DRAFT FOR THE DRINKING WATER CRITERIA DOCUMENT ON GLYOXAL AND METHYLGLYOXAL Prepared Under EPA Contract OW 68 C 98 141 CDM Work Assignment No. 2 13 for Health and Ecological Criteria Division Office of Science and Technology Office of Water U. S. Environmental Protection Agency July 7, 2003 ii DISCLAIMER This document is a preliminary draft. It has not been released by the Office of Water, U. S. Environmental Protection Agency, and should not at this stage be construed to represent Agency Policy. It is being circulated for comments on its technical merit. iii CONTENTS FOREWORD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v EXECUTIVE SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii GLYOXAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 I. PHYSICAL AND CHEMICAL PROPERTIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 II. TOXICOKINETICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 A. Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 B. Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 C. Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 D. Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 E. Bioaccumulation and Retention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 III. HUMAN EXPOSURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 A. Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 B. Fate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 C. Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 IV. HEALTH EFFECTS IN ANIMALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 A. Short Term Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 B. Longer Term Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 C. Reproductive/ Teratogenic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 D. Mutagenicity and Genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 E. Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 V. HEALTH EFFECTS IN HUMANS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 A. Sensitive Populations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 VI. MECHANISM OF TOXICITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 A. Mechanism for DNA Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 VII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS . . . . . . . . . . . . . . . . . . . . . . . . 16 A. Noncarcinogenic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 B. Carcinogenic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 METHYLGLYOXAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 I. PHYSICAL AND CHEMICAL PROPERTIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 II. TOXICOKINETICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 A. Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 B. Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 iv CONTENTS ( continued)

C. Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 D. Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 E. Bioaccumulation and Retention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 III. HUMAN EXPOSURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 A. Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 B. Fate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 C. Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 IV. HEALTH EFFECTS IN ANIMALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 A. Short Term Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 B. Longer Term Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 C. Reproductive/ Developmental Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 D. Mutagenicity and Genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 E. Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 V. HEALTH EFFECTS IN HUMANS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 A. Sensitive Populations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 VI. MECHANISM OF TOXICITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 A. Mechanistic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 VII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS . . . . . . . . . . . . . . . . . . . . . . . . 49 A. Noncarcinogenic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 B. Carcinogenic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 v FOREWORD The Safe Drinking Water Act, as amended in 1996, requires the Administrator of the U. S. Environmental Protection Agency ( EPA) to publish maximum contaminant level goals ( MCLGs) and promulgate National Primary Drinking Water Regulations for each contaminant that, in the judgment of the Administrator, may have an adverse effect on public health and that is known or anticipated to occur in public water systems. The MCLG is nonenforceable and is set at a level at which no known or anticipated adverse health effects in humans occur and which allows for an adequate margin of safety. Factors considered in setting the MCLG include health effects data and sources of exposure other than drinking water.

This document provides the health effects basis to be considered in establishing the MCLG for glyoxal and methylglyoxal, which are byproducts of water ozonation. To achieve this objective, data on pharmacokinetics, human exposure, acute and chronic toxicity to animals and humans, epidemiology, and mechanisms of toxicity were evaluated. Specific emphasis is placed on data providing dose response information. Thus, although the literature search and evaluation performed in support of this document were comprehensive, only the reports considered most pertinent in the derivation of the MCLG are cited in this document. The comprehensive literature search in support of this document includes information published up to January 2003; however, more recent information may have been added during the review process.

When adequate health effects data exist, Health Advisory values for less than lifetime exposure ( 1 day, 10 day, and longer term, approximately 10% of an individual's lifetime) are included in this document. These values are not used in setting the MCLG, but serve as informal guidance to municipalities and other organizations when emergency spills or contamination situations occur.

The Reference Dose ( RfD) provides information on long term toxic effects other than carcinogenicity. The RfD is based on the assumption that thresholds exist for certain toxic effects such as cellular necrosis, but may not exist for other toxic effects such as some carcinogenic responses. It is expressed in terms of milligrams per kilogram per day ( mg/ kg/ day). In general, the RfD is an estimate ( with uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human population ( including sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a lifetime. The RfD is used in establishing the Lifetime Health Advisory for noncancer effects.

The carcinogenicity assessment provides information on two aspects of the carcinogenic risk assessment for the agent in question: ( 1) the EPA classification and ( 2) quantitative estimates of risk from oral exposure. The classification reflects a weight of evidence judgment of the likelihood that the agent is a human carcinogen and the conditions under which the carcinogenic effects may be expressed. Quantitative risk estimates are presented in three ways. The slope factor is the result of the application of a low dose extrapolation procedure and is presented as the risk per mg/ kg/ day. The unit risk is the quantitative estimate in terms of risk per micrograms per liter (

g/ L) drinking water. The third form in which risk is presented is a drinking water concentration providing cancer risks of 1 in 10,000, 1 in 100,000, or 1 in 1,000,000. vi Development of the hazard identification and dose response assessments for glyoxal and methylglyoxal has followed the general guidelines for risk assessments as set forth by the National Research Council ( 1983) and The Presidential/ Congressional Commission on Risk Assessment and Risk Management ( 1997). Other guidelines that were used in the development of this assessment include the following: Guidelines for Carcinogen Risk Assessment ( U. S. EPA, 1986), Proposed Guidelines for Carcinogen Risk Assessment ( U. S. EPA, 1996a, 1999), Guidelines for Developmental Toxicity Risk Assessment ( U. S. EPA, 1991), Guidelines for Reproductive Toxicity Risk Assessment ( U. S. EPA, 1996b), Guidelines for Neurotoxicity Risk Assessment ( U. S. EPA, 1998), Recommendations for and Documentation of Biological Values for Use in Risk Assessment ( U. S. EPA, 1988), and Health Effects Testing Guidelines ( U. S. EPA, 1997). Draft   Do Not Cite or Quote ( July, 2003) vii EXECUTIVE SUMMARY Glyoxal and methylglyoxal are small geminal ( neighboring) dicarbonyl compounds that are produced during ozonation of potable water. Both compounds are also generated endogenously in the degradation of nutrients or xenobiotics. They are metabolized by the glyoxalase enzyme system. When present in excess, they can modify DNA, RNA, and proteins. Because of their chemical similarities, glyoxal and methylglyoxal are considered together in this criteria document.

Glyoxal Administration of single oral doses of 1000 mg/ kg of glyoxal to rats resulted in decreased protein synthesis in liver and spleen. Other endpoints of toxicity were not investigated. Exposure of rats to glyoxal in drinking water for 30 to 180 days resulted in decreased food and water intake and decreased body weight gain at doses of 239 mg/ kg/ day and above. Body weight gain was slower in exposed animals than in pair fed controls, indicating that the effect on body weight gain was not entirely due to taste aversion. Histological examination revealed no evidence of tissue injury except for a slight swelling of papillary epithelial cells in the kidney in animals exposed to 298 to 315 mg/ kg/ day for 180 days. Analysis of blood revealed decreased levels of some serum enzymes and total protein at doses of 107 mg/ kg/ day and above.

Glyoxal administered orally to rats caused cross linking between DNA and proteins in epithelial cells in the stomach. In vitro, glyoxal increased the incidence of sister chromatid exchange ( SCE) and micronuclei in human lymphocytes. In vitro studies of mutagenicity indicate that glyoxal causes both point and frame shift mutations. G: C base pairs appear to be most vulnerable to the mutagenic effects of glyoxal.

The carcinogenic potential of glyoxal has not been thoroughly studied. Exposure of rats to glyoxal ( 500 mg/ kg/ day) in drinking water for 32 weeks did not result in tumors or hyperplastic lesion of the stomach, but too few animals were exposed and exposure was too brief to draw meaningful conclusions. Several short term studies indicate that glyoxal may have cancerinitiating or cancer promoting potential. Glyoxal ( 500 mg/ kg/ day in water for 32 weeks) caused hyperplasia and increased adenocarcinomas in the stomach of rats pretreated with N methyl Nnitro N nitrosoguanidine ( MNNG).

No data were located on health effects in humans exposed to glyoxal by the oral route. Several studies found that dermal exposure to glyoxal may produce skin sensitization. Diabetics may be sensitive to glyoxal because of compromised glyoxylase activity. Glyoxal exposure may increase oxalate excretion in individuals with hyperoxaluria.

The mechanism by which glyoxal produces adverse effects is not known, but is presumably related to the reactivity of this compound and its ability to form adducts and cross links with key cellular molecules such as proteins nucleic acids.

The available data on glyoxal are inadequate for the development of an RfD or HA values. Neither the U. S. Environmental Protection Agency ( EPA) nor the International Agency for the Research of Cancer ( IARC) have evaluated the cancer weight of evidence category for glyoxal, Draft   Do Not Cite or Quote ( July, 2003) viii and no quantitative estimates of cancer potency were located. Due to limitations in the data, the carcinogenic potential for glyoxal cannot be determined at this time.

Methylglyoxal Oral LD 50 values for methylglyoxal in rats were greater than (>) 1 mg/ kg; the LD 50 value for newborn rats was less than half that for adult rats. When administered intravenously to cats and rabbits, methylglyoxal seemed to cause decreased respiration and slowed cardiac function, but this response was not observed when administered intraperitoneally. Intraperitoneal administration of methylglyoxal to rats caused mild hepatotoxic responses. Pretreatment with 10% ethanol or 1% acetone in drinking water intensified the effects of methylglyoxal in the liver.

Exposure of young mice to about 5 g/ kg/ day methylglyoxal in drinking water during gestation, lactation, through the first 2 months after weaning was associated with depletion of erythrocyte glutathione and decreased activity of glutathione S transferase. A glucose challenge after fasting led to hyperglycemia in nearly half the treated mice. Red cells from treated animals had decreased resistance to oxidation when tested in vitro.

Exposure of rats for 32 weeks to 0.25% methylglyoxal in drinking water ( a dose of about 250 mg/ kg/ day) did not result in any obvious effect on weight gain and did not cause any obvious signs of toxicity. However, histological and biochemical studies were not performed so these data do not identify a reliable NOAEL.

Numerous studies indicate that methylglyoxal is mutagenic in bacterial systems, and this effect is increased by hydrogen peroxide. Methylglyoxal was weakly clastogenic in rats, causing increased micronuclei in liver and bone marrow. Methylglyoxal also has produced clastogenicity, chromosomal aberrations, or mutagenicity in yeast, Chinese hamster ovary ( CHO) cells, Chinese hamster lung cells, and human lymphocytes exposed in vitro.

The carcinogenic potential of methylglyoxal has not been thoroughly studied. Exposure of rats to methylglyoxal ( 250 mg/ kg/ day) for 32 weeks did not result in tumors or hyperplastic lesions of the stomach, but too few animals were exposed and exposure was too brief to draw meaningful conclusions. Methylglyoxal was reported to produce no tumors in rats exposed to 20 mg/ kg/ day for 2 years, but these findings have been presented in only abstract form.

Several short term studies indicate that methylglyoxal may have cancer initiating or promoting potential. Methylglyoxal ( 250 mg/ kg/ day in water for 32 weeks) induced hyperplasia in the stomach of rats pretreated with N methyl N   nitro N nitrosoguanidine, but did not cause tumors. Exposure of rats to methylglyoxal in water at doses of 5 to 20 mg/ kg/ day for 6 weeks resulted in an increase in the number and volume of   glutamyl transpeptidase ( GGT) foci in liver, and this effect was increased by prior exposure to 2 acetylaminofluorene. Exposure to single oral doses of 50 to 500 mg/ kg resulted in increased unscheduled DNA synthesis and Draft   Do Not Cite or Quote ( July, 2003) ix ornithine decarboxylase activity in stomach mucosal cells of rats. Two studies indicate that subcutaneous injection of methylglyoxal may result in injection site tumors.

No data were located on health effects in humans exposed to methylglyoxal by the oral route. Several studies indicate that the levels of methylglyoxal are elevated in diabetic subjects and are associated with the formation of advanced glycylation endproducts. The adducts are liberated from proteins during degradation and cleared through the kidneys. Although the adducts form in healthy subjects, concentrations are increased in diabetes. Thus, diabetics may be sensitive to direct and indirect effects of methylglyoxal. Advanced glycylation endproducts are believed to play an important role in several of the complications of long term diabetes including atherosclerosis, renal problems, and changes in lens proteins. Individuals with kidney problems may not be able to excrete advanced glycylation endproducts and may also have heightened sensitivity to methylglyoxal.

The mechanism by which methylglyoxal produces adverse effects is not known, but is presumably related to the reactivity of this compound and its ability to form adducts and cross links with key cellular molecules.

The available data on methylglyoxal are inadequate for the development of an RfD or HA values. There are no bioassay data on methylglyoxal that can be used to classify this compound for its potential to cause cancer. According to the EPA 1999 guidelines, the carcinogenicity of methylglyoxal cannot be determined at this time. Draft   Do Not Cite or Quote ( July, 2003) 1 GLYOXAL I. PHYSICAL AND CHEMICAL PROPERTIES Glyoxal is a white crystalline material that melts near room temperature. It is readily soluble in water, with little tendency to evaporate. It is commercially available as a crystalline dihydrate and in 30% or 40% aqueous solutions ( HSDB, 1999). Other physical chemical properties are summarized in Table 1.

Glyoxal is prepared by oxidation of acetaldehyde with nitric or selenious acid, by vaporphase oxidation of ethylene glycol, or by hydrolysis of dichlorodioxane ( Budavari et al., 1996; HSDB, 1999). In concentrated solution it polymerizes in a highly exothermic reaction. In diluted aqueous solution, glyoxal has been shown to exist as a mixture of the fully hydrated monomer, dimer, and trimer, with the monomer and dimer being the predominant species ( Whipple, 1970, as cited in NTP, 1994). Glyoxal is used industrially as an intermediate in the synthesis of a substituted ethylene urea compound used in postcuring of permanent press fabrics, as a crosslinking and insolubilizing agent, and in the paper industry in applications such as sizing and washable wallpaper. Minor uses are found in medicine, bacteriology, and pest control, and as a substitute for formaldehyde in embalming fluids ( HSDB, 1999). There is no indication that glyoxal is a major environmental contaminant as a result of these industrial uses ( HSDB, 1999).

