Metabolism and disposition of [14C]dibromoacetonitrile in rats and mice following oral and intravenous administration.

1. Tissue distribution, metabolism, and disposition of oral (0.2-20 mg/kg) and intravenous (0.2 mg/kg) doses of [2-(14)C]dibromoacetonitrile (DBAN) were investigated in male rats and mice. 2. [(14)C]DBAN reacts rapidly with rat blood in vitro and binds covalently. Prior depletion of glutathione (GSH) markedly diminished loss of DBAN. Chemical reaction with GSH readily yielded glutathionylacetonitrile. 3. About 90% of the radioactivity from orally administered doses of [(14)C]DBAN was absorbed. After intravenous administration, 10% and 20% of the radioactivity was recovered in mouse and rat tissues, respectively, at 72 h. After oral dosing, three to four times less radioactivity was recovered, but radioactivity in stomach was mostly covalently bound. 4. Excretion of radioactivity into urine exceeded that in feces; 9-15% was exhaled as labeled carbon dioxide and 1-3% as volatiles in 72 h. 5. The major urinary metabolites were identified by liquid chromatography-mass spectrometry, and included acetonitrile mercaptoacetate (mouse), acetonitrile mercapturate, and cysteinylacetonitrile. 6.The primary mode of DBAN metabolism is via reaction with GSH, and covalent binding may be due to reaction with tissue sulphydryls.

Reactive metabolites from the bioactivation of toxic methylfurans.

An important mechanism of toxicity of furans involves the cytochrome P-450 monooxygenase-catalyzed bioactivation of the compound in situ directly within the target tissues to highly reactive electrophilic products. The unsaturated aldehydes acetylacrolein and methylbutenedial have been identified as the principal reactive intermediates of 2- and 3-methylfuran, respectively, that are produced and bound covalently to tissue macromolecules in hepatic and pulmonary microsomal systems in vitro.

Estrogenic phenol and catechol metabolites of PCBs modulate catechol-O-methyltransferase expression via the estrogen receptor: potential contribution to cancer risk.

Commercial PCB mixtures have been shown to induce liver tumors in female rats and this effect has been attributed to the effects of PCBs on estrogen metabolism. Catechol metabolites of PCBs are potent inhibitors of COMT activity and are likely to contribute significantly to reduced clearance of genotoxic catechol metabolites of estrogen. The effect of PCB metabolites on COMT expression in cultured cells was investigated to explore potential mechanisms by which PCB exposure alters catechol estrogen clearance. We hypothesize that estrogenic PCB metabolites may contribute to reduction of COMT expression via interaction with the estrogen receptor. To test this hypothesis, human MCF-7 cells were exposed to PCB analogues and the expression of COMT determined. Western blot analysis demonstrated that COMT protein levels were statistically significantly reduced by both the phenolic and the catechol compounds, an effect which was abolished by the anti-estrogen, ICI182780. The above suggests that COMT levels may be reduced by estrogenic PCB metabolites, via interactions between PCB metabolites and the ER. It supports the hypothesis that both phenolic and catechol metabolites of PCBs may contribute to PCB-mediated carcinogenesis through reduction of COMT levels and activities and subsequent reduction in clearance of endogenous and xenobiotic catechols.

Phytol metabolites are circulating dietary factors that activate the nuclear receptor RXR.

RXR is a nuclear receptor that plays a central role in cell signaling by pairing with a host of other receptors. Previously, 9-cis-retinoic acid (9cRA) was defined as a potent RXR activator. Here we describe a unique RXR effector identified from organic extracts of bovine serum by following RXR-dependent transcriptional activity. Structural analyses of material in active fractions pointed to the saturated diterpenoid phytanic acid, which induced RXR-dependent transcription at concentrations between 4 and 64 microM. Although 200 times more potent than phytanic acid, 9cRA was undetectable in equivalent amounts of extract and cannot be present at a concentration that could account for the activity. Phytanic acid, another phytol metabolite, was synthesized and stimulated RXR with a potency and efficacy similar to phytanic acid. These metabolites specifically displaced [3H]-9cRA from RXR with Ki values of 4 microM, indicating that their transcriptional effects are mediated by direct receptor interactions. Phytol metabolites are compelling candidates for physiological effectors, because their RXR binding affinities and activation potencies match their micromolar circulating concentrations. Given their exclusive dietary origin, these chlorophyll metabolites may represent essential nutrients that coordinate cellular metabolism through RXR-dependent signaling pathways.

