INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY ENVIRONMENTAL HEALTH CRITERIA 164 Methylene Chloride Second Edition) This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the United Nations Environment Programme, the International Labour Organisation, or the World Health Organization. Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization World Health Organization Geneva, 1996 The International Programme on Chemical Safety (IPCS) is a joint venture of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization. The main objective of the IPCS is to carry out and disseminate evaluations of the effects of chemicals on human health and the quality of the environment. Supporting activities include the development of epidemiological, experimental laboratory, and risk-assessment methods that could produce internationally comparable results, and the development of manpower in the field of toxicology. Other activities carried out by the IPCS include the development of know-how for coping with chemical accidents, coordination of laboratory testing and epidemiological studies, and promotion of research on the mechanisms of the biological action of chemicals. WHO Library Cataloguing in Publication Data Methylene chloride. (Environmental health criteria; 164) 1.Methylene chloride - adverse effects 2. Solvents I.Series ISBN 92 4 157164 0 (NLM Classification: QV 633) ISSN 0250-863X The World Health Organization welcomes requests for permission to reproduce or translate its publications, in part or in full. Applications and enquiries should be addressed to the Office of Publications, World Health Organization, Geneva, Switzerland, which will be glad to provide the latest information on any changes made to the text, plans for new editions, and reprints and translations already available. (c) World Health Organization 1996 Publications of the World Health Organization enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Convention. All rights reserved. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. CONTENTS ENVIRONMENTAL HEALTH CRITERIA FOR METHYLENE CHLORIDE 1. SUMMARY 1.1. Identity, physical and chemical properties, and analytical methods 1.2. Sources of human and environmental exposure 1.3. Environmental transport, distribution and transformation 1.4. Environmental levels and human exposure 1.5. Kinetics and metabolism 1.6. Effects on organisms in the environment 1.7. Effects on laboratory mammals and in vitro test systems 1.7.1. Single exposure 1.7.2. Short- and long-term exposure 1.7.3. Skin and eye irritation 1.7.4. Developmental and reproductive toxicity 1.7.5. Mutagenicity and related end-points 1.7.6. Chronic toxicity and carcinogenicity 1.8. Effects on humans 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS 2.1. Identity 2.2. Physical and chemical properties 2.3. Conversion factors 2.4. Analytical methods 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1. Natural occurrence 3.2. Anthropogenic sources 3.2.1. Production 3.2.2. Uses 3.2.3. Consumer applications 3.2.4. Sources in the environment 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION Appraisal 4.1. Transport and distribution between media 4.1.1. Water/air 4.1.2. Soil/air 4.1.3. Water/soil 4.1.4. Multicompartment distribution 4.2. Abiotic degradation 4.2.1. Atmosphere 4.2.2. Water 4.2.3. Soil 4.3. Biotransformation 4.3.1. Aerobic 4.3.2. Anaerobic 4.3.3. Bioaccumulation 4.4. Interaction with other physical, chemical or biological factors 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE Appraisal 5.1. Environmental levels 5.1.1. Atmosphere 5.1.1.1 Ambient air 5.1.1.2 Precipitation 5.1.2. Water 5.1.3. Aquatic organisms 5.1.4. Soil and sediment 5.2. Human exposure 5.2.1. General population 5.2.1.1 Indoor air 5.2.1.2 Drinking-water 5.2.1.3 Foodstuffs 5.2.1.4 Consumer exposure 5.2.2. Occupational exposure 5.2.2.1 Production 5.2.2.2 Paint stripping and related activities 5.2.2.3 Aerosol production and use 5.2.2.4 Use as a process solvent 5.2.2.5 Cleaning and degreasing 5.2.3. Occupational exposure limits 5.3. Human monitoring data 5.3.1. Body burden 5.3.2. Occupational exposure studies 5.3.3. Biological exposure indices 6. KINETICS AND METABOLISM 6.1. Absorption 6.1.1. Inhalation exposure 6.1.1.1 Human studies 6.1.1.2 Animal studies 6.1.2. Oral exposure 6.1.3. Dermal exposure 6.2. Distribution 6.2.1. Inhalation exposure 6.2.1.1 Human studies 6.2.1.2 Animal studies 6.2.2. Oral exposure 6.2.3. Dermal exposure 6.3. Metabolism 6.3.1. In vitro studies 6.3.2. In vivo studies 6.4. Elimination and excretion 6.4.1. Inhalation exposure 6.4.1.1 Human studies 6.4.1.2 Animal studies 6.4.2. Oral exposure 6.4.3. Dermal exposure 7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT 7.1. Microorganisms 7.1.1. Bacteria 7.1.1.1 Aerobic bacteria 7.1.1.2 Anaerobic bacteria 7.1.2. Protozoa 7.1.3. Algae 7.2. Aquatic organisms 7.2.1. Plants 7.2.2. Invertebrates 7.2.2.1 Insects 7.2.2.2 Crustaceans 7.2.2.3 Molluscs 7.2.3. Fish 7.2.3.1 Acute toxicity 7.2.3.2 Chronic toxicity and reproduction 7.2.4. Amphibians 7.3. Terrestrial organisms 7.4. Population and ecosystem effects 7.4.1. Soil microorganisms 7.4.2. Sediment microorganisms 7.4.3. Microcosms and mesocosms 8. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS 8.1. Single exposure 8.1.1. Acute toxicity data 8.1.2. Oral administration 8.1.3. Inhalation administration 8.1.3.1 Rat 8.1.3.2 Mouse 8.1.3.3 Other animals 8.1.4. Dermal administration 8.1.5. Intraperitoneal administration 8.1.6. Intravenous administration 8.1.7. Subcutaneous administration 8.1.8. Appraisal 8.2. Short-term exposure 8.2.1. Oral administration 8.2.2. Subcutaneous administration 8.2.3. Inhalation administration 8.2.3.1 Rat 8.2.3.2 Other animals 8.3. Long-term exposure 8.3.1. Rat 8.3.1.1 Inhalation exposure 8.3.1.2 Oral exposure 8.3.2. Mouse 8.3.2.1 Inhalation exposure 8.3.2.2 Oral exposure 8.3.3. Other animals 8.3.4. Appraisal 8.4. Skin and eye irritation; skin sensitization 8.4.1. Skin irritation 8.4.2. Eye irritation 8.4.3. Sensitization 8.4.4. Appraisal 8.5. Developmental and reproductive toxicity 8.5.1. Developmental toxicity 8.5.2. Reproductive toxicity 8.5.3. Appraisal 8.6. Mutagenicity and related end-points 8.6.1. In vitro 8.6.1.1 Bacteria 8.6.1.2 Fungi and yeasts 8.6.1.3 Mutation in mammalian cells 8.6.1.4 Chromosomal effects 8.6.1.5 DNA damage 8.6.1.6 DNA binding in vitro 8.6.1.7 Cell transformation 8.6.2. In vivo 8.6.2.1 Chromosome damage 8.6.2.2 Drosophila 8.6.2.3 DNA damage 8.6.2.4 DNA binding 8.6.2.5 Dominant lethal assay 8.6.2.6 Replicative DNA synthesis 8.6.3. Appraisal 8.7. Chronic toxicity and carcinogenicity 8.7.1. Inhalation exposure 8.7.1.1 Rat 8.7.1.2 Mouse 8.7.1.3 Hamster 8.7.2. Oral administration 8.7.2.1 Rat 8.7.2.2 Mouse 8.7.3. Appraisal 8.8. Mechanistic studies 8.8.1. In vitro metabolic studies 8.8.2. In vivo metabolic studies 8.8.3. Pulmonary effects 8.8.4. Studies on oncogene activation 8.8.5. The use of mechanistic studies in extrapolation 8.8.6. Mammary tumour promotion 8.8.7. Appraisal 8.9. Interspecies and dose extrapolations by kinetic modelling 9. EFFECTS ON HUMANS 9.1. General population exposure 9.1.1. Environmental exposure 9.1.2. Oral exposure 9.2. Occupational exposure 9.2.1. Short-term exposure 9.2.1.1 Case studies 9.2.1.2 Skin and eye effects 9.2.1.3 Laboratory studies 9.2.2. Long-term exposure 9.2.2.1 Case studies 9.3. Appraisal of human effects 10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT 10.1. Evaluation of human health risks 10.2. Evaluation of effects on the environment REFERENCES RESUME RESUMEN NOTE TO READERS OF THE CRITERIA MONOGRAPHS Every effort has been made to present information in the criteria monographs as accurately as possible without unduly delaying their publication. In the interest of all users of the Environmental Health Criteria monographs, readers are requested to communicate any errors that may have occurred to the Director of the International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland, in order that they may be included in corrigenda. * * * A detailed data profile and a legal file can be obtained from the International Register of Potentially Toxic Chemicals, Case postale 356, 1219 Chatelaine, Geneva, Switzerland (Telephone No. 9799111). * * * This publication was made possible by grant number 5 U01 ES02617- 15 from the National Institute of Environmental Health Sciences, National Institutes of Health, USA, and by financial support from the European Commission. Environmental Health Criteria PREAMBLE Objectives In 1973 the WHO Environmental Health Criteria Programme was initiated with the following objectives: (i) to assess information on the relationship between exposure to environmental pollutants and human health, and to provide guidelines for setting exposure limits; (ii) to identify new or potential pollutants; (iii) to identify gaps in knowledge concerning the health effects of pollutants; (iv) to promote the harmonization of toxicological and epidemiological methods in order to have internationally comparable results. The first Environmental Health Criteria (EHC) monograph, on mercury, was published in 1976 and since that time an ever-increasing number of assessments of chemicals and of physical effects have been produced. In addition, many EHC monographs have been devoted to evaluating toxicological methodology, e.g., for genetic, neurotoxic, teratogenic and nephrotoxic effects. Other publications have been concerned with epidemiological guidelines, evaluation of short-term tests for carcinogens, biomarkers, effects on the elderly and so forth. Since its inauguration the EHC Programme has widened its scope, and the importance of environmental effects, in addition to health effects, has been increasingly emphasized in the total evaluation of chemicals. The original impetus for the Programme came from World Health Assembly resolutions and the recommendations of the 1972 UN Conference on the Human Environment. Subsequently the work became an integral part of the International Programme on Chemical Safety (IPCS), a cooperative programme of UNEP, ILO and WHO. In this manner, with the strong support of the new 14 partners, the importance of occupational health and environmental effects was fully recognized. The EHC monographs have become widely established, used and recognized throughout the world. The recommendations of the 1992 UN Conference on Environment and Development and the subsequent establishment of the Intergovernmental Forum on Chemical Safety with the priorities for action in the six programme areas of Chapter 19, Agenda 21, all lend further weight to the need for EHC assessments of the risks of chemicals. Scope The criteria monographs are intended to provide critical reviews on the effect on human health and the environment of chemicals and of combinations of chemicals and physical and biological agents. As such, they include and review studies that are of direct relevance for the evaluation. However, they do not describe every study carried out. Worldwide data are used and are quoted from original studies, not from abstracts or reviews. Both published and unpublished reports are considered and it is incumbent on the authors to assess all the articles cited in the references. Preference is always given to published data. 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Content The layout of EHC monographs for chemicals is outlined below. * Summary - a review of the salient facts and the risk evaluation of the chemical * Identity - physical and chemical properties, analytical methods * Sources of exposure * Environmental transport, distribution and transformation * Environmental levels and human exposure * Kinetics and metabolism in laboratory animals and humans * Effects on laboratory mammals and in vitro test systems * Effects on humans * Effects on other organisms in the laboratory and field * Evaluation of human health risks and effects on the environment * Conclusions and recommendations for protection of human health and the environment * Further research * Previous evaluations by international bodies, e.g., IARC, JECFA, JMPR Selection of chemicals Since the inception of the EHC Programme, the IPCS has organized meetings of scientists to establish lists of priority chemicals for subsequent evaluation. Such meetings have been held in: Ispra, Italy, 1980; Oxford, United Kingdom, 1984; Berlin, Germany, 1987; and North Carolina, USA, 1995. The selection of chemicals has been based on the following criteria: the existence of scientific evidence that the substance presents a hazard to human health and/or the environment; the possible use, persistence, accumulation or degradation of the substance shows that there may be significant human or environmental exposure; the size and nature of populations at risk (both human and other species) and risks for environment; international concern, i.e. the substance is of major interest to several countries; adequate data on the hazards are available. If an EHC monograph is proposed for a chemical not on the priority list, the IPCS Secretariat consults with the Cooperating Organizations and all the Participating Institutions before embarking on the preparation of the monograph. 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It is accepted that the following criteria should initiate the updating of an EHC monograph: new data are available that would substantially change the evaluation; there is public concern for health or environmental effects of the agent because of greater exposure; an appreciable time period has elapsed since the last evaluation. All Participating Institutions are informed, through the EHC progress report, of the authors and institutions proposed for the drafting of the documents. A comprehensive file of all comments received on drafts of each EHC monograph is maintained and is available on request. The Chairpersons of Task Groups are briefed before each meeting on their role and responsibility in ensuring that these rules are followed. WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR METHYLENE CHLORIDE Members Dr L.A. Albert, Consultores Ambientales Associados, Xalapa, Veracruz, Mexico Mr D. Farrar, ICI Chemicals and Polymers, Runcorn, Cheshire, United Kingdom (Rapporteur) Dr R. Fransson-Steen, Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden Dr S. Henry, US Food and Drug Administration, Washington, DC, USA Mr P. Howe, Institute of Terrestrial Ecology, Monks Wood Experimental Station, Huntingdon, United Kingdom Dr P. Standring, Health and Safety Executive, Bootle, Merseyside, United Kingdom Dr L. Stayner, Division of Standards Development and Technology Transfer, National Institute for Occupational Safety and Health, Cincinnati, Ohio, USA Dr T. G. Vermeire, Toxicology Advisory Centre, National Institute of Public Health and Environmental Hygiene, Bilthoven, The Netherlands (Chairman) Dr Ruqiu Ye, National Environmental Protection Agency, Beijing, China Observers Dr C. De Rooij, Solvay & Cie S.A., Brussels, Belgium Dr T. Green, ICI Chemicals & Polymers Ltd., Runcorn, Cheshire, United Kingdom Secretariat Dr M. Gilbert, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland (Secretary) Dr P. Demers, Unit of Analytical Epidemiology, International Agency for Research on Cancer, Lyon, France ENVIRONMENTAL HEALTH CRITERIA FOR METHYLENE CHLORIDE A WHO Task Group on Environmental Health Criteria for Methylene Chloride met at the Institute of Terrestrial Ecology, Monks Wood, United Kingdom from 16 to 20 August 1993. Dr S. Dobson welcomed the participants on behalf of the host institution, and Dr M. Gilbert opened the meeting on behalf of the three cooperating organizations of the IPCS (ILO/UNEP/WHO). The Task Group reviewed and revised the draft monograph and made an evaluation of the risks for human health and the environment from exposure to methylene chloride. The first draft of this monograph was prepared by Mr D. Farrar, ICI Chemicals and Polymers, Runcorn, United Kingdom. Dr M. Gilbert, IPCS, was responsible for the overall scientific content of this monograph. After his death in July 1994, this responsibility was transferred to Dr P.G. Jenkins, IPCS, who also dealt with the technical editing. The efforts of all who helped in the preparation and finalization of the monograph are gratefully acknowledged. ABBREVIATIONS ALT alanine aminotransferase AST aspartate aminotransferase BEI Biological Exposure Index CO-Hb carboxyhaemoglobin GST glutathione transferase LEV local exhaust ventilation MATC maximum acceptable toxicant concentration NADPH reduced nicotinamide adenine dinucleotide phosphate NIOSH National Institute for Occupational Safety and Health (USA) SCE sister-chromatid exchange SGOT serum glutamic-oxaloacetic transaminase SGPT serum glutamic-pyruvic transaminase TT toxicity threshold TWA time-weighted average UDS unscheduled DNA synthesis 1. SUMMARY 1.1 Identity, physical and chemical properties, and analytical methods Methylene chloride (dichloromethane) is a clear, highly volatile, non-flammable liquid with a penetrating ether-like odour. The pure dry compound is very stable. Methylene chloride hydrolyses slowly in the presence of moisture, producing small quantities of hydrogen chloride. Commercial methylene chloride normally contains small quantities of stabilizers to prevent decomposition. Analytical methods are available for the determination of methylene chloride in biological media and environmental samples. All methods involve gas chromatography in combination with a suitable detector. In this way, very low detection limits have been reached (e.g., in food: 7 ng/sample; water: 0.01 µg/litre; air: 1.76 µg/m3 (0.5 ppb); blood: 0.022 mg/litre). 1.2 Sources of human and environmental exposure World production of methylene chloride is estimated to be 570 000 tonnes/year. Most applications are based on its solvent capacity for grease, plastics and paint binding agents, in combination with its volatility and stability. The worldwide usage pattern comprises aerosols (20-25%), paint remover (25%), process solvent in the chemical and pharmaceutical industry (35-40%), miscellaneous uses (e.g., polyurethane foam manufacturing) and metal cleaning (10-15%). The usage of methylene chloride is tending to decrease, at least in western Europe. More than 99% of the atmospheric releases of methylene chloride result from its use as an end-product by various industries and the use of paint removers and aerosol products at home. 1.3 Environmental transport, distribution and transformation Due to its high volatility, most of the methylene chloride released to the environment will partition to the atmosphere, where it will degrade by reaction with photochemically produced hydroxyl radicals with a lifetime of 6 months. Abiotic degradation in water is slow compared to evaporation. Methylene chloride has been shown to disappear rapidly from soil and ground water. The aerobic and anaerobic degradation of methylene chloride has been established by a variety of different test systems. Complete biodegradation, especially by acclimated bacterial cultures under aerobic conditions, is rapid (e.g., 49-66% mineralization in 50 h with acclimated municipal sludge). In bioreactors, up to 10% degradation per hour is achievable. There is no evidence of significant bioaccumulation or biomagnification. 1.4 Environmental levels and human exposure Methylene chloride has been detected in the ambient air of rural and remote areas at concentrations of 0.07-0.29 µg/m3. In suburban areas, the average concentration is < 2 µg/m3 and in urban areas < 15 µg/m3. In the vicinity of hazardous waste sites up to 43 µg/m3 has been found. Precipitation may also contain methylene chloride. Methylene chloride enters the aquatic environment through waste water discharge from various industries, and methylene chloride has been found in surface water, ground water and sediment. Exposure of members of the general public to methylene chloride will occur from its use in consumer products such as paint removers, which can result in relatively high levels being found in indoor air. Occupational exposure during production arises primarily during filling and packaging (manufacturing is in closed systems). Because of its use in paint strippers, occupational exposure to methylene chloride occurs during formulation of paint-remover, original equipment manufacture, and in commercial furniture refinishing. Methylene chloride is widely used as a process solvent in the manufacture of a variety of products, in particular in the industries mentioned in section 1.2. Biological monitoring of methylene chloride exposure can be based on measurement of the solvent itself in exhaled air or blood. However, as production of carbon monoxide with exposure for more than 3-4 h/day appears to be the limiting factor in regard to health risk, biological monitoring based upon either analysis of carbon monoxide in exhaled air or of carboxyhaemaglobin (CO-Hb) in blood is to be preferred. However, this can only be used for non-smoking subjects. Sampling should be done at about 0-2 h post-exposure, or after 16 h, i.e. on the following morning. Post-exposure CO-Hb levels 2 h after exposure ceases are not expected to exceed 2-3%, and at 16 h 1%, in the case of an 8-h exposure to less than 350 mg methylene chloride/m3 in non-smokers. 1.5 Kinetics and metabolism Methylene chloride is rapidly absorbed though the alveoli of the lungs into the systemic circulation. It is also absorbed from the gastrointestinal tract, and dermal exposure results in absorption but at a slower rate than via the other routes of exposure. Methylene chloride is quite rapidly excreted, mostly via the lungs in the exhaled air. It can cross the blood-brain barrier and be transferred across the placenta, and small amounts can be excreted in urine or in milk. At high concentrations, most of the absorbed methylene chloride is exhaled unchanged. The remainder is metabolized to carbon monoxide, carbon dioxide and inorganic chloride. Metabolism occurs by either or both of two pathways, whose relative contribution to the total metabolism is markedly dependent on the dose and on the animal species concerned. One pathway involves oxidative metabolism mediated by cytochrome P-450 and leads to both carbon monoxide and carbon dioxide. This pathway appears to operate similarly in all rodents studied and in man. Whilst this is the predominant metabolic route at lower doses, saturation occurs at a relatively low dose (around 1800 mg/m3). Increasing the dose above the saturation level does not lead to extra metabolism by this route. The other pathway involves a glutathione transferase (GST), and leads via formaldehyde and formate to carbon dioxide. This route seems only to become important at doses above the saturation level of the "preferred" oxidative pathway. In some species (e.g., the mouse) it becomes the major metabolic pathway at sufficiently high doses. In contrast, in other species (e.g., hamster, man) it seems to be used very little at any dose. Species difference in GST metabolism correlates well with the observed species difference in carcinogenicity. The extent of metabolism by this pathway in relevant species has been used as the basis for a kinetic model to describe the metabolic behaviour of methylene chloride in various species. 1.6 Effects on organisms in the environment Algae and aerobic bacteria show no inhibition of growth below 500 mg/litre. Bacteria have been identified that are able to grow in the presence of methylene chloride at much higher concentrations including a saturated solution in water (section 4.2.4.1). Anaerobic bacteria are more sensitive; growth inhibition has been observed at 1 mg/litre in anaerobic biological sludge. In soil a concentration of 10 mg/kg strongly decreased the ATP content of the biomass including fungi and aerobic bacteria, and induced transient inhibition of enzyme activity. The no-observed- effect level was 0.1 mg/kg. In earthworms methylene chloride is moderately toxic (100-1000 µg/cm2) in the filter-paper contact toxicity test. In sediment no toxic effects were observed even at very high levels. In higher plants no effects were found after exposure for 14 days to 100 mg/m3. Adult fish seem to be relatively insensitive to methylene chloride even after prolonged exposure (14-day LC50 > 200 mg per litre). The effect of methylene chloride on Daphnia is difficult to assess given the large variation in the outcome of the studies performed. The lowest reported EC50 was 12.5 mg/litre. In the aquatic environment, fish and amphibian embryos have been shown to be the most sensitive with effects on hatching from 5.5 mg/litre. 1.7 Effects on laboratory mammals and in vitro test systems 1.7.1 Single exposures The acute toxicity of methylene chloride by inhalation and oral administration is low. The inhalation 6-h LC50 values for all species are between 40 200 and 55 870 mg/m3. Oral LD50 values of 1410-3000 mg/kg were recorded. Acute effects after methylene chloride administration by various routes of exposure are primarily associated with the central nervous system (CNS) and the liver, and these occurred at high doses. CNS disturbances were found at concentrations of 14 100 mg/m3 or more, with slight changes in EEG at 1770 mg/m3. Slight histological changes in the liver were found at 17 700 mg/m3 or more. Occasionally other organs were affected such as the kidney or respiratory system. In mice, effects on the lungs were restricted to the Clara cells after exposure to 7100 mg/m3. Cardiac sensitization to adrenaline-induced arrhythmia has been reported. Cardiovascular effects have been seen but the effects were inconsistent. 