to give the corresponding quinoxaline prior to HPLC/ultraviolet (UV) detection has been described (Barros et al., 1999).

As described for gaseous and liquid samples, glyoxal is usually derivatized either directly in suspended samples or after extraction by using o-phenylenediamine with subsequent GC/ECD detection or 2,4-DNPH with HPLC/UV detection. Kawata et al. (1980) found a detection limit of 0.02 mg/kg analysing sediment samples for the presence of glyoxal. No specific method for the analysis of soil is available (BUA, 1997).

The concentration of glyoxal in whole-blood samples was determined by derivatization with 1,2-diamino-4,5-dimethoxybenzene, solid-phase extraction, and HPLC of the resulting quinoxaline adduct with fluorometric detection (Thornalley et al., 1996). The interbatch coefficient of variation was 20%, the limit of detection 40 pmol, and the recovery 99%. Odani et al. (1999) employed a similar method for plasma, using quantitative derivatization of glyoxal present in plasma with 2,3-diaminonaphthalene prior to organic extraction followed by subsequent analysis employing HPLC resolution and detection by electrospray ionization/MS. Lapolla et al. (2003) quantified glyoxal in plasma using GC/MS after derivatization with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride.

There are several natural sources of glyoxal. Thus, glyoxal can be produced biologically as a useful by-product (i.e., for the generation of hydrogen peroxide required by manganese-dependent peroxidase enzymes; Kersten, 1990) or non-enzymatically by autoxidation of lipids (Hirayama et al., 1984). Furthermore, it can be produced from a range of abiotic reactions with aromatic compounds in the presence of ozone and/or hydroxyl radicals. Accordingly, Mopper & Stahovec (1986) detected the formation of glyoxal from humic acids by photochemical reactions in seawater. Mopper et al. (1991) estimated the photochemical glyoxal formation rates in Sargasso seawater (0-4000 m) to be in the range of 0.4-1.1 nmol carbon/h. In addition, one can safely assume that natural fires — in analogy with results reported for domestic and residential log fires (Kleindienst et al., 1986; McDonald et al., 2000) — will release glyoxal in addition to other aldehydes. Ozone can — for example, when applied as a water disinfectant — catalyse the formation of glyoxal from organic carbon present in water (Glaze et al., 1989; Le Lacheur et al., 1991; Lopez et al., 1999).

Two well established processes employed for the production of glyoxal are the gas-phase oxidation of ethylene glycol with air in the presence of copper or silver catalysts at elevated temperature (about 300 °C) and the liquid-phase oxidation of acetaldehyde with nitric acid (Chumbhale & Awasarkar, 2001). In Germany, less than 10 000 tonnes of glyoxal (40%) were produced in 1992 (BUA, 1997). However, in 2002, BASF started up a new production plant with an annual capacity of about 60 000 tonnes (BASF AG, personal communication, 2003). The Japanese production figure for glyoxal was 13 000 tonnes in 1999 (J. Sekizawa, personal communication, 2001). The world production volume of glyoxal is about 120-170 kilotonnes (OECD, 2002).

Glyoxal is used as a chemical intermediate in the production of pharmaceuticals and dyestuffs. It is also used in the industrial production of alpha-hydroxyalkylureas (the addition of glyoxal to urea) and is industrially employed as a cross-linking agent in the production of a range of different polymers, such as textiles (e.g., permanent press fabrics) (Hoechst AG, 1984a; Choi et al., 1998, 1999; Choi, 2002), paper (Xu et al., 2002), and proteins (Marquié, 2001). It is used as a biocide and as a disinfecting agent and is present in many products, such as cleansers used for the disinfection of surfaces (BPI, 1993; OECD, 2002; BASF AG, personal communication, 2003).

Generally, glyoxal might be released during its manufacture or its application (BUA, 1997; see also section 4.3). A well recognized source of glyoxal is automotive emissions and the subsequently formed photochemical smog, which gives rise to the formation of this compound (California State Air Resources Board, 1984; Jing et al., 2001). In addition, emissions from cigarettes have been shown to contain trace amounts of glyoxal (Moree-Testa & Saint-Jalm, 1981). Another potential source of glyoxal is domestic and residential log fires (Kleindienst et al., 1986; McDonald et al., 2000). Using an irradiated smog chamber, Kleindienst et al. (1986) and McDonald et al. (2000) detected glyoxal concentrations of up to about 110 µg/m³. Interestingly, glyoxal was detected as a minor species in turbulent flames of acetylene and ethylene under atmospheric pressure (Tichy et al., 1998).

The predominant target compartments for glyoxal in the environment are the hydrosphere and soil (about 46% and 54%, respectively) and, to a lesser extent, air (<1%; Level III fugacity calculation, using EPI v.3.1). According to Thomas (1982), the reported Henry's law constant of <3.38 × 10^-4 Pa·m³/mol (Betterton & Hoffmann, 1988) indicates that glyoxal is essentially non-volatile with regard to the aqueous phase. Therefore, a noteworthy transfer of glyoxal from the aqueous to the gas phase is not expected. This is supported by the findings of Harke & Höffler (1984), who used Hela cells as an indicator for the presence of inhibitory compounds (i.e., a range of biocides) in the gas phase. Their results clearly indicated that glyoxal — as opposed to other biocides tested, such as formaldehyde — was not transferred from solution to the gas phase. However, glyoxal has to be regarded as a highly mobile compound in soil due to the low log K_oc value (<1) reported (BUA, 1997). Due to its excellent solubility in water and its low log K_ow, it is not expected to bioaccumulate.

Atkinson (2000) calculated a lifetime of 1.1 days for glyoxal in the presence of hydroxyl radicals (assuming an average 12-h daytime concentration of 2 × 10⁶ molecules/cm³). With respect to photolytic transformation (overhead sun), a lifetime of 5 h was calculated by the same author. Li & Schlegel (2001) showed that the photofragmentation of glyoxal proceeded — under collision-free conditions — by internal conversion to a vibrationally excited state, which dissociates to yield H₂ + CO + CO (28%), H₂CO (formaldehyde) + CO (65%), and HCOH (hydroxycarbene) + CO (7%). Hence, glyoxal released into the atmosphere will undergo a rapid degradation in this environmental compartment. According to Yadav & Gupta (2000), the hydrolysis of glyoxal with sodium hydroxide as catalyst proceeded with a second-order rate constant of 9.3 × 10^-6 cm³/mol·s at 25 °C to yield glycolic acid. Brunet et al. (1984) showed the transformation of glyoxal to oxalic acid via glyoxylic acid in the presence of ozone.

In biodegradation tests corresponding to Organisation for Economic Co-operation and Development (OECD) guideline 301C, glyoxal was readily biodegradable (65% biochemical oxygen demand [BOD] of theoretical oxygen demand [ThOD], incubation for 14 days; MITI, 1992). Similar results were obtained by applying the method of Zahn Wellens, yielding an elimination of >70% of dissolved organic carbon in 7 days (Hoechst AG, 1991a). Conway et al. (1983) observed a significant bio-oxidation of glyoxal by sewage inocula (76% of ThOD after 20 days), while Gerike & Gode (1990) showed that glyoxal was biodegradable (i.e., 90% of ThOD within 28 days) by employing the OECD 301D closed bottle test. Furthermore, using both the oxygen consumption inhibition test and the OECD confirmatory test, they established an inhibition limit of 500 mg/litre. Hence, water or sewage treatment plants should be impacted detrimentally only at high influent concentrations. In fact, a large number of microbial enzymes catalyse the transformation of glyoxal to common intermediates in microbial catabolism. Thus, Sakai et al. (2001) reported the efficient transformation of glyoxal to glycolaldehyde by glyoxal reductase from Bacillus subtilis. Furthermore, glyoxal can be effectively oxidized by enzymes such as the fungal glyoxal oxidase (Kersten, 1990) and its bacterial counterpart (Whittaker et al., 1999) to yield glyoxylic acid, which, in turn, is a common intermediate of many metabolic sequences (i.e., glyoxylate cycle) present in microorganisms. Finally, the microbial glyoxalase system (Cooper, 1984) should yield glycolate in analogy to the reaction with methylglyoxal, whereas microbial aldehyde dehydrogenases with sufficient activity for 2-oxoaldehydes should yield glyoxylate.

Even under physiological conditions, glyoxal reacts quickly with arginine, leading to the formation of 1-(4-amino-4-carboxybutyl)-2-imino-5-oxo-imidazolidine (Schwarzenbolz et al., 1997). Thus, arginine residues present in proteins can act as scavengers for glyoxal. Further, glyoxal is able to oxidize an amino acid such as phenylalanine to a plethora of products, such as Strecker aldehydes and O- and N-heterocycles (Adamiec et al., 2001), and can produce amides from amino acids such as lysine (Glomb & Pfahler, 2001) and arginine (Glomb & Lang, 2001).