Differential expression of CYP1A, 2B, and 3A genes in the F344 rat following exposure to a polybrominated diphenyl ether mixture or individual components.

Polybrominated diphenyl ethers (PBDEs), used as flame retardants, have been detected in the environment and in mammalian tissues and fluids. Evidence indicates that PBDE mixtures induce CYPs through aryl hydrocarbon receptor (AhR)-dependent and -independent pathways. The present work has investigated the effects of individual components of a commercial PBDE mixture (DE71) on expression of CYP1A1, a biomarker for activation of the AhR (dioxin-like), and CYP2B and CYP3A, biomarkers for activation of the constitutive androstane and pregnanexreceptors (CAR and PXR), respectively, in the rat. Male F344 rats were dosed orally on three consecutive days with either DE71, PBDE components, 2,2',4,4'-tetraBDE (BDE47), 2,2',4,4',5-pentaBDE (BDE99), 2,2',4,4',5,5'-hexaBDE (BDE153), representative polybrominated dibenzofurans (PBDFs) present in DE71, or reference PCBs. Differential expression of target genes was determined in liver 24 h after the last dose. Quantitative PCR analysis indicated up-regulation of CYP1A1 by DE71; however, the response was weak compared to that for dioxin-like PCB126. Individual PBDE components of DE71 up-regulated CYP1A1 only at the highest administered dose (100 micromol/kg/day). Representative PBDFs efficiently up-regulated CYP1A1; therefore, they, along with other PBDFs and polybrominated dibenzodioxins detected in DE71 and individual PBDE components, may be responsible for most, if not all, dioxin-like properties previously observed for PBDEs. Conversely, PBDEs appear capable of up-regulating CYP2B and CYP3A in rats at doses similar to that for non-dioxin-like PCB153. These results indicate that in vivo PBDE-mediated toxicity would be better categorized by AhR-independent mechanisms, rather than the well-characterized AhR-dependent mechanism associated with exposure to dioxin-like chemicals.

Identification of five hemoglobins in B6C3F1 mice by mass spectrometry and sequence analysis.

The aim of the work is to identify and characterize the hemoglobins found in B6C3F1 mice using mass spectrometry. The primary structures are compared to those reported for BALB/c mice. Individual hemoglobin chains were isolated by reverse-phase high performance liquid chromatography (RP-HPLC). The molecular masses of the globins were determined using electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). The purified globin chains were enzymatically cleaved and the resulting peptides were separated by RP-HPLC. The chains were identified by N-terminal sequencing and mass spectrometry (MALDI). Selected peptides were analysed by Edman degradation. ESI analysis indicates that B6C3F1 mice have two alpha-globin chains (alpha-1 and alpha-2) and at least three beta-globin chains, beta-1, beta-2 and beta-3. This is one additional alpha- and one additional beta-globin chain than reported in the literature for BALB/c mice. Mass and sequence analysis of enzymatically generated peptides showed variations in the amino acid sequence in the alpha-1, alpha-2, beta-2 and beta-3 chains compared to the BALB/c mouse hemoglobins (alpha, beta (minor) and beta (major)). The study showed that mass spectrometry in combination with traditional protein chemistry is able to identify and locate minor protein sequence variations.

Enzyme inhibition and the toxic action of moniliformin and other vinylogous alpha-ketoacids.

The inhibition of two thiamine-requiring enzymes by the potent mycotoxin, moniliformin (1-hydroxycyclobutene-3,4-dione), was investigated. Rat brain transketolase and pyruvate dehydrogenase were inhibited 25 percent by 10-9 M moniliformin. Studies carried out to determine if moniliformin causes enzyme inhibition by reaction with thiamine were negative. Varying the hydroxycyclobutenedione structure by substitution or ring expansion resulted in loss of toxicity and inhibition.

Purpose and guidelines for toxicokinetic studies within the National Toxicology Program.