1.7.2 Short- and long-term exposure Prolonged exposure to high concentrations of methylene chloride (> 17 700 mg/m3) caused reversible CNS effects, slight eye irritation and mortality in several laboratory species. Body weight reduction was observed in rats at 3500 mg/m3 and in mice from 17 700 mg/m3. Slight effects on the liver were noted in dogs continuously exposed to 3500 mg/m3 for up to 100 days. After intermittent exposure, effects on the liver were observed in rats at 3500 mg/m3 and in mice at 14 100 mg/m3. Other target organs are the lungs and the kidneys. No evidence of irreversible neurological damage was seen in rats exposed by inhalation to concentrations up to 7100 mg/m3 for 13 weeks. Oral administration of methylene chloride to rats caused effects on the liver from about 200 mg/kg per day. 1.7.3 Skin and eye irritation Methylene chloride is moderately irritant to the skin and eyes of experimental animals. 1.7.4 Developmental and reproductive toxicity Methylene chloride is not teratogenic in rats or mice at concentrations up to 16 250 mg/m3. No evidence of an effect on the incidence of skeletal malformations or other developmental effects were observed in three animal studies. Small effects on either fetal or maternal body weight were reported at 4400 mg/m3, and on postnatal weight gain of male rats at 0.04% in the diet. A two- generation reproductive toxicity study in rats exposed to methylene chloride by inhalation at concentrations up to 5300 mg/m3, 6 h/day, 5 days/week for 17 weeks did not show evidence of an adverse effect on any reproductive parameter, neonatal survival or neonatal growth in either the F0 or F1 generation. 1.7.5 Mutagenicity and related end-points Under appropriate exposure conditions, methylene chloride is mutagenic in prokaryotic microorganisms with or without metabolic activation (Salmonella or Escherichia coil). In eukaryotic systems it gives either negative or, in one case, weakly positive results. In vitro gene mutation assays and tests for unscheduled DNA synthesis (UDS) in mammalian cells were uniformly negative. In vitro assays for chromosomal aberrations using different cell types gave positive results, whereas negative or equivocal results were obtained in tests for sister chromatid exchange (SCE) induction. The majority of the in vivo studies reported provided no evidence of mutagenicity of methylene chloride (e.g., chromosome aberration assay, micronucleus test or UDS assay). Marginal increase in frequencies of SCEs and micronuclei in mice has been reported following inhalation exposure to high concentrations of methylene chloride. There was no evidence of binding of methylene chloride to DNA or DNA damage in rats or mice given high doses of methylene chloride. These studies are potentially the most sensitive in vivo studies, the best of which are capable of detecting one alkylation in 106 nucleotides. Within the limitations of the short-term tests currently available, there is no conclusive evidence that methylene chloride in genotoxic in vivo. 1.7.6 Chronic toxicity and carcinogenicity Methylene chloride is carcinogenic in the mouse, causing both lung and liver tumours, following exposure to high concentrations (7100 and 14 100 mg/m3) of methylene chloride. The incidence of both lung and liver tumours was increased in mice exposed to 7100 mg/m3 for 26 weeks and maintained for a further 78 weeks. There was no substantial evidence of associated toxicity or hyperplasia in the target organs. Syrian hamsters exposed to methylene chloride by inhalation at concentrations up to 12 400 mg/m3 for 2 years showed no evidence of a carcinogenic effect related to exposure to methylene chloride. Rats exposed to methylene chloride via various routes have shown increased incidences of tumours at certain sites. An excess of tumours in the region of the salivary gland was reported in female rats exposed to either 5300 or 12 400 mg/m3 for 2 years. This excess was only evident when the tumours, which were all of mesenchymal origin, were grouped together for statistical analysis. As the tumours arose from a variety of different cells, the statistical approach adopted was inappropriate. Furthermore, it was reported that the rats in the study had been infected with a common viral disease (sialoda- cryoadenitis) early in the study, an infection that affects primarily the salivary gland. It is likely that these tumours were not causally related to exposure to methylene chloride but that the exposure had exacerbated the response of the infection in the region of the salivary gland. The response was not seen in a second study in which rats were exposed to either 3500, 7100 or 14 100 mg/m3 for their lifetime. A further inhalation study on rats exposed to methylene chloride at concentrations up to 1770 mg/m3 for their lifetime showed no evidence of carcinogenicity. Rats exposed to methylene chloride via their drinking-water or by gavage similarly showed no substantive evidence of carcinogenicity. An increased incidence of benign mammary tumours in rats exposed to methylene chloride has been reported in three studies, two following exposure by inhalation and the third by gavage. There are no reports of increases in mammary tumour incidence in hamsters or in mice receiving methylene chloride at comparable dose levels. The dependence of mammary tumours upon pituitary hormones in both male and female rats has been established unequivocally. In the rat, prolactin acts as both an initiator and promoter of mammary carcinogenesis. There is good evidence that increased prolactin levels increase the incidence of mammary tumours (e.g., the grafting of multiple pituitary glands into Sprague-Dawley rats increases the incidence of mammary tumours and there is a positive correlation between elevated blood prolactin levels and mammary tumours in aged R-Amsterdam female rats). Treatments that induce hyperprolactinaemia in female rats that have received carcinogens produce a dramatic increase in tumour incidence. These treatments include adrenalectomy, pituitary homografts and high dietary fat. The mechanism by which methylene chloride induces mammary adenomas in the rat is important for human hazard assessment. Female Sprague- Dawley rats receiving methylene chloride have a high blood level of prolactin. In common with the response to other agents which act via hyperprolactinaemia, the methylene chloride-induced response is of benign neoplasms only. There is no evidence for the binding of methylene chloride to the DNA of other tissues and hence it seems unlikely that it will bind to mammary tissue when the primary site of metabolism is the liver. It seems most likely, therefore, that the increased incidence of mammary adenomas is the result of an indirect mechanism operating via hyperprolactinaemia. In humans, there is conflicting evidence on whether or not mammary tumours are as responsive to prolactin as is the case in the rat. The rat has elevated levels of prolactin when fed ad libitum in comparison to a restricted dietary regimen and this may explain why the mammary tumour incidence is so responsive to a variety of environmental and other effects. In the rat, however, prolactin is luteotrophic. An increase in the circulating levels of prolactin will lead to an increase in progesterone and exogenous oestrogen levels. It is the presence of all three factors that causes tubular-alveolar growth of the mammary glands, which ultimately leads to tumour development. Prolactin is not luteotrophic in primates. It is unlikely, therefore, that this mechanism of tumour development is of relevance to man. The mechanism of production of mammary tumours in the rat involving hyperprolactinaemia will occur only at doses of methylene chloride which affect prolactin levels. There is no direct information on prolactin levels in rats receiving low doses of methylene chloride, but no increase in mammary adenomas has been observed following the administration of low doses in either inhalation or drinking-water studies (i.e. below 250 mg/kg body weight). 1.8 Effects on humans Methylene chloride irritates the skin and eyes especially when evaporation is prevented. In these circumstances, prolonged contact may cause chemical burns. A case of serious pulmonary oedema has been reported after excessive inhalation. Fatalities due to accidental inhalation and skin contamination have been reported. The main toxic effects of methylene chloride are reversible CNS depression and CO-Hb formation. Liver and renal dysfunctions and effects on haematological parameters have also been reported following exposure to methylene chloride. Neurophysiological and neurobehavioural disturbances have been observed in human volunteers exposed to methylene chloride at concentrations of 694 mg/m3 for 1.5-3.0 h. No evidence of neurological effects was seen in men with exposure for several years to methylene chloride at concentrations ranging from 260 to 347 mg/m3. Similarly, a group of retired airplane strippers with a long history of exposure to methylene chloride (22 years) at high but unspecified levels performed a battery of neurophysiological and psychological tests within the "normal" range, when compared with a control group who had a history of either no or only low exposure to methylene chloride. An increased rate of spontaneous abortion in employees in Finnish pharmaceutical industries has been attributed to exposure to methylene chloride. A causal relationship was not established because of insufficiencies in the design of the study. Several mortality studies in relevant cohorts show an inconsistent pattern in the causes of death. Excesses in mortality from specific diseases (e.g., pancreatic cancer, ischaemic heart disease) were not consistently increased, but confined to single studies. These effects cannot be attributed to exposure to methylene chloride. 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS 2.1 Identity Formula: CH2CI2 CI ' Structure: CI - C - H ' H Relative molecular mass: 84.93 Common name: Methylene chloride Synonyms: DCM; dichloromethane; methane dichloride; methylene bichloride; methylene dichloride; methylenum chloratum Tradenames: Aerothene MM; Freon 30; Narkotil; Solaestin; Solmethine CAS name (9 CI): Methane, dichloro- CAS registry number:75-09-2 EC registry number: 602-004-00-3 EINECS registry number: 200-838-9 RTECS registry number: PA 8050000 Purity of technical 99.9% (analytical grade) product: Impurities of technical Mostly C1- and C2-chlorinated product hydrocarbons (up to 200 mg/kg) (ECETOC, 1984) Stabilizer: Typically 0.005-0.2% (w/w) methanol, ethanol, amylene (2-methyl-but-2-ene), cyclohexane or tertiary butylamine (ECSA, 1989) 2.2 Physical and chemical properties Methylene chloride is a clear, colourless, highly volatile, non- flammable liquid with a penetrating ether-like odour. Pure dry methylene chloride is a very stable compound and is non-corrosive. In the presence of water, it undergoes very slow hydrolysis to produce small quantities of hydrogen chloride, which can lead to corrosion, e.g., to mild steel. This reaction is accelerated by elevated temperatures and the presence of alkalis or metals. In the vapour phase under abnormal conditions (elevated temperatures, high UV light exposure, flame, sparks, red hot surfaces), methylene chloride may be decomposed to give small amounts of hydrogen chloride, carbon monoxide and phosgene (ECSA, 1989). Other physical and chemical properties are given in Table 1. Commercial methylene chloride is normally stabilized (section 2.1) to prevent decomposition. Applications in aggressive conditions, such as special metal cleaning operations may require more sophisticated stabilizer technology. Poorly stabilized methylene chloride can react violently with aluminium or other light metals. 2.3 Conversion factors Conversion factor for methylene chloride concentrations in air, calculated at 20°C and 1.013 hPa are: 1 mg/m3 = 0.28 ppm 1 ppm = 3.53 mg/m3 and for carbon monoxide: 1 mg/m3 = 0.86 ppm 1 ppm = 1.16 mg/m3 2.4 Analytical methods Details of sampling and methods of analysis used in biological media and environmental samples are given in Tables 2 and 3. Table 1. Physical and chemical properties Parameter, units Value Reference Boiling temperature (°C at 1.013 hPa) 40 Weast et al. (1988) Melting temperature (°C at 1.013 hPa) -95.1 Weast et al. (1988) Relative density of liquid D (20) 1.3266 Weast et al. (1988) (water at 4°C = 1 kg/m3) 4 Vapour pressure (hPa at 20°C) 470 ECSA (1989) Saturation concentration in air 1.7 Calculated (kg/m3 at 20°C) Vapour density at 20°C (air = 1) 2.93 IPCS (1984) Threshold odour concentration 743 Leonardos et al. (mg/m3) (1969) (odour: ether-like) 700-1060 DFG (1983) 880 Amoore & Hautala (1983) 540-2160 Ruth (1986) Solubility in water (g/kg at 20°C) 20 Verschueren (1983) 13.0 Horvath (1982) Solubility in alcohol, ether, acetone Weast et al. (1988) and benzene Partition coefficients, at 20°C 1.25 IPCS (1984) log Pow (octanol/water) 1.3 Hansch & Leo (1979) log Koc 0.89 calculated from Kow (Karickhoff, 1981) Henry's Law constant, Pa.m3/mol at 380 20°C Smith (1989) Flash point, closed cup (°C) None ECSA (1989) Explosion limits in aira (%) 13-22 ECSA (1989) Auto-flammability, ignition temp. (°C) 605 ECSA (1989) a This is with a high energy source; these conditions are unlikely to arise in normal operations. Table 2. Analytical methods for determining methylene chloride in biological monitoring (ATSDR, 1991) Sample matrix Preparation method Analytical Sample detection Percentage Reference methoda limit recovery Blood Heat sample, collect GC/FID 0.022 mg/litre 49.8±1.33 Di Vincenzo et al. headspace vapour (1971) Urine Heat sample, collect GC/FID No data 59±2.75 Di Vincenzo et al. headspace vapour (1971) Breath Heat sample, inject into gas GC/FID 0.706 ± 0.353 No data Di Vincenzo et al. sample loop mg/m3 (1971) (0.2 ± 0.1 ppm) Adipose tissue Hydrolyse with acid, heat GC/FID 1.6 mg/kgb No data Engström & Bjurström sample, collect headspace (1977) vapour Human milk Purge with helium, trap on GC/MS No data No data Pellizzari et al. (1982) sorbent trap, desorb thermally a FID = flame ionisation detector; GC = gas chromatography; MS = mass spectrometry b Lowest reported concentration Table 3. Analytical methods for determining methylene chloride in environmental samples (ATSDR, 1991) Sample Preparation method Analytical Sample detection Percentage Reference matrix methoda limit recovery Air Adsorb on charcoal, desorb with GC/FID 88.25µg/m3 90-110c APHA (1977) carbon disulfide (25 ppb)b Air Adsorb on charcoal, desorb with GC/FID 0.01 mg 95.3 NIOSH (1987) carbon disulfide Air Adsorb on charcoal, desorb with GC/ECD approx. 1.76 µg/m3 No data Woodrow et al. benzyl alcohol (approx. 0.5 ppb) (1988) Water Purge with inert gas, trap on sorbent GC/HSD No data 85 US EPA (1989c) trap, desorb thermally Water Purge with inert gas, trap on sorbent GC/ELCD 0.01 µg/litre 97-100 US EPA (1989) trap, desorb thermally Water Purge with inert gas, trap on sorbent GC/MS 1.0 µg/litre 99 US EPA (1989b) trap, desorb thermally Water Purge with inert gas, trap on sorbent HRGC/MS 0.03-0.09 µg/litre 95-97 US EPA (1989a) trap, desorb thermally Water Purge with inert gas, trap on sorbent HRGC/ELCD 0.01-0.05 µg/litre 97±28 APHA (1989a) trap, desorb thermally Table 3 (Cont'd) Sample matrix Preparation method Analytical Sample detection Percentage Reference methoda limit recovery Water Purge with inert gas, trap on sorbent HRGC/MS 0.02-0.2 µg/litre 95±5 APHA (1989b) trap, desorb thermally Water Purge with helium, trap on sorbent GC/MS No data 99-105 Michael et al. trap, desorb thermally (1988) Waste Purge with inert gas, trap on sorbent GC/HSD 0.25 µg/litre 97.9±2.6 US EPA (1982a) water trap, desorb thermally Waste Purge with inert gas, trap on sorbent GC/MS 2.8 µg/litre 89±28 US EPA (1982b) water trap, desorb thermally Soil/solid Purge with inert gas, trap on sorbent GC/MS 5 µg/kg D-221 US EPA (1986a) waste trap, desorb thermally Soil/solid Purge with inert gas, trap on sorbent GC/HSD No data 25-162 US EPA (1986b) waste trap, desorb thermally; or inject directly into GC Food Equilibrate in heated sodium sulfate GC/ELCD 0.05 ppm No data Page & Charbonneau solution, collect headspace vapour (1984) Food Isolate solvent by closed system GC/ELCD 7 ng 94 Page & Charbonneau vacuum distillation with toluene as (1977) carrier solvent Table 3 (Cont'd) Sample matrix Preparation method Analytical Sample detection Percentage Reference methoda limit recovery Food Isolate solvent by closed system GC/ECD 7 ng 100 Page & Charbonneau vacuum distillation with toluene as (1977) carrier solvent Food Purge with nitrogen, trap on sorbent GC/ELCD 1.2 mg/kgd 84-96 Heikes (1987) trap, elute with hexane Food Extract with acetone-water, back GC/ELCD 4 µg/kg 66 Daft (1987) extract with iso-octane a ECD = electron capture detector; ELCD = electrolytic conductivity detector; FID = flame ionisation detector; GC = gas chromatography; HRGC = high resolution gas chromatography; HSD = halogen-specific detector; MS = mass spectrometry b Lowest value for various compounds reported during collaborative testing of this method c Estimated accuracy of the method when the personal sampling pump is calibrated with a charcoal tube in the line d Lowest reported concentration 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1 Natural occurrence Methylene chloride is not known to occur naturally in the environment. 3.2 Anthropogenic sources 3.2.1 Production Methylene chloride is produced almost exclusively by the Stauffer process. Methyl chloride is first produced by the reaction of methanol with hydrogen chloride and is then reacted with chlorine. Chloroform and, to a lesser extent, carbon tetrachloride are also produced. Historically the direct route to methylene chloride by chlorination of methane was also used; this also produced the other three chloromethanes in varying proportions depending on the conditions used (CEC, 1986; ICI, personal communication to the IPCS). World production of methylene chloride in 1980 was estimated to be 570 000 tonnes (Edwards et al., 1982); a similar figure is considered to apply currently (ECSA, 1992). USA production was 229 000 tonnes in 1988, the demand being 207 000 tonnes. The total amount produced in western Europe ranged from 331 500 tonnes in 1986 to 254 200 tonnes in 1991 (ECSA, 1992). 3.2.2 Uses The usage of methylene chloride in Western Europe shows a decrease from 200 000 tonnes/year in 1975-1985 (CEFIC, 1986) to 175 000 tonnes/year in 1989 and to 150 000 tonnes/year in 1992 (CEFIC, 1993). Most of the applications of methylene chloride are based on its considerable solvent capacity, especially for grease, plastics and various paint-binding agents. Other important properties are its volatility and stability; it is also non-flammable. Among its uses are (CEFIC, 1983): - a component of paint and varnish strippers, and adhesive formulations - a solvent in aerosol formulations - an extractant in food and pharmaceutical industries - a process solvent in cellulose ester production and fibre and film forming - a process solvent in polycarbonate production - a blowing agent in flexible polyurethane foams - the extraction of fats and paraffins - plastics processing, and metal and textile treatment - a vapour degreasing solvent in metal-working industries An estimated breakdown of usage worldwide before 1985 is given in Table 4. Table 4. Estimated usage patterns (BUA, 1986) USA (1985) Western Europe (1984) Aerosols 25 10 Paint strippers 23 50 Degreasing agent 8 13 Film, electronics industries 7 15 Blowing agent 5 Others 35 12 It should be noted that these data apply to the situation approximately 10 years ago and may have changed since. Reliable reports on present trends are not available. 3.2.3 Consumer applications The main use in consumer products is in paint strippers, where methylene chloride is the main constituent (70-75%). The second important use is in hairspray aerosols, where it acts as a solvent and vapour pressure modifier. In the European Community (EC) it may be used in such products at concentrations of up to 35% w/w (European Council, 1982). The US Food and Drug Administration has banned the use of methylene chloride in cosmetic products. It is also used in aerosol paints. Other types of methylene chloride-containing products are household cleaning products and lubricating, degreasing and automotive products, some of which may be in aerosol form. Chemical products containing methylene chloride were banned from sale or transfer to consumers for their private use in 1993 according to the Swedish Code of Statutes. Furthermore, it may not be used for working purposes after 1st January 1996 (National Chemical Inspectorate, Sweden, personal communication to the IPCS). 3.2.4 Sources in the environment Most of the methylene chloride released to the environment results from its use as an end-product by various industries, and the use of paint removers and aerosol products in the home. Methylene chloride is mainly released to the environment in air and, to a lesser extent, in water and soil. Methylene chloride is released to the atmosphere during its production, storage and transport, but more than 99% of the atmospheric releases result from industrial and consumer uses (US EPA, 1985). It has been estimated that 85% of the total amount of methylene chloride produced in the USA is lost to the environment, of which 86% is released to the atmosphere (US EPA, 1985). Data reported to the US EPA for the 1988 Toxic Chemical Release Inventory indicate that approximately 170 000 tonnes of the USA production volume for 1988 (230 000 tonnes) was lost to the atmosphere; of this, 60 000 tonnes resulted from industrial methylene chloride emissions and 110 000 tonnes from the use of consumer products and from other sources such as hazardous waste sites. Estimates of annual global emissions of 500 000 tonnes have been reported for methylene chloride (WMO, 1991). The short atmospheric lifetime of methylene chloride (see section 4.2.1) implies that emissions quantities given on a seasonal as well as on a regional basis are more relevant for comparison with atmospheric measurements. The total emission into the air in western Europe was estimated to be 173 000 tonnes for 1989 and 180 000 tonnes in 1991. 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION Appraisal Due to its high volatility, most of the methylene chloride released to the environment will partition to the atmosphere, where it will degrade by reaction with photochemically produced hydroxyl radicals with a lifetime of 6 months. Given an intra-hemispheric mixing time of approximately 1 month, transport can occur to regions far removed from the emission source. The atmospheric lifetime is fairly short relative to the inter-hemispheric transport time of 1 to 1.5 years, resulting in higher concentrations of methylene chloride in the northern hemisphere, where most of the emissions occur at present. Methylene chloride is expected to have no significant impact on stratospheric ozone depletion. It will not contribute significantly to photochemical smog formation. Hydrolysis and photolytically induced degradation in water are slow compared to evaporation. Methylene chloride has been shown to disappear rapidly from soil and ground water due to bio- transformation. The aerobic and anaerobic degradation of methylene chloride has been proven by a variety of different test systems. Complete biodegradation by acclimated bacterial cultures under aerobic conditions is rapid. There is no evidence that significant bioaccumulation or biomagnification of methylene chloride along the food chain will occur. 4.1 Transport and distribution between media 4.1.1 Water/air Methylene chloride enters the hydrosphere either directly, via aqueous effluents, or indirectly from the atmosphere by dissolution in sea water and in rain water. Due to its high volatility (Henry's Law constant 380 Pa.m3/mol at 20°C) and low liquid-film transfer coefficient (Kp = 0.005 m/h), methylene chloride is rapidly transferred from the hydrosphere to the atmosphere. Under laboratory conditions, the estimated half-life for volatilization of methylene chloride from water at 25°C was 18-25 min (when present at 1 mg/litre and stirred at 200 rpm). Removal of 90% of the methylene chloride required 60-80 min. When stirring was minimal (15 seconds every 5 min), the time required for 50% reduction in the concentration was about 90 min. The presence of 3% sodium chloride (as in sea water) decreased the evaporation rate by 10% (Dilling et al., 1975; Dilling, 1977). Various factors have been shown to affect the rate of volatilization. For example, the half-life for volatilization of methylene chloride from a depth of 1 m has been shown to be 3 h (Lyman, 1982). The application of wind across the surface of the water caused an increase of 17% in volatilization over a period of 20 min compared to the presence of still conditions (Dilling et al., 1975). A decrease in the water temperature decreased the rate of volatilization. For example, over a period of 30 min, a 28% decrease in rate was seen at 1-2°C compared to that at 25°C (Dilling et al., 1975). When measured under field conditions in experimental ponds, half- lives for methylene chloride of 26-28 h have been reported (Merlin et al., 1992). Its half-life for evaporation from the river Rhine has been estimated to be 33-38 clays (Zoeteman et al., 1980). Further estimates of the half-life for its evaporation are between 3 and 48 h depending on wind and mixing conditions (Halbartschlager et al., 1984). In a further study, methylene chloride was not detected at a point 4-8 km from the point of release into an estuarine bay (Helz & Hsu, 1978) or at 25 km below its discharge point in a river basin (De Walle & Chain, 1978). Rain-out is considered to be a limited process for removal of methylene chloride from the troposphere. If it is assumed that its aqueous-phase concentration is in equilibrium with the background concentration in the northern hemisphere of about 123-134 ng/m3 (35-38 ppt) (Cox et al., 1976; WMO, 1991), the total amount of methylene chloride rained out in the northern hemisphere will be 700 tonnes/year (assuming a rain fall of 2.5x1014 tonnes/year containing 9.9 ng/m3 (2.8 ppt) at 10°C). The same calculation performed at 20°C (Henry's constant is 1.57 times higher) would lead to a value of 445 tonnes methylene chloride rained out annually in the northern hemisphere. For the southern hemisphere, rainout quantities of 390 and 248 tonnes methylene chloride can be calculated. The half- life for removal by wet deposition is 550 years (Cupitt, 1980). In 1978, it was estimated that 2.5% of releases at ground level may reach the stratosphere (Derwent & Eggleton, 1978). 