Table 1. Physical and Chemical Properties of Glyoxal Parameter Data CASRN 107 22 2 Empirical Formula C2H2O2 ( OHCCHO)

Structure CHO CHO Molecular Weight 58.04 Vapor Pressure 255 mm Hg at 25

C Water Solubility Very Soluble Conversion Factors ( air) 1 ppm = 1 mg/ L = 2.4 mg/ m3 at 25

C 422 ppm at 25

C Melting Point 15

C Boiling Point 51

C Density 1.14 g/ mL Sources: Budavari et al., 1996; HSDB, 1999. Draft   Do Not Cite or Quote ( July, 2003) 2 II. TOXICOKINETICS A. Absorption No data were located regarding oral absorption of glyoxal. However, based on its chemical structure, it is expected that glyoxal will be well absorbed from the gastrointestinal tract.

B. Distribution No data were located regarding tissue distribution of glyoxal following oral exposure. However, it is expected that glyoxal would be widely distributed throughout the body. The aversge concentration of glyoxal in human blood samples is about 200 pmol/ g ( Thornalley, 1998).

C. Metabolism Glyoxal is a substrate for the glyoxalase I ( GLO I) and glyoxalase II ( GLO II) system ( Thornalley, 1998). GLO I conjugates glyoxal to glutathione ( GSH) forming S glycolglutathione, and GLO II catalyzes the hydrolysis of S glycolglutathione to glycolate ( hydroxyacetic acid) and GSH ( Thornalley, 1996). Glycolate can be oxidized to glyoxylate and then converted to glycine through a transamination reaction ( Montgomery et al., 1990).

Arnold et al. ( 1983) reported that glyoxal reacts in vitro with the enediol portion of ascorbic acid to yield a five membered cyclic acetal. This condensation is reversible under alkaline conditions. No studies were performed to determine whether this reaction occurs in vivo.

Glyoxal can be produced endogenously from slow oxidation of glucose, degradation of glycosylated proteins and lipid peroxidation ( Thornalley, 1998; Thornalley et al., 1999). It is also produced during the metabolism of diethylene glycol and alloxan ( HSDB, 1999).

D. Excretion No data were located on the route or degree of glyoxal excretion. It is expected that glyoxal or its metabolites ( glycolate glyoxylate, oxylate) will be excreted mainly in urine.

E. Bioaccumulation and Retention No data were located on the bioaccumulation or retention of glyoxal. Glyoxal is not expected to accumulate in the body. Draft   Do Not Cite or Quote ( July, 2003) 3 III. HUMAN EXPOSURE A. Occurrence Air There was little information in the available literature on the distribution of glyoxal in ambient air. It is apparently generated during the combustion of gasoline and has been reported to occur in the exhaust of automobiles. Glyoxal was identified in air samples collected at the toll plaza for the San Francisco Bay bridge ( Destaillats et al., 2002) and in air samples collected in Los Angeles at concentrations  1ppb ( Kawamura et al., 2000). The glyoxal samples from downtown Los Angeles were higher than those from west Los Angeles. Glyoxal could also be extracted from fine particulate matter collected from the Caldicott Tunnel near Oakland, CA over a four day period in 1997 ( Rao et al., 2001) Gasoline combustion appeared to produce more glyoxal than diesel fuel. Glyoxal is present in cigarette smoke ( NTP, 1994) and can gain access to the atmosphere from this source.

Food Small amounts of glyoxal may be present in foods as metabolic intermediates. Nagao et al. ( 1986a) reported that glyoxal was present in coffee ( both instant and brewed) and other beverages ( Table 2). Glyoxal has also been detected in toast and soy sauce ( Nagao et al., 1986a). The level of glyoxal in toast was 0.5

g/ g. In soy sauce, there was 4.9

g/ mL glyoxal. Small amounts of glyoxal can be produced when sugar containing foods are cooked at high temperatures ( Nishi et al., 1989); it is found in alcoholic beverages.

Table 2. Concentrations of Glyoxal in Selected Beveragesa Beverage Glyoxal (

g/ mL)

Beer b 0.07 0.21 Bourbon whiskey a 0.39 Apple brandy a 0.33 Wine a, b 0.4 1.5 Japanese sake a, 0.29 Instant coffee a, c 0.34 Brewed coffeea, d 0.87 Black teaa trace aNagao et al., 1986a. bBarros et al., 1999 CPrepared by dissolving 1.5 g powder in 100 mL water. dPrepared from 10 g ground coffee beans and 150 mL water. Draft   Do Not Cite or Quote ( July, 2003) 4 Water Glyoxal is formed as a byproduct from ozonation of water ( Glaze et al., 1989; Langlais et al., 1991). Ozonation of water from the Los Angeles Aqueduct Filtration Plant resulted in a significant increase of several aldehydes and carboxylic acids compared with their level in source water. For example, glyoxal was not detectable in raw water, but was detected at concentrations of 13

g/ L following ozonation ( Langlais et al., 1991).

No trace of glyoxal was reported by Glaze et al. ( 1989) in surface water treatment plant influents. However, glyoxal can be formed during the ozonation of water containing humic acids. Following ozonation, the concentration of glyoxal in potable water was about 7

g/ L after 10 minutes and about 2 to 8

g/ L after 2 to 10 minutes for wastewater ( Sayato et al., 1989). Lower concentrations are expected to form from ground water sources due to their lower organic carbon content. Ueno et al. ( 1989) studied ozonated water containing concentrated humic acids isolated from three different sources and found that glyoxal was produced in all three cases. Following ozonation of surface waters to which odor causing compounds ( 2 methylesoborneal and geosmin) had been added, glyoxal concentrations ranged from 0.3 to 0.8

g/ L. Ozonation was carried out for 16 minutes at pH 7.6 or 7.7. Odorant concentrations were decreased with time as they were oxidized to simpler compounds ( Yamada and Somiya, 1989).

After a 40 minute ozone reaction time, the concentration of glyoxal was increased when ozonation was carried out at a basic pH rather than an acid pH. At pH 10.5, the glyoxal concentration was 1.0

g/ L, while at pH 6.6, the concentration was 0.3

g/ L ( Yamada and Somiya, 1989).

In the work by Yamada and Somiya ( 1989), there was no glyoxal in the postfiltration water influent to the ozone contractors at a demonstration plant. After ozone treatment, the concentration of glyoxal was 0.3

g/ L. Complete removal of glyoxal was accomplished using a granular activated filter as postozonation treatment. Glaze et al. ( 1989) showed that glyoxal is also removed from potable water by reaction with chloramines used as secondary disinfectants.

As part of the Information Collection Rule for Disinfection By Products, quarterly data on the presence of glyoxal were collected from ozonation water treatment plants. In more than 50% of the samples, no glyoxal was detected. The maximum concentration detected in quarterly samples from each of 20 plants was 22

g/ L ( McLain and Blank, 2000).

Soil There was no information in the available literature concerning levels of glyoxal in soils. There is no reason to suspect that it is present in any significant concentrations except as breakdown products of organic materials. Draft   Do Not Cite or Quote ( July, 2003) 5 B. Fate Glyoxal undergoes addition and condensation reactions with amines, amides, aldehydes, and hydroxyl containing materials ( Ueno et al., 1989). Due to its reactivity, the environmental persistence of glyoxal is expected to be minimal.

C. Exposure The available information on exposure to glyoxal is sparse. Most of the daily exposure is likely to be from foods. Exposure from drinking water is expected to be minimal. Glyoxal can be generated endogenously during the catabolism of energy nutrients, particularly carbohydrates. Exposure from endogenous glyoxal production is likely to exceed that from exogenous sources. Draft   Do Not Cite or Quote ( July, 2003) 6 IV. HEALTH EFFECTS IN ANIMALS A. Short Term Exposure Smyth et al. ( 1942) reported an oral acute LD 50 of 2020 mg/ kg in albino rats and 760 mg/ kg in guinea pigs ( Ueno et al., 1991c). Oral LD 50 values of 400 to 800 mg/ kg for mice and 1500 to 1800 mg/ kg in rats are cited in NTP ( 1994). An LD 50 of 200 to 400 mg/ kg in rats is also cited by NTP ( 1994) and is not consistent with the other values reported.

In order to examine the effects of glyoxal on protein synthesis, Ueno et al. ( 1991c) administered single oral doses of 0 or 1000 mg/ kg of glyoxal to groups of four male Sprague Dawley rats followed by an intraperitoneal injection of 3H leucine 4 hours later. The animals were sacrificed 2 hours after the leucine injection, and the rate of protein synthesis in liver, kidney, and spleen was estimated by measuring incorporation of 3H leucine into trichloroacetic acid insoluble tissue protein. Exposure to 1000 mg/ kg glyoxal resulted in a significant ( p< 0.01) decrease in protein synthesis in liver and spleen. A slight decrease was noted in kidneys, but this was not statistically significant. Comparable effects on the liver were noted following an i. v. injection of 150 mg/ kg.

B. Longer Term Exposure Subchronic Takahashi et al. ( 1989) exposed groups of 10 male Wistar rats to 0.5% glyoxal for 32 weeks. Assuming water intake of 0.10 L/ kg/ day, this corresponds to a dose of about 500 mg/ kg/ day. Animals were weighed weekly. Exposure to glyoxal clearly depressed weight gain. There was very little change in average weight of the treated animals between weeks 8 and 40, the period of glyoxal administration. In fact, there was a decrease in average body weight over the first 4 weeks of glyoxal administration. Gross inspection of internal organs at the end of exposure did not reveal any signs of toxicity. Histological evaluations were not performed.

A study by Ueno et al. ( 1991c) examined the effects of glyoxal exposure via drinking water on groups of five to seven male Sprague Dawley rats. The study was conducted in two phases. In Phase 1, test animals received drinking water ( ad libitum) containing glyoxal at 0, 2000, 4000, or 6000 mg/ L for 30, 60, or 90 days. The authors indicated that average associated doses were 188, 407, and 451 mg/ kg/ day at the 30 day termination; 135, 239, and 344 mg/ kg/ day at the 60 day termination; and 107, 234, and 315 mg/ kg/ day at the 90 day termination.

There was a statistically significant ( p< 0.01) dose related decrease in average daily food intake, which was not generally significant when converted to g/ kg/ day. There was a statistically significant ( p< 0.01) but relatively mild decrease in mean daily water consumption per rat for all dose groups at 30, 60, and 90 day terminations. Terminal body weight was significantly reduced ( p< 0.01) at the two highest doses at all three termination times. The calculated percent change in terminal body weight ranged from 12% to 14% at 4000 mg/ L and from 26% to 31% at 6000 mg/ L. Absolute organ weights decreased significantly in all exposed groups at all three termination times and there was a significant increase in relative kidney weight at 90 days for the Draft   Do Not Cite or Quote ( July, 2003) 7 high dose group. Analysis of blood revealed no significant effects on BUN, creatine, or creatinine. Serum levels of aspartate amino transferase, alanine amino transferase and lactate dehydrogenase, albumin, and total protein were all reduced in a dose dependent manner. Several effects ( alanine amino transferase, total protein) were statistically significant at all doses.

At the 30 day termination but not for the other durations, there was an increase in GLO I activity in erythrocytes at all three doses, in liver at the two highest doses, and in the kidneys only at the highest dose. GLO II also tended to increase in liver and erythrocytes, but the effects were not as clear. The authors identified the lowest dose ( 107 mg/ kg/ day over the 90 day period) as a LOAEL based on its effects on the total serum protein.

In Phase 2, a diet limited control group was run concurrently with the 6000 mg/ L exposure group and a control group that was not diet limited. The diet limited control group was fed the same amount of food as the 6000 mg/ L exposure group in order to distinguish effects of reduced food consumption from glyoxal toxicity. This study was carried out to two time points, 90 days and 180 days. Based on measured body weights and water intakes, the average intake was 315 mg/ kg/ day at 90 days and 298 mg/ kg/ day at 180 days. Reduced food consumption could account for only about half of the body weight effect, indicating glyoxal exposure was suppressing growth.

Two of the animals exposed to glyoxal died within 30 days. Necropsy revealed hemorrhage of the glandular stomach. Food intake and body weight gain were significantly reduced in the exposed animals. Inhibition of weight gain was greater in the exposed animals than in the group of pair fed controls, suggesting that part of the effect on body weight was caused by systemic toxicity. Absolute weights of organs except testes and brain were reduced, and an increase in the organ to body weight ratio was observed in liver, kidney, and heart at both 90 and 180 days. Gross examination revealed stomach lesions ( hemorrhage, polyps) in two high dose animals, but the authors did not consider these to be necessarily treatment related. Histological examination of liver, kidney, spleen, stomach, thymus, and lymph nodes did not reveal any treatment related effects except for a slight swelling of papillary epithelial cells in the kidneys. Examination of blood revealed significant decreases in serum aspartate amino transferase and total protein. Based on body weight, serum protein levels, and histological effects in kidney, this study identifies a LOAEL of 298 to 315 mg/ kg/ day.

C. Reproductive/ Developmental Effects NTP ( 1991) as cited in NTP ( 1994) conducted a pilot developmental study using groups of eight Sprague Dawley rats and doses of 0, 200, 800, 1200, 1600, or 2000 mg/ kg/ day on gestation days 6 15. All dams in the two highest dose groups and five animals in the 1200 mg/ kg/ day dose group died or had to be euthanized on or before gestation day 17. Six of eight litters in the 800 mg/ kg/ day group and two of three litters in the 1200 mg/ kg/ day group were completely resorbed. The NOAEL in the pilot study was 200 mg/ kg/ day and the LOAEL was 800 mg/ kg/ day based on complete fetal resorption for six of eight litters.