6.1.1 Atmosphere

Residential wood combustion was reported to release up to about 600 mg glyoxal/kg hardwood used in the fireplace (McDonald et al., 2000). Borrego et al. (2000) detected glyoxal in ambient air sampled in the Giesta area (a rural location 20 km south-east of Aveiro, Portugal) and reported an average level of 3.7 µg/m³. Kawamura et al. (2000) showed the presence of glyoxal in all ambient air samples taken at four different locations (modest to high degree of urbanization and traffic) in the Los Angeles region (USA) and found glyoxal at concentrations ranging from about 0.096 to 2.3 µg/m³. More recently, Ho & Yu (2002) analysed the ambient air at a roadside location near a bus stop (Clear Water Bay, Kowloon) in Hong Kong for 24 h and showed that the lowest levels of glyoxal (about 1.2 µg/m³) were present in the early hours (01:00-05:00), whereas the maximum levels (about 9.9 µg/m³) were obviously correlated with increasing traffic (09:00-13:00). Jing et al. (2001) found glyoxal present in urban air samples taken in Las Vegas (USA) in both summer (range 0.29-0.99 µg/m³) and winter (range 0.22-0.51 µg/m³).

6.1.2 Hydrosphere

Steinberg & Kaplan (1984) detected glyoxal in fog samples collected near Los Angeles (Topanga Canyon), USA, at concentrations up to about 1.9 mg/litre.

The presence of glyoxal was reported at 4-12 µg/litre in water of the river Elbe sampled near Brunsbüttel, Germany (Edelkraut & Brockmann, 1990), and in samples from the Sargasso Sea (Mopper et al., 1991; no values given). Le Lacheur et al. (1991) detected glyoxal at low µg/litre concentrations in raw drinking-water sampled in Fort Dix, USA. More recently, Nawrocki et al. (1996) detected glyoxal in distilled (0.9 µg/litre) and double-distilled (0.1 µg/litre) reagent water. The same authors (Dabrowska et al., 2003) demonstrated the presence of glyoxal at concentrations up to 1.9 µg/litre in groundwater (Mosina water intake serving Poznañ) and surface water samples (river Bogdanka) from Poland. These levels did not significantly increase upon chlorine dioxide treatment of the raw water samples. IPCS (2000) reported the median concentration of glyoxal in ozone-treated drinking-water to be 9 µg/litre.

6.1.3 Sediment

Kawata et al. (1980) reported the presence of glyoxal in sediment samples from Japanese rivers at up to 13 mg/kg dry weight.

6.1.4 Food

Glyoxal is a substance frequently detected in fermented food and beverages. This is mainly due to microbial activity as well as non-enzymatic browning reactions such as caramelization and Maillard reactions of saccharides (Hollnagel & Kroh, 1998; Glomb & Tschirnich, 2001; Hollnagel & Kroh, 2002). Accordingly, Barros et al. (1999) found glyoxal present in different brands of beer and wine on sale in Portugal. Sampling three different brands of white wine, they detected glyoxal at concentrations of 6.2, 8.7, and 26 µmol/litre (about 360, 464, and 1509 µg/litre). De Revel & Bertrand (1993) evaluated a range of French wines and detected glyoxal in one white wine (mean of 125 µg/litre), red wines (151-368 µg/litre), and five sherry wines (lowest level of glyoxal in a Seco with 435 µg/litre and highest level in an Olorosso with 1556 µg/litre). Palamand et al. (1970) detected glyoxal levels ranging from about 230 to 1000 µg/litre in eight different beers. Nagao et al. (1986) detected glyoxal in Bourbon whiskey (390 µg/litre), wine (970 µg/litre), and apple brandy (33 µg/litre), as well as in black tea (20 µg/litre) and instant (340 µg/litre) and brewed coffee (870 µg/litre). Yamaguchi et al. (1994) detected glyoxal in beverages such as beer (20-40 µg/litre) as well as white (510 µg/litre) and red wine (740 µg/litre).

Nagao et al. (1986) found glyoxal in soybean paste (4.2 mg/kg), soy sauce (4.9 mg/litre), toast (0.5 mg/kg), and bread (0.3 mg/kg). Markianova et al. (1971) reported glyoxal levels in bread ranging from 0.07 to 0.31 mg/kg, depending on the yeast type employed. However, Roiter & Borovikova (1972) showed that using amylase in the baking process led to glyoxal levels of up to 1.4 mg/kg in the bread crust and of up to 1.6 mg/kg in the bread crumbs. Plant materials used for brewing (rice — about 14 mg/kg; barley — about 3 mg/kg; malt — about 7 mg/kg) might contain glyoxal as well (Palamand et al., 1970). Yamaguchi et al. (1994) detected glyoxal in fermented food such as yoghurt (about 0.63-0.92 mg/kg). Due to heat-induced autoxidation, edible oils might contain glyoxal, as was shown for sardine oil, containing up to 6.5 mg/kg (Hirayama et al., 1984).

6.2.1 General population

The main route of exposure of the general population to glyoxal is probably via intake of water and food containing glyoxal. Glyoxal is present in a broad range of different food products. However, due to the lack of quantitative data on the presence of glyoxal in food products such as meat, dairy products, or fish, an exact value cannot be given. The general population might also be exposed to glyoxal via cigarette or residential log fire smoke or vehicle exhaust containing glyoxal.

An exposure scenario has been compiled as a hypothesized worst case. A food/drink intake of 10 mg/day has been calculated from foods with a known glyoxal content (see section 6.1.4). About three cups of brewed coffee per day (>400 µg glyoxal), toast (>50 µg glyoxal), a stir-fried meal containing rice (>4 mg glyoxal), oil (>500 µg glyoxal), soy sauce (>200 µg glyoxal), a pint of beer (500 µg glyoxal), one yoghurt (>130 µg glyoxal), and one glass of sherry (>30 µg glyoxal) leads to an intake of about 6 mg/day. A further intake of 3-4 mg glyoxal/day might come from other fermented products (dairy products or vegetables), from other popular roasted or fried products (meat, fish, mushrooms, sausages), or from additional bakery products.

Assuming a daily intake of 20 m³ air containing about 4 µg glyoxal/m³ (Borrego et al., 2000), a daily consumption of 2 litres of water containing 9 µg glyoxal/litre (median for ozone-treated drinking-water; IPCS, 2000), and an estimated daily intake of 10 mg glyoxal via food, an intake of about 160 µg of glyoxal per kg body weight (using 64 kg as the value for body weight) per day can be calculated. This intake is almost totally from food.

Modifying this calculation by including a 2-h daily exposure to traffic exhaust containing glyoxal (at 9.9 µg/m³ — from Ho & Yu (2002) — instead of 4 µg/m³) while still using the other values as stated above (water, food) does not yield a significantly higher value.

Glyoxal has been reported as being present in some household cleaners up to a concentration of 4% (product databanks, Switzerland, Denmark, and Germany; R. Hertel, personal communication, 2003). People can therefore be exposed to glyoxal during its use as a household cleaner.

6.2.2 Occupational exposure

Glyoxal does not appear to evaporate from solution (Harke & Höffler, 1984). Further, the reported Henry's law constant of <3.38 × 10^-4 Pa·m³/mol (Betterton & Hoffmann, 1988) indicates that glyoxal is essentially non-volatile with regard to the aqueous phase. Therefore, occupational exposure by inhalation will probably take place only in situations where aerosols containing glyoxal are released. Such an exposure situation might be the spray application of biocidal products containing glyoxal.

A model calculation has been made using an aerosol droplet simulation programme for a worst-case exposure via inhalation of aerosol droplets — for example, of a farmer disinfecting his stable by spray application of a commercial product (see Appendix 5 for details). The model calculation using the given assumptions predicts an exposure concentration of 24 µg glyoxal/m³ for a 6-min exposure and 32 µg glyoxal/m³ for 15 min.

Exposure via skin (i.e., unprotected use of disinfectant solution) may be estimated using DermWin v.1.43 (US EPA, 2000). A typical brand of disinfectant (7.5 g in 100 g = 7.5% glyoxal) recommends a dilution of 1% for disinfection and cleaning of surfaces (i.e., 0.075% glyoxal). Using a rounded-up figure of 0.1% glyoxal solution and a K_p value (estimated from the following equation: log K_p = -2.72 + 0.71 log K_ow — 0.0061 MW) of 5.63 × 10^-5 cm/h (given in DermWin v.1.43 taken from US EPA, 2000; where K_p is the permeability coefficient from water and MW is molecular weight) for glyoxal, the dermally absorbed dose per event (assuming a final concentration of glyoxal in the aqueous solution used for cleaning surfaces of 1 mg/cm³ [0.1%] and an event duration of 30 min) yields a potential uptake of 2.8 × 10^-2 µg/cm² per event (using Fick's first law) with regard to exposed, unprotected skin. Taking a worst case of 10 events/day, a surface area of hands of 840 cm² (US EPA, 1997), and assuming 100% uptake through the skin, this would mean 235 µg glyoxal/day, which equals 3.7 µg/kg body weight, assuming a body weight of 64 kg.