4.1.2 Soil/air Methylene chloride present in the soil is predicted to evaporate from the near-surface layer into the atmosphere because of its high vapour pressure (470 hPa at 20°C). 4.1.3 Water/soil The adsorption coefficient sediment/water for methylene chloride is 8-10 (log Koc = 0.89-1.05). Methylene chloride has a low tendency to adsorb to soil (adsorption coefficient 0.25 for a soil containing 1% organic carbon, Giger et al., 1983). Therefore there is a potential for it to leach to ground water. The amount of adsorption of methylene chloride to dry granular bentonite clay added at a concentration of 375-750 mg/litre was found to be 10-22% within 10-30 min. In the presence of 500 mg/litre peat moss, about 40% of methylene chloride was absorbed after 10 min. Some adsorption by dry-powdered dolomitic limestone was observed, but not with silica sand (Dilling et al., 1975). 4.1.4 Multicompartment distribution The regional distribution of methylene chloride over water, soil and air compartments may be estimated by means of the fugacity model developed by MacKay (Slooff & Ros, 1988). Application of this model suggests that over 98% of the total emissions of the chemical will be found in air, 1 to 2% in water and far less than 1% in soil and ground water (BUA, 1986; Slooff & Ros, 1988). 4.2 Abiotic degradation 4.2.1 Atmosphere The principal process by which methylene chloride is scavenged from the atmosphere is the reaction with hydroxyl rate of methylene chloride can be calculated from the rate constant for the initiating breakdown reaction with HO. and the varying concentration of these radicals in the troposphere. Determination of the rate constant for the reaction of methylene chloride with hydroxyl radicals has been the subject of various investigations. WMO (1991) recommends the following value: kOH = 5.8 × 10-12 exp(-1100/T) cm3 molecule-1 s-1 Other reactive species (e.g., ozone, oxygen atoms, chlorine atoms and nitrate radicals) are not thought to contribute significantly to the primary attack on methylene chloride (Table 5). As methylene chloride does not absorb in the visible or near ultraviolet light region (> 290 nm), direct homogeneous gas-phase photolysis in the troposphere is of negligible importance. Table 5. Primary tropospheric reactions of methylene chloride (other than with .OH) Reaction k (at 25°C) Global average [X] Lifetime with: (cm3 molecule-1 s-1) (molecule cm-3) (years) .Cl 4.1 × 10-13 103 77 (IUPAC, 1992) (estimated) (estimated) .NO3 <3.2 × 10-17 1.2 × 108 > 8.3 .O(3p) 6.44 × 10-16 2.5 × 104 approx. 2000 (Barassin & Cambourieu, 1973) .O(1D) < 5 × 10-10 0.5 > 120 (estimated) Carbon dioxide and hydrogen chloride are the major breakdown products and minor quantities of carbon monoxide and phosgene are formed (Sanhueza & Heicklen, 1975; Rayez et al., 1987). The breakdown reaction can be described as follows: CH2Cl2 + HO. --> .CHCl2 + H2O .CHCl2 + O2 --> .CHCl2O2 .CHCl2O2 + NO --> .CHCl2O + NO2 .CHCl2O --> .Cl + HCOCl or .CHCl2O + O2 --> COCl2 + HO2 (minor reaction) Formyl chloride may be taken up by cloud droplets, hydrolysed to formic acid and wet deposited as such, or dry deposited to the ocean or land surfaces and then hydrolysed. The overall lifetime for wet or dry deposition is unlikely to exceed a few months and may be much shorter. On the other hand, degradation in the troposphere by photolysis or reaction with HO. may possibly be a more rapid process. The reaction products would be carbon oxides (CO, CO2) and HCl (Libuda et al., 1990). Phosgene is known to hydrolyse slowly in the gas phase, but rapidly once dissolved in liquid water, to give CO2 and HCl. HCl is removed from the troposphere by wet deposition (dissolution in atmospheric water droplets and subsequent rain-out) or dry deposition (direct uptake by the oceans, land surfaces, vegetation etc.) with an average lifetime of about 1 week. The amount of chloride deposited in this manner is completely negligible compared to the natural atmospheric chloride flux of around 1010 tonnes/year primarily from sea-salt aerosols (WMO, 1991). In the stratosphere methylene chloride will rapidly degrade by photolysis and reaction with chlorine radicals (Derwent et al., 1976). 4.2.2 Water Sunlight absorption of water results in the formation of HO. and hydrated electrons (e-aq). The near surface concentrations of HO. and e-aq are 4 × 10-16 mol/litre and 5 × 10-17 mol/litre, respectively, which corresponds to theoretical half-lives for methylene chloride of 400 and 33 days. In water systems these reactions are very limited, the reaction with hydroxyl radicals being dominant. The total rate constant for the sunlight-induced transformation in surface water (with a depth of 2.5 m, a DOC content of 4 mg/litre, a chlorophyll a content of 10 µg/litre and a suspended matter content of 40 mg/litre) was estimated to be 2.8 × 10-5 day-1 (half-life 68 years). The HO. causes 90% of this transformation (Slooff & Ros, 1988). No direct photolysis of methylene chloride was found after visible and UV irridiation for 5 days at 22°C (Chodola et al., 1989). The half-life of a 1 mg/litre aqueous solution of methylene chloride was found to be about 1.5 years when measured in sealed glass tubes in the dark at 25°C and pH 7 (Dilling et al., 1975). No significant hydrolysis was found at 50°C and pH 4 or 9.2 after 7 days in the dark (Chodola et al., 1989). Under acidic and basic conditions in the temperature range of 80-150°C, the hydrolysis of methylene chloride results in the formation of formaldehyde and HCl (Fells & Moelwyn-Hughes, 1958). Extrapolation of these data to 25°C gives a long half-life of about 680-704 years (Dilling et al., 1975; Radding et al., 1977). As the activation energy for hydrolysis of methylene chloride varies with temperature, the extrapolation of rate data from 80-150°C may not be valid. No reductive dehalogenation of methylene chloride in water was observed in the presence of sodium sulfide and haematein, a common iron porphyrin (Klecka & Gonsior, 1984). 4.2.3 Soil As is the case in aqueous systems, hydrolysis is probably not an important process in the removal of methylene chloride from soil (see section 4.2.2). In a lysimeter experiment, a 90% decrease over 2.5 m soil column was obtained (Nellor et al., 1985). In the report of a spillage, the concentrations of methylene chloride were up to 802 mg/m3 and 26 900 mg/m3 near the point of leakage. In both cases, methylene chloride could not be detected some hundred metres away from the points of contamination even in the direction of the groundwater flow (ECSA, 1989). In the neighbourhood of polluted areas, an increase of bacterial activity has been found. In well-documented cases of accidental spills to soils, methylene chloride disappeared rapidly from ground water, probably due to (bio)degradation (Baldanf, 1981; Leitfaden für die Beurteilung, 1983). 4.3 Biotransformation 4.3.1 Aerobic Negligible oxygen consumption was found in a biochemical oxygen demand (BOD) test (Klecka, 1982), and methylene chloride was considered to be degradation resistant in a degradation test following the Japanese MITI standards (Kawasaki, 1980). However, complete degradation occurred during a static-culture flask test (Tabak et al., 1981). In laboratory studies methylene chloride was almost completely transformed within days by bacteria enriched from a primary sewage sludge, municipal activated sludge (with or without acclimitization) and industrial waste water (Rittmann & McCarty, 1980; Davis et al., 1981; Klecka, 1982; Stover & Kincannon, 1983; Halbartschlager et al., 1984). In field studies it has been shown that methylene chloride is efficiently removed from water treatment works (Namkung & Rittmann, 1987). Certain strictly aerobic, facultative methylotrophic bacteria, like Pseudomonas DMI and Hyphomicrobium DM2, both readily isolated from contaminated soil and waste-water treatment plants, are capable of using methylene chloride as a sole carbon source for growth (Brunner et al., 1980; Stucki et al., 1981). Secondary substrate utilisation of methylene chloride was demonstrated by Pseudomonas sp. strain LP. This strain showed a preference towards degrading methylene chloride over acetate, whether it was the primary or the secondary substrate (Lapat-Polasko et al., 1984). In Hyphomicrobium DM2, a glutathione (GSH)-dependent, strongly inducible enzyme (a glutathione S-transferase) was found to be responsible for the degradation of methylene chloride. It converted methylene chloride to formaldehyde via the nucleophilic displacement of chloride and the formation of S-chloromethyl glutathione and S-hydroxymethyl glutathione. This enzymic dehalogenation in extracts of methylene-chloride-grown cells amounted to 1160 mg/g protein per h under alkaline (pH 8-9) conditions (Stucki et al., 1981; Leisinger, 1983). Eight other bacteria (mainly Pseudomonads ), capable of growing on methylene chloride as their sole carbon source, were isolated from enriched cultures. Maximum degradation rates for methylene chloride (up to 860 mg/litre per h) were found for an initial saturated solution of 14.5 g/litre in a pH-controlled fermenter (flow rate 10 ml/h). Further increases in degradation rate were limited by the high salt concentration resulting from the neutralization of the degradation products. In a fluidized bed reactor with bacteria immobilized on silica, a degradation rate of methylene chloride of up to 1600 mg/litre per h was observed (Gälli and Leisinger, 1985; Stucki, 1990). Ubiquitous soil- and water-dwelling nitrifying bacteria such as Nitrosomonas europaea, which depends for growth on the oxidation of ammonia, were able to degrade 1 mg methylene chloride/litre completely within 24 h in the presence of ammonia and by 67% in the absence of ammonia (Vannelli et al., 1990). The removal of methylene chloride from aerobic soil was significantly increased following exposure to methane (Henson et al., 1988). Flathman et al. (1992) described the remediation of ground water contaminated with dichloromethane after a leak. Air stripping was used initially on water pumped out from the contaminated site, and 97% of the contamination was removed in this way. This was followed by the first phase of bioremediation, in which contaminated water was withdrawn from the site and added to a bioreactor containing bacteria acclimated to DCM. The treated water was reinjected on the site together with the bacteria. This phase decreased the concentration by 97% over a period of 40 days. A second phase of bioremediation followed some 3 years later, dealing with a subsection of the original site. In this case, the indigenous bacteria were used and nutrients were added to the site. Concentrations before treatment were up to 5200 mg/litre; after 10 months these had reduced to < 2 mg/litre. At this point active treatment ceased, but the levels of DCM continued to decrease, falling below 10 µg/litre at all but one of the sampling sites. The biodegradation of methylene chloride in contaminated ground water can be strongly inhibited in the presence of other contaminants such as 1,2 dichloroethane, xylene and ethylbenzene (Scholz-Muramatsu et al., 1988). Aerobic biodegradation of methylene chloride was observed in a variety of surface soils including sand, a sandy loam and a sandy clay loam, as well as in subsurface clay soil. Degradation occurred over concentrations ranging from approximately 0.1 to 5 mg/litre. The time required for 50% disappearance of the parent compound varied between 1.3 and 191.4 days. 4.3.2 Anaerobic Details of studies on the anaerobic biodegradation of methylene chloride are given in Table 6. Methylene chloride was degraded at a concentration of 200 µg/litre in the aqueous phase of natural sediment. Degradation was observed to proceed via methyl chloride, although accumulation was not observed (Wood et al., 1981). After a varying acclimation period using anaerobic digestion in waste water, 86-92% conversion to CO2 will occur (Gossett, 1985). The half-life of methylene chloride in an anaerobic water/sludge system is 11 days (Bayard et al., 1985). Methylene chloride degradation was observed under anaerobic conditions in sandy loam soil (Davis & Madsen, 1991). 4.3.3 Bioaccumulation The n-octanol/water partition coefficient for methylene chloride is 18 (log Pow = 1.25-1.3). As a consequence, its bioaccumulation is not expected to be significant. Moreover, its high depuration and degradation rate will reduce the probability of bioaccumulation. No experimental bioconcentration factor (BCF) for methylene chloride is available. Its theoretical BCF ranges between 0.91 and 7.9 (Veith et al., 1980; Lyman et al., 1982; Veith and Kosian, 1983; Bayard et al., 1985). Further data indicative of bioaccumulation in aquatic organisms and human breast milk can be found in sections 5.1.3 and 5.3.1, respectively. There is no evidence of biomagnification. 4.4 Interaction with other physical, chemical or biological factors The ozone-depletion potential (ODP) of methylene chloride, as compared to the standard ODP of CFC11, can be estimated from the numbers of chlorine atoms (2 as compared to 3 for CFC11) and the atmospheric lifetime (0.7 years as compared to 60 years). This results in an ODP for methylene chloride of 0.4% of that of CFC11. Table 6. Aerobic biodegradation of methylene chloride Test system Condition Duration Degradation Initial concentration Reference Laboratory studies Unknown aerobic, BOD 20 days none Klecka (1982) Domestic waste aerobic 28 days none Kawasaki (1980) water (MITI) Domestic waste aerobic, static, 7 days for each 100% 5, 10 mg/litre, loss by Tabak et al. (1981) water subcultures taken at days culture transformation volatilization 6.25% 14 and 21 Enriched primary aerobic, static, closed 24 h almost 25 mg/litre Rittmann & McCarty sewage effluent complete (1980) transformation Industrial waste aerobic 6 h 92% 50 mg/litre Davis et al. (1981) water, municipal transformation, activated sludge no metabolites Activated sludge aerobic, continuous-flow 2-6 days > 99% 180 mg/litre, loss by Stover & Kincannon reactor volatilization 5% (1983) Municipal activated aerobic 50 h 49-66% 1, 10, 100 mg/litre Klecka (1982) sludge (9-11 days mineralization acclimatization) Activated sludge aerobic 20-28 mg//litre 264-1300 mg/litre Halbartschlager et al. (6 weeks per hour (1984) acclimatization) transformation Table 6 (Cont'd) Test system Condition Duration Degradation Initial concentration Reference Field studies Water treatment aerobic 30-55% removal 50-150 µg/litre Loehr (1987) works Conventional aerobic 5-6 h 96.0-96.3% Namkung & Rittmann activated sludge transformation (1987) plant At the current estimated total emission rate of 500 000 tonnes per year, the calculated tropospheric chlorine loading due to methylene chloride is 35 ppt, i.e. approximately 1% of the total chlorine loading of 3600 ppt (WMO, 1991). As methylene chloride has a low photochemical ozone creation potential in the troposphere (0.9), when compared with chemicals such as ethanol (27) or ethylene (100), it will not contribute significantly to photochemical smog formation (Derwent & Jenkin, 1991). 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE Appraisal As a consequence of release during its production and use, methylene chloride is found in biota, water and air. Levels in water and air tend to be higher in industrial and urban areas than in rural areas. Improved control of emissions has led to lower environmental levels of methylene chloride. For the general population, air is the major source of exposure to methylene chloride. In indoor air, higher levels may result from the use of consumer products which contain methylene chloride. High levels of methylene chloride may occur for short periods of time when paint strippers and aerosols are used. Exposure to methylene chloride can occur during its production and use as a paint stripper. cleaner, degreaser, process solvent and as an aerosol. 5.1 Environmental levels Environmental levels measured before 1980 were summarized in EHC 32: Methylene Chloride (IPCS, 1984). This monograph therefore focuses on levels measured after 1980. 5.1.1 Atmosphere 5.1.1.1 Ambient air In the ambient air of rural and remote areas, mean background levels of methylene chloride are 0.07-0.29 µg/m3 (Table 7). The average concentrations in suburban and urban areas, respectively, are reported to be < 2 µg/m3 and < 15 µg/m3. In the vicinity of hazardous waste sites, up to 43 µg/m3 have been round. 5.1.1.2 Precipitation Rain water sampled in Koblenz (Germany) in 1982-1983 was found to contain up to 4 µg methylene chloride/litre (Hellmann, 1984). 5.1.2 Water Data on the levels of methylene chloride in water are presented in Table 8. Table 7. Methylene chloride levels in ambient air Country/ Location Year of Concentration Reference region measurement (µg/m3) Germany urban area: Frankfurt 1980 2.1-4.2 Arendt et al. (1982) Italy northern part 1983-1984 < 14 De Bortoli et al. (1986) Netherlands Delft, Vlaardingen (urban 1980-1981 14.1 (max. annual mean) Guicherit & Schulting (1985) area) Isle of Terschelling (rural, 1980-1981 1.4 (max. annual mean) Guicherit & Schulting (1985) suburban area) mean concentration in 1980-1981 9 Guicherit & Schulting (1985) the country USA rural, suburban areas - 0.18-2.1 Shah & Heyerdahl (1988) San Francisco Bay area 1984 3.2-9.1 Levaggi et al. (1988) urban areas 1980 0.8-6.7 Shah & Heyerdahl (1988) Shikiya et al. (1984) 1980-1981 1.35-6.76 Singh at al. (1982) 1981 0.8-2.5 Harkov (1984) 1982 2.4-4.2 Harkov (1984) 1987 0.95-1.64 Pleil & McClenny (1990) 1988 0.62-1.80 Pleil & McClenny (1990) 1989 0.48-1.68 Pleil & McClenny (1990) hazardous waste sites 1983-1984 0.3-43 Harkov et al. (1985) Arctic Spitzbergen July 1982 0.26±0.04 Hov et al, (1984) March 1983 0.29±0.06 Hov et al. (1984) Northern eastern Pacific 1981 0.12±0.15 Singh et al. (1983) hemisphere Southern eastern Pacific 1981 0.07 Singh et al, (1983) hemisphere Table 8. Methylene chloride levels in water Country Location Year of Concentration Reference measurement (µg/litre) Ground water Italy Milan 1983 4.5 CEFIC (1986) USA Iowa 128 wells 1984-1985 1-5 (4 wells) Kelley (1985) Surface water Germany Mosel 1983 1.5-2.0 Hellmann (1984) Neckar 1983 0.6-1.0 Hellmann (1984) Elbe 1983 0.7-2.1 Hellmann (1984) Elbe 1988 11 (max) LWA (1990) Weser 1982-1983 < 0.5 Hellmann (1984) Weser 1988 6 (max) LWA (1990) Rhine at various sites 1981-1983 < 1 LWA (1981,1982,1983) Rhine at Koblenz 1983 5.35-171 (monthly mean) Hellmann (1984) Rhine at the Wesel 1983 < 2.0 Hellmann (1984) Rhine at Duisburg 1984 1.5 (max) LWA (1984) Rhine at various sites 1988 3.3 (max) LWA (1989) Table 8 (Cont'd) Country Location Year of Concentration Reference measurement (µg/litre) 1989 1.0 (max) LWA (1990) 1990 1.1-3.9 (90th percentile) LWA (1991) 1991 < 0.1 (max) LWA (1992) 1986 0.1 (mean) BUA (1986) Main 1985 ± 0.2 Van de Graaff (1986) Emscher 1988 8.5 (max) LWA (1989) 1989 2.5 (max) LWA (1990) 1990 3.9 (max) LWA (1991) 1991 < 0.1 (max) LWA (1992) Lippe 1988 5.5 (max) LWA (1989) 1989 < 1 (max) LWA (1990) 1990 2.4 (90th percentile) LWA (1991) 1991 < 0.1 (max) LWA (1992) Wupper 1988 2.3 (max) LWA (1989) 1989 13.6 (90th percentile) LWA (1990) 1990 3.0 (90th percentile) LWA (1991) Table 8 (Cont'd) Country Location Year of Concentration Reference measurement (µg/litre) USA Susquehanna river, 1987 10 (mean) Smith (1989) Columbia Lancaster 1987 4.7 (mean) Smith (1989) Ohio river basin (11 1980-1981 > 1 (238 samples) Howard et al. (1990) stations, 4972 samples) > 10 (19 samples) Sea and estuarine East Pacific Ocean 1981 0.002 (mean) Singh et al. (1983) water (30 samples) East Sea (German Coast) 1983 1.3-2.6 Hellmann (1984) North Sea (German 1983 0.06-0.20 Hellmann (1984) Coast) In surface water, levels of methylene chloride have been reported to vary from not detectable to 10 µg/litre. According to data recorded in the US EPA STORET database, 30% of the samples showed methylene chloride levels above the detection limits. A median concentration of 0.1 µg/litre was estimated (Staples et al., 1985). Limited information concerning the contamination of sea water and estuaries by methylene chloride is available. It appears that methylene chloride can be found at up to 2.6 µg/litre in coastal waters of the Baltic Sea. Levels of up to 0.20 µg/litre have been found in North Sea coastal waters. Methylene chloride is generally not detected in open oceans. A mean concentration of 2.2 ng/litre has been reported in the South Pacific Ocean. Methylene chloride enters the aquatic environment primarily through waste water discharge. An estimated amount of 0.2% of the total methylene chloride production is released in waste water (Dequinze et al., 1984). The input from air rain-out has been estimated for the northern and southern hemisphere (section 4.1.1). Waste water from certain industries has been reported to contain methylene chloride at average concentrations in excess of 1000 µg/litre, these being coal mining, aluminium forming, photographic equipment and supplies, pharmaceutical manufacture, organic chemical/plastics manufacture, paint and ink formulation, rubber processing, foundries and laundries. The maximum concentration measured was 210 mg/litre in waste water from the paint and ink industry and the aluminium-forming industry (US EPA, 1981). In the US EPA STORET database on industrial effluents, 38.8% of the samples recorded contained methylene chloride with a median concentration of 10 µg/litre (Staples et al., 1985). Samples from the outfalls of four municipal treatment plants in Southern California, USA, with both primary and secondary treatment, contained < 10 to 400 µg methylene chloride/litre (Young et al., 1983). In 30 Canadian water-treatment facilities, average concentrations of methylene chloride in summer and winter were found to be 10 µg/litre and 3 µg/litre, respectively (maximum, 50 µg/litre) (Otson et al., 1982). In leachate from industrial and municipal landfills, methylene chloride concentrations were reported to range from 0.01 to 184 000 µg/litre (Sabel & Clark, 1984; Brown & Donnelly, 1988; Sawhney, 1989). Background data on ground water contamination by methylene chloride are limited. It is the sixth most frequently detected organic contaminant in ground water at hazardous waste disposal sites in the CERCLA database (178 sites), the detection frequency being 19% (Plumb, 1987). In contaminated ground water in Minnesota, USA, up to 250 µg/litre has been detected (Sabel & Clark, 1984). Levels of up to 110 µg/litre were found in percolation water from a waste-disposal site in Germany. However, methylene chloride was not found (< 1 µg/litre) in the ground water below the site (Heil et al., 1989). 5.1.3 Aquatic organisms Concentrations of methylene chloride in freshwater organisms have been reported for oyster and clams from Lake Ponchartrain, Louisiana, USA. Levels ranging from 4.5 to 27 µg/kg (wet weight) could be detected (Ferrario et al., 1985). No methylene chloride was detected in fish taken from the River Rhine in 1981 (Binnemann et al., 1983). Levels of methylene chloride up to 700 µg/kg wet weight were found in marine bottom fish taken from Commencement Bay in the state of Washington, USA (Nicola et al., 1987). Data on biota collected in the US EPA STORET data base show an average level of 660 µg/kg in the 28% of the samples in which methylene chloride was detected (Staples et al., 1985). 5.1.4 Soil and sediment No data are available on the levels of methylene chloride in soil. The levels of methylene chloride found in sediment from Lake Pontchartrain, Louisiana ranged from not detectable to 3.2 µg/kg wet weight (Ferrario et al., 1985). Data recorded in the US EPA STORET database revealed a median concentration of 13 µg/kg in 20% of 338 sediment sampling data (Staples et al., 1985). The levels of methylene chloride found in sediments from the river Rhine in 1987-1988 varied from non-detectable to 30-40 µg/kg. At one site maximum concentrations of 220-2200 µg/kg were measured (BUA, 1993, personal communication to the IPCS). 5.2 Human exposure 5.2.1 General population 5.2.1.1 Indoor air In buildings where products containing methylene chloride are used, air levels of methylene chloride much higher than outdoor levels (< 15 µg/m3, see section 5.1.1.1) may be found (Table 7). Relatively high levels (mean 670 µg/m3, peak level 5000 µg/m3) have been found in the indoor air of residential houses (De Bortoli et al., 1986). 5.2.1.2 Drinking-water Methylene chloride has been detected in drinking-water supplies (estimations made before 1980) in numerous cities in the USA (Dowty et al., 1975; Coleman et al., 1976; Kopfler et al., 1977; Kool et al., 1982), the mean concentrations reported being generally less than 1 µg/litre. An average of 3-10 µg/litre and a maximum of 50 µg/litre were observed in a Canadian study of 30 drinkable water treatment facilities (Otson et al., 1982). Samples from 128 drinking-water wells in the USA showed that 3.1% of them had methylene chloride levels of 1-5 µg/litre (Kelley, 1985). Rodruigez Rojo et al. (1989) sampled the drinking-water of Santiago de Compostela, Spain, in 1987. Methylene chloride was found in 98.4% of the samples; the average concentration was 14.1 µg/litre, with a range of 1.2-93.2 µg/litre. Other halomethanes were also found and measured in the samples at average concentrations ranging from 9 to 25 µg/litre. A wide sampling exercise involving 630 public community water supplies (serving 6.9 million people in New Jersey, USA) was carried out in 1984 and 1985 by McGeorge et al. (1987). The percentage of positive results for methylene chloride ranged from 2.6 to 7.1%. The median concentration ranged from 1.1 to 2.0 µg/litre and the range for the whole sampling period was 0.5 to 39.6 µg/litre. 