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Dichloromethane (DCM) is a high production volume chemical (>1000 t/a) mainly used as an industrial solvent. Carcinogenicity studies in rats, mice and hamsters have demonstrated a malignant tumor inducing potential of DCM only in the mouse (lung and liver) at 1000-4000 ppm whereas human data do not support a conclusion of cancer risk. Based on this, DCM has been classified as a cat. 2 carcinogen. Dose-dependent toxicokinetics of DCM suggest that DCM is a threshold carcinogen in mice, initiating carcinogenicity via the low affinity/high capacity GSTT1 pathway; a biotransformation pathway that becomes relevant only at high exposure concentrations. Rats and hamsters have very low activities of this DCM-metabolizing GST and humans have even lower activities of this enzyme. Based on the induction of specific tumors selectively in the mouse, the dose- and species-specific toxicokinetics in this species, and the absence of a malignant tumor response by DCM in rats and hamsters having a closer relationship to DCM toxicokinetics in humans and thus being a more relevant animal model, the current classification of DCM as human carcinogen cat. 2 remains appropriate., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Exposure to dichloromethane (DCM), a commonly used chlorinated solvent in industrial settings and for the production of many household products, reportedly elicits detrimental effects in animals and humans. The present study investigated the protective role of dietary quercetin on DCM-induced hepatorenal damage in rats. Experimental rats were orally administered with DCM (150 mg/kg) and 30 min later with quercetin at 10, 20 and 40 mg/kg or none for 7 consecutive days. The results indicated that DCM-mediated significant (p<0.05) increases in serum alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transferase and alkaline phosphatase activities as well as urea and creatinine levels were dose-dependently normalized to the control values in rats co-treated with quercetin. Further, quercetin co-treatment ameliorated DCM-mediated decrease in the hepatic and renal activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferase as well as glutathione level in the treated rats. Moreover, quercetin co-treatment markedly reduced lipid peroxidation level and protected against histological changes in liver and kidney of the treated rats. Taken together, quercetin abrogated hepatorenal oxidative damage in DCM-treated rats via improvement of antioxidant status and suppression of oxidative damage.

Objective: The cytoprotective enzyme heme oxygenase 1 (HO-1) is highly up-regulated in acute pancreatitis (AP). In this study, we tested its metabolites as potential therapeutic agents for AP in rats., Methods: Acute necrotizing pancreatitis was induced by retrograde intraductal injection of sodium taurocholate in rats. Biliverdin hydrochloride (BV HCl) (50 μmol/kg subcutaneously), the carbon monoxide, donor methylene chloride (MC) (500 mg/kg orally), or iron-chelating desferrioxamine (DFO) (125 mg/kg subcutaneously) were administered in a therapeutic manner starting with the first dose 4 hours after taurocholate injection to mimic the effects of HO-1 metabolites., Results: Administration of BV HCl, MC, or DFO showed significant reduction of inflammatory activity in comparison to controls leading to lower myeloperoxidase activity in the pancreas, less edema, lower ascites volumes, and preservation of tissue integrity (P < 0.05). Administration of either BV HCl or MC markedly increased 5-day survival rate (70% and 75% vs 40%; P < 0.05), whereas DFO had no significant effect on survival (60%). When given in therapeutic manner, all 3 substances led to diminished nuclear factor κB activity in the pancreas (P < 0.05)., Conclusions: Therapeutic use of BV HCl and MC led to marked reduction of mortality in experimental pancreatitis. Thus, HO-1 metabolites may present a novel therapeutic approach in AP treatment.

Kielmeyera coriacea Mart. (Calophyllaceae) is known popularly as "Pau Santo". The hydroethanolic extract (HE) of Kielmeyera coriacea stems and its semi-pure dichloromethane (DCM) constituent produced an antidepressant-like effect in rats. The purpose of this study was to investigate the effects of repeated administration (21 days) by gavage of the DCM fraction (5, 10 or 15mg/kg) in rats submitted to the elevated T-maze (ETM), a model of generalized anxiety and panic disorders. The tricyclic antidepressant imipramine (15mg/kg) was used as a positive control. Rat locomotion was assessed using the open field test (OFT) following each drug treatment. The 2-hydroxy-1-methoxyxanthone (1), aucuparin (2), swertinin (3), 1,3,7-trihydroxy-2-(3-methylbut-2-enyl)-xanthone (4) and 1,3,5-trihydroxy-2-(3-methylbut-2-enyl)-xanthone (5) were identified in DCM fraction, and suggest that the xanthone (4) is related with the antidepressant-like profile of this plant. Pharmacological evaluation showed that DCM fraction (10 and 15 mg/kg) decreased the inhibitory avoidance latency from the closed arm and increased the one-way escape latency from the open arm in the ETM, which is indicative of anxiolytic and panicolytic effects, respectively, as occurs with the positive control, imipramine (15 mg/kg), when compared to their control group (vehicle). Locomotor activity was not significantly altered by the different treatments. This study suggests that the DCM fraction from stems of Kielmeyera coriacea can be an important therapeutic alternative in the treatment of anxiety disorders, such as generalized anxiety and panic disorders., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Recent studies suggest that exogenously administered CO is beneficial for the resolution of acute pulmonary inflammation. In this study, we assessed the role of CO donor, methylene chloride (MC), on modulation of lung inflammation during sepsis. Acute lung injury in Sprague-Dawley rats was induced by cecal ligation and perforation (CLP). MC (100mg/kg) was intragastrically administered 2h before CLP induction. Lung tissues and lavage samples were isolated for biochemical determinations and histological measurements 10h after CLP operation. In addition, we investigated survival rate with the other 40 rats. Intragastric administration with MC significantly decreased morbidity and mortality of CLP-induced ALI as confirmed by blinded histological changes, myeloperoxidase activity, mortality, and the content of TNF-alpha and IL-10. This protective effect could be abolished by an MC inhibitor, disulfiram. These results suggested that MC has obvious protective effects against CLP-induced ALI in rats. The mechanism of the protective effects partly involves modulating inflammatory mediators., ((c) 2010 Elsevier B.V. All rights reserved.)