NTP ( 1994) conducted a developmental toxicity study of glyoxal trimeric dihydrate in Sprague Dawley CD ® rats. Glyoxal trimeric dihydrate was chosen because it is more stable than glyoxal and because the human population is as likely to be exposed to the trimeric dihydrate as a Draft   Do Not Cite or Quote ( July, 2003) 8 constituent of other mixtures as to glyoxal. The test compound was administered by gavage in water on gestational days 6 through 15 ( the period of organogenesis) to time mated groups of 26 animals. Doses were 0, 50, 150, or 300 mg/ kg/ day. The major effect noted during exposure was rooting ( digging in the bedding) behavior around the dosing time for the 150 and 300 mg/ kg/ day doses.

Animals were sacrificed on gestation day 20 and examined for maternal body and organ weights, implant status, fetal weight, fetal sex, and fetal morphological development. Maternal body weight of the exposed animals was consistently slightly below the control body weight but the difference was only statistically significant for the highest dose on gestation day 12. Maternal body weight gain of animals was also significantly reduced ( p< 0.05) at the highest dose during gestation days 6 to 15, which corresponded to a reduction in relative food consumption. Maternal relative water consumption of dosed animals was generally above the consumption of control animals, and was significantly increased at all dose levels on gestation days 15 to 20, increasing dramatically after cessation of exposure. There were no maternal deaths in this study, and maternal toxicity at 300 mg/ kg/ day was reflected only as a small decrease in body weight and food consumption. No dose related developmental toxicity was observed in the dose range tested. For the dams, the NOAEL was 150 mg/ kg/ day and the LOAEL 300 mg/ kg/ day for minimal maternal toxicity in CD rats. The highest dose ( 300 mg/ kg/ day) was a NOAEL for developmental toxicity.

The NOAEL/ LOAEL data from the NTP ( 1991, 1994) studies suggest that there is less than a threefold difference between the dose that produces no effect on reproduction ( 300 mg/ kg/ day) and the dose that produces almost complete toxicity ( 800 mg/ kg/ day).

In a study of the effects of ethylene glycol and its metabolites on development, 9.5 day old embryo were harvested from pregnant Wistar rats and exposed to glyoxal concentration of 0, 3 or 6 mM in a whole embryo culture system ( Klug et al., ( 2000). At the 3 mM concentration the number of dysmorphic embryos ( 6%) fell within the range for historic controls.. At the 6 mM concentration 78 % of the embryos were dysmorphic.

D. Mutagenicity and Genotoxicity The mutagenicity and genotoxicity data on glyoxal are predominantly positive. Glyoxal is mutagenic in the Ames assay with many strains of Salmonella typhimurium ( Kato et al., 1989) and has been shown to cause base pair substitutions and some frameshift mutations at G: C base pairs ( Murata Kamiya et al., 1997a). It has also been found to damage DNA by a variety of tests, including causing single strand breaks in vivo and in vitro ( Ueno et al., 1991b).

Gene Mutation Assays Numerous gene mutation assays have been conducted on glyoxal, most using S. typhimurium, not only to determine its mutagenicity but also to study its mechanism of action and its contribution to the mutagenicity of complex mixtures to which the human population is routinely exposed. However, no Ames assay using all of the standard recommended strains was located. Draft   Do Not Cite or Quote ( July, 2003) 9 The role of intercellular enzymes and active oxygen species associated with the mutagenic activity of glyoxal was evaluated in S. typhimurium strains TA100 and TA104 ( Ueno et al., 1991a). TA100 and TA104 were exposed to glyoxal in the presence of various active oxygen scavengers, or with suppression of intercellular levels of superoxide dismutase ( SOD), catalase ( CAT), or GSH. The mutagenic activity of glyoxal was reduced by addition of GSH in both strains and was further reduced by the addition of GLO I and/ or GLO II with GSH ( Ueno et al., 1991a). The mutagenic activity in TA100 was not reduced by addition of SOD, CAT, SOD+ CAT, or D mannitol; however, addition of CAT resulted in a slight inhibition of mutagenic activity in TA104. Mutagenic activity of glyoxal did not vary with the suppression of SOD activity in the cells but was enhanced by the GSH depleters, with about a 40% decrease of GSH level in TA100.

Dorado et al. ( 1992) examined the mutagenicity of glyoxal ( and eight other dicarbonyl compounds) against S. typhimurium strain TA100 using the standard plate incorporation Ames assay without preincubation and without metabolic activation. Glyoxal was found to be mutagenic, whereas sodium glyoxylate ( CAS 2706 75 4) was found not to be mutagenic to strain TA100. The authors suggested that the difference in mutagenicity between glyoxal and sodium glyoxylate was that glyoxal forms adducts with the guanine residues of nucleic acids, whereas sodium glyoxylate cannot form stable adducts with guanine. Because strain TA100 reverts to His+ from base pair mutations, this study demonstrated that the mutagenic activity of glyoxal resulted from base pair transversions, as opposed to frame shift mutations.

Mellado and Montoya ( 1994) examined equilibrium constants of the adduct formation reactions with guanine and guanosine for several dicarbonyl compounds in order to determine a mutagenic mechanism. Glyoxal, methylglyoxal, and phenylglyoxal were evaluated. The values of K F and   H F related to the stability of the adduct formation and the chemical reactivity. The higher the K F, the greater the stability, and the more negative the   H F the greater the reactivity of the compound. The order of compounds with the highest K F and lowest   H F was found to be: glyoxal > methylglyoxal > phenylglyoxal.

The correlation coefficients between K F and mutagenicity and   H F and mutagenicity in S. typhimurium strain TA100 were determined. A strong correlation was found between adduct formation with the purinic bases guanine and guanosine, and mutagenicity ( average r = 0.94). The authors concluded that the mutagenic activity for glyoxal was directly related to reactions with the guanine.

Murata Kamiya et al. ( 1997b) used S. typhimurium strains TA7001, TA7002, and TA7003, which detect base pair substitutions at A: T pairs; strains TA7004, TA7005, and TA7006, which detect base pair substitutions at G: C pairs; and strain TA98, which detects frameshift mutations at G: C pairs. The plate incorporation method was used, with preincubation used to increase sensitivity. Strain specific positive controls were used. Glyoxal was tested at 20 100

g/ plate, with no mention of metabolic activation, and was mutagenic in strains TA7004, TA7005, TA7006, and TA98, the strains that detect base pair substitutions at G: C pairs plus frameshift mutations at G: C pairs. This reference supported results of others that glyoxal reacts with guanine, and provided insight into the mechanism of genotoxicity. Draft   Do Not Cite or Quote ( July, 2003) 10 Murata Kamiya et al. ( 1997b) attempted to gain a better understanding of glyoxal's mutagenic mechanisms by analyzing the mutations induced in the chromosomal lacI gene of wildtype E. coli. This study strongly suggested that glyoxal is mutagenic to E. coli. Cell death and mutation frequency increased with dose, and glyoxal induced base pair reversions predominately at G: C base pairs.

Chromosomal Aberration Assays Nishi et al. ( 1989) described the clastogenic activity of thermally decomposed products of carbohydrates in cultured Chinese hamster V79 cells. Products included glyoxal and methylglyoxal. The mitotic index was calculated as the percentage of metaphases among more than 2000 interphase nuclei. Glyoxal and methylglyoxal induced a significant number of chromosome aberrations and lowered the mitotic index significantly.

Glyoxal was tested at doses of 100, 200, 300, and 400

g/ mL. The highest dose showed evidence of cytotoxicity. The percentage of aberrant cells was 6% in the solvent control, and 17%, 41%, 48%, and 32% for the four increasing doses. Chromatid gaps and breaks and exchanges predominated. Glyoxal had only approximately one tenth the potency of methylglyoxal.

Other Assays A series of chlorinated chemicals and aldehydes, representing chlorination and ozonation byproducts, was tested by Matsui et al. ( 1989) using the liquid Bacillus subtilis/ microsome recassay to detect DNA damage. Glyoxal was classified as strongly DNA damaging both with and without metabolic activation. Only acrolein and formaldehyde produced stronger responses without activation, and only formaldehyde produced a stronger response with activation.

Hellmer and Bolcsfoldi ( 1992) employed the differential DNA repair test by using DNA repair deficient and proficient strains of E. coli K12/ 343/ 113 in an in vitro test. Results were considered positive if the DNA repair deficient strain viability was significantly reduced at a lower concentration than the DNA repair proficient strain. However, if both strains were reduced at the same concentration, then a genotoxic mechanism was not likely. The glyoxal result was positive with and without metabolic activation. Results for 49 out of the 61 compounds were in agreement with the Ames test, indicating an 80% concordance between the two tests. Thus, the study found these bacterial strains, and differential DNA repair as an endpoint, to be sufficiently accurate as an indicator of genotoxicity.

Ueno et al. ( 1991b) applied the alkaline elution technique to evaluate the damage to rat hepatic DNA following in vitro and in vivo exposure to glyoxal. For in vitro exposures, primarycultured hepatocytes were exposed to 0.1 to 0.6 mg/ mL glyoxal for 60 minutes. For in vivo exposures, male Sprague Dawley rats were administered 200, 500, or 1000 mg/ kg body weight of glyoxal via gastric intubation. After 1 to 24 hours of exposure, animals were sacrificed and hepatocytes prepared for analysis. After 60 minutes of exposure, the in vitro study found dosedependent DNA single strand breaks while no DNA cross link was observed. Within 2 hours, DNA lesions were detected in the liver following in vivo exposure to glyoxal at 200 to 1000 Draft   Do Not Cite or Quote ( July, 2003) 11 mg/ kg body weight. The DNA lesions reached a maximum around 9 hours after exposure and returned to control levels 24 hours after exposure to all doses. This study strongly suggested that glyoxal is genotoxic to rat hepatocytes, and able to cause DNA lesions by single strand breaks. However, it also suggested that the DNA damage was readily repaired.

Furihata and Matsushima ( 1989) and Furihata et al. ( 1989) applied the alkaline elution technique to evaluate the DNA damage of rat pyloric mucosa of the stomach following in vivo exposure to glyoxal. Male F344 were administered glyoxal via gastric intubation at doses of 5, 50, 500, and 550 mg/ kg body weight. The results of this study showed a dose dependent increase in the elution rate constant after administration of glyoxal, indicating glyoxal induced DNA damage to the pyloric mucosa of rat stomach after in vivo administration. Pyloric mucosal DNA damage was observed with a dose of 50 mg/ kg body weight but no effects were seen with the 5 mg/ kg dose.

Glyoxal and kethoxal were tested in human peripheral lymphocytes to determine whether the responses were typical of mammalian cells or were unique to AUXB1 cells ( Tucker et al., 1989). Although glyoxal produced small but significant SCE increases at every dose, it lacked a dose response. On the other hand, kethoxal produced a linear SCE response. Neither of these agents induced ERCs.

E. Carcinogenicity Takahashi et al. ( 1989) exposed 10 male Wistar rats to glyoxal [ 5000 mg/ L; 617 mg/ kg/ day using EPA ( 1988 conventions)] in drinking water for 32 weeks. Assuming water intake of 0.10 L/ kg/ day, this corresponds to a dose of about 500 mg/ kg/ day. At termination, all internal organs were examined macroscopically, and the stomachs were fixed and examined histologically. No gastric carcinomas or hyperplastic lesions were detected in animals exposed to glyoxal. This negative finding is limited by the small number of animals tested, the relatively short exposure period, and the absence of a follow up observation period.

Takahashi et al. ( 1989) tested the cancer promoting activity of glyoxal in rats. Groups of 30 male Wistar rats were exposed for 8 weeks to N methyl N' nitro N nitrosoguanidine ( MNNG) in drinking water ( 100 mg/ L). Following this, animals were exposed to glyoxal ( 5000 mg/ L) in drinking water for 32 weeks. Assuming consumption of 0.10 L/ kg/ day, this corresponds to a dose of about 500 mg/ kg/ day. Control animals were exposed to MNNG for 8 weeks and then given untreated water for 32 weeks. At termination, animals were sacrificed, internal organs were examined macroscopically, and the stomach was fixed and examined histologically. Exposure to glyoxal increased the incidence of hyperplasia and adenocarcinoma of the stomach, especially in the pylorus. No toxic or neoplastic changes were noted in any other organs. These findings were taken to indicate that glyoxal exerts a cancer promoting effect in the stomach.

Furihata et al. ( 1985b) studied the initiating and promoting effects of glyoxal in rat glandular stomach. Groups of five male F344 rats were given single oral doses of 300 to 1500 mg/ kg of glyoxal by stomach tube, and the rates of unscheduled DNA synthesis ( UDS) and ornithine decarboxylase ( ODC) activity were measured in gastric mucosa over the next 45 to 68 hours. Glyoxal increased both UDS and ODC, with a maximal effect about 16 hours after exposure. The Draft   Do Not Cite or Quote ( July, 2003) 12 authors took the increases in UDS and ODC to suggest that glyoxal had potential tumor initiating and  promoting activities in the glandular stomach.

Miyakawa et al. ( 1991) tested pyrolysates of carbohydrates, including glyoxal and methylglyoxal, in a two stage mouse skin carcinogenesis model. Initiation involved treatment with 0.1 mL aliquots of test chemical or positive control on the shaved backs of female CD 1 mice twice weekly for 5 weeks. One week after the last initiation treatment, 12 Otetradecanoylphorbol 13 acetate ( TPA) in 0.1 mL acetone or acetone alone was applied twice weekly for 47 weeks, as a promoter. Glyoxal produced an average of 0.2 skin tumors per mouse, which was not statistically significant.