5.2.1.3 Foodstuffs Although methylene chloride is used in food processing (solvent extraction of coffee, spices, hops), there is little information on its residual levels in food. In the USA, residues of methylene chloride were found in decaffeinated coffee beans (0.32 to 0.42 mg/kg) whilst a major coffee processor reported levels of 0.01 to 0.1 mg/kg (ATSDR, 1992). No methylene chloride was detected in ice-cream and yoghurt (BUA, 1986). In seven types of decaffeinated ground coffee the methylene chloride content ranged from < 0.05 to 4.04 mg/kg; in eight instant coffee samples <0.05 to 0.91 mg/kg was found (Page & Charbonneau, 1984). Heikes & Hopper (1986) analysed samples of grains and intermediate grain-based foods for a range of fumigants using a purge-and-trap method. Methylene chloride was not found in any of the grain samples, nor in uncooked rice or dried lima beans. It was found in some of the intermediate foods such as bleached flour (30 µg/kg), yellow corn meal (4.7 µg/kg), lasagne noodles (5.4 µg/kg) and yellow cake mix (4.6 µg/kg). One of the authors (Heikes, 1987) investigated levels of methylene chloride in table-ready foods, taken from the US Food and Drug Administration's Total Diet Study. Of the 19 foods examined, eight contained methylene chloride above the quantification limit (not given). Detailed results for six of the foods are given in Table 9. Table 9. Dichloromethane content of table ready foods (Heikes, 1987) Food Number of Number Range of samples positive concentration (µg/kg) Butter 7 7 1.1-280 Margarine 7 7 1.2-81 Ready-to-eat cereal 11 10 1.6-300 Cheese 8 8 3.9-98 Peanut butter 7 4 26-49 Highly processed foodsa 12 10 5-310 a e.g., frozen chicken dinner, fish sticks, pot pie 5.2.1.4 Consumer exposure Consumers are exposed to methylene chloride via the use of a number of formulated products such as aerosols or paint strippers. A USA survey found that 78% of paint removers and 66% of aerosol spray paints sold as household products contained methylene chloride (US EPA, 1987). Over 100 consumer products in Sweden contain methylene chloride (National Chemical Inspectorate, Sweden, personal communication to the IPCS). In Norway the number is around 140, including 45 paint removers (AKZO, personal communication to the IPCS). Methylene chloride does not appear to be subject to widespread volatile substance abuse. Statistics on deaths resulting from substance abuse in the United Kingdom were collected over the period 1971-1991 and analysed by product type. Of the 1221 deaths recorded, five were assigned to the group "paint thinners and paint strippers". Methylene chloride is used only in the latter products, the former containing solvents such as toluene and xylene which are known to be substances of abuse (Flanagan et al., 1990). A large do-it-yourself consumer population uses paint strippers containing methylene chloride on furniture and woodwork. Formulations are available mainly in liquid form, but also, occasionally, as an aerosol. Exposures have been estimated on the basis of USA investigations of household solvent products. The estimated levels ranged from less than 35 mg/m3 to a few short-term exposures of 14 100 to 21 200 mg/m3. The majority of the concentration estimates were below 1770 mg/m3 (US EPA, 1990). Methylene chloride exposure was estimated while using a number of formulations of paint stripper in a small room. Various ventilation conditions were evaluated and a worst possible case was simulated, with doors and windows closed. In one test, involving furniture stripping in a room with through ventilation, the operator exposure was found to be 289 mg/m3 on a 2-h TWA. Peaks of exposure were observed during application (460 mg/m3) and during scraping-off (710-1410 mg/m3) (ICl, 1988, personal communication to the IPCS). A series of paint-stripping exercises were performed in a small room. Various ventilation conditions were evaluated while using a number of formulations of paint stripper. A worst possible case was simulated with doors and windows closed. Concentrations of methylene chloride in the room rose to 14.1-17.6 g/m3 (4000-5000 ppm), although it is questionable whether anyone could work in such conditions without breathing apparatus. Further exercises with the door and windows open (as recommended by suppliers) reduced atmospheric concentrations by more than a factor of 10. Exposure to methylene choride resulted in an 8-h TWA of 187-226 mg/m3 (53-64 ppm). The actual stripping operations took between 1 and 1.5 h. Maximum exposures occurred during initial application and scraping- off. These exposures were of a few minutes duration and concentrations never exceeded 3.53 g/m3 (1000 ppm) (ICI, 1990, personal communication to the IPCS). The effect of variation in the formulation was also investigated. During paint stripping, background concentrations in the room varied from 710-1410 mg/m3 to less than 350 mg/m3 depending on the formulation. However, within 5-6 min of application the level of methylene chloride fell to an equilibrium concentration of around 71 mg/m3, irrespective of the formulation used. The shortest time before reaching equilibrium was about 2-2.5 min (ICI, 1990, personal communication to the IPCS). Studies in the Netherlands have measured peak concentrations in salons of 21-106 mg/m3 (6-30 ppm), with an 8-h TWA of 3.53-17.65 mg/m3 (1-5 ppm) (CEC Scientific committee of Cosmetology). The same study measured a peak concentration of 265 mg/m3 (75 ppm) arising from home use of a hairdressing aerosol containing 35% methylene chloride. This equates to a TWA of 2.65 mg/m3 (0.75 ppm). Studies in the United Kingdom simulating consumer exposure during salon use yielded values well within the national Maxium Exposure Limit (353 mg/m3 or 100 ppm for an 8-h TWA). The hairdresser received an exposure of 77.7 mg/m3 (22 ppm, on an 8-h TWA) during what is considered to be exceptionally heavy use (i.e. a 10-second spray every 15 min for an 8-h period). The customer exposure was found to be 106-265 mg/m3 (30-75 ppm) (10-min TWA) (ICI, 1990, personal communication to the IPCS). The same study simulated home use of personal-care aerosols containing methylene chloride. Even adverse conditions (small room, no ventilation) resulted in an exposure of 353 mg/m3 (100 ppm) (10-min TWA), equating to 7.06 mg/m3 (2 ppm) on an 8-h TWA (ICI, 1990, personal communication to the IPCS). This work was characterized by low air changes, virtually no ventilation and a more frequent rate of application than that determined by surveys of actual hairdressing work in salons. 5.2.2 Occupational exposure Exposure to methylene chloride can occur during its production and use as a paint stripper, cleaner, degreaser, process solvent and as an aerosol. Exposure concentrations that have been reported in various industries are presented in Table 10. Below is a brief description of exposure conditions in some of the reported industries. 5.2.2.1 Production Production of methylene chloride is normally carried out in a closed system, with a relatively small number of people being involved. Exposure arises primarily during filling and packing operations. Occupational exposures are listed in Table 10. Some measured ranges, e.g., 35-81 mg/m3 and 85-244 mg/m3 (HSE, 1992), indicate that engineering control techniques can bring 8-h TWA exposures below 350 mg/m3. 5.2.2.2 Paint stripping and related activities Workers in the formulation of paint removers are exposed while transferring methylene chloride from storage tanks, during mixing (blending) operations and while packaging. The extent of exposure will depend on the control measures and work practices in force. Exposure levels (8-h TWA) range from a low of 0-18 mg/m3 to over 1770 mg/m3 (US EPA, 1990). Table 10. Occupational exposure to methylene chloride Industry Activity Exposure range Commentsa Reference (8-h TWA) (mg/m3) Production Production actvities 219-374 Maintenance activity with RPE; HSE (1987) Results obtained at one plant Aircraft Paint stripping 35-81 RPE provided HSE (1992) Paint stripping 35-289 Submission to OSHA in 1987. RPE Air Transport Association provided (USA) Various industries Painting 21-299 See IARC (1986) Chrostek & Levine (1981) Paint stripping 18-1765 US EPA (1990) Used aerosol < 0-494 Some work areas were congested Fleeger & Lee (1988) adhesives Pharmaceuticals - 7.1-3749 NCI feasibility study Zahm et al. (1987) Production work 0-18 Enclosed process HSE (1992) - Aerosol products Aerosol filling 95-628 ICI (UK) (1984) Rubber products Fabrication 208-304 LEV HSE (1992) Fibre glass Cleaning and mould 187-6693 intermittent exposure, RPE may be HSE (1992) manufacture preparation worn; may not be representative of the industry Cleaning, mixing etc. 0-350 Small factory units Post et al. (1991) Printing 3.5-558 NCI feasibility study Zahm et al. (1987) Table 10 (Cont'd) Industry Activity Exposure range Commentsa Reference (8-h TWA) (mg/m3) Triacetate Production 180-350 ECSA (1989) fibre/film 237-3442 NCI feasibility study Zahm et al. (1987) manufacturing 180-2440 Products contain acetone Ott et al. (1983) Furniture Paint stripping 25-3810 Many without adequate controls HSE (1992) Paint stripping 201-1292 Variable degrees of control McCammon et al. (1991) Washing/refinishing 53-780 Variable degrees of control McCammon et al, (1981) Spraying adhesive 219-1490 Many without adequate controls HSE (1992) General Cleaning, degreasing, 53-141 See IARC (1986) Ruhe et al. (1981) manufacturing, etc. < 0-460 See IARC (1986) Ruhe et al. (1982) cleaning and degreasing Foam industry Glue spraying 85-244 LEV HSE (1992) Moulding 88-1090 High exposure due to HSE (1992) insufficient/inadequate LEV < 247 Jernelov & Antonsson (1987) Unknown 7.1-251 NCI feasibility study Zahm et al. (1987) Various jobs 18-580 High exposure experienced by Boeniger (1991) sprayers Motor vehicle Paint spraying, 7.1-247 LEV and RPE HSE (1992) manufacture stripping Table 10 (Cont'd) Industry Activity Exposure range Commentsa Reference (8-h TWA) (mg/m3) Quarry Laboratory work, 71-1370 High exposures due to inadequate HSE (1992) mineral processing control Metal treatment 7.1-790 NCI feasibility study Zahm et al. (1987) Nutrition Extraction < 0-106 See IARC (1986) Cohen et al. (1980) a RPE = respiratory protection equipment; LEV = local exhaust ventilation Paint strippers are widely used in a number of industries: automotive, rubber products, furniture and fixtures, plastic, and electronic industries. Exposure to methylene chloride takes place during application, removal of the substrate soaked in methylene chloride, and the disposal of the spent paint remover. Typical exposures (8-h TWA) range from 18 mg/m3 to about 1770 mg/m3 (US EPA, 1990). Exposure of commercial furniture refinishers to methylene chloride occurs when stripping involves either the dipping of furniture into a tank containing a mixture of solvents including methylene chloride (typically 65%) or coating it manually with a brush. Exposure levels are highly variable and greatly influenced by the size of the organization, engineering controls in place and work practices. Some refinishers may operate on a part-time basis and from their homes. In some instances where the worker was leaning over the tank or using a brush to scrub the surface coating, concentrations of > 7100 mg/m3 have been recorded (McCammon et al., 1991; HSE, 1992). Better work stations and work practices have helped to reduce exposures greatly. 5.2.2.3 Aerosol production and use In the packaging of aerosol cans, exposure arises primarily during filling and packing. Levels observed are generally below 180 mg/m3 (ECSA, 1989). Potential occupational exposure to methylene chloride as a result of aerosol products varies according to the use and the work undertaken. Consumer exposure due to the use of cosmetic and paint spray aerosols is discussed in section 5.2.1.4. 5.2.2.4 Use as a process solvent Methylene chloride is widely used as a process solvent in the manufacture of a variety of products. Most of the processes are carried out in closed systems, with the exception of triacetate fibre and film manufacture. Normally, exposure levels are low, but occasionally high exposures (> 350 mg/m3; 10-min TWA) may occur in such operations as filter changing, charging and discharging. Some industrial processes involve somewhat higher exposure levels; in particular, the manufacture of cellulose triacetate fibres and films can involve exposure up to 350 mg/m3 (8-h TWA) even when good engineering controls are installed (Zahm et al., 1987; ECSA, 1989). As part of an epidemiological study of cellulose fibre production workers, Ott et al. (1983) reported exposure levels ranging from 177 to 2436 mg/m3 in the processing area, and exposure ranging from 18 to 1341 mg/m3 in the preparation area based on sampling performed in 1978. The range for the entire plant was later reported to be from below detectable limits to 6000 mg/m3 (Lanes et al., 1990). As part of an epidemiological study of photographic film workers, Friedlander et al. (1978) reported exposures ranging from 0 to 1236 mg/m3 based on sampling carried out between 1959 and 1975. The highest mean exposures at this plant were reported for group leaders (402 mg/m3) (Hearne et al., 1987). In the pharmaceutical industry, methylene chloride is used as a solvent and extraction medium. Sealed processes, high recovery rates and careful handling of discharges have helped to keep the exposure levels below around 106 mg/m3 (Zahm et al., 1987; HSE, 1992). Methylene chloride is also used as an extraction medium in the nutrition industry, where the exposure levels are generally low when the processes are adequately controlled (Cohen et al., 1980). Methylene chloride is used in the foam industry for cleaning process equipment, purging spray guns, and as an auxiliary blowing agent. It is also used as a releasing agent in the moulding of polyurethane products. Exposure levels ranging from a few mg/m3 to short-term exposures of over 1770 mg/m3 have been reported (Jernelov & Antonsson, 1987; Boeniger, 1991; HSE, 1992). The use of methylene chloride as a solvent in adhesives can result in occupational exposure, during the application of the adhesive, to short-term levels in excess of 350 mg/m3 (Fleeger & Lee, 1988; HSE, 1992). Processes involving the formulation of adhesives are likely to be well controlled. Methylene chloride is also used as a solvent in the analysis of bitumen samples. This work is normally carried out in small laboratories and exposure levels will be high unless adequate control measures are used. 5.2.2.5 Cleaning and degreasing In the manufacture of metal products, cleaning (degreasing) is required before painting, plating, plastic coating, etc. The degree of exposure to methylene chloride will be influenced by many factors, including the age of the equipment, type of engineering controls available, their maintenance, handling, and drying methods. In general, it is possible to reduce exposure levels to below 124 mg/m3 (Swedish National Board of Occupational Safety & Health, personal communication to the IPCS). 5.2.3 Occupational exposure limits A listing of some national occupational exposure limits is given in Table 11. Table 11. Occupational exposure limit valuesa Country TWA STEL TWA STEL Remarks Reference (mg/m3, 20°C)b (ppm) (ppm) (ppm) Australia 350 - 100 - Suspected carcinogen ILO (1991) Austria 360 1800c 100 500 Suspected of carcinogenic DFG (1991) potential Belgium 174 - 50 - Suspected human carcinogen ACGIH (1992) Czechoslovakia 500 2500 - - ILO (1991) Denmark 174 - 50 - Absorption through skin may be ILO (1991) significant Suspected carcinogen Finland 350 870 100 250 ILO (1991) France 360 1800 100 500 ILO (1991) Germany 360 1800c 100 500 Suspected of carcinogenic DFG (1991) potential Italy 174 - 50 - Suspected human carcinogen ACGIH (1992) Japan 350 - 100 - ILO (1991) Netherlands 350 1750 100 500 Arbeidsinspectie (1991) Norway 125 - 35 - Carcinogen Arbeidstilsynet (1990) Table 11 (Cont'd) Country TWA STEL TWA STEL Remarks Reference (mg/m3, 20°C)b (ppm) (ppm) (ppm) Portugal 174 - 50 - ILO (1991) Sweden 120 250d 35 70 Classified as a low potent AFS (1990) carcinogen Switzerland 360 1800 100 500 Classification for teratogenic ILO (1991) effects not possible; biological monitoring required United Kingdom 350 870 100 250 Maximum exposure limit USA - ACGIH 174 50 Suspected human carcinogen ACGIH (1992) a TWA = time-weighted average concentration (8-h working period); STEL = short-term exposure limit (15 min, unless specified) b Official values; some countries use different conversion factors and/or other ambient temperature c 30 min d 15 min 5.3 Human monitoring data 5.3.1 Body burden Methylene chloride was detected in all eight samples of human milk from four urban areas (Pellizzari et al., 1982). Hardin & Manson (1980) could still find methylene chloride in mother's milk 17 h after the end of exposure. In 12 male volunteers exposed to 2600 mg/m3, biopsies showed adipose tissue levels of 10.2, 8.4 and 1.6 mg/kg at 1, 4, and 22 h, respectively, after a 1-h exposure (Engström & Bjurström, 1977). When Antoine et al. (1986) analysed whole blood from 250 individuals, the mean concentration of methylene chloride was 0.7 µg/litre with a range from not detected to 25 µg/litre. BUA (1986) monitored saliva and tissues from people living in industrialized areas of Beckum, Germany. They reported that no methylene chloride was detected. 5.3.2 Occupational exposure studies In a cohort of 14 furniture strippers exposed to methylene chloride at concentrations of 53 to 1290 mg/m3, post-exposure breath concentrations of methylene chloride ranged from 8.1 to 590 mg/m3 (McCammon et al., 1991). Mother's milk in Soviet women manufacturing rubber articles contained a mean of 74 µg/kg in 17 out of 28 samples approximately 5 h after start of work, the level declining after termination of work (Jensen, 1983). A group of seven non-smoking workers, who had previously been exposed to methylene chloride for several years, were exposed to a mean concentration of 635 mg/m3 (in addition, there was exposure to 154 mg/m3 (mean) chloroform). The pre-exposure average carbon monoxide level in alveolar air was 34 mg/m3, increasing to 58 mg/m3 during exposure; before the next exposure, the carbon monoxide level was 27 mg/m3: this corresponded to carboxyhaemoglobin (CO-Hb) levels of 4.9%, 8.3% and 3.9%, respectively. The biological half-life of CO-Hb was 13 h (Rathey et al., 1974). Although methylene chloride does not accumulate following repeated exposure, these data clearly indicate that CO-Hb levels will be cumulative if the periods between exposure are insufficient to allow the CO-Hb levels to return to normal. CO-Hb levels in a worker accidentally overcome by methylene chloride vapour were found to have increased to 19%. A further worker with a history of ischaemic heart disease, who had been exposed concurrently with the first patient, had a CO-Hb level of 6% on the day following the exposure (Benzon et al., 1978). Methylene chloride levels in alveolar air and blood were measured in 14 shoe-sole factory workers. The alveolar concentration, mean blood levels, and CO-Hb levels, following exposure to 74±28 mg/m3 methylene chloride, were: 49 mg/m3, 0.41 mg/litre, and 4.0%, respectively; upon exposure to 124+42 mg methylene chloride/m3: 71 mg/m3, 0.99 mg/litre, and 5.2%, respectively; and upon exposure to 339±265 mg methylene chloride/m3: 229 mg/m3, 3.07 mg/litre, and 6.5%, respectively. In this factory, the methylene chloride exposure was highly variable; the data are too limited to allow valid extrapolation (Perbellini et al., 1977). The relationship between low environmental levels of methylene chloride, carbon monoxide in alveolar air and urinary excretion of methylene chloride was studied in a group of 20 manufacturing workers (12 smokers, 8 non-smokers). A good correlation was observed between levels of methylene chloride and urinary excretion of methylene chloride. The correlation between alveolar air and levels of methylene chloride was poor except when the analysis was restricted to nonsmokers (Ghittori et al., 1993) 5.3.3 Biological exposure indices Biological Exposure Indices (BEIs) are reference values intended for guidelines for the evaluation of potential health hazards in industrial hygiene practice. The BEIs for methylene chloride at the end of a working shift have been given as: CO-Hb level 5%, blood level of methylene chloride 1 mg/litre. Biological monitoring of methylene chloride exposure can be based on measurement of the solvent itself in exhaled air or blood. However, as production of carbon monoxide with exposure for more than 3-4 h/day appears to be the limiting factor with respect to health risk, biological monitoring based upon either analysis of carbon monoxide in exhaled air or of CO-Hb in blood is to be preferred. However, this can only be applied in non-smoking subjects. Sampling should be carried out either about 0-2 h after exposure or 16 h later, i.e. on the following morning. In the case of an 8-h exposure to less than 350 mg methylene chloride/m3 in non-smokers, CO-Hb levels 2-h after exposure ceases are not expected to exceed 2-3%, and after 16 h to exceed 1%. 6. KINETICS AND METABOLISM Appraisal Methylene chloride is rapidly absorbed though the alveoli of the lungs into the systemic circulation. It is also absorbed from the gastrointestinal tract and dermal exposure results in absorption but at a slower rate than that of the other exposure routes. Distribution studies indicate that, via inhalation or dermal exposure, methylene chloride distributes to all tissues. It can cross the blood-brain barrier and it can be transferred across the placenta. Concentrations of methylene chloride rise more slowly in adipose tissue and longer exposures are required before these tissue levels equal those of the blood. Data indicate that methylene chloride and/or its metabolites do not accumulate in tissues. Methylene chloride is metabolized to carbon monoxide, carbon dioxide and inorganic chloride. Methylene chloride is eliminated from the body primarily via the lungs in expired air. Urinary excretion plays a minor role in its elimination. As exposure levels increase, a large proportion of methylene chloride is exhaled unchanged. Metabolism occurs by either or both of two pathways; their relative contribution to the total metabolism is markedly dependent on the exposure level and on the animal species concerned. One pathway involves oxidative metabolism mediated by cytochrome P-450 and leads to both carbon monoxide and carbon dioxide. This pathway appears to operate similarly in a qualitative and quantitative sense in all rodents studied and in humans. This is the predominant metabolic route, but saturation occurs at around 1800 mg/m3. Increasing the dose above the saturation level does not lead to extra metabolism by this route. The other pathway involves a glutathione transferase, and leads via formaldehyde and formate to carbon dioxide. This route seems only to become important at doses above the saturation level of the "preferred" oxidative pathway. There are marked species and dose- dependent differences in the contribution that this pathway makes to the metabolism of methylene chloride. 6.1 Absorption 6.1.1 Inhalation exposure 6.1.1.1 Human studies The principal route of human exposure to methylene chloride is inhalation. Evaluation of pulmonary uptake indicated that 70-75% of inhaled vapour was absorbed in human subjects exposed to 180, 350, 530 and 710 mg/m3. Initial absorption was rapid, as indicated by levels of methylene chloride in the blood of approximately 0.6 mg/litre in the first hour of exposure to levels of 350-710 mg/m3. At a level of 180 mg/m3, the increase in blood methylene chloride concentration was 0.2 mg/litre for the first hour. There was a direct correlation between the steady-state blood methylene chloride values and the exposure concentration, both during the exposure and for the first 2 h after the exposure in all groups. Steady-state blood levels appeared to be reached after 4 h and remained constant until the end of exposure. Once exposure ceased, methylene chloride was rapidly cleared from the blood. Seven hours after exposure, less than 0.1 mg/litre was detected following exposure to 180, 350 or 530 mg/m3. Only 1 mg/litre was detected in the highest group (710 mg/m3) 16 h after exposure. In all other dose groups the blood levels had returned to baseline concentrations (Di Vincenzo & Kaplan, 1981a,b). In common with other lipophilic organic vapours, methylene chloride absorption appears to be influenced by factors other than the vapour concentration. Prior to reaching steady state, increased physical activity increases the amount of methylene chloride absorbed by the body, due to an increase in ventilation rate and cardiac output, since these factors increase blood flow through the lungs and promote absorption (Di Vincenzo et al., 1972; Astrand et al., 1975). Uptake also increases with the body fat percentage, since methylene chloride dissolves in fat to a greater extent than it dissolves in aqueous media. Therefore, obese subjects will absorb and retain more methylene chloride than lean subjects exposed to the same vapour concentration. Under controlled conditions, there was a 30% greater absorption and retention of methylene chloride by obese subjects exposed to 2650 mg/m3 for 1 h as compared to lean subjects (Engström & Bjurström, 1977). Åstrand et al. (1975) reported that the amount of methylene chloride taken up increases with physical workload, whereas the retention decreases. With a 50-watt workload, the uptake was twice as high, whereas the retention decreased from 55% to 45%. When exposure was coupled with workload (physical exercise), the concentration in alveolar air was increased during the whole post-exposure period compared with exposure under rest conditions. 