Morphological changes in hair subjected to decontamination protocols were evaluated by scanning electron microscopy (SEM) as part of the National Laboratory Certification Program's (NLCP) efforts to develop proficiency testing materials in support of Federal Workplace Drug Testing programs. Hair from five different donors was evaluated. Hair samples were subjected to three decontamination protocols: (1) aqueous phosphate buffer, (2) methanol or (3) methylene chloride as models for aqueous, alcohol and polar organic solvent protocols, respectively. Under these protocols, samples of hair were treated for 225 min (aqueous), 15 min (alcohol), or 15 min (polar organic). After decontamination, hair strands were sputter coated with gold/palladium (AuPd) and observed by SEM. Modest lifting of cuticle scales was observed in hair treated with methanol and methylene chloride consistent with some changes to the cell membrane complex (CMC) between cuticle scales. Damage resulting from aqueous buffer treatment ranged from substantial degradation to apparent complete loss of cuticle scales. Fracture structures consistent with cuticle damage were also observed. Each decontamination protocol had a different impact on the cuticle of the hair shaft.

Experience with dose response and mechanisms of toxicity has shown that multiple mechanisms may exist for a single agent along the continuum of the full dose-response curve. It is highly likely that critical, limiting steps in any given mechanistic pathway may become overwhelmed with increasing exposures, signaling the emergence of new modalities of toxic tissue injury at these higher doses. Therefore, dose-dependent transitions in principal mechanisms of toxicity may occur, and could have significant impact on the interpretation of reference data sets for risk assessment. To illustrate the existence of dose-dependent transitions in mechanisms of toxicity, a group of academic, government, and industry scientists, formed under the leadership of the ILSI Health and Environmental Sciences Institute (HESI), developed a series of case studies. These case studies included acetaminophen, butadiene, ethylene glycol, formaldehyde, manganese, methylene chloride, peroxisome proliferator-activated receptor (PPAR), progesterone/hydroxyflutamide, propylene oxide, vinyl acetate, vinyl chloride, vinylidene chloride, and zinc. The case studies formed the basis for technical discourse at two scientific workshops in 2003.

Background: Modeling of pharmacokinetic parameters and pharmacodynamic actions requires knowledge of the arterial blood concentration. In most cases, experimental measurements are only available for a peripheral vein (usually antecubital) whose concentration may differ significantly from both arterial and central vein concentration., Methods: A physiologically based pharmacokinetic (PBPK) model for the tissues drained by the antecubital vein (referred to as "arm") is developed. It is assumed that the "arm" is composed of tissues with identical properties (partition coefficient, blood flow/gm) as the whole body tissues plus a new "tissue" representing skin arteriovenous shunts. The antecubital vein concentration depends on the following parameters: the fraction of "arm" blood flow contributed by muscle, skin, adipose, connective tissue and arteriovenous shunts, and the flow per gram of the arteriovenous shunt. The value of these parameters was investigated using simultaneous experimental measurements of arterial and antecubital concentrations for eight solutes: ethanol, thiopental, 99Tcm-diethylene triamine pentaacetate (DTPA), ketamine, D2O, acetone, methylene chloride and toluene. A new procedure is described that can be used to determine the arterial concentration for an arbitrary solute by deconvolution of the antecubital concentration. These procedures are implemented in PKQuest, a general PBPK program that is freely distributed http://www.pkquest.com., Results: One set of "standard arm" parameters provides an adequate description of the arterial/antecubital vein concentration for ethanol, DTPA, thiopental and ketamine. A significantly different set of "arm" parameters was required to describe the data for D2O, acetone, methylene chloride and toluene - probably because the "arm" is in a different physiological state., Conclusions: Using the set of "standard arm" parameters, the antecubital vein concentration can be used to determine the whole body PBPK model parameters for an arbitrary solute without any additional adjustable parameters. Also, the antecubital vein concentration can be used to estimate the arterial concentration for an arbitrary input for solutes for which no arterial concentration data is available.

Background: Lichen simplex chronicus is a troublesome intractable itchy dermatosis, which may persist despite intensive topical treatments. Recently it has been demonstrated that topical aspirin solution with dichloromethane has a significant antipruritic effect in an experimentally induced itch., Objective: The aim of this double-blind, crossover placebo trial was to evaluate the efficacy of this solution in the treatment of lichen simplex chronicus., Methods: Twenty-nine patients with lichen simplex chronicus of at least 3 months' duration that did not respond to topical corticosteroids were randomized in a double-blind fashion to receive aspirin/dichloromethane solution in treatment period 1 for 2 weeks followed by placebo in treatment period 2 or placebo followed by aspirin in period 2 with a crossover design after a 2-week washout. The patients rated the pruritus intensity before and during therapy with a visual analog scale; a blinded investigator performed photographic assessment., Results: A significant therapeutic response was achieved in 11 (46%) of the patients who completed the study compared with 3 patients (12%) receiving placebo. Overall, aspirin-treated patients experienced an average decrease in the visual analog scale of 2.18 +/- 2.86 versus 0.69 +/- 2.31 of those receiving placebo. The difference between the 2 treatments for week 2 was significant (P =.03)., Conclusion: The study suggests that topical aspirin/dichloromethane might be a practical treatment for lichen simplex chronicus.

A 22-year-old white male was found dead at his working place in a car lacquering company. He had removed lacquer residues by using a solvent containing dicloromethane (DCM) without using a gas mask. Pathology revealed signs of asphyxiation with obvious petechial bleedings and expressed microthrombosis of the pulmonary arteries. Toxicological analysis showed excessive concentrations of DCM which are inhaled due to exposure of extreme air concentrations.

We have studied the effect of the solvent system during preparation on the morphology, encapsulation efficiency, and release characteristics of enoxacin microparticles intended for localized delivery to the bone for the treatment of bone infections. Microparticles of enoxacin were formulated using poly(glycolic acid-co-DL-lactic acid) (PGLA) of different viscosity grades by the solvent-evaporation technique. Microparticles prepared with pure dichloromethane had smoother surfaces and less tendency to aggregate than microparticles prepared with dichloromethane-acetone solvent mixtures, which had porous surfaces. Approximately 65% of the microparticles prepared with pure dichloromethane were < 125 microm in diameter compared with 16% (approx.) of microparticles prepared with dichloromethane-acetone mixtures. Increasing the proportion of acetone from dichloromethane-acetone, 10:0, to dichloromethane-acetone, 1:1, resulted in an increase in encapsulation efficiency from 25 to 37%, and an increase in the yield of microparticles harvested from 39 to 51%. Although a further increase in the amount of acetone to dichloromethane-acetone, 1:9, had no significant effect on the yield, aggregation, or fraction of microparticles below 125 microm in diameter, the encapsulation efficiency increased to 56%. Approximately 55% of enoxacin was released in 24 h for microparticles prepared with dichloromethane-acetone, 1:9, compared with 100% release in 10h and 2h for microparticles of the same size range prepared with dichloromethane-acetone, 1:1, and dichloromethane-acetone, 10:0, respectively. The results suggest that the composition of the dichloromethane-acetone solvent system significantly influences the encapsulation efficiency and the rate of release of enoxacin from microparticles. This is important for the formulation of sustained-release enoxacin microparticles for the localized treatment of osteomyelitis.