Several intermediate term rat liver bioassays using similar protocols have been reported for glyoxal. Hasegawa and Ito ( 1992) made use of the two step theory of hepatocarcinogenesis, giving F344 male rats a single intraperitoneal initiating dose of diethylnitrosamine, followed 2 weeks later by 6 weeks of exposure to the test chemical. At week 3, rats had a two thirds partial hepatectomy to stimulate growth of new liver tissue. Results were considered to be positive if there was a significant ( p< 0.05) increase in either the number or area of GST P foci. The results for glyoxal were negative. In fact, glyoxal ( 5000 mg/ L in drinking water) caused a statistically significant decrease ( p< 0.05) in both the number and area of glutathione S transferase placental form ( GST P) positive foci when compared with the diethylnitrosamine treated control. The results reported indicated that glyoxal exposure after diethylnitrosamine treatment resulted in 6.06 foci per square centimeter, with an area of 0.40 square millimeter per square centimeter of liver tissue. The corresponding results reported for dimethylnitrosamine alone were 7.83 foci per square centimeter and an area of 0.55 square millimeter per square centimeter of liver tissue. The significance of the role glyoxal may have played in inhibiting the formation of GST P foci from diethylnitrosamine is unknown.

Hasegawa et al. ( 1995) evaluated the hepatocarcinogenicity potential of coffee and its constituent compounds ( methyl xanthines and glyoxal) using the same procedure employed in their 1992 study above. The test group was initially exposed to diethylnitrosamine ( DEN) intraperitoneally to initiate hepatocarcinogenesis followed by dietary exposure to the test compound. Glyoxal was administered at a concentration of 0.2% ( w/ v) in drinking water and again in this experiment exerted an inhibitory effect on both the number and areas of GST P positive foci. The results reported indicated that glyoxal exposure after diethylnitrosamine treatment resulted in 6.06 foci per square centimeter, with an area of 0.40 square millimeter per square centimeter of liver tissue. The corresponding results reported for dimethylnitrosamine alone were 7.83 foci per square centimeter and an area of 0.55 square millimeter per square centimeter of liver tissue.

The results reported in the two Hasegawa papers were identical, although the concentrations of glyoxal tested were reported to be 5000 mg/ L in the first study and 0.2% ( 2000 mg/ L) in the second study. It is difficult to determine if the results reported are from one experiment with an error in the glyoxal concentration in one case or two different experiments with identical results. Draft   Do Not Cite or Quote ( July, 2003) 13 V. HEALTH EFFECTS IN HUMANS No data were located on health effects in humans exposed to glyoxal by the oral route.

Hindson and Lawlor ( 1982) described a case of a woman who developed eczema on her hands while working in an occupation that involved wrapping pipe with fiberglass and coating it with a polyvinyl resin. The resin contained vinyl acetate, glyoxal, formaldehyde, and ammonium persulfate. Patch testing of the exposed woman revealed a positive response to 1% glyoxal, whereas 10 other people did not respond. This finding suggests that repeated dermal contact with glyoxal may lead to skin sensitization, but the presence of the other chemicals in the resin limits the confidence in this conclusion.

Schnuch et al. ( 1998) reviewed the results of 31,849 patch tests for allergic sensitization collected over a three year period by the German Information Network of Departments of Dermatology. The results for different dermal sensitizing agents were compared for health care workers and the rest of the population. The percentage of health care workers responding to glyoxal was 4.2% compared to 1.4% for individuals from other occupations. Glyoxal is an ingredient in some disinfectants applied to the surfaces of medical equipment and instruments. Dermal sensitization is caused by exposure of a sensitive individual to a sensitizing dose. Thus, the increased incidence for positive patch test results among health care workers is more likely a consequence of increased exposure than biological sensitivity. However, once a person has been sensitized, that person is more likely to have a dermatological response at a lower dose than the remainder of the population.

In a similar study, 2689 patients were tested for their reaction to 1% glyoxal in petrolatum at 33 centers of the German Austrian Information Network of Departments of Dermatology ( Uter et al., 2001) Allergic reaction were observed in 1.6% of the patients and irritant reaction in 0.3%. Readings were equivocal in 0.6 % of the patients. Many of the patients that were sensitive to glyoxal worked in the health care professions.

Uter et al. ( 2001) also reported on the testing of 189 patients at a dermatitis clinic in Osnabruk, Germany. Eleven ( 5.8%) had positive reactions to 1% glyoxal in water and/ or petrolatum; 81% of those tested were women. Three people had what were classified as irritant reactions and two had equivocal reactions.

A. Sensitive Populations Possible Childhood Susceptibility No references were located related to childhood susceptibility to glyoxal.

Possible Gender Differences No references were located related to gender differences in susceptibility to glyoxal.

Other Draft   Do Not Cite or Quote ( July, 2003) 14 Diabetics may be sensitive to glyoxal because of their compromised glyoxalase activity. A study by Shinohara et al. ( 1998) focused on chronic hyperglycemia and its role in diabetic microvascular disease. The autoxidation of glucose results in glyoxal, which can further react to produce advanced glycation end products by condensation with protein ( Thornalley et al., 1999). Formation of advanced glycation end products has been associated with low catalytic efficiency of aldehyde reductase in the diabetic rat kidney. This study not only points out the importance of glyoxal in normal metabolism but also may identify diabetics as a sensitive subpopulation for glyoxal exposure. However, there was no increase in cellular glyoxal levels in vitro in cultures of human fetal endothelial cells exposed to 30 mM glucose.

Carboxymethyllysine ( CML) is the reaction product of glyoxal with lysine and is characterized as an advanced glycation endproduct. Dedenhardt et al. ( 1998) found that CML in human lens proteins increased linearly with age and Dyer et al. ( 1993) observed a similar phenomenon in skin collagen. The amount of CML in the skin collagen of diabetic rats was about twice that for controls when the animals ( 12 13 female Sprague Dawley rats) were evaluated about 28 weeks after induction of diabetes ( Dedenhardt et al., 1998). In humans, the amounts of CML are increased in diabetics as compared to the non diabetic populations ( Dyer et al., 1993) and the concentrations of CML are significantly correlated to the duration of diabetes, and the incidence of retinopathy and nephropathy ( McCance et al., 1993). However the CML is believed to result from the slow Maillard modification of fructoslysine, the initial product from the reaction of glucose with protein ( McCance et al., 1993), rather than from direct reaction of glyoxal with protein.

CML dimerizes to form bis( lysyl) imidazolium crosslink ( glyoxal lysyl dimer: GOLD), an advanved glycation endproduct. Endothelial cells, monocytes, and macrophages carry receptor sites for advanced glycation endproducts through which they can take up the modified proteins for degradation ( Thornalley et al., 1999). Odani et al. ( 1998 found that the te levels of GOLD in the serum of uremic patients ( n= 15) was increased four to five fold over that for controls ( n 15).

Individuals with the rare genetic disorder ( hyperoxaluria Type 1) would be susceptible to elevated exposures to glyoxal. In this condition, the inability to convert glyoxalate to glycine leads to the formation and excretion of oxalate produced by oxidation of glyoxalate ( Montgomery et al., 1990).

Glyoxal, previously identified as a radiosensitizer at the cellular level, was studied in solution by UV spectroscopy with and without DNA and with and without irradiation ( Peinado et al., 1989). The presence of DNA did not alter the radiosensitivity of glyoxal. Draft   Do Not Cite or Quote ( July, 2003) 15 VI. MECHANISM OF TOXICITY The mechanism of action of glyoxal is not known. However, aldehydes and   ketoaldehydes are relatively reactive compounds, and form adducts with DNA, RNA, proteins, or thiols ( Conroy, 1979; Krymkiewicz, 1973). It seems likely that the toxic and mutagenic activities of glyoxAL is ultimately attributable to ITS reaction with nucleophilic groups ( amines, thiols, alcohols) present in key cellular molecules. Glyoxal can also participate in aldol condensation reactions with other carbonyl compounds A. Mechanism for DNA Effects It has been hypothesized that glyoxal can be produced endogenously within the cell nucleus as a result of free radical reaction damage to DNA. A study by Murata Kamiya et al. ( 1995) found that glyoxal was produced during DNA exposure to an oxygen radical forming system. Calf thymus DNA was exposed to 5 mM FeSO 4 EDTA, a metal containing oxygen radical forming system, at 37  C for 60 minutes. Glyoxal was identified as a reaction product and the amount produced was 17 times higher than 8 hydroxy deoxyguanosine, a common biomarker for free radical induced DNA damage. Glyoxal produced from DNA will readily react with neighboring DNA bases, especially those with amino functional groups ( adenine, guanine, and cytosine) causing mutations.

Thornalley et al. ( 1999) incubated 50 mM glucose in phosphate buffer ( 100 mM) at pH 7.4 and 37 C and measured the production of glyoxal over 20 days. Glyoxal was formed at a rate of 1.72 +/ 0.05 mmol/ day. Methylglyoxal and 3 deoxyglucosone were other products of the reaction but were produced at slower rates and at lower concentrations. In a second part of this same study, 50 mM glucose and NtBOC lysine were incubated under the same pH and temperature conditions. Fructosameine, glyoxal, methylglyoxal and 3 deoxyglucosone were formed. Among the alpha oxoaldehydes produced, glyoxal was present at the highest concentration. The rates of formation for the alpha oxoaldehydes were increased in the presence of the N t BOC lysine; there was a five fold enhancement of glyoxal production. This study provides support for the hypothesis that the formation of glyoxal is involved in the process of the glycation of lysine. However there were no actual measurements for carboxymethyllysine or GOLD Draft   Do Not Cite or Quote ( July, 2003) 16 VII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS Quantification of toxicological effects of a chemical consists of separate assessments of noncarcinogenic and carcinogenic health effects. Chemicals that do not produce carcinogenic effects are believed to have a threshold dose below which no adverse, noncarcinogenic health effects occur. Carcinogens are assumed to act without a threshold unless there are data elucidating a nonmutagenic mode of action and demonstrating a threshold for the precursor events that commit a cell to an abnormal tumorigenic response.

A. Noncarcinogenic Effects 1. Reference Dose In the quantification of noncarcinogenic effects, a Reference Dose ( RfD) ( formerly called the Acceptable Daily Intake [ ADI]) is calculated. The RfD is " an estimate ( with uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human population ( including sensitive subgroups) that is likely to be without appreciable risk of deleterious effects during a lifetime" ( U. S. EPA, 1993). The RfD is derived from a no observed adverse effect level ( NOAEL), lowest observed adverse effect level ( LOAEL), or a NOAEL surrogate such as a benchmark dose identified from a subchronic or chronic study, and divided by a composite uncertainty factor( s). The RfD is calculated as follows:

RfD = NOAEL ( LOAEL) UF . MF where:

NOAEL = No observed adverse effect level: A highest exposure level at which there are no statistically or biologically significant increases in the frequency or severity of adverse effects between the exposed population and its appropriate control.

LOAEL = Lowest observed adverse effect level: The lowest exposure level at which there are statistically or biologically significant increases in frequency or severity of adverse effects between the exposed population and its appropriate control group.

UF = Uncertainty factor chosen according to EPA/ NAS guidelines MF = Modifying factor Selection of the uncertainty factor to be employed in the calculation of the RfD is based on professional judgment, while considering the entire database of toxicological effects for the chemical. To ensure that uncertainty factors are selected and applied in a consistent manner, the Office of Water ( OW) employs a modification to the guidelines proposed by the National Academy of Sciences ( NAS, 1977, 1980). According to the EPA approach ( U. S. EPA, 1993), Draft   Do Not Cite or Quote ( July, 2003) 17 uncertainty is broken down into its components and each dimension of uncertainty is given a quantitative rating. The total uncertainty factor is the product of the component uncertainties. The individual components of the uncertainty are as follows:

UF H A 1, 3, or 10 fold factor used when extrapolating from valid data in studies using long term exposure to average healthy humans. This factor is intended to account for the variation in sensitivity ( intraspecies variation) among the members of the human population.

UF A An additional factor of 1, 3, or 10 used when extrapolating from valid results of long term studies on experimental animals when results of studies of human exposure are not available or are inadequate. This factor is intended to account for the uncertainty involved in extrapolating from animal data to humans ( interspecies variation).

UF S An additional factor of 1, 3, or 10 used when extrapolating from less than chronic results on experimental animals when there are no useful long term human data. This factor is intended to account for the uncertainty involved in extrapolating from less than chronic NOAELs to chronic NOAELs.

UF L An additional factor of 1, 3, or 10 used when deriving an RfD from a LOAEL, instead of a NOAEL. This factor is intended to account for the uncertainty involved in extrapolating from LOAELs to NOAELs.

UF D An additional 1, 3 or 10 fold factor used when deriving an RfD from an " incomplete" database. This factor is meant to account for the inability of any single type of study to consider all toxic endpoints. The intermediate factor of 3 ( approximately ½ log 10 unit, i. e., the square root of 10) is often used when there is a single data gap exclusive of chronic data. It is often designated as UF D.

On occasion, EPA also uses a modifying factor in the determination of the RfD. A modifying factor is an additional uncertainty factor greater than zero and less than or equal to 10. The magnitude of the MF reflects the scientific uncertainties of the study and database not explicitly treated with standard uncertainty factors ( e. g., the number of species tested). The default value for the MF is 1.

In establishing the UF or MF, it is recognized that professional scientific judgment must be used. The total product of the uncertainty factors and modifying factor should not exceed 3000. If the assignment of uncertainty results in a UF/ MF product that exceeds 3000, then the database does not support development of an RfD.

2. Drinking Water Equivalent Level The drinking water equivalent ( DWEL) is calculated from the RfD. The DWEL represents a drinking water specific lifetime exposure at which adverse, noncarcinogenic health effects are not anticipated to occur. The DWEL assumes 100% exposure from drinking water. The DWEL Draft   Do Not Cite or Quote ( July, 2003) 18 provides the noncarcinogenic health effects basis for establishing a drinking water standard. For ingestion data, the DWEL is derived as follows:

DWEL = ( RfD) × BW WI where:

BW = 70 kg adult body weight WI = Drinking water intake ( 2 L/ day)

3. Health Advisory Values In addition to the RfD and the DWEL, EPA calculates health advisory ( HA) values for noncancer effects. HAs are determined for lifetime exposures as well as for exposures of shorter duration ( 1 day, 10 day, and longer term). The shorter duration HA values are used as informal guidance to municipalities and other organizations when emergency spills or contamination situations occur.