6.1.1.2 Animal studies Studies of the relationship between inhalation exposures of animals and their blood methylene chloride concentrations indicate that absorption is proportional to the magnitude and duration of the exposure over a methylene chloride concentration range of 350 to 28 200 mg/m3. This conclusion is based on the monitoring of blood methylene chloride concentrations following inhalation exposure in dogs and rats (Di Vincenzo et al., 1972; MacEwen et al., 1972; McKenna et al., 1982). As is the case with humans, blood methylene chloride levels reach a steady-state value which does not increase further as the duration of exposure increases (McKenna et al., 1982). The data from studies of blood methylene chloride values during a 6-h exposure of rats to between 180 and 5300 mg/m3 suggest that the steady-state blood/air concentration ratio increases as the exposure concentration increases. The ratio of the steady-state methylene chloride concentrations in plasma to the exposure concentration was 0.001, 0.005 and 0.006 at levels of 180, 1800 and 5300 mg/m3, respectively (McKenna et al., 1982). It is postulated that the increased ratio at steady state results from saturation of metabolic pathways as exposure increases, rather than from an increased absorption coefficient. Kim & Carlson (1986)conducted experiments to compare the effects of a 12-h exposure schedule to those of an 8-h schedule on the CO-Hb formation resulting from methylene chloride inhalation. Rats and mice were exposed to 710, 1800 or 3500 mg/m3 8 h/day for 5 days, or 12 h/day for 4 days. No significant difference in carboxyhaemoglobin levels was found. The peak blood methylene chloride level was found to be dependent upon the methylene chloride exposure concentration, but the half-life was independent of the duration of exposure or the concentration of methylene chloride, 6.1.2 Oral exposure No data are available on oral absorption of methylene chloride in humans. Treatment of mice and rats with methylene chloride in water or in corn oil suggests that methylene chloride is easily absorbed from the gastrointestinal tract. Methylene chloride levels were measured in gut segments up to 40 min after rats were administered single oral doses in water. The amounts measured were similar at both dose levels (50 or 200 mg/kg). Of the administered dose (200 mg/kg), 60% was recovered from the upper gastrointestinal tract < 10 min after treatment (20% recovery after 40 min). The amount of methylene chloride in the lower gastrointestinal tract accounted for < 2% of the administered dose up to the 40-min test interval (Angelo et al., 1986b). In mice administered oral doses of non-radioactive methylene chloride at 10 or 50 mg/kg in water, approximately 25% of the administered dose was detected in the upper gastrointestinal tract within < 20 min. Similarly, after treatment with methylene chloride at 10, 50, or 1000 mg/kg in corn oil, approximately 55% of the administered dose was detected in the upper gut segment and remained there for 2 h (Angelo et al., 1986a). 6.1.3 Dermal exposure Methylene chloride has been shown to be absorbed through human skin (Stewart & Dodd, 1964). In this study, a volunteer immersed a thumb in methylene chloride for 30 min under conditions which precluded the inhalation of vapour. The subsequent alveolar air concentration was 3.1 ml/m3; 2 h later it had fallen to 0.69 ml/m3. Various studies on the rate of absorption through animal skin and subsequent pharmacokinetics have been reported. Tissue concentrations of methylene chloride were measured in various organs (lung, liver, brain, kidney, heart and fat) of 128 white rats, using gas chromatography, following immersion of two-thirds of their tails in the solvent for 1, 2, 3 or 4 h. Small increases were seen in most tissues after 1 or 2 h of exposure, and methylene chloride concentrations in fatty tissues increased markedly after 3 h of exposure. After 4 h of exposure, methylene chloride concentrations remained elevated in fatty tissues and were increased in all other tissues studied (Makisimov & Mamleyeva, 1977). The dermal absorption rate for methylene chloride through mouse skin in vivo has been measured to be 6.58 mg/h per cm2 (Tsuruta, 1975). The dermal permeability constant for the absorption of methylene chloride vapour through rat skin in vivo has been measured following exposure to 106, 212 and 353 g/m3 for 4 h. Blood levels of methylene chloride were shown to reach steady state levels after 1 h of exposure to the two lower concentrations and after 3 h of exposure to 353 g/m3. The mean dermal permeability constant was calculated to be 0.28 mg/h per cm2 (McDougal et al., 1986). 6.2 Distribution 6.2.1 Inhalation exposure 6.2.1.1 Human studies Engström & Bjurström (1977) exposed 12 male subjects (six slim and six obese) to 2650 mg methylene chloride/m3 for 1 h. The total uptake of methylene chloride in the slim group was 1116 ± 34 mg and in the obese group 1446 ± 110 mg/kg. Estimation of methylene chloride in needle biopsies showed that the adipose tissues contained approximately 8 to 35% of the average total amount absorbed. The amount or methylene chloride absorbed was highly correlated with the degree of obesity and body weight. In the slim subjects, the concentration in the adipose tissue during the 4-h period after exposure was approximately twice that in the obese subjects. However, despite a lower concentration, the total amount of methylene chloride calculated to be in the body fat was greater in obese subjects. A survey of the levels of methylene chloride in certain tissues from pregnant or nursing women has been reported. The study was conducted following observations of disturbances in the pattern of pregnancy and lactation in female operatives in an industrial rubber article manufacturing facility. The survey was conducted in an unspecified number of women who had been exposed to several chemicals during their work for at least 3 years. The chemicals included gasoline, ethylene dichloride and methylene chloride. An estimate of the average workplace concentration of methylene chloride was reported to be 85.6 mg/m3. A control group (number unspecified) was constituted from women working in the same facility but who had had no direct contact with the chemicals. The tissues examined were the blood, the fetal membranes and the fetus, all tissue samples being obtained at the time of abortion of the fetus. The mean tissue concentrations of methylene chloride (54 observations) were reported to be 0.66 ± 0.21, 0.34 ± 0.10 and 1.15 ± 0.20 mg/kg for the blood, fetal membranes and fetus, respectively, compared to 0.12 ± 0.07, 0.013 ± 0.01 and 0.016 ± 0.001 mg/kg in the controls. Methylene chloride was also detected in 17 out of 28 specimens of breast milk taken from exposed nursing women. An average concentration of 0.074 ± 0.04 µg/litre (n = 40) was found in the breast milk 5-7 h after the start of the exposure; an insignificant quantity of methylene chloride was reported 17 h after cessation of exposure (Vosovaja et al., 1974). 6.2.1.2 Animal studies Distribution studies in rats demonstrate that methylene chloride (and/or its metabolites) is present in the liver, kidney, lungs, brain, muscle and adipose tissues after inhalation exposures (Carlsson & Hultengren, 1975; McKenna et al., 1982). One hour after exposure, the highest concentration of radioactive material was found in the white adipose tissue, followed by the liver. The concentration in the kidney, adrenal and brain were less than half that in the liver. Radioactivity in the fat deposits declined rapidly during the first 2 h after exposure (Carlsson & Hultengren, 1975). Concentrations in the other tissues declined more slowly. Whole body autoradiography in mice at one hour after inhalation of 10 µl 14C-methylene chloride for 10 min showed a rapid and even distribution immediately after exposure. A high uptake was noted in brain, body fat, blood, liver and kidney. Evaporated sections showed a high retention of non-volatile radioactivity, presumably representing metabolites, in the liver, bronchi and kidneys. Thirty minutes after inhalation, radioactivity started to appear in tissues with a high cell turnover such as bone marrow, thymus and gastrointestinal mucosa, and in tissues with a high rate of protein synthesis such as the spleen, exocrine pancreas and salivary glands (Bergman, 1979). On the other hand, after 5 days of exposure to 710 mg/m3 for 6 h/day, the concentration of methylene chloride in the perirenal fat was 6-7 times greater than that in the blood and liver (Savolainen et al., 1977). It has been suggested that methylene chloride first saturates the blood and extravascular fluid compartment before entering the fatty deposits (Di Vincenzo et al., 1972). Thus, concentrations of methylene chloride will rise slowly in adipose tissues, and longer exposures to methylene chloride will be required before adipose tissue levels equal those in the blood. The animal data are therefore consistent with the human adipose tissue data discussed above. Exposure of pregnant rats to methylene chloride leads to exposure of the fetus to both methylene chloride and carbon monoxide (Anders & Sunram, 1982). 6.2.2 Oral exposure No studies are available regarding distribution of methylene chloride in humans following oral exposure. In animals, radioactivity from labelled methylene chloride was detected in the liver, kidney, lung, brain, epididymal fat, muscle, and testes after exposure of rats to a single gavage dose of 1 or 50 mg/kg. The tissue samples were taken 48 h after dosing. At that time, the lowest concentration of radioactivity was found in the fat. The highest concentrations were in the liver and kidney. This was true for both doses (McKenna & Zempel, 1981). Similar results were observed in rats administered methylene chloride doses of 50-1000 mg/kg for 14 days. At each dose tested, and in each tissue, the label was rapidly cleared during the 240 min following each exposure (Angelo et al., 1986b). 6.2.3 Dermal exposure No information is available regarding distribution in humans or animals following dermal exposure to methylene chloride. 6.3 Metabolism Species differences in metabolism and their relevance to carcinogenicity are described in section 8.8.2. 6.3.1 In vitro studies In vitro experiments using liver fractions, homogenates, slices and hepatocytes, mainly from the rat, confirmed the presence of the two metabolic pathways. The primary reaction, first described by Kubic & Anders (1975), appears to be an oxidative dehalogenation giving carbon monoxide and chloride ion. The reaction is catalysed by rat liver microsomal fractions and is dependent upon NADPH and molecular oxygen. The presence of a binding spectrum and inducers confirmed the involvement of the cytochrome P-450 mixed function oxidase system. More recent studies have identified the cytochrome P-450 isoenzyme as cytochrome P-450 IIEl (Pankow et al., 1991; Guengerich et al., 1992; Pankow & Jagielki, 1993). The highest activity was found in liver microsomes, which were five times more active than lung microsomes and thirty times more active than kidney microsomes. The proposed metabolic route involves rearrangement of the primary hydroxylation product to formyl chloride followed by decomposition to carbon monoxide (Kubic & Anders, 1978). Although the transient intermediates have not been isolated or identified, their formation is consistent with the enzyme involved and the products formed. The effect of pyrazole on methylene chloride metabolism in male Wistar rats was investigated by Pankow et al. (1991). Rats received a single methylene chloride close of 6.2 mmol/kg (0.4 ml/kg) orally. Pyrazole was administered by intraperitoneal injection. The metabolism of methylene chloride to carbon monoxide can be stimulated or inhibited by pyrazole; the effect depends on the interval between pyrazole and methylene chloride administration, and on the dose. Stimulation of methylene chloride metabolism to carbon monoxide is due to inducers of the isoenzyme cytochrome P-450 (CYP2El) such as isoniazid, ethanol and other solvents. The inhibition was observed following pre-treatment with high pyrazole doses or following simultaneous administration of pyrazole and methylene chloride. The inhibition may reflect the competition between pyrazole and methylene chloride for oxidation by CYP2El as long as pyrazole is present in the blood, or may also reflect the hepatotoxic effect of pyrazole. Hepatic cytochrome P-450 levels were not increased in rats exposed by inhalation to methylene chloride (5.29-10.59 g/m3 (1500 or 3000 ppm)) 6 h/day for 3 clays (Toftgard et al., 1982), nor in rats exposed to 1.76 or 3.53 g/m3 (500 or 1000 ppm) 6 h/day for 2 weeks (Kurppa & Vainio, 1981). Marginal increases were seen in a third study (Norpoth et al., 1974) in which rats were exposed to 0.176-17.6 g/m3 (50-5000 ppm), 5 h/day for 10 days. In the study by Kurppa & Vainio (1981) an increase in renal ethoxycoumarin de-ethylase activity was reported. The second metabolic pathway occurring in rat liver is localized in the soluble (cytosolic) fraction (Ahmed & Anders, 1976, 1978). It does not require oxygen but is dependent upon glutathione and a glutathione- S-transferase enzyme, the products in vitro being formaldehyde and chloride ion. The rapid and almost quantitative conversion of formaldehyde to formic acid and then carbon dioxide known to occur in vivo (Neely, 1964) is consistent with this pathway being the source of carbon dioxide exhaled after exposure to methylene chloride. The intermediates involved in the metabolism of methylene chloride to formaldehyde are unknown, but the nature of the enzyme involved and the dependence upon glutathione suggest that S-chloromethyl-glutathione is formed and rapidly hydrolysed and degraded to glutathione and formaldehyde (Ahmed & Anders, 1978). The isoenzyme involved in the metabolism of methylene chloride has been identified as a member of glutathione- S-transferase class theta (Meyer et al., 1991). The chemistry of the S-chloromethyl thioethers (Bohme et al., 1949) and the lack of depletion of glutathione during this reaction are consistent with these conjugates being extremely transient. Formaldehyde, in addition to its metabolism to carbon dioxide, becomes incorporated into the C-1 metabolic pool via formic acid. Therefore, exposure to radiolabelled methylene chloride results in the incorporation of radioactivity into macromolecules including nucleic acids. Hallier et al. (1993) described an apparent polymorphism in the metabolism of methylene chloride in human blood. The metabolic activity was reported to be localized in erythyrocytes (Thier et al., 1991) and to be due to the presence of a glutathione- S-transferase enzyme (Schroeder et. al., 1992). The work by Schroeder describes the detection of enzyme activity in erythrocytes using methyl bromide as a substrate, not methylene chloride. Furthermore, in experiments investigating the influence of cofactors on enzyme activity, glutathione could be replaced by L-cysteine, suggesting that this enzyme is not a glutathione- S-transferase. The work by Thier et al. (1991) identified metabolic activity in plasma and not, as reported by Hallier et al. (1993), in erythrocytes. 6.3.2 In vivo studies The metabolism of methylene chloride in various animal species and in humans has been studied extensively (e.g., Fodor et al., 1973; Kubic et al., 1974; Roth et al., 1975; Lee Rodkey & Collison, 1977; Peterson, 1978; McKenna & Zempel, 1981; McKenna et al., 1982; Angelo et al., 1986a,b). Methylene chloride and the other dihalomethanes are unique in being the only class of industrial chemicals known to be metabolized to carbon monoxide. This metabolic pathway (dependent on cytochrome P-450), first discovered in humans (Stewart et al., 1972), results in elevated levels of CO-Hb and in increased levels of carbon monoxide in expired air. Subsequent studies in experimental animals and in humans established that this pathway is rate-limited by enzyme saturation, so that at high doses the levels of CO-Hb become constant and independent of dose (Lee Rodkey & Collison, 1977). Later experiments in animals using radiolabelled methylene chloride identified carbon dioxide as the other major metabolite (Di Vincenzo & Hamilton, 1975). Although carbon dioxide is a known metabolite of carbon monoxide, the amount of carbon dioxide formed from the monoxide was thought unlikely to account for the levels found during exposure to methylene chloride. This suggested the presence of a second pathway (dependent on glutathione- S-transferase), which was subsequently confirmed in experimental animals. Two reports have described the effects of pretreatment or co- administration of other organic solvents on the metabolism of methylene chloride to carbon monoxide. Pankow et al. (1991) described increases in CO-Hb levels in rats pretreated with a single gavage dose of benzene, toluene or isomers of xylene, up to 32 h prior to a 6-h exposure to methylene chloride. CO-Hb levels increased from 9.3%, in rats exposed to methylene chloride alone, to 22.7% in rats pretreated with m-xylene. Similar increases were seen in rats pretreated with a single garage dose of methanol (Pankow & Jagielki, 1993). In both studies, the levels of CO-Hb were reduced when the solvents were co- administered with methylene chloride. The results of both studies were considered to be consistent with the metabolism of methylene chloride by cytochrome P-450 IIE1. At first sight it might appear that the relative molar amounts of carbon monoxide and carbon dioxide exhaled in vivo provide an index of the activity of the two metabolic pathways. Studies using metabolic inhibitors suggest that significant amounts of carbon dioxide are also derived from the oxidative P-450 pathway (Gargas et al., 1986; Reitz et al., 1986). Similar studies in mice using metabolic inhibitors have confirmed these findings, leading to the conclusion of the authors that the cytochrome P-450 pathway is the major route of metabolism of methylene chloride within species (Ottenwalder et al., 1989). This finding is consistent with either hydrolysis of formyl chloride to formic acid or with formyl chloride reacting with glutathione to form S-formyl glutathione. The rapid enzymatic and chemical breakdown of this conjugate (Uotila & Koivusalo, 1974a,b) would yield formic acid and hence carbon dioxide. Thus, a quantitative correlation between the amount of carbon monoxide and carbon dioxide exhaled and the activity of the two pathways no longer appears to be valid. Levels of CO-Hb in the blood, following exposure to methylene chloride, are both dose- and time-dependent. Human subjects exposed to concentrations of 1770 mg/m3 or less for 1 h have CO-Hb levels of 1-4%. These levels rose to an average of 10% saturation within 1 h after exposure to 3500 mg/m3 for 2 h (Stewart et al., 1972). Hake et al. (1975) reported CO-Hb levels in excess of 8% following exposure to 880 mg/m3 for 7.5 h. Human volunteers were exposed to 350 or 1240 mg/m3 for 6 h and levels of methylene chloride in blood and exhaled air, CO-Hb and exhaled CO were measured. At the end of the 6-h exposure, the CO-Hb concentration of the group exposed to 1240 mg/m3 was 1.4 times higher than that of the group exposed to the lower dose. Likewise, the concentration of exhaled CO in the high-dose group was 2.1 times higher than that of the low-dose group. The authors concluded that their finding of non-linearity between administered dose and the CO-Hb and CO levels is an indication of saturation of the metabolic pathway (McKenna et al., 1980). Physical exercise performed during exposure to methylene chloride will produce higher blood CO-Hb levels than those found in sedentary workers (Åstrand et al., 1975; Di Vincenzo & Kaplan, 1981b). Under a moderate workload, an exposure to 350 mg/m3 for 7.5 h may cause a CO-Hb saturation of about 5% at the end of the exposure period (Di Vincenzo & Kaplan, 1981b). Other factors, including smoking and exposure to combustion and automobile exhaust, will increase CO-Hb levels. 6.4 Elimination and excretion 6.4.1 Inhalation exposure 6.4.1.1 Human studies Methylene chloride is removed from the body mainly in expired air and urine. In four human subjects exposed to methylene chloride (350 mg/m3) for 2 h, an average of 22.6 µg methylene chloride was excreted in the urine within 24 h after the exposure. In seven subjects exposed to 710 mg/m3 for 2 h, the corresponding value was 81.5 µg (Di Vincenzo et al., 1972). These data show that the amount excreted in the urine is insignificant. Methylene chloride excretion in expired air was most evident during the first 30 min after exposure. Initial post-exposure concentrations of methylene chloride in expired breath following 2-and 4-h exposure periods were about 71 mg/m3 and fell to about 18 mg/m3 at the end of 30 min. Small amounts of methylene chloride remained in the expired air at 2.5 h. A detailed study of the relationship between the measurements of methylene chloride in expired air or blood, carbon monoxide in expired air and CO-Hb in blood was undertaken by Di Vincenzo & Kaplan (1981a,b). At the end of exposure of non-smoking, sedentary volunteers for 7.5 h to methylene chloride vapour concentrations of 180-710 mg/m3, the mean concentration of the solvent in alveolar air and in blood, and the percent CO-Hb saturation were measured, as shown in Table 12. By 7 h after exposure to any concentration, the expired air contained less than 3.5 mg/m3 methylene chloride; at 16 h, only negligible levels were detected (Di Vincenzo & Kaplan, 1981a). These data suggest that, due to its rapid elimination, measurements of methylene chloride in expired air are unsuitable for use as a marker of occupational exposure. Table 12. Methylene chloride in expired air and blood, and carboxyhaemoglobin (CO-Hb) levels of human volunteers following 7.5 h exposure (from Di Vincenzo & Kaplan, 1981a) Methylene Methylene choride Methylene chloride CO-Hb chloride exposure in expired air in blood levels (mg/m3) (mg/m3) (mg/litre) 180 53 0.3 1.9% 350 124 0.8 3.4% 530 194 1.2 5.3% 710 282 1.8 6.8% Di Vincenzo & Kaplan (1981a) reported that, in a human volunteer study, exposure to 180, 350, 530 or 710 mg/m3 for 7.5 h/day (for 5 days) resulted in peak CO-Hb levels of 1.9, 3.4, 5.3 and 6.8%, respectively (Table 12). It was estimated that an 8-h exposure to about 530 mg methylene chloride/m3 is equivalent to an 8-h exposure to 124 mg carbon monoxide/m3, in as much as either exposure under sedentary conditions will increase blood CO-Hb levels to about 5% of saturation by the end of the exposure. Di Vincenzo & Kaplan (1981b) also investigated the effects of exercise and cigarette smoking on the uptake, metabolism and excretion of methylene chloride. The effects of smoking and methylene chloride exposure on CO-Hb saturation levels were found to be additive. Exercise was found to increase the absorption of methylene chloride and CO-Hb levels. However, the effects of exercise on CO-Hb were not observed to increase with heavy workloads beyond the level achieved with moderate work-loads, suggesting a saturation of this effect (see also section 5.3.2). Engström & Bjurström (1977) found that, during the first 2 h after exposure, the concentration in alveolar air tended to be lower and declined more rapidly in obese subjects than in slim ones. After this, the concentration dropped more slowly in the obese group. During the late phase of elimination, the obese subjects tended to have a higher concentration in expired air. 6.4.1.2 Animal studies In rats, methylene chloride was excreted in the expired air, urine, and faeces following a single 6-h exposure to 180, 1800 or 53 000 mg methylene chloride/m3 (McKenna et al., 1982). At 180 mg/m3, only 5% of the exhaled label was found as methylene chloride. The remainder was exhaled as CO and CO2. As the exposures increased, so did the exhalation of non-metabolized methylene chloride. Methylene chloride accounted for 30% of the label from the 1800 mg/m3 dose and 55% of the label for the 53 000 mg/m3 dose. A combination of exhaled methylene chloride, CO2 and CO accounted for 58%, 71% and 79% of the inhaled methylene chloride dose for the 180, 1800 and 53 000 mg/m3 doses, respectively. Urinary excretion accounted for 7.2-8.9% of the dose and 1.9-2.3% of the dose was in the faeces. 6.4.2 Oral exposure Expired air accounted for 78-90% of the excreted dose in rats in the 48-h period following a 1 or 50 mg/kg dose of methylene chloride in aqueous solution (McKenna & Zempel, 1981). The radiolabel was present in the exhaled air as CO and CO2, as well as in expired methylene chloride. The amount of methylene chloride in the expired air increased from 12% to 72% as the dose was increased from 1 to 50 mg/kg. Radiolabel in the urine accounted for 2-5% of the dose under the above exposure conditions, while 1% or less of the dose was found in the faeces. These data indicate that the lungs are the major organ of methylene chloride excretion even under oral exposure conditions. Mice excreted 40% of the administered dose (100 mg/kg) unchanged in expired air within 96 h (Yesair et al., 1977). 6.4.3 Dermal exposure No information is available regarding excretion and elimination in humans or animals following dermal exposure to methylene chloride. 7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT Appraisal Algae and aerobic bacteria show no inhibition of growth below 500 mg/litre. Bacteria which are able to grow in the presence of methylene chloride at much higher concentrations (including a saturated solution in water) have been identified. Anaerobic bacteria are more sensitive; growth inhibition has been observed at 3 mg/litre in anaerobic biological sludge. In the aquatic environment, fish and amphibian embryos are the most sensitive with effects on hatching from 5.5 mg/litre. Adult fish are relatively insensitive to methylene chloride. even after prolonged exposure (14-day LC50 > 200 mg/litre). The effect of methylene chloride on Daphnia is variable; the lowest reported EC50 was 135 mg/litre in a closed system. In soil, 10 mg/kg strongly decreased the ATP content of the biomass, adversely affected the growth of fungi and aerobic bacteria. and induced transient inhibition of enzyme activity. The no-observed- effect level was 0.1 mg/kg. In earthworms, LC50 values were in the range of 300 to more than 1000 µg/cm2 ). In sediment, no toxic effects were observed even at very high levels. In higher plants, no effects were found after exposure for 14 days to 100 mg/m3. 