Several descriptive studies have reported the occurrence of infra-additive and supra-additive toxic interactions in rodents given high doses of chemicals by routes different from anticipated human exposures. In order to assess the relevance of such rodent data on chemical interactions for humans, the route, species, and dose extrapolations need to be conducted on the basis of proven/hypothetical interaction mechanisms. The present study initially developed a physiologically based model of the toxicological interaction reported in rats receiving high oral doses of dichloromethane (DCM) and toluene (TOL). This predictive model was then used to asses the relevance of DCM-TOL interaction for humans exposed to threshold limit values (TLVs) of these chemicals, following the conduct of the various, essential extrapolations (i.e., rat to human, oral to inhalation, high dose to low dose). The interaction modeling approach involved (i) obtaining validated rat and human physiologically based pharmacokinetic (PBPK) models for TOL and DCM from the literature, and (ii) linking them via the modified Michaelis-Menten equation accounting for hypothetical mechanisms of interactions (no interaction, competitive inhibition, noncompetitive inhibition, and uncompetitive inhibition). Of the various interaction mechanisms investigated, the noncompetitive and uncompetitive metabolic inhibitions were found to adequately describe the reduction of carboxyhemoglobinemia (COHB) observed in rats during combined exposures (18.8 mmol/kg TOL, +6.2 mmol/kg DCM, po; 0.005 mmol/kg TOL, ip +5000 ppm DCM, 1 hr). The simulation model, based on noncompetitive and uncompetitive inhibition mechanisms, suggests that only < 10% reduction in the area under the COHB vs time curve (AUCCOHB) is likely to occur in humans exposed to the current TLVs of DCM and TOL (compared to AUCCOHB resulting from an 8-hr exposure to TLV of DCM alone). The present modeling approach, based on hypothetical mechanisms of interaction, then indicates that rodent data on DCM-TOL interaction are not relevant for humans, particularly with respect to the COHB effect. The application of this kind of a predictive modeling approach should be useful in screening the available reports on chemical interactions for identifying those of greater concern at relevant human exposure levels (RfD, RfC, TLV).

Previous research showed that acute exposure to dichloromethane (DCM) produced a selective reduction in peak N30 of flash evoked potentials (FEPs) in rats. In contrast, acute exposures to p-xylene or toluene selectively reduced FEP peak N160. The present experiments compared the effects of DCM (log P = 1.25; oil:water partition coefficient), 1,3-dichloropropane (DCP; log P = 2.00), and 1,2-dichlorobenzene (DCB; log P = 3.38) on FEPs recorded from adult Long-Evans rats. Before administration of test compounds, FEPs were recorded for five daily sessions to develop FEP peak N160. Test compounds were dissolved in corn oil and administered i.p. at doses based on proportions of their LD50 values. The doses were: DCM, 0, 57.5, 115, 230, or 460 mg/kg; DCP, 0, 86, 172, 343, or 686 mg/kg; and DCB, 0, 53, 105, 210, or 420 mg/kg. Testing times after dosing varied among compounds and were based on pilot studies to measure both the times of peak effect and recovery. Each solvent produced significant changes in the latency and amplitude of multiple components of the FEP waveforms. However, the predominant effect of DCM was to reduce the amplitude of peak N30 (ED50 = 326.3 mg/kg), that of DCP was to reduce both peaks N30 (ED50 = 231.0 mg/kg) and N160 (ED50 = 136.8 mg/kg), and that of DCB was to reduce peak N160 (ED50 = 151.6 mg/kg). There was no consistent relationship between log P values and the potency of the compounds to alter FEP peaks N30 and N160. The results suggest that organic solvents have multiple acute effects on the function of the central nervous system, which are not predictable solely by the compound's lipid solubility.

B6C3F1 mice exposed to high dose levels of methylene chloride by inhalation for 2 years had an elevated incidence of liver and lung tumours. These tumours were not increased in rats or hamsters exposed under the same or similar conditions. This paper gives an overview of research conducted over the last 10 years into the mechanism of action of methylene chloride as a mouse carcinogen and into the relevance of the mouse data to humans exposed to this chemical. Data are presented on the comparative metabolism and pharmacokinetics of methylene chloride in mice, rats, hamsters and humans, on the toxicity of methylene chloride to the target organs in the mouse, and on the genotoxicity of methylene chloride in vitro and in vivo. The enzyme which activates methylene chloride to its carcinogenic form has been isolated, sequenced, and cloned, and its distribution studied within cells, organs and between species. Evidence has been obtained to show the methylene chloride caused cancer in mice as a result of interactions between metabolites of the glutathione S-transferase pathway and DNA. Damage to mouse lung Clara cells and increased cell division are believed to have influenced the development of the lung tumours. The species specificity was a direct consequence of the very high activity and specific cellular and nuclear localisation of a theta class glutathione S-transferase enzyme which was unique to the mouse. Consequently, DNA damage was not detectable in rats in vivo, or in hamster and human hepatocytes exposed to cytotoxic dose levels of methylene chloride in vitro. These results provide evidence that the mouse is unique in its response to methylene chloride and that it is an inappropriate model for human health assessment.