The shorter term HAs are calculated using an equation similar to the RfD and DWEL; however, the NOAELs or LOAELs are derived from acute or subchronic studies. The HAs are derived as follows:

HA = NOAEL or LOAEL × BW UF × WI where:

NOAEL or LOAEL = No or lowest observed adverse effect level in mg/ kg bw/ day BW = Assumed body weight of a child ( 10 kg) or an adult ( 70 kg)

UF = Uncertainty factor in accordance with EPA or NAS/ OW guidelines WI = Assumed daily drinking water intake of a child ( 1 L/ day) or an adult ( 2 L/ day)

Using the above equation, the following drinking water HAs are developed for noncarcinogenic effects:

° 1 day HA for a 10 kg child ingesting 1 L water per day ° 10 day HA for a 10 kg child ingesting 1 L water per day ° Longer term HA for a 10 kg child ingesting 1 L water per day ° Longer term HA for a 70 kg adult ingesting 2 L water per day Draft   Do Not Cite or Quote ( July, 2003) 19 Each of these shorter term HA values assumes that the total exposure to the contaminant comes from drinking water.

The lifetime HA is calculated from the DWEL and takes into account exposure from sources other than drinking water. It is calculated using the following equation:

Lifetime HA = DWEL × RSC where:

DWEL = Drinking water equivalent level RSC = Relative source contribution. The fraction of the total exposure allocated to drinking water. The default value is 20% of the total exposure.

The lifetime HA becomes the MCLG for a chemical that is not a carcinogen.

The following paragraphs evaluate the data available to support development of HA values for glyoxal.

1 Day Health Advisory The 1 day HA calculated for a 10 kg child assumes a single acute exposure to the chemical and is generally derived from a study of fewer than 7 days' duration.

Ueno et al. ( 1991c) reported that a single oral dose of glyoxal caused inhibition of tissue protein synthesis in rats, but a NOAEL was not identified. In the absence of suitable data on the acute NOAEL for glyoxal, no 1 day HA value can be derived at present. LD 50 data for the oral route indicate that glyoxal has low to moderate acute toxicity.

10 Day Health Advisory The 10 day HA assumes a limited exposure period of 1 to 2 weeks and is generally derived from a study of fewer than 30 days' duration. Because no data were located on the short term oral toxicity of glyoxal, no 10 day HA values can be derived at present.

Longer Term Health Advisory A longer term HA is derived for both a 10 kg child and a 70 kg adult and assumes an exposure period of approximately 7 years ( or 10% of an individual's lifetime). A longer term HA is generally derived from a study of subchronic duration ( exposure for 10% of animal's lifetime).

Takahashi et al. ( 1989) reported that exposure of rats to 500 mg/ kg/ day of glyoxal for 32 weeks resulted in suppression of normal weight gain. Ueno et al. ( 1991c) reported that exposure of rats to glyoxal at doses of 298 to 315 mg/ kg/ day for 90 to 180 days resulted in decreased weight gain and mild histological changes in kidney. Decreased levels of serum aspartate amino Draft   Do Not Cite or Quote ( July, 2003) 20 transferase and total serum protein were detected at a LOAEL of 107 mg/ kg/ day averaged over 90 days. None of these studies identifies a NOAEL for glyoxal. The effects detected at 107 mg/ kg/ day appear to be mild, suggesting that the NOAEL is not far below this dose. However, given the limitation of the data ( one sex; 5 to 7 animals/ dose group, lack of a NOAEL), the data are inadequate for risk assessment and thus were not used to derive a longer term HA. A more complete description of the database limitations is provided below.

Reference Dose, Drinking Water Equivalent Level, and Lifetime Health Advisory Table 3 summarizes studies that provide quantitative data on the noncancer adverse effects associated with oral exposure to glyoxal. The suitability of these studies for derivation of an RfD value and lifetime HA is evaluated below.

No lifetime data were located on the oral toxicity of glyoxal, and there are data from only two subchronic toxicity studies: Takahashi et al. ( 1989) and Ueno et al. ( 1991c). Both studies are limited by the small number of animals evaluated ( 5 to 10) and the use of male rats only.

The Takahashi et al. ( 1989) study did not evaluate either tissue histopathology or biomarkers of adverse organ effects. The toxicological evaluation conducted by Ueno et al. ( 1991c) was more complete ( organ weights, histopathology, serum markers for biochemical abnormalities) and identified decrements in hepatic protein synthesis as the critical effect at a LOAEL of 107 mg/ kg/ day averaged over the 90 day intake period. Higher doses had adverse effects on weight gain and were associated with marginal pathological changes in the kidney epithelium.

Data from a developmental study using doses of 0, 5, 150, or 300 mg/ kg/ day in groups of 26 Sprague Dawley rats showed a deficit in weight gain and food intake at the highest dose over the 9 day administration period ( NTP, 1994). The data from this study support the observations of Takahashi et al. ( 1989) and Ueno et al. ( 1991c).

Each of these studies identified decrements in weight gain following exposures to 300 to 500 mg/ kg/ day glyoxal for periods of 9 days to 32 weeks. Other effects observed included Table 3. Summary of Quantitative Studies on the Oral Toxicity of Glyoxal Reference Duration Species Route LOAEL ( mg/ kg/ day) Effect Ueno et al. ( 1991c) Single exposure Rat Gavage 1000 Decreased protein synthesis in liver and spleen Ueno et al. ( 1991c) 30   90 days Rat Water 107   188 Decreased serum proteins Decreased weight gain Ueno et al. ( 1991c) 90   180 days Rat Water 298   315 Decreased body weight, mild swelling in kidneys, decreased serum protein Takahashi et al. ( 1989) 32 weeks Rat Water 500 Decreased weight gain NTP ( 1994) 9 days Rat Gavage 300 mg/ kg/ day Decreased weight gain ( maternal) Draft   Do Not Cite or Quote ( July, 2003) 21 apparent deficits in protein synthesis at a LOAEL of 107 mg/ kg/ day and mild effects on the kidney. However, the number of animals evaluated was small, only males were tested, and there was no monitoring of hematological parameters. There are no chronic data and two subchronic studies, neither of which monitored all standard subchronic endpoints. Accordingly, the database is not considered adequate to support development of an RfD for lifetime glyoxal exposures.

B. Carcinogenic Effects In 1986, EPA established a five category, alphanumeric system for carcinogen with the publication of Guidelines for Carcinogen Risk Assessment ( U. S. EPA, 1986). The five categories were as follows:

Group A: Human Carcinogen. Group B: Probable Human Carcinogen. Group B1: Limited evidence in humans. Group B2: inadequate evidence in humans; sufficient evidence in animals. Group C: Possible Human Carcinogen. Group D: Not classified as to Human Carcinogenicity. Group E: Evidence of Noncarcinogenicity for Humans.

In 1996 the Agency issued proposed revisions to Guidelines for Carcinogen Risk Assessment for public comment. The 1996 proposal was later refined and released as a revised draft in 1999. Although the 1999 version of the Guidelines for Carcinogen Risk Assessment has not yet been formally adopted by the agency, use of the 1986 guidelines ceased in 2000 with the publication of a directive from the administrator ( Federal Register, 2001 ) specifying that the 1999 guidelines are to be used on an interim basis.

Under the U. S. EPA ( 1999) Guidelines for Carcinogen Risk Assessment: Review Draft, the U. S. EPA presents the carcinogenic potential of a chemical compound in a narrative fashion, and uses one of the following five standard descriptors to express the conclusion regarding the weight of evidence for carcinogenic hazard potential:

° Carcinogenic to humans ° Likely to be carcinogenic to humans ° Suggestive evidence of carcinogenic potential, but not sufficient to assess human carcinogenic potential ° Inadequate information to assess human carcinogenic potential ° Not likely to be carcinogenic to humans Each standard descriptor is presented only in the context of a chemical specific, weight ofevidence narrative. Additionally, more than one conclusion may be reached for an agent ( e. g., an agent is " likely to carcinogenic" by inhalation exposure and " not likely to be carcinogenic" by oral exposure.

In cases where the toxicological evidence leads to the classification of the contaminant as a carcinogen or likely to be a carcinogen, mathematical models are used to calculate the estimated 1 A" point of departure" ( POD) marks the beginning of extrapolation to lower doses. The POD is an estimated dose ( expressed in human equivalent terms) near the lower end of the observed range, without significant extrapolation to lower doses ( U. S. EPA, 2003).

2LED10 = Lower bound on the dose associated with an increased tumor incidence of 10%

Draft   Do Not Cite or Quote ( July, 2003) 22 excess cancer risk associated with ingestion of the contaminant in drinking water. The data input to the models usually come from lifetime exposure studies in animals. In order to predict the risk for humans, animal doses must be converted to equivalent human doses. The conversion can include corrections for noncontinuous exposure, less than lifetime studies, and allometric scaling of the animal body weight. The dose response assessment is performed in two stages. A mathematical assessment of experimental dose data is used to derive a point of departure ( POD) 1. Extrapolation from the POD may assume either linearity or nonlinearity of the dose response relationship, or both. The linear approach ( slope factor) is used for mutagenic carcinogens, i. e. those with linear mode of action, or where the mode of action cannot be determined. For carcinogens with a well substantiated nonlinear mode of action, an RfD approach is used. In both cases a range of models are available.

With the linear approach the slope of the line from the point of departure ( LED 10) 2 to the origin is calculated. The slope factor ( q 1*) is a reflection of the cancer potency. It is used to calculate the concentration in drinking water that is equivalent to a specific risk to the population. Risk estimates are generally presented for the one in ten thousand risk ( 1/ 10.000; E 4), the onein one hundred thousand ( 1/ 100,000; E 5) risk and the one in a million ( 1/ 1,000,000; E 6) risk using the following equation:

Concentration in Drinking Water ( mg/ L) = Risk x Body Weight ( Kg) Slope Factor ( risk/ mg/ kg/ day) x 2 L/ day It is assumed that the average adult human body weight is 70 kg and that the average water consumption of an adult human is two liters of water per day. Drinking water regulations target the E 4 to E 6 risk range as determined from the lower confidence bound on the POD.

The data base used to calculate and support the setting of cancer risk rates has an inherent uncertainty due to systematic and random errors in scientific measurement. Thus, there is uncertainty when the risk is extrapolated from epidemiological or animal data to the entire humans population. When developing cancer risk rates, some of the uncertainties that exist, include incomplete knowledge concerning the health effects of contaminants in drinking water, the impact of the experimental animal's age, sex and species, the nature of the target organ system( s) examined, and the actual rate of exposure of the internal targets in experimental animals or humans. Dose response data usually are available only for high levels of exposure, not for the lower levels at which a standard may be set. When there is exposure to more than one contaminant, additional uncertainty results from a lack of information about possible synergistic or antagonistic effects. The true risk to humans, while not identifiable, is not likely to exceed the upper limit estimate and, in fact, may be lower or even zero. Draft   Do Not Cite or Quote ( July, 2003) 23 Categorization of Carcinogenic Effects Numerous studies indicate that glyoxal is genotoxic ( see Section V D), but there are only minimal data on its carcinogenicity. Several groups of researchers have reported that glyoxal has initiating and/ or promoting activity in various short term tests ( Takahashi et al., 1989; Martelli et al., 1988; Furihata et al., 1985a, b).

The carcinogenic potential of glyoxal cannot be determined using the proposed Guidelines for Carcinogen Risk Assessment ( U. S. EPA, 1999) because no adequate epidemiological studies exist and because no properly conducted long term animal bioassay has been conducted. Draft   Do Not Cite or Quote ( July, 2003) 24 METHYLGLYOXAL I. PHYSICAL AND CHEMICAL PROPERTIES Methylglyoxal is a yellow liquid. It is readily soluble in water, with little tendency to evaporate. Aqueous solutions are a mixture of anhydrous, monohydrated and dihydrated forms in a 1: 71: 28 ratio ( Thornalley, 1996). Other physical chemical properties are summarized in Table 4.

Methylglyoxal is obtained by warming isonitrosoacetone with dilute sulfuric acid ( Budavari et al., 1996). It does not have widespread industrial use. It is used as a reagent in organic synthesis and is an intermediate in the metabolism of acetone and acetone derivatives in various biological systems ( Inoue and Kimura, 1995). It is found naturally in several beverages such as coffee and tea, in foods ( IARC, 1991), and is also listed, under its synonym pyruvaldehyde, as a direct food additive in 21 CFR 172.515 " Synthetic flavoring substances and adjuvants" ( FDA, 1997).

Table 4. Physical and Chemical Properties of Methylglyoxal Parameter Data CASRN 78 98 8 Empirical Formula C3H4O2 Structure CH3COCHO Molecular Weight 72.06 Water Solubility Very soluble Boiling Point 72

C Density 1.05 g/ mL Sources: Budavari et al., 1996; Patnaik, 1992. Draft   Do Not Cite or Quote ( July, 2003) 25 II. TOXICOKINETICS A. Absorption No data were located regarding oral absorption of methylglyoxal. However, based on its chemical structure and aqueous solubility, it is expected that methylglyoxal will be well absorbed from the gastrointestinal tract.

B. Distribution Few data were identified regarding tissue distribution of methylglyoxal following oral exposure. Disposition studies by Phillips et al. ( 1993), as cited by Thornalley ( 1996), identified methylglyoxal in the liver, kidney, lungs, lens, blood, and sciatic nerve of rats. The highest concentrations were found in the lens and sciatic nerve ( 2.1 and 2.4 nmol/ g, respectively). Concentrations in the liver and kidney were about 1 nmol/ g. The level of methylglyoxal in the blood was 0.16

mol/ L.

Diabetics form higher levels of endogenous methylglyoxal than nondiabetic subjects. McLellan et al. ( 1994) found that the median serum methylglyoxal concentration was 80 pmol/ g blood ( range 25 to 892 pmol/ g) in 21 normal subjects. The difference between the median and the high end of the range demonstrates wide variability among individuals. In a group of 42 Type I ( juvenile) diabetics, the median value was 471 pmol/ g with a range of 86 to 1040 pmol/ g. In a group of 105 Type II ( adult onset) diabetics, the median value was 287 pmol/ g with a range of 55 to 2370 pmol/ g. These distributions are obviously skewed with some individuals having rather high levels of serum methylglyoxal.