Toxicokinetic studies undertaken within the National Toxicology Program are intended to aid the design of toxicology and carcinogenicity studies, help interpret the results of toxicology and carcinogenicity studies with respect to the relationship between toxic effects and external exposure, and define the parameters of dose, distribution, metabolism, and elimination that can be used in human risk assessment. Descriptions of two study designs presented here represent the possible extremes in approaches to toxicokinetic studies. The comprehensive approach is geared toward the development of physiology based pharmacokinetic models that relate external exposure to target organ dosimetry and addresses the questions: Is the chemical absorbed? How is the chemical metabolized? Where are the chemical and/or metabolites distributed in the body? What are the elimination rate and route of the chemical? What is the effect of dose on absorption, distribution, metabolism, and elimination? The minimal study design is more limited in scope than the comprehensive design and addresses primarily the issues of absorption, distribution, and elimination of the parent chemical. Study protocols for most chemicals lie somewhere between these two extreme approaches. An increased understanding of the relationships between external exposure, target organ dosimetry, and adverse effects should provide greater confidence in making low-dose extrapolations of human risk. This paper focuses on the collection of data from animal toxicokinetic studies. The construction of comparable models to characterize target organ dosimetry in exposed humans would certainly require the use of human parameter values obtained from human tissue samples and volunteers.

Metabolism and disposition of phenolphthalein in male and female F344 rats and B6C3F1 mice.

A recent 2-year carcinogenicity/toxicology study determined that phenolphthalein (PHTH) is a multisite carcinogen in both mice and rats at all doses evaluated. In response to this finding the metabolism and disposition of PHTH has been evaluated in both F344 rats and B6C3F1 mice at a single oral dose of 800 mg/kg. This dose fell within the range previously found to be carcinogenic in rats and mice. Studies were also performed using 1 and 50 mg/kg doses. At 800 mg/kg recovery of [14C]PHTH after 72 h was near 100% in females but closer to 75% in males. Radioactivity was primarily recovered in the feces in rats (> 90%), while mice excreted 30-40% of administered activity in the urine. There was no significant retention of radioactivity in tissues by 72 h and no significant accumulation of radioactivity in any tissue at any time point. Covalent binding to protein in target tissues, bone marrow and ovary, was at or less than the pmol/mg protein range. The major metabolite was PHTH glucuronide. Three minor metabolites were detected. A sulfate conjugate and and a hydroxylated metabolite were identified by comparison of retention times and 1H NMR and/or mass spectra with synthetic standards. A diglucuronide conjugate was tentatively identified. Biliary elimination was extensive in rats (35% of dose within 6 h); the only product detected in bile was phenolphthalein glucuronide.

Comparative xenobiotic metabolism between Tg.AC and p53+/- genetically altered mice and their respective wild types.

The use of transgenic animals, such as v-Ha-ras activated (TG:AC) and p53+/- mice, offers great promise for a rapid and more sensitive assay for chemical carcinogenicity. Some carcinogens are metabolically activated; therefore, it is critical that the altered genome of either of these model systems does not compromise their capability and capacity for metabolism of xenobiotics. The present work tests the generally held assumption that xenobiotic metabolism in the TG:AC and p53+/- mouse is not inherently different from that of the respective wild type, the FVB/N and C57BL/6 mouse, by comparing each genotype's ability to metabolize benzene, ethoxyquin, or methacrylonitrile. Use of these representative substrates offers the opportunity to examine arene oxide formation, aromatic ring opening, hydroxylation, epoxidation, O-deethylation, and a number of conjugation reactions. Mice were treated by gavage with (14)C-labeled parent compound, excreta were collected, and elimination routes and rates, as well as (14)C-derived metabolite profiles in urine, were compared between relevant treatment groups. Results of this study indicated that metabolism of the 3 parent compounds was not appreciably altered between either FVB/N and TG:AC mice or C57BL/6 and p53+/- mice. Further, expression of CYP1A2, CYP2E1, CYP3A, and GST-alpha in liver of naive genetically altered mice was similar to that of corresponding wild-type mice. Thus, these results suggest that the inherent ability of TG:AC and p53+/- mice to metabolize xenobiotics is not compromised by their altered genomes and would not be a factor in data interpretation of toxicity studies using either transgenic mouse line.

Benzyl acetate carcinogenicity, metabolism, and disposition in Fischer 344 rats and B6C3F1 mice.