6.2.3 Human plasma and urine

Glyoxal is produced endogenously and is commonly present in blood plasma of healthy subjects, with one study giving values of about 67 ng/ml (corresponding to about 1.16 µmol/litre; Odani et al., 1999) and other studies reporting 0.23 µmol/litre (Agalou et al., 2002) and 0.3 µmol/litre (Lapolla et al., 2003). Higher levels are found in patients with diabetes or renal diseases (see section 7.1). The urine of patients without these diseases contained glyoxal at about 132 µmol/litre (Espinosa-Mansilla et al., 1998). This value is in apparent conflict with the low levels found in tissues and body fluids and with the assumed efficient glyoxalase activities in these patients.

Glyoxal is endogenously produced during normal cellular metabolism by a multitude of enzyme-independent pathways, such as the spontaneous reaction of amino groups in proteins with reducing sugars (Maillard reaction), sugar autoxidation, DNA oxidation, peroxidation of polyunsaturated fatty acids, and UV photodamage, and in conditions of oxidative stress and depletion of GSH (Loidl-Stahlhofen & Spiteller, 1994; Shibamoto, 1994; Murata-Kamiya et al., 1995, 1997a; Wells-Knecht et al., 1995; Fu et al., 1996; Mlakar & Spiteller, 1996; Abordo et al., 1999; Miyata & Kurokawa, 1999; Thornalley et al., 1999; Kasper & Funk, 2001; Ulrich & Cerami, 2001; Kasai, 2002; Thornalley, 2002; Wondrak et al., 2002a) (see Figure 1). Furthermore, glyoxal is a product of the metabolism and microsomal oxidation of compounds such as glycolaldehyde, ethylene glycol, and beta-hydroxy-substituted N-nitrosamines and possibly contributes to the toxic, genotoxic, and tumorigenic action of these substances (Loeppky & Goelzer, 2002; Loeppky et al., 2002).

In biological materials, less than 10% of the glyoxal present is in unbound forms in aqueous solution (free glyoxal and hydrates), as most of the reactive carbonyl groups are reversibly bound to cysteinyl, lysyl, and arginyl residues of proteins (Thornalley, 1995).

The endogenous concentrations of glyoxal in human tissues and body fluids, as with other alpha-oxoaldehydes, are limited by the high catalytic efficiency of the glyoxalase system (Thornalley, 1995) as well as by the rapid reaction of glyoxal with proteins (Sady et al., 2000).

During certain pathological conditions (e.g., diabetes mellitus, uraemia), raised concentrations of glyoxal have been measured. The concentration of glyoxal in blood samples from normal human subjects (n = 19) was 0.21 ± 0.14 µmol/kg (Thornalley et al., 1996). For blood plasma, a value of approximately 0.1 µmol/litre was estimated for normal healthy subjects, which can double in diabetics (Thornalley, 1998; Thornalley et al., 2000).

Fig. 1: Scheme of formation, detoxification, and protein and DNA adduct formation of glyoxal

The concentration of glyoxal in blood plasma was 0.23 ± 0.13 µmol/litre in controls (n = 6), 0.4 ± 0.16 µmol/litre in patients with mild/moderate uraemia (n = 10), and 0.76 ± 0.21 µmol/litre in patients with end-stage renal disease on haemodialysis (n = 5) (Agalou et al., 2002). Similar levels in plasma samples were reported by Lapolla et al. (2003) (n = 3 persons/group): a mean of 17.3 µg/litre (0.3 µmol/litre) for healthy subjects, 26.4 µg/litre (0.45 µmol/litre) for badly controlled diabetics, and 27.2 µg/litre (0.47 µmol/litre) for those affected by chronic renal failure. Another working group reported higher levels of glyoxal in plasma (n = 15-20 subjects): 67 µg/litre in normal control subjects and 78 µg/litre in patients with non-insulin-dependent diabetes, corresponding to approximately 1 µmol/litre. Chronic renal failure resulted in accumulation of glyoxal, with a mean plasma level of 221 µg/litre (about 4 µmol/litre), which was possibly caused by accelerated autoxidation of glucose in uraemic patients (Odani et al., 1999). A possible higher non-physiological production of glyoxal leading to local accumulation was assumed in patients with hyperglycaemia associated with diabetes (Akhand et al., 2001).

In porcine ischaemic heart tissue, glyoxal levels in the lipid fraction (determination of "free glyoxal") increased 4-fold after up to 4 h of ischaemia and 24-fold after 6 h of ischaemia (0.2 µg/g lipid) in comparison with extraischaemic heart tissue (Dudda et al., 1996).

In cultures of P388D₁ cells (murine macrophage cell line), the intracellular background concentration was 31.2 pmol glyoxal/10⁶ viable cells (sum of free glyoxal and glyoxal reversibly bound to proteins). Intracellularly produced glyoxal readily crosses cell membranes, possibly by passive diffusion. Despite reversible binding to cellular peptides and proteins, accumulation of glyoxal in the extracellular medium could be demonstrated, with concentrations in the culture medium rising from below the detection limit to 61 nmol/litre (P < 0.01) during a 3-h culture period (Abordo et al., 1999).

There are limited qualitative and no quantitative data on the absorption and distribution of glyoxal in humans and experimental animals. Acute and subacute inhalation exposure resulted in local effects on eyes and respiratory organs, the extent of systemic absorption being unclear. After acute and chronic oral administration, there is evidence of systemic absorption, with distribution to erythrocytes, liver, lung, kidney, pancreas, and adrenal glands (BUA, 1997; see also section 8; e.g., Ueno et al., 1991a). There is some qualitative evidence that glyoxal is absorbed after dermal exposure. Granular and vacuole degeneration in liver, kidney, and pancreas have been observed along with a distinct increase in blood glucose levels following dermal application (Ito, 1963). Further, data on skin sensitization (see sections 8.7 and 9) provide supportive qualitative evidence that glyoxal is absorbed across the skin.

In normal human urine, a glyoxal concentration of 132 µmol/litre was found by HPLC analysis (Espinosa-Mansilla et al., 1998). However, this could either be produced endogenously or stem from an exogenous source, such as food intake.

The cytosolic GSH-dependent glyoxalase system is the major pathway for the detoxification of glyoxal (see Figure 1). Glyoxal reacts non-enzymatically with GSH with formation of a hemithioacetal, which is subsequently converted to S-glycolylglutathione by glyoxalase I. Glyoxalase II catalyses the hydrolysis of S-glycolylglutathione to glycolate, re-forming the GSH from the first reaction. The activity of glyoxalase I in situ is approximately proportional to the cytosolic concentration of GSH. When GSH is severely depleted (e.g., under conditions of oxidative stress), however, 2-oxoaldehyde dehydrogenase and aldose reductase may also metabolize glyoxal. Imbalances in intracellular redox systems may impair these detoxification mechanisms, resulting in higher levels of glyoxal (Thornalley, 1995, 1998; Abordo et al., 1999; Miyata et al., 1999, 2001). A further GSH-independent route of detoxification via glyoxalase III exists. Glyoxalase III is reported to be the most abundant glyoxalase in Escherichia coli (MacLean et al., 1998; Okada-Matsumoto & Fridovich, 2000).

The glyoxalase I concentration in human tissues and blood cells was about 0.2 µg/g protein. In human tissues, the specific activity was highest in pancreas, lung, kidney, and brain and lowest in adipose tissue and liver. Specific activities in fetal tissues were about 3 times higher than in corresponding adult tissues. Human glyoxalase I was found to exhibit genetic polymorphism, with three phenotypes resulting from a diallelic gene. The frequency of the GLO¹ allele in various populations on average ranges from 0.046 to 0.853 (Thornalley, 1993).

Exposure to glyoxal induced the activity of glyoxalase I in Salmonella typhimurium strains TA 100 and TA 104 (0.1 mg glyoxal/ml) (Ueno et al., 1991b) as well as in erythrocytes, liver, and kidney of male Sprague-Dawley rats (4000 or 6000 mg glyoxal/litre drinking-water for 30 days, no increased activity for longer exposure periods; for details, see section 8.3) (Ueno et al., 1991a).

Glyoxal attacks the amino groups of proteins, nucleotides, and lipids with its highly reactive carbonyl groups. A sequence of non-enzymatic reactions, called glycation, yields stable AGEs with a background extent of 0.1-1% of lysine and arginine residues in proteins and 1 in 10⁷ nucleotides in DNA.

AGEs originating from the reaction of glyoxal with lysine and arginine residues of proteins identified so far are N^ε-(carboxymethyl)lysine (CML), imidazolium cross-links as glyoxal-lysine dimer and imidazolysine, arginine-derived imidazolium products, and arginine-lysine cross-links. Cyclic imidazolidones may be formed on reaction with arginine residues.

Glyoxal forms stable adducts with guanosine by reaction with the N-1 as well as with the exocyclic nitrogen of guanine. The rate of glyoxal-guanine adduct formation is rapid under physiological conditions (Loeppky et al., 1999). A stable tricyclic glyoxal-DNA adduct is formed by covalent binding to two nitrogens of guanine under physiological conditions in vitro (for details, see BUA, 1997). Besides 8-hydroxy-deoxyguanosine, the glyoxal-deoxyguanosine (dG) adduct is one of the major deoxyguanosine oxidation products, being formed by oxygen radicals, lipid peroxidation systems, various types of oxidative stress, and UV irradiation and after in vivo exposure to beta-hydroxy-substituted N-nitrosamines (Murata-Kamiya et al., 1997a,b; Loeppky et al., 1999; Mistry et al., 1999; Cooke et al., 2000; Kasai, 2002).