7.1 Microorganisms 7.1.1 Bacteria 7.1.1.1 Aerobic bacteria No inhibition of growth was observed at 19.6-19 600 mg/litre methylene chloride in Bacillus subtilis, Pseudomonas cepacia and Aeromonas hydrophylia (Schubert, 1979). Inhibition of bioluminescence of Photobacterium phosphoreum by 50% occurred after a 15-min exposure to 2880 mg/litre (Hermens et al., 1985). In a standard 16-h growth-inhibition test with Pseudomonas putida, a threshold of 500 mg/litre for methylene chloride was determined (Bringmann & Kühn, 1977b). The glycolysis of Pseudomonas putida was inhibited after a 16-h exposure to 1000 mg/litre (Bringmann & Meinck, 1964). Nenzda & Seydel (1988) report minimum inhibitory methylene chloride concentrations for the bacteria Escherichia coli and M. smegmatis of 1049 and 1468 mg/litre, respectively. For heterotrophs, 50% inhibition of oxygen consumption occurred at 320 mg/litre after 24 h (Blum & Speece, 1991). With other bacteria (Acinetobacter, Alcaligenes, Flavobacterium, Pseudomonas cepacia, Aeromonas hydrophila), stimulation of growth was observed at 200 mg/litre (Davis et al., 1981). The IC50 for inhibition of multiplication of Escherichia coli was 37.2 mg/litre (Nendza & Seydel, 1988). In the OECD activated sludge respiration-inhibition test (method 209) using sealed vessels, the EC50 value for methylene chloride was more than 1000 mg/litre after 30 min (Volskay & Grady, 1988). Concentrations up to 1000 mg/litre had no effect on the oxygen consumption or glucose metabolism of activated sludge acclimated to methylene chloride for 3 days (Klecka, 1982). In methylene chloride-utilizing bacteria, e.g., Hypho-microbium, up to 1700 mg/litre did not interfere with growth (Stucki et al., 1981). Blum & Speece (1991) found that the IC50 for reduction of ammonia was 1.2 mg/litre after 24 h for Nitrosomonas. 7.1.1.2 Anaerobic bacteria Anaerobic bacteria are more sensitive than aerobic bacteria. Methanogenesis of mixed rumen microflora was inhibited from 136 mg/litre (Bauchop, 1967). At 93 mg/litre, the growth of a mixed bacterial population from an anaerobic digester was inhibited by 50% (Thiel, 1969). Addition of methylene chloride to anaerobic sludge from an operating municipal digester showed, after 48 h, a 20% inhibition of gas production at 3 mg/litre and a 50% inhibition at 50 mg/litre (Hayes & Bailey, 1977). Addition of methylene chloride to the feed of a mixed anaerobic culture, developed in the laboratory from seed from a sewage treatment plant, decreased the gas production to such an extent that at 3.3 mg/litre it had virtually ceased after 5 days, compared with 15 days in controls (Vargas & Ahlert, 1987). Blum & Speece (1991) determined the toxicity of methylene chloride to methanogenic bacteria and found an IC50 for inhibition of gas production of 7.2 mg/litre. Stuckey et al. (1980) used a batch system in which methylene chloride was added in ethanol to sludge from a laboratory digester in a Warburg apparatus. Some inhibition was noted at the lowest concentration tested (3.16 mg/litre); the concentration for 50% inhibition over 60 h was estimated to be 14 mg/litre. 7.1.2. Protozoa The bacteriovorous ciliated protozoan Uronema parduczi Chatton- Lwoff was not affected after a 20-h exposure to 16 000 mg/litre (EC5 inhibition cell proliferation) (Bringmann & Kühn, 1980). Also, no effects were observed in Microregma heterostoma after a 28-h exposure to 1000 mg/litre (Bringmann & Meinck, 1964). 7.1.3 Algae In several freshwater green algae (Selenastrum capricornutum, Scenedesmus subspicatus. Scenedesmus quadricauda, Chlorella vulgaris, Chlamydomonas angulosa), photosynthesis (chlorophyll a content, CO2 uptake) and cell number were only affected by methylene chloride from 1450 mg/litre (Bringmann & Kühn, 1978; Hutchinson et al., 1978; US EPA, 1980). The threshold (7-day EC3) for effects in the cyanobacterium (blue-green alga) Microcystis aeruginosa was 550 mg/litre (Bringmann & Kühn, 1978). In the marine diatom Skeletonema costatum methylene chloride exposure had no effect on chlorophyll a content or cell number at 662 mg/litre (US EPA, 1980). 7.2 Aquatic organisms The volatility of methylene chloride presents difficulties in aquatic toxicity testing. Therefore, care should be taken when interpreting results based on nominal concentrations and open static systems. Flow-through systems or closed static systems are necessary to conduct adequate toxicity studies on volatile substances. However, these systems were not always used (Tables 14 and 15). 7.2.1 Plants The EC50 for Lemna minor growth was 2000 mg/litre, whilst both growth and photosynthesis of the plant Groenlandia densa were totally inhibited at this concentration after 7 days (Merlin et al., 1992). 7.2.2 Invertebrates 7.2.2.1 Insects The toxicity of methylene chloride for insects was investigated in adult Tribolium confusum and grain weevil (Calandra granaria). The LC50 for a 5-h exposure in fumigation vessels was 82 and 380 mg/litre, respectively (Ferguson & Pirie, 1948; Negherbon, 1959). Table 13. Acute aquatic toxicity of methylene chloride to algae Organism Description Method Parametera Concentration Reference (mg/litre) Diatom Skeletonema costatum Chlorophyll a content, 96-h EC50 > 662 US EPA (1980) (salt water) cell number Green alga Selenastrum Chlorophyll a content, 96-h EC50 > 662 US EPA (1980) capricornutum cell number Green alga Scenedesmus Cell number 8-day TT 1450 Bringmann & Kühn quadricauda (1978) Green alga Chlorella vulgaris CO2 uptake 3-h EC50 2292 Hutchinson et al. (1978) Green alga Chlamydomonas CO2 uptake 3-h EC50 1477 Hutchinson et al. angulosa (1978) Cyanobacterium Microcystis aeruginosa Cell number 8-day TT 550 Bringmann & Kühn (blue-green alga) (1978) a TT = toxicity threshold (i.e. the concentration at which cell multiplication was inhibited by more than 3%) Table 14. Acute aquatic toxicity of methylene chloride in fresh water pH/dissolved Hardness Organism Temperature oxygen (mg CaCO3 Flow/ Parameter Concentration References (°C) (mg/litre) per litre) Static (mg/litre) and remarks Crustacean Water flea 22 7.4-9.4/ 173 Static 48-h LC50 220 Le Blanc (1980) (Daphnia magna) 6.5-9.1 48-h NOEC 68 (nominal concentration Water flea 20-22 7.6-7.7 Unknown Static 24-h EC50 2100-2270 Bringmann & Kühn (Daphnia magna) 24-h NOEC 1550-1707 (1977a, 1982) (nominal concentration) Water flea Unknown Unknown Unknown 48-h LC50 1250 Bringmann & Meinck (Daphnia magna) (1964) Water flea Unknown Unknown Unknown Static 24-h EC50 1959 Kühn et al, (1989) (Daphnia magna) 48-h EC50 1682 (closed system) Water flea Unknown Unknown Unknown Static 48-h EC50 135 Abernethy et al. (1986) (Daphnia magna) (closed system) Water flea 18-20 8/8.7-8.8 11.7 Unknown 48-h LC50 480 RIVM (1986) (Daphnia magna) 48-h NOEC 100 (nominal concentration closed system) Fish Goldfish Unknown Unknown Unknown Static 24-h LC50 420 Jenson (1978) (Carrassius (nominal concentration) auratus) Table 14 (Cont'd) pH/dissolved Hardness Organism Temperature oxygen (mg CaCO3 Flow/ Parameter Concentration References (°C) (mg/litre) per litre) Static (mg/litre) and remarks Fathead minnow 12 7.8-8.0/ 67 Static 98-h LC50 310 Alexander et al, (1978) (Pimephales > 5 Flow 96-h LC50 193 (static test nominal, flow- promelas) (adult) 96-h NOEC 66.3 through measured concentrations; aquarium covered with plastic film for the first 24-h) Fathead minnow 25 Unknown 73-82 Flow 96-h LC50 502 Dill et al. (1987) (Pimephales (analysed concentration) promelas) (juvenile) Bluegill, 21-23 6.5-7.9/ 32-48 Static 96-h LC50 220 Buccafusco et al. (1981) (Lepomis unknown (nominal concentration, macrochirus) aquarium not capped) Table 15. Acute aquatic toxicity of methylene chloride in salt water Temperature pH/dissolved Hardness Flow/ References Organism (°C) oxygen (mg CaCO3 Stat Parameter Concentration and remarks (mg/litre) per litre) (mg/litre) Crustacean Mysid shrimp Unknown Unknown Unknown Static 96-h LC50 260 US EPA (1980) (nominal (Mysidopsis bahia) concentration) Grass shrimp 20±2 6.1-8.0 8-12 Static 48-h LC50 108.5 Burton & Fischer (1990) (Palaemonetes pugia) > 4 Fish Golden orfe Unknown Unknown Unknown Static 48-h LC50 521-528 Juhnke & Lüdemann (1978) (Leuciscus idus) Killifish (juvenile) 20±2 6.1-8.0/ Unknown Static 48-h LC50 97.0 Burton & Fischer (1990) (Fundulus > 4 heteroclitus)a Sheepshead minnow 25-31 Unknown 10-31 Static 98-h LC50 330 Heitmuller et al. (1981) (Cyprinodon 98-h NOEC 130 (nominal concentration) variegatus) a fish died within 1 hour; the measured 1-h LC50 was 135 mg/litre. The 48-h value was the average of the initial and final concentrations. 7.2.2.2 Crustaceans Data on the toxicity of methylene chloride to crustaceans are presented in Table 14. Daniels et al. (1985) and Knie (1988) reported a 48-h LC50 of 27 mg/litre and a 24-h LC50 of 12.5 mg/litre; such values are lower than those presented in Table 14 by almost one order of magnitude. However, no experimental details were given and, therefore, the validity of the data cannot be assessed. 7.2.2.3 Molluscs In seawater, metamorphosis was induced in up to 63% of the larvae of the nudibranch mollusc (Phestilla sibogae) when exposed to 8.5-25.5 mg/litre (Pennington & Hadfield, 1989). 7.2.3 Fish 7.2.3.1 Acute toxicity Data on the acute toxicity of methylene chloride to fish are presented in Table 14. The acute toxicity of methylene chloride to adult fathead minnows (Pimephales promelas) has been studied both in a static and a flow- though system. The observed effects (loss of equilibrium, melanization, narcosis and swollen, haemorrhaging gills) were reversible at a sublethal level (Alexander et al., 1978). 7.2.3.2 Chronic toxicity and reproduction Data on the chronic and embryo-larval toxicity of methylene chloride to fish are summarized in Table 16. In a 32-day embryo-larval test with fathead minnow (Pimephales promelas), the larval survival and weight was affected from 209 and 142 mg/litre, respectively. The maximum acceptable toxicant concentration (MATC) based on body weight was calculated to be 108 mg/litre. The ratio between the acute 8-day LC50 value and the 32-day embryo-larval MATC is 4.6, indicating a small difference between acute and chronic effects of methylene chloride (Dill et al., 1987). 7.2.4 Amphibians In closed flow-through systems, short-term embryo-larval tests were carried out, from 2 to 6 h post-spawning to 4 days post-hatch, on amphibian eggs of Rana catesbeiana. R. palustris and Bufo fowleri (hatching times ranged from 3 to 4 days). After combining frequencies for lethality and teratogenesis, the analytically determined post- hatching LC50s were > 32 mg/litre for the pickerel frog Table 16. Chronic and embryo-larval toxicity of methylene chloride to fish Dissolved Hardness Flow/ References and Description Temperature pH oxygen (mg CaCO3 Static Parameter Concentration remarks (°C) (mg/litre) per litre) (mg/litre) Chronic toxicity Guppy 22±1 ? > 5 25 daily LC50, 14 days 295 Könemann (Poecilia reticulata) renewal (1981) (covered with glass, nominal) Fathead minnow 25±1 6.8-8.6 > 9 73-82 flow LC50, 8 days 471 Dill et al. (Pimephales promelas) NOEC, (1987) (juvenile) 8 days 357 (analytical concentration) Embryo-larva toxicity Fathead minnow 20.4±0.6 7.8 6.5 95 flow LC50a 34 Black et al. (Pimephales promelas) (1982) (embryo-larva) Fathead minnow 25±1 6.8-8.6 > 9 73-82 flow LOEC, 32 days 209 Dill et al. (Pimephales promelas) (survival) (1987) (embryo-larva) LOEC, 32 days 142 (analytical (weight) concentration) Rainbow trout 13.3±0.3 7.8 9.4 106 flow LC50a,b 13.1 Black et al. (Salmo gairdneri) (1982) (embryo) Table 16 (Cont'd) Dissolved Hardness Flow/ References and Description Temperature pH oxygen (mg CaCO3 Static Parameter Concentration remarks (°C) (mg/litre) per litre) (mg/litre) Rice fish 25±1 7.6-8.4 4.5-8.8 11.7 renewal LC50, 3 weeks 106 RIVM (1986) (Oryzias latipes) 23±2 3 times/ NOEC, 3 weeks 75 (analytical (egg-larva) week concentration) a Eggs were exposed from 30 min after fertilization to 4 days post-hatch b Teratogenic effects were observed at 5.5 mg/litre (R. palustris) and Fowler's toad (Bufo fowleri) and 17.78 mg/litre for the bullfrog (R. catesbeiana). In the latter, anomalous larvae and 16% decreased hatching were observed at 6.73 mg/litre. For the pickerel frog and Fowler's toad, hatching was decreased by 14 and 20% at 10 and 32 mg/litre, respectively. In the hatched populations slightly higher incidences of teratogenic effects were observed (Birge et al., 1980). Black et al. (1982) exposed several amphibian species to methylene chloride from 30 min after fertilization to 4 days post-hatch. Post- hatching LC50 values ranging from 16.9 to > 48 mg per litre were found for the European common frog (Rana temporaria), Northwestern salamander (Arabystoma gracile), African clawed frog (Xenopus laevis) and the Leopard frog (R. pipiens). The European common frog and the Northwestern salamander were the most sensitive to methylene chloride. 7.3 Terrestrial organisms The toxicity of methylene chloride to higher plants (Phaseolus vulgaris, Raphanus sativus radicula, Lepidum sativum, Trifolium pratense, Saintpaula ionatha, Petunia hybrida) was evaluated, using the LIS (Landesanstalt für Immissionsschutz, Essen) test; no effect was observed at 100 mg/m3 exposure over 14 days (Van Haut & Prinz, 1979). In leaves of alfalfa (Medicago sativa), the effect of methylene chloride vapour on the photosynthetic fixation of 14CO2 was tested; photosynthesis appeared to be reduced from 388 000 mg/m3 (Lehman & Paech, 1972). In a 48-h filter-paper contact toxicity test on the earthworm Eisenia fetida, the LC50 was 304 µg/cm2 in one study and > 1000 µg/cm2 in another. Therefore, methylene chloride was classified as moderately toxic (100-1000 µg/cm2) (Roberts & Dorough, 1984; Neuhauser et al., 1985). In embryos of White leghorn chicken, the LD50 for injection of methylene chloride in the yolk sac is 14 mg/egg (Verrett et al., 1980). 7.4 Population and ecosystem effects 7.4.1 Soil microorganisms When added to brown soil at 10 mg/kg (dry weight), methylene chloride decreased the ATP content of the soil biomass by 80-85%, compared to controls, and adversely affected the growth of soil fungi and aerobic bacteria after 3 days. A slight recovery was observed by the end of the 56-day experiment. Anaerobic bacteria were hardly influenced and, in the case of the obligate anaerobic Clostridium sp., the growth was even increased. The replacement of oxygen probably explained the stimulation of growth in the latter case (Kanazawa & Filip, 1987). Incubation of soil for 2 months with 1-10 mg/kg (dry weight) methylene chloride reduced the activity of ß-glucosidase, ß-acetylglucosaminidase and proteinase during the first 28 days, with recovery after 2 months; no effect was observed at 0.1 mg/kg (Kanazawa & Filip, 1986). 7.4.2 Sediment microorganisms In sediment from a freshwater stream, methylene chloride did not significantly affect the electron transport system (ETS) activity during a 1-h enzymatic assay at 66 500 mg/kg. When assayed over an 11-day period, 1330-66 500 mg/kg caused a fluctuating stimulation of ETS activity, which may indicate a marked alteration of the stability of the biological activity in the sediment. Microbial respiration, measured by CO2 evolution, was inhibited (EC50) after 7 days at 15 500 mg/kg. However, when measured by oxygen uptake, it was stimulated at up to 26 500 mg/kg (Trevors, 1985). 7.4.3 Microcosms and mesocosms Microcosms composed of water plants (Elodea canadensis, Lemna minor), algae (Scenedesmus subspicatus) and snails (Physa sp.) were exposed to 500 or 1000 mg/litre for 21 days. At 1000 mg/litre a decrease in oxygen content of the water was observed, together with mortality in snails and algae, as well as necrosis on fronds of Lemna minor. The photosynthesis of the plants was inhibited. These effects were less at 500 mg/litre, but this concentration was still lethal to snails. In outdoor mesocosms, containing a large diversity of species, initial concentrations of 137-156 mg/litre did not induce any toxicity (Merlin et al., 1992). 8. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS 8.1 Single exposure 8.1.1 Acute toxicity data See Table 17. 8.1.2 Oral administration Musculoskeletal disturbances were found in Sprague-Dawley rats at doses of 530 mg/kg or more. Hypotension, hypothermia and haematuria were also noted (dose threshold not reported). The gastrointestinal tract was found to be congested with micro-haemorrhages or partial destruction from doses of 530 mg/kg or more. Blood CO-Hb increased in a dose-related manner (Laham, 1978). Various effects have been reported following the acute administration of large oral doses of methylene chloride. The effects include a decreased cytochrome P-450 content in liver microsomes of Sprague-Dawley rats receiving 1000 mg/kg (Moody et al., 1981), CNS effects and evidence of pathological changes in the liver and kidney of Wistar rats receiving 2000 mg/kg (Janssen & Pott, 1988a), evidence of liver necrosis and increased glucose-6-phosphatase activity in male rats (strain unspecified) receiving 2210 mg/kg (Reynolds & Yee, 1967), and decreased hepatic secretion of triglycerides followed by an increased hepatic triglyceride content in male mice receiving 2700 mg/kg (Selan & Evans, 1982). No liver toxicity was found in male Wistar rats receiving up to 4400 mg/kg by oral administration (Danni et al., 1981). Liver damage was investigated in Sprague-Dawley rats exposed to up to 1275 mg/kg given orally. Serum ALT activity was unaffected, as was liver cytochrome P-450 and glutathione content. However, increased ornithine decarboxylase activity and DNA damage were found in the liver (Kitchin & Brown, 1989). 8.1.3 Inhalation administration 8.1.3.1 Rat Behavioural changes and CNS disturbances were found in several studies. Decreased running activity was found in rats exposed to 17 700 mg/m3 for 1.5 h (Heppel & Neal, 1944); hypothermia, hypotension and convulsion in Sprague-Dawley rats from 28 200 mg/m3 (6-h exposure) (Laham, 1978); CNS depression in Alderley-Park rats at 31 800 mg/m3 (10-min EC50) (Clark & Tinston, 1982) and in rats at 40 000 mg/m3 (2-h exposure) (Ulanova & Yonovskayo, 1959); and dyspnoea and anaesthesia in rats from 53 000 mg/m3 (30-min exposure) (Schumacher & Grandjean, 1960; Kashin et al., 1980). Table 17. Acute toxicity of methylene chloride Species Route Vehicle Parameter Concentration Reference Rat (Wistar, male) oral none LD50 1710-2250 mg/kg Klimmer (1988) Rat CDF (F-344) oral none LD50 1530-2524 mg/kg Carreon (1981) Rat (Sprague-Dawley) oral none LD50 2120 mg/kg Kimura et al, (1971) young male Rat (Sprague-Dawley) - male oral none LD50 2280 mg/kg Laham (1978) - female oral none LD50 1410 mg/kg Mouse (CF-1, male) oral unknown LD50 1987 mg/kg Aviado et al. (1977a,b) Dog oral mucillage of LD50 3000 mg/kg Barsoum & Saad acacia (1934) Rat (Alderley Park) inhalation 15-min LC50 197 790 mg/m3 Clark & Tinston (1982) Rat (Sprague-Dawley) inhalation 6-h LC50 52 000 mg/m3 Bonnet et al. (1980) male Rat (Sprague-Dawley) inhalation 6-h LC50 > 28 000 mg/m3 Landry et al, (1981) Mouse (CF-1, male) inhalation 20-min LC50 92 680 mg/m3 Aviado et al. (1977a,b) Mouse (LF1, female) inhalation 6-h LC50 49 100 mg/m3 Gradiski et al, (1978) Mouse (ICR, male) inhalation 6-h LC50 55 870 mg/m3 Scott et al. (1979) Guinea-pig inhalation 6-h LC50 40 200 mg/m3 Balmer et al. (1976) Table 17 (Cont'd) Species Route Vehicle Parameter Concentration Reference Rat (Sprague-Dawley) intratracheal ALDa 350 mg/kg McCarty et al. (1992) male Mouse (CF-1, male) intraperitoneal unknown LD50 448 mg/kg Aviado et at. (1977a,b) Mouse intraperitoneal unknown LD50 500 mg/kg Schumacher & Grandjean (1960) Mouse (Swiss-Webster, intraperitoneal corn oil LD50 1990 mg/kg Klaassen & Plaa (1966) male) Dog intraperitoneal corn oil LD50 1260 mg/kg Klaassen & Plaa (1967) Mouse subcutaneous olive oil LD50 6500 mg/kg Kutob & Plaa (1962) a "ALD = Approximate Lethal Dose, the lowest dose causing death within 3 days Studies on sleeping patterns in Wistar rats by measuring electroencephalographic (EEG) and electromyographic (EMG) activity have shown a dose-related increase in total sleeping time and intervals between rapid eye movement (REM) sleep when the rats were exposed to 1770, 3500 or 10 600 mg/m3 (500, 1000 or 3000 ppm) methylene chloride for 3 h. During exposure to 3500 or 10 600 mg/m3, the percentage of sleep defined as REM decreased by nearly 20% (Fodor & Winneke, 1971; Fodor et al., 1973). Studies using similar techniques on the narcotic effects of methylene chloride in rats (strain unspecified) have been reported by Berger & Fodor (1968). Rats were exposed to a range of concentrations of methylene chloride from 9900 to 99 000 mg/m3 (2800 to 28000 ppm) for unspecified periods of time. There was an initial period of excitation followed by deep narcosis with a decrease in muscle tone and EEG activity and subsequent breathing difficulties. Cessation of electrical activity was noted after a 1.5-h exposure to 88 000 or 99 000 mg/m3 (25 000 or 28 000 ppm) and after a 6-h exposure to 54 000 or 64 000 mg/m3 (16000 or 18000 ppm). Following exposure to 17 700 to 31 800 mg/m3 (5000 to 9000 ppm) methylene chloride, long periods of sleep occurred without desynchronization phases. Following exposure to concentrations of methylene chloride below 17 700 mg/m3 (5000 ppm), there were no measurable effects on either EEG or EMG activity (Berger & Fodor, 1968). Exposure of F-344 rats to 7100 mg/m3 for 2.5 h caused statistically significant changes in somatosensory evoked responses and EEG. The lack of effect of 157 mg carbon monoxide/m3 (which induces a CO-Hb level of 10%, comparable with that produced by exposure to 7100 mg methylene chloride/m3) on evoked responses, indicated that the effects were probably due to methylene chloride itself and not to its principal metabolite carbon monoxide (Mattsson et al., 1988). Alterations in somatosensory evoked potentials were also observed after a 1-h exposure of F-344 rats to dose levels of 17 700 mg/m3 or more (Rebert et al., 1989). No macroscopic lesions were found in rats at the 6-h LC50 of 53 000 mg/m3 (Bonnet et al., 1980). Congestion of various organs, as well as oedema of the brain, heart, lungs and hip region, was noted following exposure to 71 000 mg/m3 (6-h exposure) in Sprague-Dawley rats (Laham, 1978). Increased CO-Hb levels were found from 1770 mg/m3 in various strains of rats (MacEwen et al, 1972; Laham, 1978; Dill et al., 1978; Kurppa et al., 1981). Ascorbic acid content of the liver was increased in rats exposed to 40 000 mg/m3 for 2 h (Ulanova & Yonovskayo, 1959), but no effect on cytochrome P-450 or specific liver enzymes was noted in Wistar rats after a 3-h exposure to 3500 mg/m3 or in Sprague-Dawley rats after a 5-min (repeated 5 times) exposure to 350 mg/m3, except for increased microsomal and decreased lysosomal ß-glucuronidase activity (Kurppa et al., 1981). Intratracheal administration of methylene chloride in Sprague- Dawley rats showed lethal levels at 350 mg/kg (corresponding to 17.5% of the oral LD50), death occurring in a few seconds. This result emphasises that aspiration of methylene chloride may present more of a hazard than oral ingestion (McCarty et al., 1992). 8.1.3.2 Mouse CNS depression resulting in reversible narcosis was reported in mice exposed to between 14 100 and 52 200 mg/m3 methylene chloride for 2-6 h (Flury & Zernik, 1931) or to 47 700 mg/m3 for 128 min (EC50) (Kashin et al., 1980). This effect was also noted in CF-1 mice exposed to 35 300 mg/m3 for 20 min (Aviado et al., 1977a,b) or in Swiss mice exposed to 45 900 mg/m3 for 7 h (Svirbely et al., 1947). Exposure at 35 000 mg/m3 for 2 h was the minimal CNS effective concentration found in the mouse (Lazarew, 1929). Ability to learn a simple passive avoidance task, 1-4 days after exposure, was reduced in Swiss-Webster mice exposed to 168 000 mg/m3 for 20 seconds (Alexeef & Kiglore, 1983). Fatty changes were noted in the liver and, less frequently, in the kidney from 56 500 mg/m3 after a 7-h exposure (Svirbely et al., 1947). Microscopic lesions in the liver, kidneys, lungs and adrenal were found (no dose-response relationship) in mice exposed to lethal doses for 6 h (Gradiski et al., 1978). Cardiac sensitization to the effects of adrenaline was reported in Swiss mice exposed to 710 000 mg/m3 for 6 min) (Aviado & Belej, 1974). Male NMRI mice were exposed to methylene chloride by inhalation in a study of tolerance, i.e. decreased responsiveness to a chemical that arises as a result of previous exposure to the same chemical (Kjellstrand et al., 1990). Motor activity measured with Doppler radar units was used to monitor the behavioural reactions of the animals. Motor activity increased on exposure to methylene chloride and decreased on constant exposure. Termination of exposure was followed by hypoactivity. 