An inhalation study utilizing over 1400 female B6C3F1 mice was undertaken to study mechanistic factors associated with liver and lung tumor induction following exposure to 2000 ppm of methylene chloride. Mice were exposed to methylene chloride (treated) or chamber air (controls) 6 h per day, for varying durations up to 104 weeks. Several interim sacrifices and 'stop exposures' were included. Exposure to 2000 ppm methylene chloride caused an increase in liver and lung neoplasia in the absence of overt cytotoxicity. Measurement of replicative DNA synthesis done after 13, 26, 52 and 78 weeks of exposure showed a significant decrease in the hepatocyte labeling index at 13 weeks. Replicative DNA synthesis in pulmonary airways after 1, 2, 3, 4, 13 and 26 weeks of exposure to methylene chloride was significantly lower than in air-exposed controls. Likewise, the increase in tumor induction in treated mice was not associated with increased replicative DNA synthesis in liver foci or in alveolar parenchyma. The frequency and pattern of H-ras gene activation were similar in control and methylene chloride-induced liver neoplasms. Similarly, the frequency and pattern of K-ras activation in lung neoplasms were not altered by exposure to methylene chloride. Early exposure to methylene chloride for only 26 weeks was sufficient to cause an increase in lung tumors by 2 years, suggesting that methylene chloride may cause early and persistent loss of growth control in lung cells. This implies that risk management strategies should be aimed at minimizing or eliminating exposure to methylene chloride. Liver neoplasms continued to increase in incidence and multiplicity as exposure continued, suggesting that methylene chloride-induced hepatocarcinogenesis is facilitated by continuing exposure to methylene chloride. Since methylene chloride is a more potent inducer of lung than liver neoplasia, it is recommended that health risk assessment be based on the lung data. While no novel molecular lesions have been found to explain the induction of lung and liver neoplasia in mice, ongoing studies may identify other molecular changes that are important in the genesis of these neoplasms. Hence, it may be necessary to revise risk assessment and management strategies in light of future research findings.

Single exposures of mice to methylene chloride (MC) cause vacuolation and necrosis of the bronchiolar Clara cells which subsequently recover normal morphology on continued exposure. Both cytochrome P-450 (CYP)- and glutathione S-transferase (GST)-dependent metabolism of MC are known to occur. The current studies have investigated the metabolism of MC in mouse lung using inhibitors of both GST and CYP-dependent routes of metabolism, the consequences of metabolic inhibition on the Clara cell vacuolation, and any changes in cell proliferation, assessed in vitro, in Clara cells cultured from exposed individuals. Vacuolated bronchiolar cells were seen in mice exposed to 2000 and 4000 ppm MC but were not seen at lower concentrations, while addition of the CYP inhibitor, piperonyl butoxide, significantly reduced the bronchiolar cell vacuolation seen following exposure to 2000 ppm MC. Treatment of mice with the glutathione depletor, buthionine sulphoximine, had no effect on the number of vacuolated bronchiolar cells following MC. Exposure of mice to 1000 ppm MC and above for 6 h caused a burst of DNA synthesis in bronchiolar Clara cells cultured in vitro from the lungs of exposed animals. The results suggest that the Clara cell vacuolation following MC exposure is mediated via CYP metabolism, that depression of the CYP metabolic pathway occurs following exposure, and that Clara cell vacuolation may have a priming role in stimulating cell proliferation in the unaffected cell population.

The parameters in a physiologically based pharmacokinetic (PBPK) model of methylene chloride were varied systematically, and the resulting variation in a number of model outputs was determined as a function of time for mice and humans at several exposure concentrations. The importance of the various parameters in the model was highly dependent on the conditions (concentration, species) for which the simulation was performed and the model output (dose surrogate) being considered. Model structure also had a significant impact on the results. For sensitivity analysis, particular attention must be paid to conservation equations to ensure that the variational calculations do not alter mass balance, introducing extraneous effects into the model. All of the normalized sensitivity coefficients calculated in this study ranged between -1.12 and 1, and most were much less than 1 in absolute value, indicating that individual input errors are not greatly amplified in the outputs. In addition to ranking parameters in terms of their impact on model predictions, time-dependent sensitivity analysis can also be used as an aid in the design of experiments to estimate parameters by predicting the experimental conditions and sampling points which will maximize parameter identifiability.

In conducting reviews or meta-analyses, epidemiologists frequently must reconcile conflicting results. This paper addresses heterogeneity in nonexperimental studies. The emphasis is on simple exploratory methods rather than formal approaches. Five examples illustrate how quantitative concordance among studies is possible, even when measured effects appear discrepant. The examples concern ethylene oxide and leukemias, methylene chloride and liver cancer, saccharin and bladder cancer, prenatal lead exposure and birthweight, and aspirin and bleeding tendencies in labor and delivery. Data examined here indicate that differences in dose levels frequently explain heterogeneous effect measures, often outweighing other sources of variability among studies. We present simple methods for combining dose information from the study of interest with dose-response data from other epidemiologic studies or animal studies to derive plausible hypothesized effect levels. These plausible effect sizes are the measures of association that would be predicted, for the actual exposures, by extrapolating from other studies with possibly differing exposure levels. Post hoc power calculations and comparisons of confidence intervals for overlap to reconcile "positive" and "null" studies may be misleading, since these approaches assume a uniform true association obscured by random fluctuations only. Whenever it can be estimated, a plausible effect size should be the starting point to assess findings of either positive or null studies. Without such calculations, comparisons among conflicting studies may not be meaningful.

Andersen et al. and Reitz et al. have developed physiologically based pharmacokinetic models for the human metabolism of methylene chloride (dichloromethane; DCM) and have advanced the hypothesis that the carcinogenicity of DCM is related to target organ metabolism of DCM by glutathione S-transferase (GST). The models included physiological parameters appropriate for humans at rest and metabolic parameters based on average rates of DCM metabolism. Increasing the model parameters describing cardiac output, alveolar ventilation, and blood flows to tissues from resting values to values consistent with light work conditions, and assuming a 25 ppm exposure for an 8-hr work day, increases the estimated GST-metabolized dose for human liver by a factor of 2.9 compared to the GST-metabolized does estimated of Reitz et al. These modifications also increase the GST-metabolized dose to the lung by 2.4-fold. If the model is also modified to reflect individual variation in DCM metabolism (in addition to the modifications for light work conditions), the estimated GST-metabolized dose for human liver ranges from 0 to as much as 5.4-fold greater than the dose estimated by Reitz et al. The GST-metabolized dose to the lung ranges from 0 to as much as 3.6-fold greater than the dose estimated by Reitz et al. These results indicate that some occupationally-exposed individuals may receive GST-metabolized doses several-fold greater than the Reitz et al. human dose estimates.