Reaction of glyoxal with deoxycytidine (dC) yields 5-hydroxyacetyl-deoxycytidine or, by deamination, deoxyuridine. Deamination of 5-methyl-deoxycytidine is also possible, forming deoxythymidine. The analysis of DNA bases involved in DNA cross-links formed in vitro showed cross-linking by deoxyguanosine-glyoxal-deoxycytidine adducts and deoxyguanosine-glyoxal-deoxyadenine adducts (Kasai et al., 1998).

Incubation of rat retinal organ culture with glyoxal (<300 µmol/litre for 9 h) increased apoptotic events in all layers. After 800 µmol glyoxal/litre, approximately 50% of the cells in all layers of the retina were apoptotic. The glyoxal-induced rapid formation of CML showed the ability of the retina model to simulate AGE-related events in vitro. The neurotoxicity of glyoxal-induced AGE formation was shown by the significantly increased rate of cell death in the retina (Reber et al., 2003).

The acute toxicity of glyoxal in experimental animals is low to moderate, depending on the actual concentration of glyoxal in the tested product. However, from the documentation in the study reports, it is not always clear if the values given for the LC₅₀ or LD₅₀ refer to the tested product with its specified concentration or if the values were converted to a concentration of 100% glyoxal. A detailed compilation of acute toxicity data is given in the source document (BUA, 1997).

An LC₅₀ value of 2440 mg/m³ (2410 mg/m³ for females, 2470 mg/m³ for males) was calculated from single 4-h inhalation exposures of rats to aerosols of 40% glyoxal (Hoechst AG, 1984b). All 10 rats exposed by inhalation to an atmosphere containing dust of 80% glyoxal in the highest technically feasible concentration of 1300 mg/m³ survived (Hoechst AG, 1984c). All rats survived 7- and 8-h exposures to concentrated atmospheres (concentration not further specified) of 30% (Mellon Institute, 1958, 1965) or 40% glyoxal (Hoechst AG, 1984d,e). After inhalative uptake, observations reported included local irritations of the eyes and respiratory organs as well as hyperaemia and foamy secretion in the lungs. No macroscopic organ changes were reported in those rats surviving the 14-day post-observation period (Hoechst AG, 1984d,e).

After oral administration to rats, LD₅₀ values ranging from 2960 mg/kg body weight (lowest value in females) to 8979 mg/kg body weight (highest value in males) were reported in several studies using products containing 40% glyoxal, demonstrating a higher sensitivity of female rats. In mice (sex not given), the LD₅₀ of 40% glyoxal was 4064 mg/kg body weight. For a preparation containing 80% glyoxal, oral LD₅₀ values of 2000 mg/kg body weight in rats and 900 mg/kg body weight in guinea-pigs were found. Macroscopic observations reported after oral uptake include irritations of the gastrointestinal tract and congestions in the gastrointestinal tract, lung, kidney, and adrenal glands (BUA, 1997).

After dermal administration of 40% glyoxal, the LD₅₀ values were >2000 mg/kg body weight for the rat, 12 700 mg/kg body weight for the rabbit, and >5000 mg/ kg body weight for the guinea-pig (for details, see BUA, 1997).

In the 1940s to 1960s, histopathological findings in studies with acute application of glyoxal pointed to a connection between effects induced by glyoxal and those induced in the course of diabetes; this has been confirmed by recent intensive studies on the mechanism of action of endogenous glyoxal and its involvement in the development of diabetic complications (see section 8.8).

Pancreas and kidney were identified as the prominent target organs of the toxic action of glyoxal; severe degenerative changes in these organs were attributed to an inhibition of glyoxalase activity in these tissues. Changes in the pancreas were dominated by the observation of necrotic areas containing B-cells of the Langerhans islets in rabbits (105 mg glyoxal/kg body weight intracardial or two administrations of 320 mg/kg body weight subcutaneous) and in cats (227 mg/kg body weight, application not specified). A simultaneous increase of blood glucose levels was demonstrated in rabbits and cats, comparable to alloxan-induced diabetes (Doerr et al., 1948). The pancreas is a prominent target organ of alloxan toxicity, too, which is mediated by free radicals (Younes, 1997). Rats responded to intravenous injection of 100-200 mg glyoxal/kg body weight with a dose-dependent, reversible, and reproducible reduction of the blood glucose level, which was attributed to a glyoxal-stimulated secretion of insulin secondary to oedematous changes of the pancreas. At higher dosage (175 mg/kg body weight intravenous), more severe changes, such as irreversible necroses and degranulation of B-cells, were observed in connection with visible changes in other organs. However, the B-cells of the pancreas showed the highest sensitivity to the toxic action of glyoxal (Helge, 1959). The nephrotoxic action of glyoxal is characterized by vacuole degeneration in the kidney (460 mg glyoxal per cat subcutaneous) (Doerr, 1957a,b). Acute effects noted in the pancreas in several studies all seemed to arise when glyoxal was administered parenterally, compared with other routes. This may be due to toxicokinetic reasons.

A further study in rabbits described histopathological changes in liver, kidney, and pancreas 40 days after a single dermal application of a 40% glyoxal solution (leading to severe necrotic dermatitis at application site; dose not specified). Granular and vacuole degeneration in liver, kidney, and pancreas and atrophy and fibrous change of Langerhans islets were assessed to show a close resemblance to changes in these tissues in the course of diabetes. In glucose tolerance tests performed 5 and 10 days after dermal application of glyoxal, a distinct increase of blood glucose levels was observed in comparison with a constant level in control rabbits (Ito, 1963).

In an inhalation study conducted according to OECD guideline 412, groups of five male and five female Wistar rats inhaled aerosols containing glyoxal (40% in water) at 0, 0.4, 2.0, or 10 mg/m³ (analytical concentrations 0, 0.6, 2.3, and 8.9 mg/m³; mass median aerodynamic diameter 0.8-1.2 µm) for a period of 29 days (nose only, 6 h/day, 5 days/week). Exposure was tolerated by all dose groups without any systemic effects (examination of body weight, haematological and biochemical parameters, urine analysis, macroscopic and histological examination). The only local effect found in the larynx was a minimal squamous metaplasia of the epiglottal epithelium accompanied by a minimal submucosal infiltration of lymphocytes in the mid- and high-dose groups. Consequently, for local effects, a NOEL of 0.6 mg/m³ (nominal concentration 0.4 mg/m³) resulted for subacute inhalation exposure of rats to glyoxal (Hoechst AG, 1995).

In a 28-day oral toxicity study conducted according to OECD guideline 407, six male and six female Sprague-Dawley rats per dose group were exposed to glyoxal (40% in water) at dosages of 0, 100, 300, or 1000 mg/kg body weight per day via the drinking-water. A dose-dependent retardation of body weight gain in the mid-dose group (slight effect) and the high-dose group (significant effect) was accompanied by reduced food intake. A dose-dependent reduction of water intake was observed in male rats at the lowest dose and in female rats at the mid- and high doses (glyoxal concentrations were adjusted to water intake). Changes in mid- and high-dose groups, such as increased erythrocyte number and reduced urine volume, were attributed to reduced water intake; changes of various organ weights in the high-dose group were attributed to reduced body weight. No changes were found at macroscopic and histological examination. The NOAEL for this study is 100 mg glyoxal/kg body weight per day (Société Française Hoechst, 1987). (More details were not available to the authors of this CICAD. It is not known whether these concentrations are adjusted to 100% glyoxal. If not, the NOAEL would be about 40 mg/kg body weight adjusted to 100% glyoxal.)

In a 90-day feeding study, Wistar rats (10 males and 10 females per dose group) were exposed to glyoxal (40% preparation). The study gives the dosages converted to 100% glyoxal content as corresponding to about 32, 63, 125, and 250 mg/kg body weight per day for male and female rats. Males of the high-dose group showed a reversible significant retardation of body weight gain during the first 2 weeks of exposure without a concomitant reduction of food intake. Significant increases of liver and kidney weights were observed in the high-dose group (these are the only organ weights examined). No relevant macroscopic or micropathological changes were observed in thoracic and abdominal organs (pancreas not examined). Haematological and biochemical parameters were not analysed. From these investigations, a NOAEL of 125 mg (corresponding to 100% glyoxal)/kg body weight per day was estimated (Mellon Institute, 1966).

Beagle dogs (three per dose group) were also exposed to the same preparation of glyoxal by feeding dosages of 31, 65, or 115 mg/kg body weight per day (dosages corresponding to 100% glyoxal). Up to the high dosage, no substance-related changes of body weight, food consumption, liver or kidney weight, or haematological or serum clinical chemistry parameters and no macroscopic or histopathological changes were observed in thoracic and abdominal organs (pancreas not examined). The NOEL for 90-day feeding of glyoxal to dogs was >115 mg/kg body weight per day (dosage corresponding to 100% glyoxal) (Mellon Institute, 1966).