Carcinogenesis studies of benzyl acetate (a fragrance and flavoring agent) were conducted in F344 rats and B6C3F1 mice. The chemical was given in corn oil by gavage once daily, 5 days/week for 103 weeks, to groups of 50 animals of each sex and species. For rats the doses were 0, 250, and 500 mg/kg body weight and for mice the doses were 0, 500, and 1000 mg/kg. Mean body weights of control and dosed rats and mice were not affected adversely by benzyl acetate. The survival of control and low dose female mice was lower than that of the high dose group. A genital tract infection may have contributed to the reduced survival. No other significant difference in survival was observed for dosed rats or mice. Benzyl acetate was absorbed from the gastrointestinal tract of rats and mice, with approximately 90% of the administered dose recovered as various metabolites in the urine within 24 h. The primary metabolite was hippuric acid, with minor amounts of a mercapturic acid, and one or more unidentified metabolites. This capacity for absorption, metabolism, and disposition was unaffected by the amount or number of doses administered. Under the conditions of these studies, benzyl acetate administration was associated with an increased incidence of acinar cell adenoma of the exocrine pancreas in male F344/N rats. No evidence of carcinogenicity was found for female F344/N rats. For male and female B6C3F1 mice there was evidence of carcinogenicity, in that benzyl acetate caused an increased incidence of hepatocellular neoplasms (particularly adenomas) and squamous cell neoplasms of the forestomach.

Structure-activity relationships for the in vitro hematotoxicity of N-alkoxyacetic acids, the toxic metabolites of glycol ethers.

Ethylene glycol mono-n-alkyl ethers are a major class of industrial chemicals which cause a wide range of toxic effects in laboratory animals including reproductive and developmental toxicity, as well as hematotoxicity. Alkoxyacetic acids are the major metabolites of ethylene glycol ethers and are considered to be the proximate toxic metabolites. The structure-toxicity relationships of these acids are well documented in the reproductive and developmental systems. Therefore, current studies were conducted to investigate the structure-activity relationships of these acids for hematotoxicity in rat blood in vitro. Results presented here indicate that the effects of various alkoxyacetic acids on rat erythrocytes are qualitatively similar and comprise early swelling followed by hemolysis. The ranking of the activity of these acids was as follows: butoxyacetic acid (BAA) greater than propoxyacetic acid approximately equal to pentoxyacetic acid greater than ethoxyacetic acid greater than methoxyacetic acid. Furthermore, this effect of alkoxyacetic acids was associated with a parallel decrease in blood ATP levels. It is currently unknown if swelling or ATP depletion is the primary effect of these acids. In addition, at equimolar concentrations neither heptanoic, butoxypropionic, nor propoxypropionic acids caused any significant effect on rat erythrocytes in vitro. This suggests that the presence and position of the ether linkage, as it is in BAA, are critical for the development of hematotoxicity. Studies of the relationship between the toxic effect of BAA and its partitioning between erythrocytes and plasma showed that the concentration of [14C]BAA in plasma remained relatively constant while that in the erythrocytes increased as a function of time. This pattern of BAA distribution between plasma and erythrocytes was parallel to erythrocyte swelling. Incubation of BAA with rat blood for 30 min followed by removal of BAA by washing the erythrocytes twice and then continuing the incubation revealed that erythrocyte swelling was not reversible, however, the rate of swelling declined significantly.

Activity of hepatic drug metabolizing enzymes following oxazepam-dosed feed treatment in B6C3F1 mice.

Oxazepam has been determined to be a potent hepatocarcinogen in mice. Evidence in the literature indicates that oxazepam is capable of inducing drug metabolizing enzymes in rodents and an association between enzyme induction and carcinogenesis has been proposed for other compounds such as phenobarbital. We examined the pattern of enzyme induction that occurs under bioassay conditions in male B6C3F1 mice. The results indicate that oxazepam is capable of inducing multiple drug metabolizing enzymes under bioassay conditions. Closer examination of the most induced samples suggests that oxazepam is a phenobarbital-type enzyme inducer.

Tris(2-chloroethyl) phosphate pharmacokinetics in the Fischer 344 rat: a comparison of conventional methods and in vivo microdialysis coupled with tandem mass spectrometry.

The pharmacokinetics of tris(2-chloroethyl) phosphate (TRCP, 20 mg/kg, iv) were investigated in awake male and female and anesthetized male Fischer 344 (F344) rats by conventional (CONV) sampling/detection methods (blood withdrawal with sample workup and analysis for TRCP). TRCP pharmacokinetics were also investigated in anesthetized male F344 rats using a new sampling/detection technique, in vivo microdialysis coupled with tandem mass spectrometry (MD/MS/MS). The concentration of free TRCP in plasma versus time profiles were analyzed using noncompartmental methods to estimate pharmacokinetic parameters. Comparisons of mean parameter estimates were made for (1) awake males versus females in CONV studies (t test, no significant differences, p < or = 0.05) and (2) awake and anesthetized males in CONV studies and anesthetized males in MD/MS/MS studies. There were significant differences (Scheffe's test) for the three groups of male rats, most notably the free TRCP concentration in plasma at early time points in CONV versus MD/MS/MS studies. The contributions of an indwelling jugular cannula, the blood sampling regimen, and the in vitro MD/MS/MS standard calibration curve were investigated. It appears that quantitation of TRCP by mass spectrometry using an in vitro standard calibration is responsible for the difference.