In the National Toxicology Program 2-year inhalation study of dichloromethane (DCM), there was a significant increase in pulmonary neoplasms in female B6C3F1 mice exposed to 2000 ppm (overall rates of 30/48 versus 5/50 in control). Replicative DNA synthesis was examined to evaluate the potential role of treatment-induced lung cell proliferation on pulmonary carcinogenicity. Tritiated thymidine incorporation was assessed in methacrylate plastic sections after 1, 2, 3, or 4 weeks of inhalation exposure to 2000 ppm or 8000 ppm DCM. Similar measurements of labeling indexes were made after 13 and 26 weeks of exposure to 2000 ppm DCM using bromodeoxyuridine as the labeling agent. In all cases the labeling agent was delivered over a 6-day period using osmotic minipumps. The labeling index (LI) of bronchiolar epithelium (two branches proximal to the terminal bronchiole) of mice exposed to 2000 ppm DCM for 2-26 weeks decreased to 40-60% of the control. Terminal bronchioles showed a similar decrease in LI. Mice exposed to 8000 ppm DCM had a less dramatic decrease in LI. No pathological change was found in the exposed lungs. It is concluded that inhalation exposure to DCM for up to 26 weeks reduces cell turnover of bronchiolar cells in female B6C3F1 mice.

Methylene chloride is a high production chemical used in a variety of applications resulting in estimated occupational and consumer exposures of at least one million people per day. Results of previously reported chronic evaluations of inhaled methylene chloride indicated that it caused mammary tumors in Fischer 344 rats and neoplasia in the lungs and liver of B6C3F1 mice. Mechanism(s) for methylene chloride-induced carcinogenesis have not been adequately elucidated. In this paper we describe the histologic evaluation of animals at a number of intermittent times for the purposes of assessing the progressive development of liver and lung neoplasia. Additionally, a series of stop-exposure treatments was conducted to evaluate the role of different methylene chloride exposure durations on the induction of hepatic and pulmonary neoplasia in female mice. Inhalation exposure to 2000 p.p.m. methylene chloride for 6 h per day, 5 days per week, for 104 weeks resulted in an 8-fold increase in the incidence of exposed animals having a lung adenoma or carcinoma (63 versus 7.5%; P < 0.01) and a 13-fold increase in the total number of pulmonary adenomas and carcinomas per animal at risk (0.97 versus 0.075; P < 0.01). This exposure also caused a 2.5-fold increase in the incidence of mice having liver tumors (69 versus 27%; P < 0.01) and a 3-fold increase in the total number of hepatic adenomas and carcinomas per animal at risk (1.34 versus 0.46; P < 0.01). Methylene chloride exposure hastened the first appearance of lung tumors (by 1 year) compared to that observed in control animals; chemical-induced and spontaneous liver tumors first occurred simultaneously. A shorter exposure duration was sufficient to attain maximal numbers of lung tumors than that needed for a maximal liver tumor burden. Lung tumor multiplicity was substantially increased by having additional time after cessation of the chemical treatment. This contrasts with the findings in liver, where additional post-exposure latency time did not effect tumor multiplicity compared to that of mice evaluated immediately after cessation of exposure. The incidence of lung alveolar hyperplasia in methylene chloride exposed animals was very low, even in tumor-bearing animals and the hyperplasias were not seen until at least 13 weeks after appearance of adenomas and carcinomas. Thus, the genesis of methylene chloride induced lung tumors in B6C3F1 mice is not preceded by overt cytotoxicity, enhanced cell proliferation nor observed hyperplasia.(ABSTRACT TRUNCATED AT 400 WORDS)

We have used methylene chloride as a model to study cellular and molecular processes responsible for liver tumor induction by chlorinated hydrocarbons. Because of current interest in the role of enhanced cell proliferation in tumor induction, measurement of S-phase hepatocytes was incorporated into recently conducted toxicity and carcinogenicity studies. In prechronic studies, female B6C3F1 mice were exposed to 0, 1000, 2000 or 8000 p.p.m. methylene chloride by inhalation, 5 days per week, for up to 4 weeks followed by a 1 and 2 week recovery period. Mice exposed to concentrations of 2000, 4000 or 8000 p.p.m. methylene chloride had sustained increased liver weight commencing after 1 week of exposure and returning to normal after the 1 or 2 week recovery period. The increased liver weight was attributed to hepatocellular hypertrophy secondary to intracellular glycogen accumulation. Tritiated thymidine was administered by osmotic minipumps to label S-phase hepatocytes over a 6 day period. At most intervals examined there was decreased hepatocyte labeling in mice exposed to methylene chloride. However, there was a transitory increased number of S-phase hepatocytes observed at the 2 week interval in the 1000, 4000 and 8000 p.p.m. methylene chloride groups. In a chronic study, female mice were exposed to 2000 p.p.m. methylene chloride for up to two years. Following labeling with BRDU using 6 day minipumps, a statistically significant decrease in S-phase hepatocytes was observed after 13 weeks of methylene chloride exposure. A minor increased labeling index (LI) observed at 52 weeks was not considered to be a methylene chloride treatment-related effect. Retrospective immunohistochemical staining for proliferating cell nuclear antigen (PCNA) in liver sections containing foci of cellular alteration allowed demonstration of S-phase hepatocytes in these clonally expanded preneoplastic lesions. While foci frequently had higher LI's than surrounding normal hepatocytes, there was no difference in the mean LI of foci from methylene chloride-treated mice versus foci occurring spontaneously in control mice. The absence of a sustained increase in S-phase hepatocytes in female B6C3F1 mice suggests that enhanced cell proliferation is not a major mechanistic factor associated with the observed hepatocarcinogenicity of methylene chloride.

Male B6C3F1 mice were exposed to 4000 ppm methylene chloride (MC) for 6 hr/day, 5 days/week for up to 13 weeks. Groups of mice were killed at intervals from Day 2 to Week 13. Whole lungs were examined morphologically, immunocytochemically, and biochemically. Biochemical and morphological examination was also performed on isolated Clara cells. The major initial morphological effect seen in lungs was acute Clara cell damage after one exposure to MC. However, this damage appeared to resolve after five consecutive daily exposures to MC. After a 2-day interval the Clara cell lesion reappeared on subsequent reexposure to MC. However, the severity of the lesion decreased over the duration of the study. The appearance and disappearance of the lesion in the Clara cell correlated well with the activity of cytochrome P450 monooxygenase in the Clara cell as assessed immunocytochemically (cytochromes P450IIB 1 and 2) in the whole lung and biochemically in the freshly isolated Clara cell (determined by ethoxycoumarin O-dealkylation and aldrin epoxidation). When there was a marked decrease in cytochrome P450 monooxygenase activity the lesion was not present. This suggested that with time the lung (Clara cell) has developed tolerance to MC possibly due to the inactivation of a cytochrome P450 isozyme. The glutathione S-transferase metabolism of MC by the lung cytosol remained virtually unaltered throughout the study. Events accompanying the discussed changes include (1) a significant increase in nonprotein sulfhydryl in the lungs of all exposed animals, (2) altered plating characteristics of the isolated Clara cells from exposed lungs after 24 hr in culture, and (3) an increase in the number of bronchiolar cells in the S-phase after the first exposure to MC. The study also demonstrates the advantages of target cell isolation and study over whole lung biochemical investigation alone.