Five male Sprague-Dawley rats per group were treated with glyoxal (98.7% purity) in drinking-water at concentrations of 2000, 4000, or 6000 mg/litre for periods of 30, 60, or 90 days (Phase I study) (Ueno et al., 1991a). Due to a decrease in food intake, the actual dosages decreased with increasing time of exposure (30, 60, and 90 days) and corresponded to 188, 135, and 107 mg/kg body weight per day for the low-dose groups, 407, 239, and 234 mg/kg body weight per day for the mid-dose groups, and 451, 344, and 315 mg/kg body weight per day for the high-dose groups, respectively. The study design included observations of clinical signs, body weights, major organ weights (liver, kidneys, spleen, heart, testes, brain), serum clinical chemistry, and biochemical examinations of glyoxalase activity and extent of lipid peroxidation (content of GSH and 2-thiobarbituric acid-reactive substances) in liver, kidneys, and erythrocytes.

There was a dose-dependent retardation of body weight gain, which was significant for the mid- and high-dose groups, and also a dose-dependent decrease of food and water intake. From Phase II of this study (see below), it was concluded that body weight reduction did not correspond to decreased food intake but was a reflection of the systemic effects of glyoxal. Absolute weight of liver, kidneys, spleen, and heart significantly decreased in all dosed groups at all time points. A significant increase of relative kidney weight in the high-dose group resulted after 90 days. There was no indication of increased lipid peroxidation.

Glyoxalase I activity was significantly increased in liver and erythrocytes at the mid- and high doses and in the kidneys at the high dose at the 30-day termination, but not for longer exposure periods. In contrast, the serum clinical parameters aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, albumin, and total protein were significantly reduced by the mid- and/or high-dose exposures for all examination time points. In the low-dose group, alanine aminotransferase and total protein were significantly decreased, so that it was not possible to derive a NOAEL for this study. Consequently, a dosage of 107 mg/kg body weight per day (99% glyoxal) corresponds to the LOAEL for a 90-day exposure of rats (Ueno et al., 1991a). The decrease of serum protein levels was attributed to a decrease of protein synthesis, which was demonstrable after acute exposure to glyoxal (Ueno et al., 1991a) and is explainable by the mode of action of glyoxal (see section 8.8).

In Phase II of the study, five rats received 6000 mg glyoxal/litre drinking-water (highest test concentration from Phase I) for 90 or 180 days. One control group received food ad libitum, whereas a second diet-limited control group received the same amount of food as consumed by the dosed animals. Dosages were 315 and 298 mg/kg body weight per day (glyoxal 98.7% purity) for the 90- and 180-day exposure, respectively. The extent of examinations was comparable to that in Phase I and was further supplemented by gross and histopathological examinations of liver, kidneys, spleen, stomach, thymus, and mesenteric lymph nodes. Terminal body weight was significantly lower than in the pair-fed control, so that weight reduction is reflective of the systemic toxicity of glyoxal. Significant decreases of absolute weights and significant increases of relative weights of liver, kidneys, and heart were observed in glyoxal-exposed rats (Ueno et al., 1991a).

Fischer 344 rats (10 per dose group and sex) were exposed daily to drinking-water containing 0, 1000, 2000, 4000, 8000, or 16 000 mg glyoxal/litre for 90 days to establish dose ranges for a chronic study. All animals of the highest dose group were sacrificed prematurely on day 12 in a moribund state. Decreased dose-related body and organ weights as well as decreased food and water consumptions were observed at the lowest dosage. For chronic exposure, the maximum tolerated dose for rats was estimated in the range of 500-2000 mg/litre for males as the more sensitive sex (decrease of water consumption up to 28%) and 1000-4000 mg/litre for females (decrease of water consumption up to 46%) (NTP, 1991a).

In a similar study in B6C3F1 mice (10 per sex per dose group) exposed daily to drinking-water containing the same doses (0, 1000, 2000, 4000, 8000, or 16 000 mg glyoxal/litre for 90 days), all animals survived. The salient features observed were decreased body weight (decrease of 7-30% from 4000 to 16 000 mg/litre) and selected organ weights, decreased food and water consumption, and, in the male mice of all dose groups, possible chemical-related salivary gland changes (secretory depletion of submandibular gland). It was felt that the decreased water consumption (dose-dependently about 10-50%) was due to unsatisfactory palatability of the dosed water, subsequently leading to lower daily dosages and decreased feed consumption (up to 24%). From this preliminary study, recommended doses for further studies with long-term exposure were estimated to be in the range of 500-2000 mg/litre for males as the more sensitive sex (decrease of water consumption up to 12%) and 1000-4000 mg/litre for females (decrease of water consumption up to 27%) (NTP, 1991b).

No studies with long-term exposure to glyoxal by inhalation or oral routes were available.

After the exposure of Sprague-Dawley rats to dosages of 6000 mg glyoxal/litre drinking-water for up to 180 days (for details, see section 8.3), there were no neoplastic changes found at the gross and histopathological examination of liver, kidneys, spleen, stomach, thymus, and mesenteric lymph nodes (Ueno et al., 1991a).

Glyoxal showed tumour-promoting activity in a two-stage glandular stomach carcinogenesis model in male Wistar rats after an 8-week initiation treatment with N-methyl-N'-nitro-N-nitrosoguanidine in the drinking-water (100 mg/litre) along with a 10% sodium chloride dietary supplement. Subsequent promotion by exposure to glyoxal (0.5% in drinking-water from week 8 to week 40) induced significantly increased incidences of adenocarcinoma and hyperplasia in the pylorus of the glandular stomach in comparison with rats with initiation treatment only. Glyoxal treatment alone induced neither neoplastic nor hyperplastic changes in the pylorus (Takahashi et al., 1989). However, genotoxic activity (induction of unscheduled DNA synthesis and strand breaks) was demonstrated in the pyloric mucosa of the rat stomach (see section 8.5; Furihata et al., 1985, 1989; Furihata & Matsushima, 1989). A tumour-promoting potential was also derived from dose-dependent induction of ornithine decarboxylase and replicative DNA synthesis in the pyloric mucosa after a single application of 150-400 mg glyoxal/kg body weight (Furihata et al., 1985; Furihata & Matsushima, 1989, 1995).

In contrast, no tumour-promoting activity was found in a short-term liver foci assay with a 6-week glyoxal exposure via drinking-water (different concentrations of 5000 and 2000 mg/litre given in the publications) after initiation with diethylnitrosamine (single intraperitoneal dose of 200 mg/kg body weight, start of glyoxal exposure after 2-week recovery period, partial hepatectomy at week 3). Relative to the initiator-treated control group, number and area of glutathione-S-transferase placental form (GST-P) positive foci in the liver, as well as body weight, absolute liver weight, and water consumption, were significantly decreased in glyoxal-treated F344 rats (Hasegawa & Ito, 1992; Hasegawa et al., 1995).

No increase of skin tumours was observed after lifetime application of 3 µl glyoxal (two commercial products, 12.5% in water) 3 times a week to the skin of C3H/HeJ mice. Survival rates of glyoxal-treated rats were higher than those of controls. Some treated rats showed skin irritation with necrotic areas (Bushy Run, 1982).

In an assay for tumour-initiating activity, the dermal application of glyoxal alone (total initiating dose 30 mg glyoxal/mouse, 37-43% in water applied 2 times weekly for 5 weeks) induced no skin tumours in CD-1 mice within 53 weeks. After promotion by 12-O-tetradecanoyl-phorbol-13-acetate treatment for 47 weeks, 2 of 10 animals had a total of four skin papillomas, showing no significant tumour-initiating activity of glyoxal by this route (Miyakawa et al., 1991).

Glyoxal is directly genotoxic in vitro in bacterial and mammalian cells. In vivo tests show various findings. A detailed overview of genotoxicity tests in bacterial test systems is published in the source document (BUA, 1997).

In the Salmonella microsomal assay, glyoxal (test substance 30-40% glyoxal) was a direct mutagen in strains TA 100, TA 102, TA 104, and TA 2638, with a weaker response in the presence of a metabolic activation system (BUA, 1997). A direct genotoxic activity of glyoxal was further evident in the L-arabinose resistance assay with S. typhimurium BA9 and BA13 (Ruiz-Rubio et al., 1985; Ariza et al., 1988) and in the SOS chromotest with E. coli PQ37 (von der Hude et al., 1988).

Furthermore, DNA repair tests yielded positive responses in both the presence and absence of metabolic activation systems, as in the SOS umu-test with S. typhimurium TA 1535/pSK 1002 (Ono et al., 1991a,b), in the rec-assay with Bacillus subtilis (also with metabolic activation; Matsui et al., 1989), and in the differential DNA repair test with E. coli K-12/343/636 uvrB⁺/recA⁺ and K-12/343/591 uvrB^-/recA^- (Hellmér & Bolcsfoldi, 1992a). When the latter test was performed as a host-mediated assay in mice, with oral application of 570 or 1700 mg glyoxal/kg body weight and intravenous application of the bacteria, a genotoxic effect was not demonstrable in bacteria isolated from blood, liver, lungs, kidneys, or testicles (Hellmér & Bolcsfoldi, 1992b), which may be explained by the high reactivity of glyoxal — for example, with proteins (see section 8.8). In Saccharomyces cerevisiae D61.M, induction of mitotic recombinations pointed to reaction of glyoxal with DNA, whereas modification of proteins was indicated by chromosome losses (in the presence of propionitrile, which is a strong inducer of chromosomal malsegregation), suggesting interference of glyoxal with microtubular function (Zimmermann & Mohr, 1992).