8.1.3.3 Other animals After 6 h of exposure to 17 700 mg/m3, the concentration of triglycerides was increased in the liver of guinea-pigs, and reduced in the serum (Bulmer et al., 1976; Morris et al., 1979). Histopathological liver changes, consisting of the appearance of lipid droplets, were first seen in guinea-pigs at 17 700 mg/m3 (Morris et al., 1979). Slight to moderate vacuolation in the liver of guinea-pigs was seen after a 6-h exposure to 38 800 mg/m3. In addition, lungs showed congestion and haemorrhage. Behavioural changes were also noted (Balmer et al., 1976). No effect on blood pressure, heart rate or EEG activity was found in rabbits exposed to 90 000 mg/m3 for 2 h (Truhaut et al, 1972). Only serum AST was significantly increased. In Beagle dogs, ECG changes as well as decreased blood pressure, heart and respiratory rate were found at 141 200 mg/m3 (7-h exposure). Behavioural effects were already noted after a 1-h exposure to 53 000 mg/m3 (reduced reflexes) (Von Oettingen et al., 1950). Increased CO-Hb values, but no change in haematocrit, haemoglobin concentration or red cell count, were reported in dogs and rhesus monkeys exposed for 24 h to 17 700 mg/m3. Slight pathological changes in the liver (fatty changes, vacuolization) could be attributed to the treatment (MacEwen et al., 1972). Cardiac effects such as arrhythmia, tachycardia and hypotension were found in monkeys, dogs and rabbits exposed for 1-5 min to levels of methylene chloride exceeding 35 300 mg/m3 (Belej et al., 1974; Adams & Erickson, 1976; Taylor et al., 1976; Aviado et al., 1977a,b; Aviado, 1978). One study on rabbits showed allergic reactions after inhalation (Shmuter & Kashin, 1978). However, the experimental protocol of this study is questionable and the result has not been confirmed. 8.1.4 Dermal administration No effect was noted on rats receiving a dermal application of methylene chloride of 710 mg/kg for 0.5 to 4 h, except for an increase in CO-Hb levels (Makisimov & Mamleyeva, 1977). Only slight behavioural effects and macroscopic changes in the liver (swelling) were found in Wistar rats receiving 2000 mg/kg under an occlusive dressing for 24 h (Janssen & Pot, 1988b). Haemoglobinuria was observed in rats when abdominal skin was immersed in methylene chloride for 2-20 min (Schutz, 1960). 8.1.5 Intraperitoneal administration A single intraperitoneal injection of 510 mg methylene chloride/kg in rats slowed down the sciatic motor conduction velocity by 11% and gave rise to a CO-Hb level of 6.8% (Pankow et al., 1979). Signs of CNS depression were found in Wistar rats and CFI mice receiving, respectively, 1060 mg/kg (with phenobarbital pretreatment) and 114 mg/kg (Aviado et al., 1977; Masuda et al., 1980). Various biological investigations in rats and mice showed a dose- related increase in serum AST and/or ALT activity at > 660 mg/kg. No effect was observed on glucose-6-phosphatase activity, cytochrome P-450 content or BSP retention (Klaassen & Plaa, 1966; Cornish et al., 1973; Masuda et al, 1980; Corsi et al., 1983). Increased ALT activity was reported in mongrel dogs receiving 800 mg/kg in corn oil (Klaassen & Plaa, 1967). No histological changes in the liver could be found in rats exposed to < 1300 mg/kg (Cornish et al., 1973; Corsi et al., 1983). However, mild hepatic inflammation was noted in Swiss Webster mice receiving lethal doses of methylene chloride (2000 mg/kg) (Klaassen & Plaa, 1966). Renal tubular changes were noted in F-344 rats administered intrapertinoneally with 1330 mg/kg in corn oil (Kluwe et al., 1982) and in Swiss-Webster mice receiving 2000 mg/kg in corn oil (Klaassen & Plaa, 1966). However, no renal histological change could be found in Swiss mice receiving 1300 mg/kg in corn oil (Plaa & Larson, 1965). Slight histological changes were noted in the liver and kidneys of mongrel dogs receiving 1300 mg/kg (Klaassen & Plaa, 1967). 8.1.6 Intravenous administration The minimum lethal concentration in anaesthetized dogs was found to be 200 mg/kg in olive oil after intravenous administration (Barsoum & Saad, 1934). Methylene chloride shortened the duration of nystagmus induced by rotation in Sprague-Dawley rats during intravenous perfusion of 5.1 mg/kg per min for 60 min (Tham et al., 1984). Following a single intravenous injection of 3.1, 6.2 or 12.4 mmol/kg, methylene chloride was found to sensitize the myocardium of rats to arrhythmia development in response to catecholamines. The release by methylene chloride of endogenous catecholamines is possibly a cause of these modified cardiovascular actions (Mueller et al., 1991). 8.1.7 Subcutaneous administration The minimum lethal concentration in the rabbit was found to be 2700 mg/kg (Barsoum & Saad, 1934). Prolongation of phenobarbital- induced sleeping time occurred at 1700 mg/kg in Swiss mice. No effect on liver function or histology was observed at up to 5000 mg/kg (Kutob & Plaa, 1962). 8.1.8 Appraisal The acute toxicity of methylene chloride by inhalation and oral administration is low. The inhalation 6-h LC50 values for all species lie between 40 200 and 55 870 mg/m3. Oral LD50 values of 1410-3000 mg/kg have been recorded. Acute effects after methylene chloride administration by various routes of exposure are primarily associated with the central nervous system (CNS) and the liver. CNS disturbances were found at 14 100 mg/m3 or more and slight changes in EEG at 1770 mg/m3. Slight histological changes in the liver were found at concentrations of 17 700 mg/m3 or more. In the mouse, but not in the rat, effects on the lungs restricted to the Clara cells were observed after exposure to 7100 mg/m3. Occasionally the kidney is affected. Cardiac sensitization to adrenaline-induced arrhythmias has been reported, and cardiovascular effects were seen at concentrations above 35 000 mg/m3. However, the effects were inconsistent. 8.2 Short-term exposure 8.2.1 Oral administration CD1 mice given doses of up to 665 mg/kg per day of methylene chloride in corn oil by gavage for 14 days did not show any effect on liver enzymes. Microscopic examination revealed slight vacuolation in the liver from 333 mg/kg. No damage to the kidneys was reported (Condie et al., 1983). 8.2.2 Subcutaneous administration Reduction of the systolic blood pressure of hypertensive Sprague- Dawley rats was reported after subcutaneous exposure to 2000 mg/kg twice a week for 17 weeks. No effect was found in normotensive rats. Very slight histopathological changes in the liver and lungs of hypertensive rats were described (Loyke, 1973). 8.2.3 Inhalation administration 8.2.3.1 Rat Changes in Sprague-Dawley rats were reported after exposure to methylene chloride (3500 mg/m3) 2 h/day for 15 days; decreased body weight, increased hepatic lipid peroxidation, and high concentrations of methylene chloride in the brain, kidney and blood immediately after inhalation were observed (Ito et al., 1990). Similar hepatic effects (hypertrophic hepatocytes, and increased lipid peroxidation and glutathione peroxidase activity) were reported in male Wistar rats exposed to 3500 mg/m3 2 h/day for 20 consecutive days (Takashita et al., 1991). Biochemical tests were performed on the serum and brain of groups of six male Sprague-Dawley rats which were exposed to 250, 1100 or 3500 mg/m3 6 h/day for 3 days and killed 16-18 h later. A selective reduction in dopamine concentration, with changes in dopamine turnover in some forebrain dopamine nerve terminal systems, was reported. A dose-dependent increase in noradrenaline turnover in the anterior periventricular hypothalamic area and dose-dependent decreased noradrenaline concentration in the posterior periventricular area were observed. No significant changes were reported in the secretion of anterior pituitary hormones (Fuxe et al., 1984). Groups of five male and five female F-344 rats were exposed to methylene chloride at concentrations of 5740, 11 500, 22 900, 45 900 or 56 500 mg/m3, 6 h/day for 19 days. Intermittent scratching, ataxia and hyperactivity were seen in all rats exposed to 22 900 mg/m3 or more. Dyspnoea and anaesthesia were observed in animals exposed to 45 900 mg/m3 or more. Some deaths were also observed at these concentrations (NTP, 1986). No, or limited, lesions were found in the liver and lungs of F-344 rats, exposed to 7100 or 14 100 mg/m3, 6 h/day for 10 days, after light and electron microscopic examination (Hext et al., 1986). No effect on the liver was found in rats (strain unspecified) after inhalation of 880 mg/m3, for 5 h/day for 28 days (Norpoth et al., 1974) Inhalation exposure of Sprague-Dawley rats to methylene chloride concentrations of 12 800 mg/m3 (5 h/day, 5 days/week) for 4 weeks revealed an inflammatory response and cell damage in the lungs as demonstrated by the increase biochemical response (enzymatic and non- enzymatic) observed in cell-free lavage effluents from the lungs (Sahn & Lowther, 1981). 8.2.3.2 Other animals Increased liver weight and increased mitotic activity in hepatocytes were observed in male B6C3F1 mice after 2 weeks of repeated exposure to 14 100 mg/m3 6 h/day for up to 3 weeks (Eisenbrandt & Reitz, 1986). Necrosis of occasional epithelial cells in the bronchi and bronchioles, together with reactive hyperplasia of adjacent lymphoid tissue, was observed in a few animals. Groups of five male and five female B6C3F1 mice were exposed to methylene chloride at concentrations of 5740, 11 500, 22 900, 45 900 or 56 500 mg/m3, 6 h/day for 19 days. Hyperactivity (dose-related) was seen in exposed mice, but no exposure-related pathological findings were observed. Some deaths occurred in mice exposed to 45 900 mg/m3 or more (NTP, 1986). CD-1 mice, Golden Syrian hamsters, Sprague-Dawley and CDF (F-344) rats were exposed to 0, 8800, 17 700 or 28 200 mg/m3, 6 h/day, 5 days/week, for a total of 19, 18, 19 or 7 exposures, respectively, over a period of 21 to 28 days. Animals exposed to 28 000 mg/m3 showed anaesthetic effects and a decrease in the body weight of rats. At 18 000 mg/m3 there was slight anaesthesia, decreased body weight in male rats, increased aspartate aminotransferase (ASAT) in female mice and Sprague-Dawley rats, and increased liver weights in female mice, hamsters and rats. At 8800 mg/m3, the animals exhibited more scratching activity than the controls, but showed no other effects attributable to exposure (Nitschke et al., 1981) Following exposure to 17 700 mg/m3, a reduction in body weight of mice was observed, and relative liver weights were increased up to the end of the 7-days of continuous exposure. Fatty infiltration, an increase in the triglyceride concentration and hydropic degeneration of the endoplasmic reticulum gradually disappeared. Protein synthesis was depressed. Necrosis was observed in a few hepatocytes (Weinstein et al., 1972). Carboxyhaemoglobin levels were raised after continuous exposure of monkeys to 88.25 mg/m3 (25 ppm) for 28 days (MacEwen & Vernot, 1972; Haun et al., 1972). A group of NMRI mice was continuously exposed to 130-1059 mg/m3 (37-300 ppm) for 30 days, while another was intermittently exposed for 1-12 h/day to 2118-25 416 mg/m3 (600-7200 ppm) for 30 days, corresponding to an average exposure (24-h mean value) of 1059 mg/m3 (300 ppm). In addition, groups of mice were continuously exposed to 1059 mg/m3 for 4, 8, 15 and 90 days. The blood level of butyrylcholinesterase was significantly increased from 265 mg/m3 (75 ppm) in continuously exposed male mice and after intermittent exposure in male rats. Moreover, liver weight was significantly increased in a dose-related manner from 265 mg/m3 (75 ppm) and 330 mg/m3 (150 ppm) in male and female mice, respectively. Finally, fatty accumulation was found in both sexes at 265 mg/m3 (75 ppm) or more. All effects were reversible (Kjellstrand et al., 1986). CO-Hb levels were raised after continuous exposure of monkeys to 88.25 mg/m3 (25 ppm) for 28 days (MacEwen et al., 1972, Haun et al., 1972). 8.3 Long-term exposure 8.3.1 Rat 8.3.1.1 Inhalation exposure In a study by Leuschner et al. 1984, 20 male and 20 female Sprague-Dawley rats were exposed to 35 g/m3, 6 h/day for 90 days. Haematological, clinical chemistry and urinary parameters were measured and histological examinations were performed. A slight redness of the conjunctiva lasting for 1-10 h was observed after each exposure. No other treatment-related signs of toxicity were reported. Groups of 10 male and 10 female F-344 rats were exposed to methylene chloride at concentrations of 1850, 3700, 7400, 14 800 and 29 700 mg/m3 for 6 h/day, 5 days/week for 13 weeks. One male and one female rat exposed to 29 700 mg/m3 died before the end of the study, whereas none of the control rats died. Foreign body pneumonia was observed in some rats exposed to > 7410 mg/m3. The mean body weight in males and females exposed to 29 700 mg/m3 was lower than in controls. Liver lipid to liver weight ratios were statistically significantly reduced in both males and females exposed to 29 700 mg/m3 and in females exposed to 14 800 mg/m3 when compared to controls (NTP, 1986). Male and female F-344 rats were exposed to 177, 710 or 7100 mg/m3 6 h/day, 5 days/week for 13 weeks. No treatment-related alterations in sensory evoked potentials (flash, auditory brainstem, somatosensory or caudal nerve) or neuropathology were observed at any of the exposure levels (Mattsson et al., 1990). CNS depression was found in rats during each daily session of repeated exposure to 35 000 mg/m3 7 h/day, 5 days/week for 6 months (Heppel et al., 1944). Rats (sex and strain unspecified) were continuously exposed to either 88 or 350 mg/m3 for 100 days. Slight cytoplasmatic vacuolization with positive fat stains in the liver and tubular degeneration in the kidney were observed (Haun et al., 1972). 8.3.1.2 Oral exposure Rats receiving methylene chloride in the drinking-water at a concentration of 125 mg/litre for 13 weeks did not show any effects on behaviour, body weight, haematology, urinalysis, blood glucose level, plasma free fatty acids, or the oestrous cycle (Bornmann & Loeser, 1967). Groups of 20 male and 20 female Fischer-344 rats were given 0.15, 0.45, and 1.50% methylene chloride in drinking-water for 3 months, equivalent to 166, 420 and 1200 mg/kg per day, respectively, for males and 209, 607, and 1469 mg/kg per day for females. Slightly decreased body weights were observed in mid-dose males and high-dose females throughout the study. There were no differences between treated and control animals with regard to mortality, physical observations, food consumption or gross necropsy results. There were no exposure-related effects observed following the histopathological evaluation of rat tissues from the 1 month interim necropsies. However, hepatocellular changes were observed following treatment for 3 months; central lobular necrosis, granulomatous foci, ceroid or lipofuscin accumulation, and cytoplasmic eosinophilic bodies were observed in high-dose males and females and in some mid-dose females. A dose- dependent increased incidence of hepatocyte vacuolation was also observed, many of the vacuoles containing lipid which was generalized or concentrated in the central lobular region (Kirschman et al., 1986). 8.3.2 Mouse 8.3.2.1 Inhalation exposure Groups of 10 male and 10 female B6C3F, mice were exposed to methylene chloride at concentrations of 1850, 3700, 7400, 14 800 or 29 700 mg/m3 for 6 h/day, 5 days/week for 13 weeks. Exposure-related deaths were seen in some mice exposed to 29 700 mg/m3. Hepatic centrilobular hydropic degeneration was observed in males and females exposed to 29 700 mg/m3 and in females exposed to 14 800 mg/m3. Both mean body weights and liver lipid to liver weight ratios were reduced in males and females exposed to 29 700 mg/m3 when compared to controls (NTP, 1986). Mice (strain and sex unspecified) were continuously exposed to either 88 or 350 mg/m3 for 100 days. Slight cytoplasmatic vacuolization was found at both dose levels, and a decrease in the microsomal cytochrome P-450 content was found in the liver of mice exposed to methylene chloride at 350 mg/m3 (Haun et al., 1972). Female ICR mice were continuously exposed to 350 mg/m3 for 10 weeks. Fatty infiltration, vacuolization and enlarged hepatocyte nuclei persisted up to the end of the exposure period. A reversible increase in plasma triglycerides was also observed (Weinstein & Diamond, 1972). 8.3.2.2 Oral exposure Groups of 20 male and 20 female B6C3F1 mice were given 0.15, 0.45 and 1.50% methylene chloride in drinking-water for 3 months, equivalent to 226, 587 and 1911 mg/kg per day, respectively, for males and 231, 586 and 2030 mg/kg per day for females. Slightly lower body weights were observed in mid-dose and high-dose males and in females from week 6 to the end of the study. Treated and control animals did not differ with respect to physical and ophthalmological observations or food consumption. There were no exposure-related effects observed following the histopathological evaluation of mice following a 1-month exposure. However, after 3 months of exposure, subtle centrilobular fatty changes in the liver were observed, these being most prominent in mice receiving either 587 or 1911 mg/kg per day. No other exposure- related changes were reported (Kirschmann et al., 1986). 8.3.3 Other animals Heppel et al. (1944) did not find organ lesions related to exposure at 17 700 mg/m3 (7 h/day, 5 days/week for 6 months) in studies on dogs, monkeys, rats, rabbits and guinea-pigs, with the exception of moderate centrilobular fatty degeneration of the liver and pneumonia in 3 out of 14 guinea-pigs. CNS depression was found in all species following exposure to 35 000 mg/m3; all animals became inactive, sometimes after initial excitement. At 35 000 mg/m3, dogs also showed fatty degeneration of the liver. Hepatic changes (slight cytoplasmatic vacuolation) and vacuolar changes in the renal tubules were found in dogs exposed continuously to 3500 mg/m3 for up to 100 days. Abnormal haematology and increased activity of serum enzymes were reported after 4 weeks. Oedema of the brain was observed at a concentration of 17 350 mg/m3 (Haun et al., 1972). Three male and three female beagle dogs were exposed to 17 700 mg/m3, 6 h/day for 90 days. Haematology, clinical chemistry and urinary parameters were measured and ECG and circulatory functions were examined. At the end of the study, histological examinations were performed. Slight sedation was induced throughout the exposure period and all dogs had slight erythema, lasting up to 10 h after exposure. No deaths and no other signs of toxicity were observed (Leuschner et al., 1984). Decreased levels of neurotransmitter amino acids were observed in gerbil brains after continuous inhalation exposure to methylene chloride (340 mg/m3) for 3 months (Briving et al., 1986). In gerbils exposed by inhalation to 1240 mg/m3 for 3 months, followed by a 4- month solvent-free period, increased brain concentrations of two astroglial proteins and decreased levels of DNA in the hippocampus and cerebellum were observed (Rosengren et al., 1986). Decreased hippocampal DNA levels were also observed in gerbils exposed to 740 mg/m3 (Rosengren et al., 1986; Karlsson et al., 1987). It was suggested by the authors that this effect may have been the result of the loss of nerve cells. 8.3.4 Appraisal Prolonged exposure to high concentrations of methylene chloride (> 17 700 mg/m3 ) caused reversible CNS effects, slight eye irritation and mortality in several laboratory species. Body weight reduction was observed in rats at 3500 mg/m3 and in mice at > 17 700 mg/m3 . After intermittent exposure, effects on the liver were observed in rats at 3500 mg/m3 and in mice at 14 100 mg/nz3 . After continuous exposure, slight cytoplasmatic vacuolization in the liver of both rats and mice were found at 88 and 350 mg/m3. No evidence of irreversible neurological damage was seen in rats exposed by inhalation to concentrations of < 7100 mg/m3 for 13 weeks. Oral administration of methylene chloride to rats caused effects on the liver with a no-observed-effect level of 125 mg/m3. 8.4 Skin and eye irritation; sensitization 8.4.1 Skin irritation Application of 0.5 ml methylene chloride to rabbits for 24 h, under a semi-occlusive patch on abraded or intact skin, caused severe erythema and oedema with necrosis and acanthosis (Duprat et al., 1976). Rabbits exposed to 0.5 ml methylene chloride for 4 h under occlusive patch test condition, either with or without simultaneous exposure to other chlorinated solvents, showed moderate skin irritation but no corrosive effect (Van Beek, 1990). 8.4.2 Eye irritation Duprat et al. (1976) and Ballantyne et al. (1976) exposed rabbits once to 0.1 ml methylene chloride by ocular instillation. Moderate to severe changes were seen in the conjunctiva, together with increased corneal thickness and intra-ocular tension. All effects were reversible. Vapour exposure of the eyes to 17 700 mg/m3 caused slight increases in corneal thickness and intra-ocular tension. 8.4.3 Sensitization No data are available. 8.4.4 Appraisal Liquid methylene chloride is moderately irritant to the skin and eyes in experimental animals. 8.5 Developmental and reproductive toxicity 8.5.1 Developmental toxicity When groups of Sprague-Dawley rats and Swiss-Webster mice were exposed to methylene chloride at a concentration of 4400 mg/m3 on days 6-15 of pregnancy for 7 h/day, maternal body weight in the mice was increased and the dams of both rats and mice had CO-Hb levels as high as 12.5% during exposure. In both species, an increased incidence of minor skeletal anomalies was observed, i.e. dilated renal pelvis in rats and extra sternebrae in mice (Schwetz et al., 1975). No significant teratogenic or fetotoxic effects were observed in either species. Groups of 18 rats were exposed before and/or during 17 days of pregnancy to a methylene chloride concentration of 16 250 mg/m3 for 6 h/day. The exposed dams exhibited increased blood CO-Hb levels, ranging from 7.1 to 10.1%, and increased relative and absolute liver weights. Fetal body weight was decreased, but no increase in the incidences of dead fetuses and/or resorptions nor any skeletal and/or visceral malformations were observed (Hardin & Manson, 1980). After exposure to methylene chloride using the same experimental conditions, litters from four groups of 10 rats were used for behavioural testing. Body weight gain, food and water consumption, wheel running activity and avoidance learning were all unaffected by the exposure. However, changes in the general activity of pups were found in both sexes starting at the age of 10 days, and were still present in male offspring at the age of 150 days. The effects cannot be definitely and directly attributed to methylene chloride, however, since elevated maternal CO-Hb- or methylene chloride-induced changes in maternal- litter interactions could have been contributing factors (Bornschein et al., 1980). Groups of seven female Wistar rats were fed methylene chloride at levels of 0.04, 0.4 and 4.0% in their diet from days 0-20 of pregnancy. Fetuses were examined on day 20 and neonatal growth was measured for 8 weeks after birth. Maternal body weight was significantly reduced in the 4.0% group. Although a reduction in the fetal weight of the females in the 0.4% group was observed, there were no differences in any group in the number of implantations and resorptions. No external malformations were observed by fetal, skeletal and visceral examination, and no differences were observed in any group in the frequency of delayed ossifications or in the dilation of the renal pelvis. A decrease in postnatal weight gain and in absolute liver weight was found in the 0.04% group males at the 8th week after birth (Nishio et al., 1984). Groups of F-344 rats (number not specified) received methylene chloride by gavage (dose level not specified) in corn oil on gestation days 6- 15. The compound was tested with at least two dose levels plus a concurrent control group, the high dose (not specified) being selected to cause maternal toxicity. The dams were allowed to deliver and their litters were examined post-natally. Although a small change in maternal weight was found, no effects on litters were reported (Narotsky et al., 1992). 8.5.2 Reproductive toxicity When rats received methylene chloride in the drinking-water at a level of 125 mg/litre during a period of 13 weeks before mating, no effects were found on the female fertility index, litter size, survival of pups at 4 weeks or the number of resorptions (Bornmann & Loeser, 1967). A two-generation inhalation study was conducted to evaluate the effects of inhaled methylene chloride on the reproductive capability, neonatal growth and survival of rats (Nitschke et al., 1988b). Groups of 30 male and 30 female 6-week-old F-344 rats (F0) were exposed to 0, 350, 1770 or 5300 mg/m3 (6 h/day, 5 days/week for 14 weeks). After this exposure, F0 animals were allowed to mate using one male and one female of the respective treatment groups to produce the F1 litters. After weaning, 30 males and 30 females (4 weeks old) from each treatment group were randomly selected and assigned to the respective exposure groups. After exposure to the relevant concentration of methylene chloride (6 h/day, 5 days/week for 17 weeks), the F1 adults were allowed to mate to produce F2 litters. Reproductive parameters examined included fertility, litter size and neonatal growth and survival. All adults and selected weanlings were examined for grossly visible lesions. No adverse effects on reproductive parameters, neonatal survival or neonatal growth were noted in animals exposed to methylene chloride in either the F0 or F1 generations. Similarly, there were no treatment-related gross pathological changes in F0 and F1 adults or F1 and F2 weanlings; histopathological examination of tissues did not reveal any lesions in F1 and F2 weanlings attributable to exposure to methylene chloride. Therefore, the results of this study indicate that exposure to concentrations as high as 5300 mg/m3 does not affect the normal reproductive function of rats (Nitschke et al., 1988b). 8.5.3 Appraisal Methylene chloride is not teratogenic in rats or mice at concentrations up to 16 250 mg/m3 . No evidence of an effect on the incidence of skeletal malformations or other developmental effects was observed in three animal studies. Small effects on either fetal or maternal body weights were reported at 4400 mg/m3 . A two- generation reproductive toxicity study in rats exposed to methylene chloride by inhalation at concentrations of up to 5300 mg/m3 , 6 h/day, 5 days/week, did not show evidence of an adverse effect on any reproductive parameter, neonatal survival or neonatal growth in either the F0 or F1 generation. 8.6 Mutagenicity and related end-points Studies on the mutagenic potential of methylene chloride have been performed on bacteria, fungi and cultured mammalian cells. Results from in vivo studies on mice and rats have also been reported. 