The percutaneous absorption of a new antifungal agent, amorolfine, has been measured through both skin and nail using an in vitro technique. Application of a 5% concentration in either an ethanol or methylene chloride lacquer resulted in permeation rates through nail in the range of 20-100 ng/cm2/h and somewhat higher through skin. Absorption was greater from the methylene chloride lacquer than the ethanol lacquer.

Development of quantitative mathematical models for the production of BRIs in target tissues can provide valuable insights into the mechanisms of toxicity for specific chemicals. Furthermore, use of mechanistic information and mathematical modeling in the hazard evaluation process should significantly reduce the uncertainty inherent in extrapolating the results of animal toxicity tests to man.

Dichloromethane (methylene chloride, CH2Cl2) has been shown to significantly increase the incidence of malignant lung and liver tumors in B6C3F1 mice inhaling high concentrations of CH2Cl2 vapor for the majority of their natural lifetime. CH2Cl2 is extensively metabolized in mammalian species through two competing pathways: (1) oxidation by the mixed function oxidase enzymes, and (2) conjugation with glutathione catalyzed by glutathione-S-transferase(s)(GST). Since elevated tumor incidences have not been observed in B6C3F1 mice exposed to 1,1,1-trichloroethane, a halogenated solvent with similar physical-chemical properties (but only minor amounts of mammalian metabolism), it appeared that biologically reactive intermediates (BRIs) from one or both of the pathways of CH2Cl2 metabolism were involved in the tumorigenic process. Development of an integrated pharmacokinetic model incorporating quantitative measures of mammalian physiology, chemical solubility, and metabolic rate constants permitted formulation of a plausible hypothesis for the tumorigenic effects of CH2Cl2: namely that BRIs formed by the CH2Cl2/GST(s) may react with critical molecules in the target organs. This hypothesis is consistent with the dose-dependency, route-dependency, and species-specificity of CH2Cl2 for the induction of lung and liver tumors. Based on this hypothesis as well as in vivo and in vitro measurements of CH2Cl2 metabolism in humans, it was possible to prepare quantitative estimates of the cancer risk in human populations. Examination of these risk estimates indicates that development of quantitative procedures for describing the production of BRI in target tissues may cause significant changes in the levels of estimated risk.

Dichloromethane sensitizes the myocardium to arrhythmia development in response to catecholamines. The effects of acute dichloromethane exposure (3.1, 6.2 or 12.4 mmol/kg) on cardiovascular actions of epinephrine (1 or 4 micrograms/kg iv) and norepinephrine (2 or 10 micrograms/kg iv) in the urethane-anaesthetized rat model was investigated. Hypertensive epinephrine effects as well as reflex bradycardia after epinephrine and norepinephrine injections were augmented in dichloromethane-exposed rats. Moreover we observed an enhanced negative dromotropic epinephrine action. The transient T-wave elevation after catecholamine injection was markedly increased in animals treated with 12.4 mmol/kg dichloromethane. The results show that dichloromethane exposure modifies cardiovascular actions after catecholamine administration. The release by dichloromethane of endogenous catecholamines could play a role in the manifestation of these effects.

Inhalation of several different halogenated solvents stimulated motor activity in mice. During prolonged exposure acute tolerance developed. The development of tolerance depended both on the schedule of exposure, and on the solvent. Exposure to trichloroethylene induced both stimulation and tolerance while the same degree of stimulation induced by 1,1,1-trichloroethane caused no tolerance. Thus the mechanisms which induce stimulation do not always initiate tolerance. Slow steady increases in the concentration of trichloroethylene could be maintained for several hours without any stimulation of motor activity. At the end of such exposures concentrations were reached which, if applied directly, would have induced considerable stimulation. Thus tolerance may develop without motor stimulation. Inhalation of ethanol also stimulated motor activity initially. During constant exposure the stimulation was followed by a considerable reduction in motor activity. This resulted in a hypoactive period, which in turn was followed by a second increase in motor activity, indicating the existence of not only two but several counteracting mechanisms. Development of metabolites with sedative effects counteracting the stimulating effect of the pure solvents seems to be one explanation for the results.

Dichloromethane (DCM) is a widely used industrial solvent that has been determined to be a carcinogen in rats and mice. In vitro and in vivo analyses of chromosome damage induced by this agent have provided conflicting results. In order to further investigate the clastogenic potential of DCM in vivo, we analyzed sister chromatid exchanges (SCEs) and chromosome aberrations (CAs) in mouse bone marrow cells following intraperitoneal exposures of 100-2000 mg kg-1 DCM. Dichloromethane failed to increase the frequencies of either SCEs or CAs.

Chromosome damage was studied in female B6C3F1 mice exposed to dichloromethane (DCM) by subcutaneous or inhalation treatments. No increase in the frequency of either sister chromatid exchanges (SCEs) or chromosome aberrations (CAs) in bone marrow cells was observed after a single subcutaneous injection of 2,500 or 5,000 mg/kg DCM. Inhalation exposure to DCM for 10 days at concentrations of 4,000 or 8,000 ppm resulted in significant increases in frequencies of SCEs in lung cells and peripheral blood lymphocytes, CAs in lung and bone marrow cells, and micronuclei (MN) in peripheral blood erythrocytes. Lung cell CAs and blood erythrocyte MN reached frequencies of approximately two times control levels. Following a 3-month inhalation exposure to 2,000 ppm DCM, mice showed small but significant increases in lung cell SCEs and peripheral blood erythrocyte MN. These findings suggest that genotoxicity may play a role in the carcinogenicity of DCM in the lungs of B6C3F1 female mice.

Spray-paint inhalation, once a common and inexpensive source of drug abuse, is now rare. Newer organic solvents, such as methylene chloride, were thought to decrease the toxicity of aerosol solvents, but they carry unique toxicities of their own. Methylene chloride toxicity is difficult to diagnose, as early symptoms are similar to those associated with many intoxicants; however, a rising carboxyhemoglobin level, despite removal of the patient from the source of exposure, is pathognomonic. In dealing with industrial exposures or organic aerosol abuse, a carboxyhemoglobin level should be part of the initial diagnostic workup, and treatment with oxygen is mandatory until toxicity resolves.