With E. coli WP2 uvrA, in both the absence and presence of metabolic activation, negative test results were found in the standard plate incorporation assay (Hoechst AG, 1984f), whereas an insufficiently documented preincubation assay reported positive test results (Kato et al., 1989). Ueno et al. (1991b) investigated the characteristics of mutagenicity by glyoxal (particularly a possible role of active oxygen species) in S. typhimurium TA 100 and TA 104. The scavengers of singlet oxygen almost completely suppressed the mutagenic action of glyoxal.

A direct genotoxic action of glyoxal was established in a variety of tests with mammalian cells without metabolic activation (see BUA, 1997): in a mutagenicity test with mouse lymphoma cells (TK assay) (Wangenheim & Bolcsfoldi, 1988), in chromosomal aberration tests with Chinese hamster ovary (CHO) cells (NOTOX, 1986) and V79 cells (Nishi et al., 1989), and in tests for the induction of unscheduled DNA synthesis in TC-SV40 cells of Syrian hamster (Cornago et al., 1989), for the induction of sister chromatid exchanges in CHO cells and human lymphocytes, for the induction of endoreduplication in CHO cells (Tucker et al., 1989), and for the induction of DNA strand breaks in mouse lymphoma cells (Garberg et al., 1988). In primary rat hepatocytes, glyoxal induced DNA single strand breaks but no DNA cross-links (Ueno et al., 1991c).

DNA damage was further demonstrated in the comet assay with TK6 human lymphoblastoid cells by the induction of concentration-dependent increases of tail moment and tail length (Henderson et al., 1998). Primary rat hepatocytes exposed to glyoxal at higher concentrations (0.5-10 mg/ml) produced different concentration-dependent types of DNA damage. Tail moment and the formation of comets with head and tail (indicative of DNA strand breakage) decreased with increasing glyoxal concentration, whereas circular DNA spots with highly condensed areas increasingly appeared at the mid- and high concentrations. Among 100 tested substances, this damage was shown to be specific for certain aldehydes and was attributed to their DNA cross-linking activity (Kuchenmeister et al., 1998). In cultures of human umbilical vein endothelial cells, addition of 100 µg glyoxal/ml caused a significant increase of formamidopyrimidine N-glycosylase (FPG)-sensitive sites (measured by the comet assay) in the absence of increased intracellular levels of hydroperoxides. FPG repairs oxidative DNA damage and abasic sites and further was supposed to repair guanine-glyoxal adducts (Shimoi et al., 2001).

A significantly increased rate of sex-linked recessive lethals reported in Drosophila melanogaster in preliminary experiments (Mazar Barnett & Muñoz, 1969) was not confirmed in later assays, showing the absence of any genotoxic effect in assays for sex-linked recessive lethals in mature sperm and in the earlier stages of spermatogenesis, as well as in assays for clastogenic activity in mature sperm (reciprocal translocation, dominant lethal, and chromosome loss). However, from the increase of radiation-induced clastogenic effects after pretreatment with glyoxal, it was concluded that glyoxal came in contact with the target cells. The possibility of detoxifying mechanisms for glyoxal or of an efficient repair of glyoxal-induced damage in Drosophila was discussed (Mazar Barnett & Muñoz, 1989).

No clastogenic activity was found in a micronucleus assay in mouse bone marrow (Société Française Hoechst, 1986; no further data available).

Glyoxal was demonstrated to be genotoxic at the site of application after administration by gastric intubation. In the pyloric mucosa of male Fischer 344 rats, both significantly increased unscheduled DNA synthesis and DNA single strand breaks were induced at dosages of 400-500 mg/kg body weight within 2 h. Cytotoxicity was not reported (Furihata et al., 1985, 1988, 1989; Furihata & Matsushima, 1989). In contrast, in rat hepatocytes, a test for unscheduled DNA synthesis was negative (CCR, 1992). Glyoxal has also been shown to cause DNA strand breaks in rat hepatocytes 2-9 h after a single oral exposure to 200-1000 mg glyoxal/kg body weight (Ueno et al., 1991b). Single strand breaks were also detected in livers of rats within 2 h following a single oral exposure at 200-1000 mg glyoxal/kg body weight. The frequency of breaks reached a maximum after 9 h of exposure. Hardly any DNA lesions were detected in other tissues following exposure to 1000 mg glyoxal/kg body weight. Glyoxal causes DNA single strand breaks in rat hepatocytes following in vitro and in vivo exposure (Ueno et al., 1991c).

Cell transformation assays in C3H/10T½ cells with three different commercial products of glyoxal (test concentrations from 0.0013 to 0.195 µl/ml) yielded negative test results (Mason 1980a,b,c).

8.6.1 Effects on fertility

There were no studies available on the effects of glyoxal on fertility.

8.6.2 Developmental toxicity

In developmental toxicity studies with Sprague-Dawley rats, glyoxal trimeric dihydrate was administered by gavage on gestation days 6-15 at doses of 0, 200, 800, 1200, 1600, or 2000 mg/kg body weight per day in the preliminary study and 50, 150, or 300 mg/kg body weight per day in the main study (NTP, 1991c, 1994a,b). In the initial range-finding study, maternal toxicity (decreased weight gain) was observed at 200 mg glyoxal dihydrate/kg body weight per day (corresponding to 123 mg glyoxal/kg body weight per day), with clinical signs of toxicity and decreased gravid uterine weight at 800 mg glyoxal dihydrate/kg body weight per day and maternal deaths at 1200 mg/kg body weight per day and above (NTP, 1991c). No maternal toxicity was observed, however, in the second study at the top dose of 300 mg/kg body weight per day (corresponding to 185 mg glyoxal/kg body weight per day). No embryotoxicity was observed at 200 mg glyoxal dihydrate/kg body weight per day in the preliminary study or at the highest dose in the main study.

In a study conducted according to OECD guideline 414, 40% glyoxal was administered to 19-24 female Wistar rats as a solution in water at doses of 0, 5, 25, or 125 mg/kg body weight per day (related to 100% glyoxal) on days 6 through 19 post-coitum. Maternal toxicity (significantly reduced food consumption and significantly lower corrected body weight gain) was observed at 125 mg/kg body weight per day. No substance-related effects were observed on gestational parameters or fetuses. NOAELs of 25 mg/kg body weight per day for maternal toxicity and 125 mg/kg body weight per day for embryotoxicity were established (BASF & Clariant, 2000).

Developmental toxicity range-finding studies in New Zealand White rabbits administered glyoxal by gavage yielded a NOEL of 200 mg glyoxal trimeric dihydrate/kg body weight per day, corresponding to 123 mg glyoxal/kg body weight per day (NTP, 1991d), and a LOEL of 400 mg glyoxal dihydrate/kg body weight per day, corresponding to 247 mg glyoxal/kg body weight per day (NTP, 1992), for both maternal toxicity and embryotoxicity. Maternal signs of systemic toxicity and decreases of weight parameters were accompanied by reduced fetal weight (NTP, 1992). The application of doses in the range of 200 mg glyoxal dihydrate/kg body weight per day was discussed as problematic due to the corrosive nature of the substance, leading to damage of gastric mucosa of pregnant rabbits (unpublished observations cited in NTP, 1993). In a subsequent study with a single dose level of 50 mg glyoxal dihydrate/kg body weight per day, corresponding to 31 mg glyoxal/kg body weight per day, there was no maternal mortality or persistent signs of toxicity, although minimal reductions in body weight gain and food consumption were noted. Glyoxal exposure did not significantly alter post-implantation loss and had no effect on fetal body weight or the incidence of external, visceral, or skeletal malformations. The authors gave a NOAEL for developmental toxicity for rabbits of 50 mg glyoxal dihydrate/kg body weight per day, corresponding to 31 mg glyoxal/kg body weight per day (NTP, 1993).

In an investigation on the effects of ethylene glycol and its metabolites, glyoxal was tested in a whole rat embryo culture test system (head-fold rat embryos, which lack biotransforming enzyme activities, such as alcohol dehydrogenase or acetaldehyde dehydrogenase) (Klug et al., 2001). At glyoxal concentrations of 3 mmol/ litre, the rate of dysmorphogenic embryos was within historical controls; at 6 mmol/litre, however, a general embryotoxic effect of glyoxal was noted, with a steep concentration-response relationship. At 10 mmol/litre, no growth or differentiation of the embryo could be determined. The relevance of this test for the in vivo toxicity of glyoxal is unclear.

8.7.1 Skin irritation

After a 4-h exposure patch test on rabbits (OECD guideline 404), glyoxal (40%) caused no irritation. However, in earlier studies (e.g., patch test on the shaven back skin of white rabbits using 30% or 40% glyoxal), slight to pronounced irritation could be seen, depending on the application period (1 min, 5 min, 15 min, and 20 h). A single dermal administration (occlusive) of 40% glyoxal to the shaven skin (dorsal, dorsolateral) of five Wistar rats per sex for 24 h caused erythemas in all animals (BUA, 1997).