C. Metabolism Methylglyoxal is produced from the triose phosphates: glyceraldehyde 3 phosphate and dihydroxyacetone phospate, by a spontaneous reaction during glycolysis ( Figure 1; Phillips and Thornalley, 1993). Methylglyoxal originates during the triose phosphate isomerase reaction by phosphate elimination from the 3 phospho 2,3 ene diol intermediate.

In bacteria, methylglyoxal is formed from dihydroxyacetone phosphate through the action of methylglyoxal synthase ( Inoue and Kimura, 1995). Thus, intestinal bacteria can contribute to the exposure of humans to methylglyoxal. Methylglyoxal synthase has not been identified in mammalian tissues.

Methylglyoxal is also produced endogenously in the metabolism of acetone and the amino acid threonine ( Figure 1) ( Peinado et al., 1986; Thornalley, 1996). Production from threonine is a two step reaction requiring threonine dehydrogenase and amine oxidase. Acetone is oxidized to methylglyoxal through the action of acetone monooxidase in reactions utilizing molecular oxygen and NADPH. Draft   Do Not Cite or Quote ( July, 2003) 26 Figure 1. Methylglyoxal metabolism. Draft   Do Not Cite or Quote ( July, 2003) 27 The catabolism of methylglyoxal occurs primarily through the action of glyoxalase I ( GLO I) and glyoxalase II ( GLO II), leading to the production of D lactate, which can be converted to pyruvate ( Argiles, 1986; Thornalley, 1990, 1996). A portion of the D lactate enters circulation and is excreted by the kidneys. Pyruvate is metabolized to carbon dioxide and water by way of the citric acid cycle and electron transport chain.

GLO I is a cytosolic enzyme ( Armeni et al., 1998) that conjugates methylglyoxal to reduced glutathione ( GSH), forming S D lactoyl glutathione ( Thornalley, 1990, 1996). GLO II is present in the cytosol and mitochondria; it removes the conjugated glutathione from S D lactoyl glutathione by hydrolysis, regenerating GSH and forming D lactate ( Thornalley, 1996). It has been hypothesized that S D lactoyl glutathione may function to transport GSH into the mitochondria ( Armeni et al., 1998).

The glyoxalase pathway for methylglyoxal disposition is least active in the kidney, where an alternate pathway seems to predominate ( Thornalley, 1996). The alternate pathway utilizes aldose reductase and NADPH to form hydroxyacetone ( acetol) and D lactaldehyde in a 95%: 5% ratio ( Vander Jagt et al., 1992). Both products are further reduced to propanediol. In humans, Aldose reductase is most active in the kidney. It is also active in the heart muscle, lens, and peripheral neurons, and its generation of alpha ketoaldehydes from sugars, along with generation of hydroxyacetone from methylglyoxal may play a role in development of the long term effects of diabetes ( Vander Jagt et al., 1992). Reaction of albumin with glucose, methylglyoxal and hydroxyacetone in vitro results in modified proteins similar to advanced glycation endproducts ( Vander Jagt et al., 1992).

There are differences in the activities of erythrocyte GLOI and GLO II in diabetic and normal subjects ( McLellan et al. 1994). The significance of the effect on GLO I activity was greater than that for GLO II in noninsulin dependant diabetics ( P< 0.001 versus P< 0.05). In the case of insulin dependant diabetics the increase in enzyme activity was only significant for GLO I ( P< 0.001). There were no significant differences in the blood levels of glutathione but concentrations of methylglyoxal metabolites were significantly elevated in the diabetic subjects.

In situations where methylglyoxal concentrations exceed the cellular capacity to metabolize it, methylglyoxal can form conjugates with DNA, RNA, and/ or protein ( Thornalley, 1996). Guanine is the DNA/ RNA base most vulnerable to modification. Adenine and cytosine are much less reactive ( Krymkiewicz, 1973, as cited by Thornalley, 1996). In each case the methylglyoxal reacts with the amino functional group of the nucleotide base. The product of the reaction with guanine, [ 3( 2' deoxyribosyl) 6,7 dihydro 6 methylimidazole [ 2,3 b] purine 9 ( 8) one], has been detected in extracts of DNA from human peripheral leucocytes incubated with methylglyoxal ( Vaca et al., 1994, as cited in Thornalley, 1996).

In proteins, the side chains of arginine, lysine, and cysteine are vulnerable to conjugation with methylglyoxal ( Thornalley, 1996). The product of the initial conjugation of methylglyoxal with the guanidino functional group in arginine proceeds to form a terminal imidazole ring, elongating and changing the acid/ base properties of the side chain. The reaction of the lysine terminal amino functional group can lead to cross linking with other lysines within the polypeptide chain or between polypeptide chains ( Thornalley, 1996). In the case of cysteine, the Draft   Do Not Cite or Quote ( July, 2003) 28 methylglyoxal adds to the sulfhydryl functional group, forming a hemithio acetate ( Thornalley, 1996). Each of these reactions modifies the conformation of the affected polypeptide, altering function and contributing to cytotoxicity.

Arnold et al. ( 1983) reported that methylglyoxal reacts in vitro with the enediol portion of ascorbic acid to yield a 5 membered cyclic acetal. This condensation is reversible under alkaline conditions. No studies were performed to determine whether this reaction occurs in vivo.

D. Excretion Few data were located on the route or degree of methylglyoxal excretion. A small amount of methyl glyoxal ( 4

mol/ day) and about 40

mol of its metabolite D lactate are excreted in urine. Other potential metabolic endproducts such as lactate or propanediol may form when concentrations of methylglyoxal are elevated but are water soluble, and should be excreted mainly in urine. Some methylglyoxal will be metabolized to carbon dioxide in the citric acid cycle via pyruvate.

E. Bioaccumulation and Retention No data were located on the bioaccumulation or retention of methylglyoxal. This compound is unlikely to accumulate in the body because of its aqueous solubility and minimal hydrophobicity. Draft   Do Not Cite or Quote ( July, 2003) 29 III. HUMAN EXPOSURE A. Occurrence Air There was little information in the available literature on the distribution of methylglyoxal in air. It is apparently generated during the combustion of gasoline and has been reported to occur in the exhaust of automobiles. Methylglyoxal was identified in three air samples collected at the toll plaza for the San Francisco Bay bridge at concentrations ranging from 0.039 to 0.101

g/ m3 ( Destaillats et al., 2002). It was also detected in air samples collected in Los Angeles at concentrations  0.5 ppb ( Kawamura et al., 2000). The methylglyoxal concentrations in samples from downtown Los Angeles were higher than those from west Los Angeles and methylglyoxal concentrations were lower than those for glyoxal. It occurs in cigarette smoke ( 50 to 100

g/ cigarette) and may be present in ambient air from this source ( IARC, 1991; Kalapos, 1999).

Food Small amounts of methylglyoxal is present in foods as a metabolic intermediate ( Table 5: Barros et al., 1999; Hayashi and Shibamoto, 1985; Nagao et al., 1986a) . It is an FDA approved synthetic flavoring agent ( 21 CFR, 172.515). Methylglyoxal was present in coffee ( both instant and brewed) and other beverages( Barros et al., 1999; Hayashi and Shibamoto, 1985; Nagao et al., 1986a). The methods of preparing the coffee and tea samples varied as did the analytical methods employed; several wine and beer products were assayed. Methylglyoxal was also detected in toast and soy sauce ( Nagao et al., 1986b). The level of glyoxal in toast was 0.5

g/ g. In soy sauce, there was 8.7

g/ mL methylglyoxal. Because methylglyoxal forms from sugars, when they are heated, it is present in cane sugar, maple syrup, and products containing these ingredients ( Hayashi and Shibamoto, 1985; IARC, 1991).

Water Methylglyoxal is formed as a byproduct from ozonation of water ( Glaze et al., 1989; Langlais et al., 1991). Ozonation of water from the Los Angeles Aqueduct Filtration Plant resulted in a significant increase of several aldehydes and carboxylic acids compared to their levels in source water. For example, methylglyoxal was not detectable in raw water, but was detected at concentrations of 28

g/ L following ozonation ( Langlais et al., 1991). Draft   Do Not Cite or Quote ( July, 2003) 30 Table 5. Concentrations of Methylglyoxal in Selected Beverages Beverage Methylglyoxal (

g/ mL)

Apple brandy c 0.32 Apple juice b 0 2.6 Beer a 0.08 0.24 Black tea c trace 2.4 Bourbon whiskey c 1.5 Brewed coffee c 7.0 25 Cocoa b 1.2 Cola b 0.23 Dry milk, nonfat b 1.4 Instant coffee a, c 1.6 23 Japanese sake c 0.26 Maple syrup b 2.5 Orange juice b 0.04 Root beer b 0.76 Soy sauce b 3.0 7.7 Tomato Juice b 0.06 Wine a, c 0.11 2.88 a Barros et al., 1999. b Hayashi and Shibamoto, 1985. c Nagao et al., 1986a.

Following ozonation of surface waters to which odor causing compounds ( 2 methylesoborneal and geosmin) had been added, methylglyoxal concentrations ranged from 0.1 to 0.2

g/ L ( Yamada and Somiya, 1989). Ozonation was carried out for 16 minutes at pH 7.6 or 7.7. Odorant concentrations decreased with time as they were oxidized to simpler compounds.

After a 40 minute ozone reaction time, the concentration of methylglyoxal was increased when ozonation was carried out at basic pHs as apposed to acid pHs. At pH 10.5, the methylglyoxal conentration was 1.5

g/ L, whereas at pH 6.6, no methylglyoxal was detected ( Yamada and Somiya, 1989).

In the work by Yamada and Somiya ( 1989), there was no methylglyoxal in the postfiltration water influent to the ozone contactors at a demonstration plant. After ozone treatment, there was 0.3

g/ L glyoxal. Complete removal of methylglyoxal was accomplished using a granularactivated filter as postozonation treatment. It has been shown that methylglyoxal is also removed Draft   Do Not Cite or Quote ( July, 2003) 31 from potable water by reaction with chloramines used as secondary disinfectants ( Glaze et al., 1989).

Concentrations of methylglyoxal in the treated water from ozonation plants were monitored under the Disinfection Byproduct Information Collection Rule over an 18 month period. Twenty ozonation plants participated in the project. One was a ground water plant, the remainder used a surface water source. After 1 year more than 50% of the sites did not detect any methylglyoxal in the finished water. The maximum concentration reported was 17

g/ L.

Soil No information was available in the literature concerning levels of methylglyoxal in soils. There is no reason to suspect that it is present in any significant concentrations except as breakdown products of organic materials.

B. Fate Methylglyoxal undergoes addition and condensation reactions with amines, amides, aldehydes, and hydroxyl containing materials ( Ueno et al., 1989). Because of its reactivity, the environmental persistence of methylglyoxal is expected to be minimal.

C. Exposure The available information on exposure to methylglyoxal is sparse. Most of the daily exogenous exposure is likely to be from foods. Exposure from drinking water is expected to be minimal. Methylglyoxal can form endogenously in humans during the catabolism of energy nutrients ( proteins, carbohydrates, triglycerides). About 120

mol/ kg/ day is generated in normal adult humans; the amounts generated daily in diabetic subjects is elevated 2 to 3 fold ( Thornalley,) Draft   Do Not Cite or Quote ( July, 2003) 32 IV. HEALTH EFFECTS IN ANIMALS A. Short Term Exposure The oral LD 50 for adult male rats was 1990 mg/ kg and in female rats it ranged from 1165 to 1623 mg/ kg depending on the age or state of pregnancy ( IARC, 1991). Neonates appeared to be more sensitive to the acute effects of methylglyoxal. The acute LD 50 of 531 mg/ kg for neonates is less than 50% of the adult value ( IARC, 1991). Conroy ( 1979) reported that the intravenous LD 50 for methylglyoxal was 108 mg/ kg/ day when given daily for 10 days. Doses of 144 mg/ kg/ day were lethal if given for 5 days or more. Clinical signs included disorientation and generalized central nervous system depression, but no detailed study of the cause of death was performed.

Jerzykowski et al. ( 1975) investigated changes in cardiovascular, respiratory, and biochemical indices resulting from methylglyoxal injection. Anesthetized cats and rabbits were given single intravenous doses of 0, 10, 25, 100, or 250 mg/ kg. Doses in excess of 25 mg/ kg resulted in diminished respiratory function, blood pressure instability, and dose related slowing of cardiac action in both rabbits and cats. In contrast, doses of up to 1000 mg/ kg administered intraperitoneally had no effect on the circulatory or respiratory parameters measured, although biochemical parameters such as lactate dehydrogenase, aspartate aminotransferase ( AST), and creatine kinase levels were elevated in rabbits at 200 mg/ kg and in rats at 500 mg/ kg.

High doses of methylglyoxal can be hepatotoxic. A single intraperitoneal injection of 400 mg/ kg led to a significantly increased relative liver weight, increased serum AST activity, and decreased levels of hepatic GSH in groups of six male CFLP mice within 24 hours of compound administration ( Kalapos, 1999). In a similar study by Chaudhary et al. ( 1997), doses of 0, 50, 100, 200, or 400 mg/ kg in groups of six male Swiss albino mice were associated with significant effects on serum AST activity ( p< 0.05) after 24 hours for all dose groups. Hepatic GSH was decreased for all doses except the lowest. More pronounced GSH depletion was seen in all dose groups at 6 and 12 hours. Lipid peroxidation was observed in the liver at 6, 12, and 24 hours: all dose groups were affected at 12 and 24 hours and all but the lowest dose group at 6 hours. When Kalapos ( 1999) examined the liver tissues microscopically, he observed increased vacuolization of cells in the centrilobular area.