Carbon 13-labeled clodronate disodium was given to healthy adult men by intravenous infusion and orally in a crossover design. Serum and urine levels were determined as a function of time by isotope-ratio mass spectrometry. Clodronate disodium (Cl2/MDP) is primarily excreted unchanged by the kidney; more than 80% of the intravenous dose was recovered within 48 hr. The serum concentrations-time curve over the first 8 hr after intravenous dosing appears biexponential with the disposition phase having a harmonic mean half-life (t 1/2) of 2 hr. The mean serum clearance was found to be 1.4 ml min-1 kg-1 and the apparent volume of distribution was approximately 25% of body weight. Simulations and computer fitting of the cumulative urinary excretion and urinary excretion rates based on the biexponential serum decay curve demonstrated the presence of a slow disposition component with a t 1/2 of 12.8 hr. Thus, the disposition kinetics of Cl2MDP appear to be triexponentials, although the slowest component is not of major significance after a single dose and could not be verified because of a lack of serum data after 8 hr. Cl2MDP is poorly absorbed with an absolute bioavailability of only 1% to 2%.

The pharmacokinetics of the solvent methylene chloride have been studied using a physiologically based model that was developed for iv administrations to mice. Subsequently, the model was expanded to simulate pharmacokinetic behavior in mice and rats following single and repeated oral exposures. Through computer simulations, how different dosing variables, such as dose vehicle and exposure route, could influence the time course of methylene chloride concentrations at potentially critical sites of toxicity was examined. With this technique, methods of quantifying tissue exposure as it relates to externally applied doses was sought. In this way pharmacokinetic models help investigators design experiments that lead to more appropriate and reliable toxicologic assessment studies.

Acute effects of inhaled dichloromethane on the spontaneous electroencephalogram (EEG) and sensory-evoked potentials (EPs) were characterized and compared to previously observed effects of toluene; both solvents are common components of abused solvent mixtures. Twelve adult male Fischer-344 rats with chronic epidural electrode implants served as subjects. Each rat was exposed for 60 min to 5,000, 10,000, and 15,000 ppm dichloromethane while held in a plastic restrainer that also served as a head-only exposure chamber. The sequence of exposures was counterbalanced across rats, and the exposures were separated by about one week. To characterize the time course of any changes, somatosensory and flash EPs were recorded every 5 min during the first 45 min of the exposures. As was the case with toluene, electrophysiologic waveforms recorded from different sensory systems, and components of these waveforms, reacted in different ways to dichloromethane. With respect to the FEP and SEP the two solvents produced quite different effects. Toluene increased the amplitudes of early FEP components, eliminated late components, induced oscillations in visual cortex, and had no discernible effects on component latencies. In contrast, dichloromethane eliminated the N1 component, at moderate exposure had little or no effects on amplitudes of the later components (N3 through N4), did not induce oscillations, and had significant effects on latencies. Whereas toluene dramatically increased SEP component amplitudes at moderate concentrations with diminishing effect at higher concentrations and exposure times, dichloromethane rather uniformly decreased SEP amplitude in a simple concentration-related way. Toluene and dichloromethane had similar effects on BAER component latencies. They both caused component (P1 through P5) latencies and the P1-P5 interwave time to increase. However, whereas toluene increased early and late (but not middle) component amplitudes, dichloromethane decreased the amplitudes of early and late components and increased the amplitudes of middle components. These results emphasize the acute pharmacologic specificity of different solvents and suggest that differences in chronic neurotoxicity might also be found; they also suggest that predictable interactions might be found with acute and chronic exposure to mixtures that contain such solvents.

Male Wistar rats were exposed to 500, 1000 or 100 ppm as time-weighted average (t.w.a.) concentrations of dichloromethane vapour. The 1000 (t.w.a.) ppm exposure consisted of two 1-h peak concentrations (2800 ppm) on a basal exposure of 100 ppm. All exposures lasted for 6 h, 5 days weekly and for 2 weeks. The solvent burdens were analyzed in the perirenal fat samples which showed a relation to the dose with the highest values in the 1000 (t.w.a.) ppm exposures. The solvent concentrations increased in the perirenal fat between the two weeks of exposure. Blood carbon monoxide concentrations did not accurately reflect the body solvent burdens. Neurochemical effects also displayed a dose relationship, and included decreased succinate dehydrogenase activity in the cerebellum at the two higher doses and increased acid proteinase activity at 1000 ppm in the cerebrum. Withdrawal of the animals for 7 days from the 2-week exposure showed that the biochemical changes were largely abolished with the exception of decreased succinate dehydrogenase activity at 1000 ppm (t.w.a.).

Dichloromethane (DCM) has been evaluated for its ability to initiate unscheduled DNA synthesis (UDS) in the livers of male mice and rats in vivo. Two types of experiment were conducted. In the first, Alpk:AP rats were exposed by oral gavage to 100, 500, or 1,000 mg/kg DCM and hepatocytes assessed for UDS via autoradiography 4 and 12 hours later. In the second, Fischer F344 rats or B6C3F1 mice were exposed by inhalation to either 2,000 or 4,000 ppm of DCM for either 2 or 6 hours, and hepatocytes assessed for UDS immediately after exposure. The dose levels and strains of rodent employed in the latter protocol correspond to those employed in a recent cancer bioassay of DCM conducted by the U.S. National Toxicology Program. DCM failed to induce UDS in any of the experiments. These data are discussed within the context of other evidence indicating DCM to be nongenotoxic in vivo, despite its reported carcinogenicity in the mouse.

A long-term study was conducted to determine the possible chronic toxicity and oncogenicity of methylene chloride. Rats and hamsters were exposed by inhalation to 0, 500, 1500, or 3500 ppm of methylene chloride for 6 hr per day, 5 days a week, for 2 years. No exposure-related cytogenetic effects were present in male or female rats exposed to 500, 1500, or 3500 ppm. Females rats exposed to 3500 ppm had an increased mortality rate while female hamsters exposed to 1500 or 3500 ppm had decreased mortality rates. Carboxyhemoglobin values were elevated in rats and hamsters exposed to 500, 1500, or 3500 ppm with the percentage increase in hamsters greater than in rats. Minimal histopathologic effects were present in the livers of rats exposed to 500, 1500, or 3500 ppm. Decreased amyloidosis was observed in the liver and other organs in hamsters exposed to 500, 1500 or 3500 ppm. While the number of female rats with a benign tumor was not increased, the total number of benign mammary tumors was increased in female rats in an exposure-related manner. This effect was also evident in male rats in the 1500- and 3500-ppm exposure groups. Finally, male rats exposed to 1500 or 3500 ppm had an increased number of sarcomas in the ventral neck region located in or around the salivary glands. Therefore, in this 2-year study, some effects were observed in male and female rats exposed to 500, 1500, or 3500 ppm of methylene chloride. In contrast, hamsters exposed to the same exposure concentrations had less extensive spontaneous geriatric changes, decreased mortality (females), and lacked evidence of definite target organ toxicity.