A 40% glyoxal solution was applied to the shaven back skin of white rabbits (no details of the time of administration). From the 3rd day, a strong reddened inflammation was observed, followed by a necrosis with tissue demarcation. Histopathological examination showed severe necrotic skin changes on the 4th day and a regeneration of the epidermis on the 18th day (Ito, 1963).

Therefore, taking into account studies where the duration of exposure was longer, glyoxal is clearly irritating to the skin (see details in BUA, 1997).

8.7.2 Eye irritation

In a study conducted according to OECD guideline 405, glyoxal (40%) led to a reversible reddening and chemosis of the conjunctiva within 8 days and thus showed an irritating effect. This confirmed older studies reporting that glyoxal caused irritations and even necrotic changes in the rabbit eye (see details in BUA, 1997).

8.7.3 Sensitization

Two studies using the Magnusson and Kligman test and one study using the Buhler test on guinea-pigs gave positive results. Glyoxal can be considered as a sensitizing substance (see details in BUA, 1997).

Glyoxal has been shown to be sensitizing in humans (see section 9).

Glyoxal, which attacks amino groups of proteins, nucleotides, and lipids, is considered an important intermediate in the formation of AGEs. AGE modification alters protein function and inactivates enzymes, resulting in disturbance of cellular metabolism, impaired proteolysis, and inhibition of cell proliferation and protein synthesis (Gan & Ansari, 1986; Ueno et al., 1991a,b; Kasper et al., 1999; Witowski et al., 2000; Bulteau et al., 2001; Kasper & Funk, 2001; Murata-Kamiya & Kamiya, 2001). The extent of AGE modification increases with the increasing life span of proteins. Consequently, AGEs are especially associated with long-lived proteins, such as collagens, lens crystallins, and neurofilaments, but also have been identified in shorter-lived proteins, including haemoglobin, plasma proteins, lipoproteins, and intracellular proteins. AGEs have a role in the pathogenesis or progression of many pathological conditions — e.g., diabetes, Alzheimer's disease and other neurodegenerative diseases, chronic inflammatory diseases, arthritis, atherosclerosis, vascular damage, cataract formation and skin changes during ageing, pulmonary fibrosis, and renal failure — as well as in peritoneal dialysis complications (Miyata et al., 1999, 2000; Thornalley et al., 1999; Cerami & Ulrich, 2001; Ulrich & Cerami, 2001; Thornalley, 2002). Although glyoxal is known to be an intermediate in the formation of AGEs, these effects have not specifically been shown for glyoxal.

Inhibition studies in bacterial mutagenicity tests demonstrated the production of the reactive oxygen species superoxide, hydrogen peroxide, and singlet oxygen from glyoxal. The mutagenic activity of glyoxal is related to singlet oxygen, as well as to the intracellular GSH level (Suwa et al., 1982; Garst et al., 1983; Yamaguchi & Nakagawa, 1983; Ueno et al., 1991b). The hydroxyl radical plays a prominent role in glyoxal-induced DNA cleavage (Roberts et al., 2003).

The sensitizing potential of glyoxal is attributed to the electron-deficient alpha,beta-dicarbonyl structure with its high electrophilic reactivity. Glyoxal easily forms Schiff bases with lysine or arginine units and so becomes bound to skin proteins (Roberts et al., 1999).

Only limited information on the effects of glyoxal in humans has been identified.

The oral ingestion of 50-300 ml of a disinfectant containing 7.5% (w/w) glyoxal, 9.5% glutaral, and 9.6% didecyldimethylammoniumchloride in suicidal intention caused severe coagulative necrosis of the mucous membranes in the upper gastrointestinal tract and whole respiratory tract in a 31-year-old female. Despite the severe effects, no perforation occurred, probably because mucous membranes were denatured and fixed through the carbonyl groups of the aldehydes, so that the substances did not penetrate deeply (Menzner et al., 1998).

Of 14 workers who had contact with 40% glyoxal, 9 exhibited a contact dermatitis with localizations mainly on the lower arms and fingers. Patch tests with a 20% glyoxal solution produced a positive reaction in 7 of 9 workers (Ito, 1963).

In a German multicentre study of dermal sensitivity, the records of 31 849 health care workers from 24 allergy departments between 1992 and 1995 were evaluated; 4.2% of the 774 female patients working in the medical profession were found to show positive reactions to glyoxal patch testing, whereas only 1.4% of the control group (1895 persons not in the medical profession) were found to be positive (Schnuch et al., 1998).

In a continuation of this multicentre study, between 1997 and 1999, 2689 patients were reported to have been patch tested with glyoxal (trimer; 1% in petrolatum). Positive (allergic) reactions were observed in 1.6% of the patients, whereas irritant (0.3%) and questionable (0.6%) (i.e., non-allergic) reactions were observed less frequently. Even prior to diagnostic patch testing, disinfectants had been suspected to be causative for dermatitis in 23 and cleaning agents in 10 of the 44 patients sensitized to glyoxal. Occupations included nursing (n = 15), room cleaning (n = 12), dental nursing (n = 5), geriatric nursing (n = 3), and some other medical associated professions (n = 5), with very few other non-medical occupations (n = 4) (Uter et al., 2001).

In a retrospective descriptive analysis of records from an occupational dermatitis clinic in Osnabrück, Germany, 189 patients with occupational dermatitis were patch tested with glyoxal (15 in water or, as trimer, 1% in petrolatum) between 1993 and 1999. Of the 11 cases with positive reactions to glyoxal, 9 were considered as being occupationally relevant — those with nursing and room cleaning jobs (Uter et al., 2001).

In another study of 280 health care workers in Poland, the frequency of allergy to aldehydes (1% formaldehyde, glutaraldehyde, or glyoxal) with allergic dermatitis was 22.8%. The majority were sensitive to only one aldehyde, indicating lack of cross-reaction; glutaraldehyde was positive in 12.4% and glyoxal in 1.9% (Kieć-Świerczyńska et al., 1998). In a further study of the causes of occupational dermatosis in 27 dental nurses during the years 1995-1999, contact sensitization by glyoxal was found in 3 cases (Kieć-Świerczyńska & Kręcisz, 2000).

In a maximization test, all 24 patients tested gave a positive reaction with 10% glyoxal solution (induction), 2% solution (challenge) showing a very strong skin sensitizing potential (Kligman, 1966).

Table 2: Aquatic and terrestrial toxicity of glyoxal.

Glyoxal may enter the hydrosphere due to its production, use, and application and can be formed, in addition, by natural processes.

A limited number of acute tests have been performed to establish the toxicity of glyoxal for aquatic organisms representing different trophic levels (data are summarized in Table 2; see BUA, 1997, for more detailed information). Assays performed by employing aerobic heterotrophic bacteria such as Pseudomonas putida, anaerobic bacteria, and Photobacterium phosphoreum showed that P. putida (end-point = inhibition of cell proliferation, 16 h) exhibited the lowest EC₅₀ value of 134 mg/litre (Hoechst AG, 1989).

The only algal species tested was Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), with a 96-h EC₅₀ value of 149 mg/litre (Bollman et al., 1990).

For the only invertebrate species tested, Daphnia magna, a 24-h EC₅₀ value of 430 mg/litre was reported (OECD, 1992).

Acute toxicity studies conducted with four different fish species established the lowest LC₅₀ value (96-h incubation) as 215 mg glyoxal/litre using Pimephales promelas (Conway et al., 1983).

Data concerning the toxicity of glyoxal for soil-bound microbial activity, terrestrial invertebrates, or vertebrates or effects upon terrestrial ecosystems are not available. For the terrestrial compartment, the only available toxicity study reported the inhibition of rhizome fragment proliferation of Helianthus tuberosus by glyoxal, with a NOEC value of 68 mg/litre and a corresponding EC₃₀ value of 136 mg/litre (BUA, 1997).

Glyoxal is endogenously produced during normal cellular metabolism by several enzyme-independent pathways. The cytosolic GSH-dependent glyoxalase system is the major pathway for the detoxification of glyoxal. When GSH is severely depleted, 2-oxoaldehyde dehydrogenase and aldose reductase also metabolize glyoxal.

Due to its highly reactive carbonyl groups, glyoxal attacks proteins, nucleotides, and lipids, followed by further reactions leading to the formation of AGEs. These adducts can interfere with normal cellular function, inducing carbonyl stress and oxidative stress and affecting protein function and signal transduction pathways of the cells; these result in a range of pathological changes, cell proliferation, genotoxicity, or programmed cell death.

During certain pathological conditions (e.g., diabetes mellitus, uraemia), raised concentrations of glyoxal have been measured in the plasma.

It is not known if acute environmental exposure to glyoxal also leads to raised concentrations in the blood or whether the high catalytic efficiency of the glyoxalase system is able to detoxify it.