The hepatic toxicity of methylglyoxal was increased in rats pretreated for 1 week prior to methylglyoxal administration with 10% ethanol or 1% acetone in their drinking water ( Kalapos, 1999). The effects of pretreatment were more severe with acetone pretreatment than with ethanol. Changes in biochemical biomarkers of toxicity ( serum AST, GSH) were more pronounced in the pretreated animals, as was the vacuolization of the centrilobular liver cells. Pretreatment with dimethylsulfoxide had little effect on methylglyoxal hepatic toxicity.

Ethanol, acetone, and dimethylsulfoxide are inducers of CYP 2E1. Since cytochrome P 450 enzymes are capable of converting acetone to methylglyoxal, the severity of the effects of acetone pretreatment likely reflect an increased total methylglyoxal internal dose. Draft   Do Not Cite or Quote ( July, 2003) 33 Kalapos ( 1999) also measured the effects of methylglyoxal and a combination of methylglyoxal and CYP 2E1 inducers on hepatic carbohydrate metabolism. Neither the methylglyoxal injection alone or in combination with the CYP 2E1 inducer pretreatment led to significant changes in blood glucose levels. However, pretreatment appeared to decrease liver glycogen. Tissue glycogen was determined by staining tissue samples with periodic acid Schiff reagent.

B. Longer Term Exposure Although methylglyoxal does not appear to be severely toxic, it can cause metabolic perturbations of concern and may disproportionally affect selected sensitive populations. Ankrah and Appiah Opong ( 1999) conducted a study in juvenile mice exposed during gestation, lactation, and weaning. This study did not evaluate standard reproductive or teratology parameters but did evaluate a series of biochemical parameters ( GSH levels, GST activity, GGT activity, fasting blood glucose, glucose tolerance, bile production, and hemoglobin oxidation) in a group of 37 juvenile DPY mice as compared with a group of 37 control mice. It is accordingly not classified as a reproductive or teratology study.

The experimental animals were exposed to 10 g/ L ( 1%) concentration of methylglyoxal in drinking water 5 days/ week. Prenatal and lactation exposures occurred through the dams. After weaning, the mice were given drinking water with the same 10 g/ L concentration for a 2 month period.

The authors reported an exposure of 1.7

mol/ mouse ( 5 mg/ kg/ day based on a 25 g body weight for the dams). However, this value is not consistent with the 10 g/ L ( 1%) methylglyoxal concentration in water because the 1.7

mol dose would be equivalent to a water intake of only 0.01 mL/ day, an unreasonably low intake. Accordingly, it appears as if the intake of 1.7

mol/ mouse should have been 1.7 mmol/ mouse and that the dose should be 5 g/ kg/ day. To achieve this dose, the water intake would have been 10 mL/ day. This value is reasonably close to the 7 8 mL water intake estimate proposed by EPA ( 1988) for B6C3F1 mice in studies with a subchronic duration. Accordingly, the dose that will be used in all discussion of this study will be 1.7 mmol or 5 g/ kg/ day rather than the dose presented in the published paper.

The methylglyoxal exposure was associated with a significant decrease in erythrocyte GSH and in the activity of GST transferase, an enzyme family responsible for conjugation of the endproducts of cytochrome P 450 oxidation with GSH for excretion ( Stipanuk, 2000). However, there was no change in the activity of   glutamyl transferase, an enzyme involved in processing glutathione conjugates.

Parallel with a decrease in erythrocyte GSH there was a loss in hemoglobin's ability to resist oxidation. When aliquots of red blood cells from exposed mice were first exposed to oxidizing conditions in vitro there were no differences in hemoglobin oxidation compared with controls. However, with continued exposure of the red cells to oxidation for a 3 hour period, the red cells of the methylglyoxal treated mice had three times more methemoglobin than the control cells. The authors suggested that individuals that carry the G6PD trait ( homozygots and heterozygots) Draft   Do Not Cite or Quote ( July, 2003) 34 may have heightened sensitivity to methylglyoxal. Additional studies will be necessary to confirm this hypothesis.

It has been proposed that diabetics and individuals with impaired glucose tolerance may be sensitive to methylglyoxal. In order to test this hypothesis, Ankrah and Appiah Opong ( 1999) drew blood samples from fasting juvenile mice exposed to 0 or 5 g/ kg/ day methylglyoxal. The animals were then given an oral bolus dose of 1.7 g glucose/ kg, and blood samples were collected after 2 hours. Fasting blood glucose levels for the exposed mice did not differ from those for the controls. However, blood glucose levels increased to levels outside the normal range after the glucose challenge for 17/ 37 of the mice given methylglyoxal compared with only 3/ 37 of the control mice. Thus, glucose tolerance was impaired in the exposed mice and they were more likely to become hyperglycemic than the controls.

The study by Ankrah and Appiah Opong ( 1999) is not suitable for dose response assessment because only a single dose was tested. However, the fact that a drinking water route of administration was used and that effects were seen with doses of about 5 g/ kg/ day makes it an important study in hazard identification. The effects observed are biologically plausible and consistent with the limited toxicology data for methylglyoxal. Thus, this study suggests the need for a validating study in laboratory animals, expanding the investigation of Ankrah and Appiah Opong ( 1999) and using a series of doses to both confirm the effects observed and delineate the dose response.

Takahashi et al. ( 1989) exposed groups of 10 male Wistar rats to 0.25% methylglyoxal for 32 weeks. Assuming water intake of 0.10 L/ kg/ day, this corresponds to doses of about 250 mg/ kg/ day. Animals were weighed weekly. The methylglyoxal treated animals gained weight during the exposure period, but because there was no comparable control group, it was not possible to determine if degree of weight gain was normal. These data are not adequate to identify a NOAEL or a LOAEL for methylglyoxal.

Fujita et al. ( 1986) exposed groups of 40 male F344 rats to 0% or 0.5% methylglyoxal in drinking water for 2 years. The average daily dose in the exposed animals was reported to be 7.7 mg/ rat ( about 20 mg/ kg/ day). [ NOTE: This corresponds to a water intake of only 1.5 mL/ day, suggesting that the authors reported either the dose or the concentration incorrectly.] Average body weight was about 15% less in the exposed groups than in the controls. These findings were reported in abstract form only, so the reliability of the data is uncertain.

C. Reproductive/ Developmental Studies No information on reproductive/ developmental effects of methylglyoxal was located from any primary source. A review paper on teratogenicity results for a number of chemicals by Schardein ( 1993) lists a negative result for methylglyoxal ( pyruvaldehyde) in rats from a study by Peters et al. ( 1977).

Clerici et al. ( 1990) found that methylglyoxal, at concentrations from 10 5 to 10 3 mol/ L ( 0.56 to 56 mg/ L), interfered with blastocyst formation in vitro in embryos recovered at the 2 cell stage from superovulated female CD 1 mice. Methylglyoxal appeared to exert its greatest effect Draft   Do Not Cite or Quote ( July, 2003) 35 when morulae became blastocysts and was classified by the authors as embryocidal. Unfortunately, the abstract did not provide many experimental details.

Amicarelli et al. ( 1988) examined the effect of exogenous methylglyoxal on Bufo bufo ( common toad) embryo development. Embryonic jelly was removed from adult Bufo bufo treated with 150, 300, or 600

M ( 11, 22, or 43 mg/ L) solutions for morphological and histological analysis. The results showed that 300 and 600

M methylglyoxal causes a marked inhibition of cell division in the early development stages that yields morulae and blastulae with cells swollen and reduced in number with respect to the controls. Stages 19, 20, 22, and 25 showed various malformations such as oedema and severe caudal and dorsal abnormalities. At 42 hours after fertilization, the treated embryos showed a significant delay in development and strong abnormalities. At 12 days following fertilization, the treated embryos displayed the complete absence of differentiated intestine, and failure to incorporate yolk. Tail abnormalities determined by fractures of notochord were also observed. The percentage of viability in the embryos developed in the presence of methylglyoxal was markedly reduced compared with controls. All effects were evident at 150

M methylglyoxal and were dose dependent.

D. Mutagenicity and Genotoxicity Gene Mutation Assays Methylglyoxal has been tested extensively for its mutagenic and genotoxic properties. The studies described below have been grouped by the type of assay and test organism. Methylglyoxal is chemically reactive in Schiff base type reactions with amine functional groups or conjugation with thiol functional groups. Accordingly, it can form adducts with several DNA bases and can also react with RNA and nuclear proteins. It can impact DNA replication and transcription through both genetic and epigenetic mechanisms.

Salmonella typhimurium Gene mutation assays using various strains of S. typhimurium ( Ames assay) have been conducted by many investigators. However, these studies were conducted to determine selective aspects of methylglyoxal mutagenicity, and no study using the full range of standard Ames tester strains was located. Most Ames assays have used strains 98, 100, 102, and 104. TA 98 detects frame shift mutations, and TA 100 detects base pair mutations as does TA104. TA102 detects changes caused by a variety of oxidants and cross linking agents ( Mitchell, 1993). The data indicate that methylglyoxal causes point mutations and/ or adducts in the presence and absence of microsomal activation ( TA100, TA104, TA102). The response is intensified in the presence of hydrogen peroxide and appears to be weakened by microsomal metabolism ( Aeschbacher et al., 1989; Bronzetti et al., 1987; Friederich et al., 1985). Frame shift changes are also possible in the absence of microsomal activation ( TA98) but are not seen after microsomal metabolism of methylglyoxal ( Fujita et al., 1985a, b; Furukawa et al., 1986; Kato et al., 1989; Migliore et al., 1990; Nagao et al., 1986a; Shane et al., 1988).

Methylglyoxal was used as the positive control in an experiment investigating the mutagenic mechanisms of four nitro group   containing aromatic amines ( Chen et al., 1997). The Draft   Do Not Cite or Quote ( July, 2003) 36 standard plate incorporation assay was used, without metabolic activation. When tested at 10

g/ plate, methylglyoxal was mutagenic in strains TA100 and TA104, but not in strains TA4001 and TA4006, which detect TA  CG and CG  GC transitions, respectively. Inhibition by the histidine analogue DL 1,2,4 triazole 3 alanine was used to distinguish histidine locus from suppressor gene mutations. In strain TA100, methylglyoxal produced 100% histidine locus mutations ( CG  AT and CG  TA) rather than suppressor gene mutations ( TA  CG). In strain TA104, methylglyoxal produced 17% histidine locus mutations ( TA  GC and TA  AT) and 83% suppressor gene mutations ( TA  AT, CG  TA, and CG  AT).

Escherichia coli Various E. coli strains are useful in evaluating the mutagenic activity of chemicals toward causing base pair substitutions. Tests of methylglyoxal in E. coli in general support the Ames assay results, although Nunoshiba et al. ( 1989, 1991) found that hydrogen peroxide weakened rather than potentiated the response. The Nunoshiba et al. study was the only one identified with E. coli that used hydrogen peroxide.

Kranendonk et al. ( 1996) described the development and characterization of strain MX100, a derivative of E. coli K12 laboratory strain AB1157. This strain has sensitivity toward the detection of base substitution mutagens. A comparison of mutagenicity results obtained in MX100 versus routinely used tester strains, namely E. coli. WP2 and Salmonella strains revealed that the response of MX100 matched that found in the other strains. Methylglyoxal was tested over a dose range of 0 to 0.349

M/ plate and showed a dose dependent increase in the number of revertants in MX100. MX100 shows sensitivity toward the detection of oxidative and carbonyl mutagens.

Murata Kamura et al. ( 2000) exposed KS40 E. coli vector plasmid PMY189 to methyl glyoxal ( 0 to 70 ug) and transvected the treated plasmid into Simian kidney COS7 cell cultures. After 24 hours the amplified plasmids were recovered from the treated COS7 cells and introduced into E. colu strain HB101. The E. coli were plated on culture media and held overnight at 37  C. The number of colonies decreased with increasing methyl glyoxal concentration indicating a partial blockage of DNA replication.

The amplified plasmid was also introduced into Ks40 PKY241 E. coli and plated on a medium that would select cells with a mutated SUP1 gene. After overnight incubation at 37  C, the colonies were counted and the mutation frequencies calculated. Mutation frequency increased with methylglyoxal dose. Fifty percent of the mutations were the result of deletions ( 8 to 320 base pairs); 35% of the mutations were base pair substitutions. Among the base pair substitutions, 89 % involved G: C pairs with transition from G: C to C: G or C: C to T: A. The may reflect the vulnerability of guanine to modification by methylglyoxal.

Other Mutagenicity Assays Bronzetti et al. ( 1987) studied the genotoxic potential of methylglyoxal in Saccharomyces cerevisiae D7 through the study of mitotic gene conversion and reverse point mutations. Yeast cells were incubated in concentrations of 10, 15, 20, and 40 mM methylglyoxal, with or without Draft   Do Not Cite or Quote ( July, 2003) 37 metabolic activation. The 40 mM dose was cytotoxic in the absence of the S9 fraction with no surviving colonies, and only 25% of colonies survived at the 20 mM dose. Methylglyoxal induced both mitotic gene conversion and reverse point mutation in S. cerevisiae D7.

Cajelli et al. ( 1987) reported that exposure of V79 Chinese hamster lung cells to methylglyoxal, at concentrations ranging from 0.16 to 1.5 mM, caused a dose dependent increase in the frequency of mutations in the hypoxanthine guanosine phosphoribosyl transferase ( HGPRT) gene locus. This effect was reduced when cells were co cultured with freshly prepared rat hepatocytes. The authors speculated that this was caused by metabolism of methylglyoxal to lactate or pyruvate.

Chromosomal Aberration Assays Chromosomal aberration assays including monitoring for strand breaks, sister chromatid exchange ( SCE) micronuclei, and cross linking have been used to study the genetic damage produced by methylglyoxal, as well as by other dicarbonyl compounds and by complex mixtures containing these compounds. Both in vitro and in vivo assays tend to be positive although the in vivo response is weaker than the in vitro response.