Acute oral and inhalation toxicity of dichloromethane was investigated in both male and female Sprague-Dawley rats. Oral LD50 was found to be 1.72 ml/kg for males and 1.06 ml/kg for females. LC50 was found to be 18 100 PPM (1 X 6 hours) for both male and female rats. Acute oral intoxication is characterized by severe vascular changes and depression of the central nervous system accompanied by gastro-intestinal hemorrhagy. A similar CNS depression is also found in acute intoxication by inhalation. The nature of the symptoms as well as the sequence of physiological (hypotension, hypothermia) and biochemical events observed (high COHb level, etc.) indicate that the combined action of dichloromethane and its metabolite, carbon monoxide, are responsible for the development of vascular and CNS disturbances and subsequently the sudden death of the animals.

Methylene chloride was administered to Sprague-Dawley rats and Swiss mice by ingestion (stomach tube), in olive oil, at the doses of 500, 100 and 0 mg/kg body weight, once daily, 4-5 days weekly, for 64 weeks, and to Sprague-Dawley rats by inhalation, at the concentration of 100 and 0 ppm, 7 hours daily, for 5 days weekly. The inhalatory treatment was started on 13-week-old breeders, and male and female offspring (12-day embryos). The breeders and part of the offspring were exposed for 104 weeks; the other part of the offspring was exposed for 15 weeks only. The most important findings were: (1) the increased incidence of pulmonary tumors in male mice treated by ingestion at 500 mg/kg body weight; (2) a not-significant increase in total malignant tumors in rats exposed by inhalation at 100 ppm for 104 weeks; and (3) a not-significant increase in total malignant mammary tumors in female rats given methylene chloride by ingestion at 500 mg/kg body weight.

Effects of methylene chloride (CH2Cl2) on blood pressure (BP), body weight, plasma renin activity, hepatic histological structure of spontaneously hypertensive rats (SHRs) and normotensive control rats (NCRs) were studied. Administration of 0.15 mL/100 g/day CH2Cl2 to SHRs for five days reduced the BP from 172 +/- 7 mm Hg (mean +/- SEM) to 155 +/- 6 mm Hg without changing the plasma renin activity. The same regimen did not change the BP of NCRs. A weekly injection of 0.15 mL/100 g/day of CH2Cl2 for five consecutive weeks failed to significantly alter the BP of either SHRs or NCRs. The polyhalogen, whether administered daily or weekly, did not affect the body weight of either SHRs or NCRs. Administration of CH2Cl2 daily for five days produced an extensive but reversible hepatic centrolobular loss of glycogen in both SHRs and NCRs. No other changes were observed in the livers.

Adult male albino rats received a single dose of dichloromethane (DCM) orally. In response to administrations of DCM, 6.2 or 9.4 mmol/kg body mass, urinary catecholamine excretion increased significantly. 4 h and 48 h following a single DCM dose of 15.6 mmol/kg body mass, morphological investigations revealed cytological changes and a distinct reduction of chromaffin reaction in the adrenal medulla. The norepinephrine contents of cells were decreased strongly 4 h after DCM administration and increased weakly again, but distinct reduced in comparison with the control animals 48 h later. The mean nuclear volumes in the norepinephrine and epinephrine cells were enhanced significantly 4 h as well as 48 h after acute DCM intoxication.

Four methods for intra- and inter-species dose extrapolation for inhalation reference doses are discussed. Dichloromethane is used as an example to illustrate quantitative differences in the methods. The methods include a procedure recommended by the U.S. EPA in 1980, 2 approaches to using physiologically-based pharmacokinetic (PB-PK) models that depend on the extent of knowledge of the values of physiological parameters, and a proposed method based on the concepts inherent to PB-PK models but requiring significantly less data on physiological parameters.

The potential of the mouse hepatocarcinogen dichloromethane (DCM) to induce hepatocellular division, as monitored by increased DNA synthesis, has been evaluated using B6C3F1 mice--the strain in which it is carcinogenic but not apparently genotoxic. Male mice were exposed to DCM either by oral gavage in corn oil (1000 mg/kg) or by inhalation of an atmosphere containing 4000 p.p.m. DCM for 2 h. Cells undergoing DNA synthesis (S-phase) were radiolabelled by means of four consecutive i.p. injections of tritiated thymidine at hourly intervals prior to killing. No evidence of S-phase activity was observed in the gavage studies. The inhalation studies resulted in some weak, but statistically significant increases in S-phase incidence, but the biological significance was unclear due to similar increases being observed in some control groups. It is concluded that DCM does not share the mitogenic properties of such presumed non-genotoxic mouse liver carcinogens as trichloroethylene, polybrominated biphenyls and carbon tetrachloride, and as such its carcinogenicity to the mouse liver remains mechanistically obscure.

Three chlorinated methanes, carbon tetrachloride, chloroform, and methylene chloride, known to cause liver tumors in rodents, were given by oral gavage to adult female rats both 21 h and 4 h before sacrifice. Then hepatic DNA damage, ornithine decarboxylase (ODC), cytochrome P-450, glutathione content, and serum alanine aminotransferase (SGPT) activity assays were performed. Carbon tetrachloride increased rat hepatic ODC activity and decreased cytochrome P-450 content at doses both below and above cytotoxicity (as measured by increased SGPT activity). At 54 and 160 mg/kg, chloroform increased hepatic ODC activity with minimal or no elevation in SGPT activity. At 480 mg/kg chloroform increased hepatic ODC and SGPT activity. A dose of 1,275 mg/kg methylene chloride caused a small, but significant amount of hepatic DNA damage. When these three compounds are compared on either an equimolar or equitoxic (1/5 LD50) basis, their ability to induce hepatic ODC or increase SGPT activity was carbon tetrachloride greater than chloroform greater than methylene chloride. The results of this biochemical study are interpreted with respect to the ability of chemicals to cause hepatic cancer by either genetic or epigenetic mechanisms.