11.1.1 Hazard identification and dose-response assessment

Studies in patients and volunteers have confirmed the sensitizing potential of glyoxal. This has been substantiated by animal studies. Glyoxal is irritating to mucous membranes. In animal studies, 30% and 40% aqueous glyoxal cause slight to definite skin irritations, depending on the application time.

There are almost no data on other toxicity end-points in humans.

Because of the limited nature of data in humans, hazard identification and dose-response analysis for glyoxal are based primarily on studies in animals.

The acute toxicity of glyoxal in experimental animals is low to moderate. After exposure by inhalation, local irritations of the eyes and respiratory organs predominate. After oral uptake of glyoxal, macroscopic observations include irritation of the gastrointestinal tract and congestion in the gastrointestinal tract, lung, kidney, and adrenal glands. In the pancreas and kidney, the toxic action of glyoxal leads to severe degenerative changes resembling those induced during diabetes.

A 29-day nose-only inhalation exposure study in rats using 40% glyoxal showed a NOEL of 0.6 mg/m³ (nominal concentration was 0.4 mg/m³) for local effects in the larynx and a NOEL of >8.9 mg/m³ (nominal concentration was 10 mg/m³) for systemic effects (Hoechst AG, 1995).

The 90-day feeding study in rats resulted in a NOAEL of 125 mg/kg body weight per day (dose corresponding to 100% glyoxal). A 28-day drinking-water study of 40% glyoxal in rats resulted in a NOAEL of 100 mg glyoxal/kg body weight per day (Société Française Hoechst, 1987). Effects at higher doses were reduced water and food intake and retardation of body weight gain (Mellon Institute, 1966). A 90-day feeding study in dogs failed to reveal any substance-related changes at the top dose of 115 mg/kg body weight per day (dose corresponding to 100% glyoxal) (Mellon Institute, 1966).

In a 90-day drinking-water study in rats examining more sensitive end-points, the lowest tested dosage of 107 mg/kg body weight per day (99% glyoxal) was given as the LOAEL for serum clinical parameters (Ueno et al., 1991a).

There are no data available on the effects of glyoxal on fertility. Fetotoxic and developmental effects occur only with doses of glyoxal that induce maternal toxicity.

Glyoxal is directly genotoxic in vitro in bacterial and mammalian cells. In vivo, a genotoxic activity of glyoxal was established at the site of application in the pyloric mucosa of rats by demonstration of unscheduled DNA synthesis and DNA single strand breaks. After oral application, DNA strand breaks were further observed in rat liver. Glyoxal forms stable adducts with proteins and DNA bases.

No carcinogenicity studies were available for exposure by inhalation. Glyoxal showed tumour-promoting activity in a two-stage glandular stomach carcinogenesis model in male Wistar rats (Takahashi et al., 1989), whereas it was inactive in a short-term liver foci assay (Hasegawa & Ito, 1992; Hasegawa et al., 1995). In a skin painting study for tumour-initiating activity and in cell transformation assays, glyoxal yielded negative test results. A lifetime skin painting study showed no increase of tumours, but some treated rats showed skin irritation with necrotic areas.

11.1.2 Criteria for setting tolerable intakes/concentrations

Due to lack of data, it is not possible to determine whether glyoxal has a carcinogenic potential. It is, however, genotoxic in vitro in bacterial and mammalian cells, and there is some evidence that this may be so in vivo. It readily forms DNA adducts, generating potential carcinogens such as glyoxalated deoxyguanosine and deoxycytidine.

Exposure to exogenous glyoxal causes local effects, probably due to formation of AGEs. Occupational exposure would be mainly due to the use of glyoxal in disinfectants and adhesives and would be via inhalation of aerosols or dermal routes, causing irritant or sensitizing effects.

A 29-day inhalation study in rats exposed to glyoxal showed a NOEL of 0.6 mg/m³ for local effects in the larynx. Use of uncertainty factors of 10 for interspecies differences and 10 for interindividual differences gives a tolerable concentration of 6 µg/m³ for local effects in the larynx for short-term exposure.

From studies on oral exposure, the NOAEL seems to be about 100 mg/kg body weight per day (adjusted to 100% glyoxal). Using uncertainty factors of 10 for interspecies differences and 10 for interindividual differences and a factor of 5 for less-than-lifetime exposure, this results in a tolerable intake of about 0.2 mg/kg body weight per day for lifetime oral exposure to glyoxal. The short- and medium-term studies seem to have similar outcomes, with no evidence of systemic effects, suggesting that exogenous glyoxal is efficiently detoxified and does not accumulate in the body. The use of the lifetime extrapolation uncertainty factor (factor of 5) is also justified on the basis of the 125 mg/kg body weight LOAEL with wide dose spacing to a NOAEL of 25 mg/kg body weight (BASF & Clariant, 2000).

11.1.3 Sample risk characterization

Example 1 — General population: An exposure scenario has been compiled as a hypothesized worst case. Using the daily intake of, maximally, 10 mg glyoxal via food given in section 6.2.1, an estimated intake of 0.16 mg glyoxal/kg body weight per day can be calculated. This is slightly less than the tolerable intake of about 0.2 mg/kg body weight per day for lifetime oral exposure to glyoxal (see section 11.1.2).

Example 2 — A nurse or hospital cleaner or consumer using disinfectant: A typical brand of disinfectant (7.5 g in 100 g = 7.5% glyoxal) is used at a dilution of 1% for disinfection and cleaning of surfaces (i.e., 0.075% glyoxal). Using a rounded-up 0.1% glyoxal solution and a calculation derived from a model gives an uptake of about 4 µg/kg body weight per day, assuming a body weight of 64 kg (see section 6.2.2).

This is much (50 times) less than the tolerable intake of about 0.2 mg/kg body weight per day for lifetime oral exposure (see section 11.1.2).

However, it should be noted that other substances (e.g., glutaral or formaldehyde) may also be present in the product.

An exposure scenario has been compiled as a hypothesized worst case. Assuming exposure to 4% glyoxal given in section 6.2.1 and using the same assumptions as above would give an uptake of about 0.15 mg/kg body weight, which is slightly less than the tolerable intake of about 0.2 mg/kg body weight per day for lifetime oral exposure (see section 11.1.2).

However, it should be noted that dermal contact to glyoxal may cause sensitization.

Example 3 — A farmer using a spray application of biocidal products containing glyoxal to disinfect a stable (see section 6 and Appendix 5): The model calculation using the given assumptions predicts a short-term exposure concentration of 24 µg glyoxal/m³ for a 6-min exposure and 32 µg glyoxal/m³ for 15 min. This can be compared with the estimated tolerable concentration of 6 µg/m³ for local effects in the larynx for a short-term exposure (see section 11.1.2). There is a perceived risk of local laryngeal effects and irritation to the skin from this spray application of glyoxal.

11.1.4 Uncertainties in the evaluation of health risks and in the sample risk characterization

Glyoxal is produced endogenously during normal cellular metabolism. Glyoxal attacks proteins, nucleotides, and lipids, followed by further reactions leading to the formation of AGEs. It is uncertain as to the effects of exogenously administered glyoxal. It is possible that the detoxification mechanisms (e.g., cytosolic GSH-dependent glyoxalase system) are sufficient to counteract this. There are, however, no data available to confirm this.

There are no data on the effects of glyoxal in humans, except for sensitization effects.

There is a lack of data on the carcinogenicity of glyoxal, in particular via inhalation and oral routes.

There is little known about the toxicokinetics of glyoxal.

There is little known about the ability of stable protein adducts to accumulate in long-life cells (e.g., retinal neurons), even at normal plasma and tissue concentrations.

There is no information on occupational exposure in the glyoxal production industry.

There are no dermal exposure data for, for example, hospital staff frequently in contact with this chemical.

There are uncertainties with regard to model calculations.

Glyoxal is present in products together with other chemicals. Therefore, the risk assessment given here is for glyoxal, but not for the product itself.

11.2.1 Aquatic environment

The main environmental target compartments of glyoxal are soil and water. Glyoxal is quickly transformed by abiotic reactions and readily biodegraded. At present, a reliable quantification of glyoxal currently released from all sources is impossible with the data available.

The reported low log K_ow values along with the high solubility in water indicate a negligible bioaccumulation potential for glyoxal.

The biocidal activity of the dialdehyde glyoxal is related to the availability of the carbonyl group. However, due to the hydration of glyoxal in the presence of water (by means of a nucleophilic addition), this activity is apparently reduced, as indicated by inhibition studies with bacteria (Eggensperger, 1977).

A sample risk characterization may be performed for glyoxal present in the hydrosphere, according to EC (1996), by calculating the ratio between a PEC (based on measured data) and a corresponding PNEC.

Due to the limited data available, a reliable quantification of glyoxal released into the environment is not possible. However, up-to-date monitoring data from Poland may be regarded as pertinent for an industrialized region. The highest reported value for surface water samples of the river Bogdanka of 1.9 µg/litre can be employed as a local PEC. A corresponding PNEC for surface water can be predicted from the lowest 96-h EC₅₀ value obtained for Pseudokirchneriella subcapitata growth inhibition (149 mg/litre) using an uncertainty factor of 1000 (EC, 1996). Thus, PNEC = 149 mg/litre / 1000 = 0.14