Tucker et al. ( 1989) studied the effect of methylglyoxal on the induction of SCEs and endoreduplicated cells ( ERCs) in CHO AUXB1 cells and human peripheral lymphocytes. SCEs were significantly elevated at methylglyoxal concentrations of 200 to 500

M. Endoreduplicated cells were significantly increased at methylglyoxal concentrations ranging from 100 to 500

M. The relative percent cell survival decreased from 100% in the control cells to 31% in cells exposed to methylglyoxal concentrations of 500

M.

Nishi et al. ( 1989) described the clastogenic activity of thermally decomposed products of carbohydrates in cultured Chinese hamster V79 cells. Products included glyoxal and methylglyoxal. The mitotic index was calculated as the percentage of metaphases among more than 2000 interphase nuclei. Glyoxal and methylglyoxal both induced a significant number of chromosome aberrations and also lowered the mitotic index. The doses of methylglyoxal that induced comparable numbers of aberrant cells were about 1/ 10 of the glyoxal doses. This assay was conducted only without metabolic activation, and positive controls were not included in the study design.

Migliore et al. ( 1990) studied the effect of methylglyoxal on chromosomal aberrations, SCEs, and micronuclei in human lymphocytes, in both the presence and the absence of metabolic activation. In the absence of metabolic activation, micronuclei were increased at methylglyoxal concentrations of 3.0 and 4.5 mM, structural chromosomal aberrations ( excluding gaps) were significantly increased at concentrations of 1.5 mM or greater, and SCEs increased significantly at concentrations greater than or equal to 0.5 mM. A significant correlation was observed between the concentration of methylglyoxal and the number of chromosome aberrations. Metabolic activation resulted in reduction in the chromosome aberrations and SCE induction, although the differences were not statistically significant. Addition of metabolic activation largely prevented the increase in micronuclei observed in the absence of activation. Draft   Do Not Cite or Quote ( July, 2003) 38 The micronucleus assay was conducted in both liver and bone marrow cells of male Sprague Dawley albino rats using two dosage protocols ( Martelli et al., 1989). In the first, 800 mg/ kg of methylglyoxal was administered by gavage to animals that had been partially hepatectomized 20 hours before treatment. In the second, rats were given methylglyoxal in drinking water at a dose of 400 mg/ kg/ day for 5 successive days, a partial hepatectomy was done 48 hours after the last dose, and liver and bone marrow cells were obtained 48 hours later. Methylglyoxal was weakly positive in terms of frequency of micronucleated hepatocytes at the single high dose, but was negative in cells of animals given successive lower doses. It was negative in bone marrow in both protocols. The authors hypothesized that methylglyoxal was positive only at doses where the glyoxalase system   which metabolizes it to lactate   and the alpha ketoaldehyde dehydrogenase system   which metabolizes it to pyruvate   became saturated.

Migliore et al. ( 1990) assessed the potential genotoxic activity of methylglyoxal using in vivo cytogenetic tests on mouse intestinal cells. Swiss CD1 mice were dosed with 400 or 600 mg/ kg methylglyoxal by oral gavage. Only a weak positive response was obtained for the induction of SCEs at the highest dose of 600 mg/ kg body weight in duodenal cells. Methylglyoxal appeared to be nongenotoxic in intestinal cells.

Other Assays for Genetic Damage DNA damage caused by methylglyoxal has been investigated in a number of studies. Much of the focus has been on strand breaks and adduct formation, although other endpoints have also been used.

Brambilla et al. ( 1985) studied the capacity of methylglyoxal to induce DNA damage and repair in CHO cells. DNA damage was evaluated using an alkaline elution technique to measure the formation of DNA protein cross links. Increasing concentrations of methylglyoxal ( 1.7 to 7.8 mM) resulted in a dose dependent decrease in cell survival. Cross linking was observed at concentrations of 1.5 and 4.5 mM methylglyoxal. CHO cells exposed to methylglyoxal in the presence of a rat liver metabolic system showed significantly reduced incidence of cross linking.

Rahman et al. ( 1990) investigated the ability of methylglyoxal to produce strand breaks in double stranded DNA by incubating calf thymus DNA with methylglyoxal in DNA base pair to methylglyoxal molar ratios of 1: 1 to 1: 16 for 2 hours, followed by incubation for 3 hours with S 1 nuclease to determine the extent of hydrolysis. The percentage of DNA hydrolyzed, suggesting destabilization possibly through strand breaks, was 22% in DNA not exposed to methylglyoxal and 25%, 25%, 36%, and 43% at DNA base pair: methylglyoxal ratios of 1: 1, 1: 2, 1: 8, and 1: 16, respectively. To determine whether the damage was really caused by strand breaks, the alkaline unwinding assay was used. The number of breaks per unit DNA increased from 0.3 at a 1: 1 DNA base pair to methylglyoxal molar ratio to 3.6 at a 1: 16 ratio, strongly suggesting the formation of single strand breaks causing increased susceptibility to hydrolysis by S 1 nuclease.

Thermal denaturation of DNA, as determined by thermal melting profiles, was also used to study the effects of methylglyoxal ( Rahman et al., 1990). The midrange melting temperature of unexposed double stranded DNA was 75oC, but was reduced to 68oC at the 1: 8 molar ratio of DNA: methylglyoxal suggesting destabilization and was increased to 80oC at the 1: 16 ratio Draft   Do Not Cite or Quote ( July, 2003) 39 suggesting stabilization, perhaps by formation of cross links. Cross links were studied by thermally degrading DNA that had been exposed to methylglyoxal ( 1: 16), and then determining its ability to reanneal, suggesting that the DNA strands were held together by covalent cross links. Some evidence of reannealing was found.

The acetylation of guanine residues in DNA by methylglyoxal was studied by Tada et al. ( 1996) using HPLC in combination with 32P postlabeling, using calf thymus DNA exposed to methylglyoxal. The adduct was detected at 2 per 106 nucleotides in double stranded DNA and at 1 per 105 nucleotides in single stranded DNA. This work supported earlier work on adduct formation.

Methylglyoxal can be formed in mammals by enteric bacteria in the intestinal system. Isolated villus and crypt cells from rat small intestine and isolated colonocytes from rat colon were studied ( Baskaran and Balasubramanian, 1990) in cell suspensions exposed to methylglyoxal in the range of 0.1 to 3.0 mM. Incorporation of radiolabeled leucine, thymidine, and uridine was used to study the effects on protein synthesis, DNA synthesis, and RNA synthesis, respectively. Methylglyoxal inhibited synthesis of all three types of macromolecules in a dose dependent manner. The thiol compounds cysteine and glutathione were not protective of this inhibition.

E. Carcinogenicity Fujita et al. ( 1986) exposed groups of 40 male F344 rats to 0% or 0.5% methylglyoxal in drinking water for 2 years. The average daily dose in the exposed animals was reported to be 7.7 mg/ rat ( about 20 mg/ kg/ day). [ NOTE: This corresponds to a water intake of only 1.5 mL/ day, suggesting that the authors reported either the dose or the concentration incorrectly.] Average body weight was about 15% less in the exposed groups than in the controls. No increase in tumor incidence was detected by either gross or histological examinations. The authors speculated that methylglyoxal might react with amines or thiols in the stomach, thereby reducing its mutagenic and carcinogenic potential. These findings were reported in abstract form only, so the reliability of these data is uncertain.

Takahashi et al. ( 1989) exposed 10 male Wistar rats to methylglyoxal ( 2500 mg/ L) in drinking water for 32 weeks. Assuming water intake of 0.10 L/ kg/ day, this corresponds to doses of about 250 mg/ kg/ day. At termination, all internal organs were examined macroscopically, and the stomachs were fixed and examined histologically. No gastric carcinomas or hyperplastic lesions were detected in animals exposed to methylglyoxal. This negative finding is limited by the small number of animals tested, the relatively short exposure period, and the absence of a followup observation period.

Takahashi et al. ( 1989) tested the cancer promoting activity of methylglyoxal in rats. Groups of 30 male Wistar rats were exposed for 8 weeks to N methyl N' nitro Nnitrosoguanidine ( MNNG) in drinking water ( 100 mg/ L). Following this, animals were exposed to methylglyoxal ( 2500 mg/ L) in drinking water for 32 weeks. Assuming consumption of 0.10 L/ kg/ day, this corresponds to doses of about 250 mg/ kg/ day. Control animals were exposed to MNNG for 8 weeks, and than given untreated water for 32 weeks. At termination, animals were sacrificed, internal organs were examined macroscopically, and the stomach was fixed and Draft   Do Not Cite or Quote ( July, 2003) 40 examined histologically. Methylglyoxal increased the incidence of hyperplasia in the pylorus, but did not increase the incidence of gastric carcinomas. No toxic or neoplastic changes were noted in any other organs. These findings were taken to indicate that methylglyoxal may exert a cancerpromoting effect in the stomach.

Furihata et al. ( 1985a) studied the initiating and promoting activity of methylglyoxal in gastric mucosal cells following oral exposure of rats. Groups of five male F344 rats were given single oral doses of 50 to 100 mg/ kg by gastric tube, and the level of ODC and UDS in gastric mucosa were measured over the next 48 to 72 hours. Methylglyoxal caused a clear dosedependent increase in both UDS and ODC activity, peaking about 16 hours after exposure. The increased rate of UDS was taken to be an indicator of possible initiating activity, and the increased level of ODC was taken to be an index of possible promoting activity.

Takayama et al. ( 1984) exposed 10 male and 10 female F344 rats to 1.3 mg of methylglyoxal ( 65.5% pure) by subcutaneous injection twice weekly for 10 weeks. Assuming a body weight of 0.25 kg, this corresponds to an average daily dose of about 0.24 mg/ kg/ day. Controls ( 10 males plus 10 females) received injections of saline solution. After 70 weeks, subcutaneous tumors were found in two experimental animals. No additional experimental details were provided.

Nagao et al. ( 1986a) exposed 8 male and 10 female F344 rats to methylglyoxal by subcutaneous injection. Doses of 2 mg were administered twice a week for 10 weeks. Assuming a body weight of 0.25 kg, this corresponds to an average daily dose of about 2.3 mg/ kg/ day. A control group of 21 males and 19 females received only saline. After 20 months, 3 males and 1 female from the treated rats had fibrosarcomas at the injection site. No tumors were observed in the control group.

Several intermediate term carcinogenesis studies have been done on methylglyoxal. Hasegawa and Ito ( 1992) made use of the two step theory of hepatocarcinogenesis, dosing male F344 rats with a single intraperitoneal dose of diethylnitrosamine, followed by a 2 week recovery period and then by 6 weeks of exposure to the test chemical. At week 3, rats had a two thirds partial hepatectomy to stimulate growth of new liver tissue. Two control groups were used. One control group received diethylnitrosamine and partial hepatectomy, but no chemical, at the same time points as the chemically exposed group. The second control group received saline instead of diethylnitrosamine, partial hepatectomy, and exposure to the test chemical. Both the numbers and areas of induced glutathione S transferase placental ( GST P) form positive foci in the livers were then determined. Methylglyoxal, when dosed at 2000 ppm in drinking water, caused a statistically significant increase ( p< 0.05) in the number of GST P positive foci, and an increase in foci area that was not statistically significant ( p> 0.05), giving overall weak results. No effects other than GST P positive foci were monitored or reported. This in vivo test did correlate with short term in vitro tests such as the Ames test for methylglyoxal.

Hasegawa et al. ( 1995) examined the potential carcinogenicity of coffee and its related compounds, at doses broadly comparable with those usually consumed by humans, to cause hepatocarcinogenicity. An 8 week medium term liver bioassay ( as described above), based on the induction of GST P form positive foci in F344 male rats was used to determine potential Draft   Do Not Cite or Quote ( July, 2003) 41 carcinogenicity. Coffee, theophylline, and caffeine exerted no effect on GST P positive foci development. Hence, these coffee related compounds examined demonstrated no obvious enhancing potential and were not considered carcinogenic for rat liver. The increase in number of foci for methylglyoxal was statistically significant at p< 0.05, but the value was within the range of historical control values. A positive result was not found for the other parameter, area of foci. Also, no foci larger than 0.2 mm in diameter were induced without prior diethylnitrosamine initiation. Therefore, the modifying effects of methylglyoxal observed were considered negligible. Because Hasegawa and Ito ( 1992) and Hasegawa et al. ( 1995) both report the number of foci per square centimeter as 7.97 ± 1.91 and the area as 0.49 ± 0.18 square millimeters per square centimeter and likely describe the same data, there has been some reinterpretation of their significance. Body weight and relative and absolute liver weight effects were also reported in Hasegawa et al. ( 1995). Body weight and absolute but not relative liver weight in the methylglyoxal experiment were significantly different from the saline with methylglyoxal controls but were not significantly different from controls that received diethylnitrosamine without methylglyoxal treatment. No effects other than body weight, liver weight, and foci were reported.

Martelli et al. ( 1988) studied the direct acting, initiating, and promoting ability of 0.05% and 0.2% methylglyoxal in drinking water in a series of experiments using male F344 rats given partial hepatectomies during the exposure period, and using the induction of gammaglutamyltranspeptidase ( GGT) positive foci for measurement as a marker of carcinogenic potential. 2 Acetylaminofluorene ( 2 AAF) was used as initiator in some experiments and as promoter in others. N Nitrosodiethylamine was used as an initiation positive control and sodium phenobarbital as a promotion positive control. The number of positive foci was reported in terms of square centimeters and cubic centimeters, and total foci volume was also reported. There were two 9 week study protocols.

To test methylglyoxal as an initiator, it was given for 6 weeks in drinking water with partial hepatectomy at day 7, followed by a 1 week recovery and 2 weeks of exposure to 0.02% 2 acetylaminofluorene in the diet with 1 mL/ kg carbon tetrachloride given by gavage on day 6. To test methylglyoxal as a promoter, the 2 week exposure to 0.02% 2 acetylaminofluorene with carbon tetrachloride at day 7 was given first, followed by the 1 week recovery period, followed by 6 weeks of exposure to methylglyoxal with partial hepatectomy at day 7 into this latter exposure ( day 28 of the experiment). Methylglyoxal was also tested without initiation or promotion.