Toxicokinetics of [14C]-saligenin cyclic-o-tolyl phosphate in anesthetized male F-344 rats.

Saligenin cyclic o-tolyl phosphate (SCOTP) has been proposed as the active metabolite of tri-o-cresyl phosphate (TOCP), a neurotoxic organophosphate. TOCP is also toxic to the testis and SCOTP mimics some of this toxicity. The stability of SCOTP in vivo and its uptake by selected tissues has been measured. Total radioactivity and SCOTP-associated radioactivity were determined in male F-344 rats treated i.v. with 1 mg of [14C]-SCOTP/kg. The half-life of SCOTP in blood was 8.0 +/- 1.1 min. Testes, brain, and muscle had lower concentrations of [14C]-SCOTP-derived radioactivity than blood. Liver and kidney had higher concentrations of radioactivity than blood. HPLC analysis of liver, kidney, testes, and blood extracts showed that 2.8, 48, 11, and 18%, respectively, of the radioactivity present at 5 min was SCOTP. The amount of SCOTP declined rapidly, and at 30 min SCOTP could be detected only in kidney. It appears that SCOTP, although reactive, has sufficient stability to be transported from organ to organ. There is no evidence, however, of active uptake of SCOTP from blood by the testes. Evidence was obtained that SCOTP may act as an alkylating agent.

The interaction of methanol, rat-liver S9 and the aromatic amine 2,4-diaminotoluene produces a new mutagenic compound.

Methanol is a widely used solvent for organic compounds and a human toxicant. In our studies of the metabolism of aromatic amines in the Ames/Salmonella assay, we observed a rapid and quantitative conversion of the mutagenic and carcinogenic aromatic amine 2,4-diaminotoluene (2,4-DAT) to a single product. This product was only produced in the presence of methanol, and not other organic solvents. Isolation of this product showed that it was highly mutagenic in Salmonella TA98 with S9 activation. Characterization of the product of the interaction of methanol and 2,4-DAT indicated that methanol is activated to a reactive intermediate, probably formaldehyde, by the 9000 X g supernatant used in the Ames/Salmonella assay. The formaldehyde subsequently reacts with 2,4-DAT to form the mutagenic product, identified as bis-5,5'(2,4,2',4'-tetraaminotolyl)methane. Results of this study demonstrate that methanol may be an inappropriate solvent for mutation and metabolism studies of aromatic amines and possibly other chemicals, and that solvent-xenobiotic interactions may in some cases lead to the misinterpretation of results.

Toxicology and carcinogenesis studies of microencapsulated trans-cinnamaldehyde in rats and mice.

trans-Cinnamaldehyde is a widely used natural ingredient that is added to foods and cosmetics as a flavoring and fragrance agent. Male and female F344/N rats and B6C3F(1) mice were exposed to microencapsulated trans-cinnamaldehyde in the feed for three months or two years. All studies included untreated and vehicle control groups. In the three-month studies, rats and mice were given diets containing 4100, 8200, 16,500, or 33,000 ppm trans-cinnamaldehyde. In rats, feed consumption was reduced in all exposed groups. In mice, feed consumption was reduced in the highest dose groups. Body weights of all treated males were less than controls. Body weights were reduced in female rats exposed to 16,500 or 33,000 ppm and female mice exposed to 8200 ppm or greater. All rats survived to the end of the study but some male mice in the highest dose groups died due to inanition from unpalatability of the dosed feed. The incidence of squamous epithelial hyperplasia of the forestomach was significantly increased in rats exposed to 8200 ppm or greater and female mice exposed to 33,000 ppm. In mice, the incidence of olfactory epithelial degeneration of the nasal cavity was significantly increased in males and females exposed to 16,500 ppm and females exposed to 33,000 ppm. In the two-year studies, rats and mice were exposed to 1000, 2100, or 4100 ppm trans-cinnamaldehyde. Body weights were reduced in mice exposed to 2100 ppm and in rats and mice exposed to 4100 ppm. In rats, hippuric acid excretion was dose proportional indicating that absorption, metabolism, and excretion were not saturated. No neoplasms were attributed to trans-cinnamaldehyde in rats or mice. Squamous cell papillomas and carcinomas of the forestomach were observed in male and female mice but the incidences were within the NTP historical control range and were not considered to be related to trans-cinnamaldehyde exposure.

Distribution and metabolism of (5-hydroxymethyl)furfural in male F344 rats and B6C3F1 mice after oral administration.

(5-Hydroxymethyl)furfural (HMF), a heat-induced decomposition product of hexoses, is present in food and drink. Recent reports have shown HMF to be an in vitro mutagen after sulfate conjugation and to be a promoter as well as a weak initiator of colonic aberrant foci in rats. In order to investigate the metabolic activation further and to provide information for HMF toxicology studies, the disposition of [14C]-HMF has been investigated in male F344 rats and B6C3F1 mice following po administration of either 5, 10, 100, or 500 mg/kg. Tissue distribution results indicated that absorption of HMF was rapid in male rats and mice and that tissue concentrations in male mice at the earliest time point are not linearly proportional to dose. Excretion was primarily via the urine in both, with 60-80% of the administered dose excreted by this route in 48 h. Tissue/blood ratios of HMF-derived radioactivity were greater than 1 for liver and kidney. Three metabolites were identified and quantitated in urine. Formation of one of the metabolites, N-(5-hydroxymethyl-2-furoyl)glycine, was inversely proportional to dose in rats but not mice. None of the metabolites were sulfate conjugates nor likely to be formed from sulfate conjugates. There were relatively low levels of nonextractable radioactivity in liver, kidney, and intestines, indicating that some reactive intermediate(s) may be formed.

Disposition of 2-methylimidazole in rats.

2-Methylimidazole (2-MI), widely used as a chemical intermediate, is also present in cigarette smoke and may form in food and forage as a result of ammoniation of simple sugars. 2-MI has been shown to be neurotoxic in several animal species and to alter serum levels of T3, T4, and thyroid-stimulating hormone (TSH) in the rat, apparently leading to hyperplasia of thyroid follicular cells. In order to better characterize 2-MI-induced toxicity, the disposition of [2-(14)C]-2-MI has been investigated following p.o. administration of either 5, 50, or 150 mg/kg to male F344 rats. Excretion data indicated that absorption of 2-MI was both rapid and proportional to dose in the range studied. Approximately 90% of the total dose was eliminated in urine within 24 h. Most of the remaining 14C was excreted in feces and as expired 14CO2. Excretion data were similar following i.v. administration of 5 mg/kg. Little or no enterohepatic circulation of compound occurred, since biliary excretion of 2-MI-derived 14C was negligible. Approximately 70% of the 14C excreted in urine, following all dosing, consisted of parent compound. High-performance liquid chromatography (HPLC) chromatograms for all treatment groups were similar, indicating that metabolism of 2-MI in rats was not affected by dose or route of administration.

Analysis of dimethyl hydrogen phosphite and its stability under simulated physiological conditions.

In a study by the National Toxicology Program, dimethyl hydrogen phosphite (DMHP) was shown to cause dose-related neoplastic lesions in the lungs and forestomachs of Fischer 344 rats. This investigation was carried out to study the stability of this carcinogen under conditions similar to those that the chemical may encounter in the physiological environment. To carry out these studies, capillary gas chromatography was utilized to analyze DMHP. The method was linear over a range of 10 to at least 1000 ng. High-performance liquid chromatography (HPLC) also was used to analyze DMHP and its degradation products. In aqueous solutions, DMHP was stable for a period of time before degradation began. Once the process began, degradation continued until 10-20% of the original concentration was reached and further decomposition was minimal. At 10% concentration in 0.1M phosphate buffer, pH 7.4, DMHP was stable for 3.6 h at 37 degrees C. This stability period increased at lower temperature, at lower DMHP concentration, and in slightly more alkaline buffer (pH 8). After the stability period, DMHP disappeared from the solution and this disappearance followed a first order kinetics with a rate dependent upon temperature, concentration of DHMP, and the pH of the solution. The half-time of disappearance of DMHP under the conditions mentioned above was 2.4 h, which increased at lower temperature, at lower DMHP concentration, and in slightly more alkaline buffer (pH 8). The decomposition products of DMHP were identified by HPLC and proton nuclear magnetic resonance (1H-NMR) spectroscopy as methanol, monomethyl hydrogen phosphite, and orthophosphorous acid.