8.6.1 In vitro 8.6.1.1 Bacteria Methylene chloride is mutagenic when tested using the Ames protocol in Salmonella typhimurium TA98, TA100 and TA 1535 (Table 18). The number of revertants increased 3- to 7-fold in a dose- related manner when plates were exposed to vapour of methylene chloride of undisclosed purity at levels ranging from 20 100 up to 201 000 mg/m3. Metabolic activation by either induced rat liver S9 fraction, cytosol fraction, or microsomal fraction increased the mutagenicity of methylene chloride (Simmon et al., 1977; Jongen et al., 1978, 1982; McGregor, 1979; Kirwin et al., 1980; Barber et al., 1980; Nestmann et al., 1980, 1981; Gocke et al., 1981; Dillon et al., 1990). In this respect the cytosolic fraction was more active than the microsomal fraction (Green, 1983). A positive result was reported in strains TA98 and TA100 with and without 30% hamster liver S9 (Zeiger et al., 1990) using the vapour phase (desiccator procedure) protocol. Negative mutagenicity results were obtained in studies not using vapour phase exposure (Rapson et al., 1980; Nestmann et al., 1980). No mutagenic activity was found when methylene chloride was tested in Salmonella typhimurium strains TA100, TA1535, TA1537, TA97 and TA98 with or without the addition of 10% or 30% rat/hamster liver S9, using the preincubation protocol (Zeiger et al., 1990). Metabolic studies of methylene chloride (see section 6.3) indicate that the conjugation of methylene chloride with glutathione (GSH), catalysed by cytosolic glutathione- S-transferase, may play a role in the observed mutagenicity of methylene chloride in Salmonella. However, the direct reaction of glutathione with methylene chloride only produced a very small increase in mutagenicity (Jongen et al., 1982). In another study, Salmonella typhimurium TA100 and the GSH- deficient strain TA100 gsh were exposed to 0-5% methylene chloride using a vapour phase protocol. The mutagenic response, with and without Aroclor-induced rat-liver S9, microsomes or cytosol, was marginally higher at the highest methylene chloride concentrations. Salmonella typhimurium TA100 gsh was slightly less responsive than TA100 at high doses in the absence of S9. This difference was not seen in the presence of S9. The addition of exogenous GSH had only a small effect on the mutagenic response in TA100 or TA100 gsh in the absence or presence of S9. According to the authors, these data suggest that if the interaction between methylene chloride and GSH is responsible for the observed mutagenicity, it occurs at extremely low levels of intracellular GSH and is not significantly affected by exogenous GSH (Dillon et al., 1992). The mutagenicity of methylene chloride has also been studied in a variety of other microbial systems using a vapour phase protocol. Mutagenic effects were observed in E. coli WP2uvrA and pKM101 which were exposed to 0-5% methylene chloride using a vapour phase protocol (Dillon et al., 1992). These strains of E. coli are deficient in glutathione, containing approximately 25% of the level of glutathione present in the strain TA100. Table 18. In vitro mutagenicity assays Assay Strain/type S9 Dose Resulta Observations Reference activation Salmonella TA100, ± 10% 100-10 000 -ve Preincubation protocol Zeiger et al. typhimurium TA1535, or 30% µg//plate (1990) TA1537, rat or TA97, TA98 hamster liver S9 Salmonella TA100, TA98 ± 30% 0.1-1.0 +ve Vapour protocol-dessicator Zeiger et al. typhimurium rat or ml/chamber procedure (1990) hamster liver S9 Salmonella TA100, ±S9 -ve Standard plate-incorporation protocol Nestmann et al. typhimurium TA1535, (1980) TA1537, TA98, TA1538 Salmonella TA100, +ve 0.5µl in open glass dish within a Nestmann et al. typhimurium TA1535 desiccator; revertants: 2-fold (1980) increase in TA1535; 6-fold in TA100 Salmonella TA1535, ± S9 up to 750/µl in +ve Active in strains TA98 and TA100 Gocke et al. typhimurium TA100, a 9 litre (1981) TA1538, desiccator TA98, TA1537 Salmonella TA100 ±S9 0-1.4% +ve Significant increases in mutagenic Jongen et al. typhimurium activity by addition of rat liver (1982) cytosol fraction; marginal increases by addition of microsomal fraction Table 18 (Cont'd) Assay Strain/type S9 Dose Resulta Observations Reference activation Salmonella TA100, ±S9 0-5% for 2, 4, 6 +ve Vapour phase protocol. Data Dillon et al. typhimurium 100gsh or 48 h suggest interaction between (1992) (glutathione methylene chloride and GSH deficient) responsible for the mutagenic activity Salmonella TA100 S9 2.8 v/v +ve Green (1983) typhimurium Salmonella TA100 -S9 0, 50-800/µl +ve Vapour phase protocol; dose-related Simmon et al. typhimurium per 9 litre increase in the number of revertants, (1977) desiccator with a mutation rate over 7-fold (approx 18-318 higher than controls at 320 mg/m3; mg/m3) for 7 h two experiments were conducted Salmonella TA98, TA100 ±S9 0, 20, 100 to +ve Vapour phase protocol; S9 prepared Jongen et al. typhimurium 201 000 mg per m3 from the livers of phenobarbital (1978) (5 concentrations) pretreated rats; a dose-related for 48 h increase of up to 5-8 fold (mean value for 3 experiments) was seen, slightly higher in the presence of S9; toxicity was noted at the highest dose level Table 18 (Cont'd) Assay Strain/type S9 Dose Resulta Observations Reference activation Salmonella TA100 Not stated Vapour phase protocol; time-course Jongen (1984) typhimurium study to evaluate the most appropriate exposure time for maximum differentiation of the methylene chloride induced reversion rates in the presence and absence of a metabolic activation system; the maximum differentation was obtained following 4-6 h exposure, and, consequently, an exposure time of 6 h was used by these workers in the study of Jongen et al. (1982) Salmonella TA100 ±S9 0 or 1 ml/9 litre +ve Study summarized in review, original Simmon & typhimurium desiccator data were not available; vapour Kauhanen (1978) (approx 390 mg phase control; S9) from livers of per m3) for 6.5 Aroclor-pretreated rats; addition of or 8 h S9 increased the mutation rate 1.5 fold; no further details are available Salmonella TA98, TA100 ±S9 0,125, 250, 500 +ve Vapour phase control; S9 from livers Rapson et al. typhimurium or 750/µl per of Aroclor pretreated rats; dose- (1980) 9 litre desiccator related increase in the mutation rate, (0-293 mg/m3) with an approximate 10-fold increase for 8 h for both strains at the highest dose used; the presence of S9 resulted in a slightly higher mutation rate Table 18 (Cont'd) Assay Strain/type S9 Dose Resulta Observations Reference activation Salmonella TA1535, Not stated +ve Same study, but no data were given typhimurium TA1537, for the strains studied; there is no TA1538 evidence for an independent confirmatory experiment Salmonella TA1535, 0.5 ml/desiccator -ve Vapour phase protocol; result Nestmann et al. typhimurium TA1537, (volume unknown) doubling of revertants for strain (1980) TA1538, TA1535 and a 6-fold increase for TA98, TA100 strain TA100 when 0.5 ml methylene chloride was added directly to the culture rather than a seperate dish Salmonella TA100 ±S9 0, 98 800, +ve Vapour phase control; S9 from livers Green (1983) typhimurium 177 000, or of Aroclor pretreated rats; a dose- 297 000 mg/m3 related increase in the mutation rate for 3 days was observed 0 or 98 800 +ve Vapour phase protocol; the presence mg/m3 for 3 of S9 enhanced the mutation rate days 1.19 fold; on dividing the S9 material into microsomal and high-speed supernatant (cytosolic) fractions, only the high-speed supernatant enhanced (1.27 fold) the mutation rate; a small isotope effect was observed when 2H-methylene chloride was substituted for 1H-methylene chloride Table 18 (Cont'd) Assay Strain/type S9 Dose Resulta Observations Reference activation Direct examination of the metabolism of methylene chloride by the bacterium indicated that radio- labelled carbon dioxide and trace amounts of radiolabelled carbon monoxide were formed, together with considerable incorporation of radioactivity into endogenous materials Salmonella TA 1535 0, 24 700, +ve Vapour phase protocol; data McGregor (1979) typhimurium 42 400, 81 200, available in summary form only; 162 400 or dose-response relationship; TA100 331 800 mg per m3; was reported to give a more marked condensation response of methylene chloride onto agar plates occurred Salmonella TA1535, ±S9 0, 38, 76, 96 or +ve Vapour phase protocol data available Barber et al. typhimurium TA98, TA100 115 µmol/plate in summary form only; dose-response (1980) (0-38 500 mg relationship (source of S9 not per m3 vapour) stated) (negative results were in gas-tight obtained in studies not employing chambers gas-tight jars) Table 18 (Cont'd) Assay Strain/type S9 Dose Resulta Observations Reference activation Salmonella TA1535, +S9 Concentration +ve Vapour phase protocol; study Longstaff et al. typhimurium TA100 range not reported only briefly, methylene (1984) stated; exposure chloride being used as a positive for 72 h control; S9 prepared from livers of Aroclor pretreated rats. Mutation rate increased 6-fold for TA1535 (with a 50% methylene chloride-air mixture) and 2.4 fold for TA100 (with a 1% methylene chloride-air mixture); the increased mutation rate was reproducible; apparently, one strain at least showed a positive dose response Salmonella TA100 0.1-1000µg -ve Plate-incorporation assays; Rapson et al. typhimurium per plate (5 insufficient information was given to (1980) concentrations), evaluate the result one plate per concentration Salmonella TA1535, ± S9 Up to 26 -ve Plate-incorporation assays; S9 from Nestmann et al. typhimurium TA1537, mg/plate, livers of Aroclor-induced rats; tested (1980) TA1538, dissolved in to limit of toxicity; positive results TA98, TA100 dimethyl-sulfoxide for 3 pro-mutagens were obtained using the same batch of S9, but not necessarily in parallel incubations; a second, independent assay was conducted over a limited concentration range; this was not a satisfactory demonstration of a negative response Table 18 (Cont'd) Assay Strain/type S9 Dose Resulta Observations Reference activation Escherichia WP2uvrA, ±S9 0-5% for 2, 4, 6 +ve Vapour phase protocol; data suggest Dillon et al. coli pKM101 or 48 h interaction between methylene (1992) chloride and GSH responsible for the mutagenic activity Microscreen E. coli WP2S ±S9 0.78-100 +ve Rossman et al. ( ) µl/well (1991) Saccharomyces D7 0-209 mM +ve Induced mitiotic gene convertants Callen et al. cerevisiae and recombinants, and, to a lesser (1980) extent, gene revertants Aspergillus Diploid P1 0-0.8 v/v +ve Crebelli et al. nidulans (1988, 1992) Sister Human +ve Thilagar et al. chromatid peripheral (1984a,b) exchange lymphocytes, CHO cells, mouse lymphoma L5178Y cells Sister Chinese ±S9 0-5000 µg/ml -ve Standard (25-29 h after treatment) Anderson et al. chromatid hamster harvest time (1990) exchange ovary (CHO) cells Table 18 (Cont'd) Assay Strain/type S9 Dose Resulta Observations Reference activation Sister CHO cells ±S9 0-15 µl/ml -ve Thilagar & chromatid Kumaroo (1983) exchange Sister Chinese 0-4% ± Marginal (< 2-fold), reproducible Jongen et al. chromatid hamster V79 increases in frequency; not dose- (1981) exchange cells related Chromosome Human Not stated +ve Thilagar et al. aberration peripheral (1984a,b) lymphocytes, CHO; mouse lymphoma L5178Y cells Chromosome CHO cells ±S9 0-15 µl/ml +ve Dose-dependent increase Thilagar & aberration Kumaroo (1983) Chromosome CHO cells ±S9 0-5000 µg/ml -ve Standard (10-14 h after treatment) Anderson et al. aberration harvest time (1990) Cells in vitro Chinese 0.5-5% v/v -ve Forward mutation to 6-thioguanine Jongen et al. hamster cells resistance; mutation rate was (1981) corrected for survival Cells in vitro Epithelial -S9 0, 35 (300- -ve Varying the expression time was Jongen et al. cells (V79) 141 000 mg/m3 reported to have no effect; cell (1981) (4 concentrations survival was reduced by about 20% for 1 h); expression at 141 000 mg/m3 time of 6 days Table 18 (Cont'd) Assay Strain/type S9 Dose Resulta Observations Reference activation Cell mutation L5178Y Not stated -ve Thilagar et al. mouse (1984a,b) lymphoma Cell mutation L5178Y ±S9 0-3000 µl/ml ± Overall questionable evaluation of Myhr et al. mouse activity (1990) lymphoma Cell Primary 0.5 ml/4.6 litre +ve Enhanced transformation by SA7 Hatch et al. transformation Syrian chamber adenovirus (1983) hamster embyro cells HGPRT- Chinese 0-4% -ve Jongen et al. deficient hamster V79 (1981) cells Micronucleus Chinese Not stated -ve Gu & Wang hamster V79 (1988) cells Unscheduled Primary rat Not stated -ve Trueman et al. DNA synthesis hepatocytes (1987) Unscheduled Primary rat Not stated ± Thilagar et al. DNA synthesis hepatocytes (1984a) Cell BALB/C-3T3 0.01% -ve Price et al. transformation mouse (1978) Table 18 (Cont'd) Assay Strain/type S9 Dose Resulta Observations Reference activation Cell C3H-10T1/2 Not stated -ve Thilagar et al. transformation CL8 mouse (1984a) Unscheduled Primary rat ± A "marginal" positive result reported Thilagar et al. DNA synthesis hepatocytes (1984a) Unscheduled Human ±S9 2.5-10.0 µl/ml -ve Perocco & Prodi DNA synthesis lymphocytes (1981) Unscheduled Chinese 0-5% -ve Jongen et al. DNA synthesis hamster V79 (1981) cells DNA repair Primary rat 0.7-16.0 mM -ve Andrae & Wolff synthesis hepatocytes (1983) a +ve = positive; -ve = negative; ± = equivocal or inconclusive A recent study demonstrated that glutathione- S-transferase 5-5 expression in Salmonella typhimurium increases mutation rates caused by methylene chloride. The plasmid pKK233-2 containing rat glutathione- S-transferase 5-5 cDNA, either in the correct or reverse direction, was transfected into TA1535. The resulting sense- transformed TA1535 (RSJ 100) expressed the enzyme and enhanced base- pair revertants as compared to the anti-sense strain (TPT 100). Mutagenicity was not seen when GSH, purified glutathione- S- transferase and a methylene dihalide such as methylene bromide were added to the pre-incubation mixture with TA1535. Formaldehyde did not produce mutations in any of the three strains (Thier et al., 1993). The nature and distribution of forward mutations in the N-terminal region of the loc I gene of excision repair-proficient (Uvr+) and excision repair-defective (Uvr B-) strains of E. coli have been described by Zielenska et al. (1993). A total of 116 locI-d mutations were characterized. 8.6.1.2 Fungi and yeasts A dose-related increase in the frequency of gene conversions, mitotic recombinations, and reversions was found for cultures of Saccharomyces cerevisiae strain D7, but not for strains D4 and D3, exposed to methylene chloride of undisclosed purity. However, the mutagenic results in D7 occurred at toxic doses (1270 g/m3) in which survival of the yeast cells was reduced to 42% (Cullen et al., 1980). When assayed for the induction of mitotic segregation in Aspergillus nidulans P1, methylene chloride significantly increased the frequency of morphologically abnormal colonies, which produced euploid whole-chromosome segregants (Crebelli et al., 1988; Crebelli et al., 1992). 8.6.1.3 Mutation in mammalian cells Methylene chloride was not mutagenic in several tests in which mammalian somatic or human cells were used (Gocke et al., 1981; Jongen et al., 1981; Perocco & Prodi, 1981; Andrue & Wolff, 1983; Burek et al., 1984). Both negative (Thilagar et al., 1984a) and questionable results (Myhr et al., 1990) were reported when methylene chloride was tested for gene mutations in a L5178Y mouse lymphoma assay at the thymidine kinuse locus. No increase in micronuclei was found when methylene chloride was tested in Chinese hamster V79 cells (Gu & Wang, 1988). 8.6.1.4 Chromosomal effects Studies on chromosome morphology in cultured mammalian cells indicate that methylene chloride is clastogenic. Chromosomni alterations (chromatid damage, chromosomni exchanges, but no increase in sister chromatid exchanges) were observed in CHO cells (Thilagar & Kumaroo, 1983), human lymphocytes and L5178Y cells (Thilagar et al., 1984a,b), both with and without metabolic activation. A small increase in sister-chromatid exchanges (SCEs), without clear evidence of a dose-response relationship, was found in V79 cells when exposed to gaseous methylene chloride at concentrations up to 5% (Jongen et al., 1981 ). A dose-related increase in SCEs was observed in CHO cells after a 24-h exposure to methylene chloride. The results were statistically significant only at the highest concentration (7%) and exposures of shorter duration (2, 4 or 6 h) were without effect (McCaroll et al., 1983). In a more recent study, Anderson et al. (1990) reported no increase in chromosomal aberrations or SCEs in CHO cells exposed to up to 5 mg/ml. Hallier et al. (1993) described an apparent polymorphism in human blood samples used to measure SCEs in lymphocytes. Those blood samples possessing metabolic activity (conjugators) were inactive in the assay, whereas those samples which were metabolically inactive (non- conjugators) produced significant increases in SCEs (see also section 6.3.1). 8.6.1.5 DNA damage Concentrations of up to 16 mM methylene chloride failed to induce unscheduled DNA synthesis (UDS) in cultured rat hepatocyte, although some reduction in replicative DNA synthesis occurred at the higher doses (Andrae & Wolff, 1983). The absence of evidence of methylene chloride-induced UDS in primary rat hepatocytes was also reported by Trueman et al. (1987). Thilagar et al. (1984a) reported a "marginal" positive result in a primary rat hepatocytes UDS assay, but details were not available. In experiments using no exogenous activation systems, exposure of hamster V79 cells or human fibroblasts (AH cells) to methylene chloride concentrations of between 0.5 and 5% did not induce UDS (Jongen et al., 1981). A non-specific but reversible inhibition of replicative DNA synthesis was observed in both cell lines, probably due to a metabolic block of synthesis. Methylene chloride in doses of 2.5, 5 or 10 µ/ml did not induce UDS in human lymphocytes in either the presence or absence of rat liver S9 (Perocco & Prodi, 1981). 8.6.1.6 DNA binding in vitro Several studies have investigated the potential of methylene chloride and its metabolites to bind covalently to DNA. Incubation of 14C-labelled methylene chloride failed to detect any DNA binding, but binding to proteins and lipids was observed (Cunningham et al., 1981). After incubation of calf thymus DNA in vitro with 14C- labelled methylene chloride (0.8 µmol/ml), together with hepatic microsomes and a NADPH-generating system, there was no evidence of any DNA alkylation (Di Renzo et al., 1982). These studies confirm those of an earlier in vitro study which failed to detect any DNA binding of methylene chloride or its metabolites (Anders et al., 1977). 8.6.1.7 Cell transformation Methylene chloride has been tested for its ability to induce transformation in a variety of cell systems. Negative results were obtained in C3H-10T1/2 CL8 mouse cells at 10 µl/ml (Thilagar et al., 1984a) and in Balb/C-3T3 mouse cells at 0.01% (Price et al., 1978). Methylene chloride significantly enhanced the frequency of transformation by SA7 virus in a dose-related manner (Hatch et al., 1983). 8.6.2 In vivo 8.6.2.1 Chromosome damage Large doses of methylene chloride (425, 850 and 1700 mg/kg) given twice intraperitoneally to NMRI mice did not increase micronuclei in the bone marrow micronucleus test (Gocke et al., 1981). Doses of up to 4 g/kg body weight (the maximum tolerated dose) administered by gavage to C57BL/6J/Alpk mice also failed to induce any increase in bone marrow micronuclei (Sheldon et al., 1987). The results of in vivo mutagenicity assays are presented in Table 19. Intraperitoneal injections of methylene chloride (100, 1000, 1500 or 2000 mg/kg) did not increase the frequencies of either SCEs or chromosome aberrations in bone marrow cells of male C57BL/6J mice (Wesforook-Collins et al., 1988; Wesforook-Collins et al., 1990). No increase in the frequency of either SCEs or chromosome aberrations was observed in bone marrow cells of female B6C3F1 mice after a single subcutaneous injection of methylene chloride (2500 or 5000 mg/kg) (Westbrook-Collins et al., 1989; Allen et al., 1990). Inhalation exposure of female B6C3F1 mice to 14 000 or 28 000 mg/m3 for 10 days (6 h/day, 5 days/week) resulted in slight increases in the frequency of SCEs in lung cells and peripheral blood lymphocytes, in chromosome aberrations in lung and bone marrow cells, and in micronuclei in peripheral blood erythrocytes. The results were statistically significant at 28 200 mg/m3 for all end-points. At 14 100 mg/m3, statistical significance was reached only for the SCE frequency in lung cells. A marginal increase in lung cell SCEs and micronuclei in peripheral blood erythrocytes was observed following a 3-month inhalation exposure to 7100 mg/m3 (Westbrook-Collins et al., Table 19. In vivo mutagenicity assays Assay Strain/type Resulta Observations Reference Chromosome Male C57BL/6 mouse bone -ve i.p., 0-2000 mg/kg Westbrook-Collins et al. (1988); aberration marrow Westbrook-Collins et al. (1990) Chromosome Female B6C3F1 mouse bone -ve s.c., 2500 or 5000 mg/kg Westbrook-Collins et al. (1989); aberration marrow Allen et al. (1990) Chromosome Female B6C3F1 mouse bone ± inhalation, 14 100 or 28 200 mg/m3 for Westbrook-Collins et al. (1989); aberration marrow 10 days Allen et al. (1990) Chromosome Rat bone marrow -ve inhalation, 1770, 3500 or 12 400 mg/m3 Burek et al. (1984) aberration 6 h/day, 5 days/week for 6 months Sister-chromatid Male C57BL/6 mouse bone -ve i.p. 0-2000 mg/kg Westbrook-Collins et al. (1988); exchange marrow Westbrook-Collins et al. (1990) Sister-chromatid Female B6C3F1 mouse bone -ve s.c., 2500 or 5000 mg/kg Westbrook-Collins et al. (1989); exchange marrow Allen et al. (1990) Sister-chromatid Female B6C3F1 mouse lung ± inhalation, 14 100 or 28 200 mg/m3 for Westbrook-Collins et al. (1989); exchange cells, peripheral blood 10 days Allen et al, (1990) lymphocytes Sister-chromatid Female B6C3F1 mouse lung ± inhalation, 7100 mg/m3 for 3 months; Westbrook-Collins et al. (1989); exchange cells small increase Allen et al. (1990) Synaptonemal Male C57BL/6 mouse ± i.p., 0-1500 mg/kg Westbrook-Collins et al. (1988) complex (damage at meiotic prophase) Table 19 (Cont'd) Assay Strain/type Resulta Observations Reference Micronucleus Female B6C3F1 mouse ± inhalation, 14 100 or 28 200 mg/m3 for Westbrook-Collins et al. (1989); peripheral blood 10 days Allen et al. (1990) erythrocytes Micronucleus Female B6C3F1 mouse ± inhalation, 7100 mg/m3 for 3 months Westbrook-Collins et al. (1988); peripheral blood Allen et al. (1990) erythrocytes Micronucleus C57BL/6 mouse bone -ve oral in corn oil, up to 4 g/kg Sheldon et al. (1987) marrow Unscheduled Male Alpk: AP rat -ve oral gavage, 100, 500 or 1000 mg/kg; Trueman et al. (1987) DNA synthesis hepatocytes autoradiography 4 and 12 h after treatment Unscheduled Male B6C3F1 mouse -ve oral gavage, 100 mg/kg in corn oil Lefevre & Ashby (1989) DNA synthesis hepatocytes Unscheduled Male Fischer-344 rat, and -ve inhalation, 7100 or 14 100 mg/m3 for 2 Trueman & Ashby (1987) DNA synthesis male B6C3F1 mouse or 6 h hepatocytes Unscheduled Male B6C3F1 mouse -ve inhalation, 14 100 mg/m3 for 2 h Lefevre & Ashby (1989) DNA synthesis hepatocytes Dominant lethal Male Swiss-Webster mouse -ve s.c., 5 ml/kg, 5% or 10% v/v in corn oil, Raje et al. (1988) 3/week for 4 weeks inhalation at 350, 530 or 710 mg/m3, Basso et al., (1987); 2 h/day, 5 days/week for 6 weeks Raje et al. (1988) no microscopic lesions of testes or brain Table 19 (Cont'd) Assay Strain/type Resulta Observations Reference Drosphila Berlin K, Basc -ve 0 to 14 260 mg/m3 for 6 h, 1 week or 2 Kramers et al. (1991) melanogaster weeks; sex-linked recessive lethal assay, and somatic mutation and recombination test Drosphila Berlin K, Basc +ve 125 or 620 mM; increased frequency of Gocke et al. (1981) melanogaster recessive lethals in Basc test a +ve = positive; -ve = negative; ± = equivocal or inconclusive 1989; Allen et al., 1990). Background data and positive control results were not available. Chromosome aberration data (excluding gaps) were not reported, and so the increases might not be significant. Increases in SCE and micronuclei were small (up to 2-fold at 28 000 mg/m3). No increase in chromosomal aberrations was observed in bone marrow cells of Sprague-Dawley rats (5 of each sex per group) following inhalation exposure to 1770, 3500 or 12 400 mg/m3 (6 h/day, 5 days/week) for 6 months (Burek et al., 1984). Inconclusive results were reported when methylene chloride was tested in C57B1/6 mice for its ability to induce damage in the synaptosomal complex, an experimental end-point which can reveal induced damage at the meiotic prophase, following intraperitoneal injection of 1500 mg/kg (Westbrook-Collins et al., 1988). 8.6.2.2 Drosophila No mutagenicity was detected in the recessive lethal test on Drosophila melanogaster fed, or injected with, 1-2% methylene chloride (Abrahamson & Valencia, 1980). A marginal increase in the number of recessive deaths was found after feeding 125 or 650 nmol methylene chloride in 2% dimethylsulfoxide to Drosophila melanogaster (Gocke et al., 1981). This study may not be reliable because control values from different solvent treatments were pooled, and because the increases seen were significant only when results from the two dose levels were combined. Methylene chloride was not active in the sex-linked recessive lethal assay or the somatic mutation and recombination test carried out with Drosophila melanogaster using inhalation exposure up to 14 260 mg/m3 (Kramers et al., 1991). 8.6.2.3 DNA damage Methylene chloride has been evaluated for its ability to initiate unscheduled DNA synthesis (UDS) in the livers of male mice and rats in vivo. Alpk:AP rats were exposed by oral garage to 100, 500 or 1000 mg/kg body weight, and hepatocytes were assessed for UDS via autoradiography 4 and 12 h later (Trueman & Ashby, 1987). In a second study, F-344 rats or B6C3F1 mice were exposed by inhalation to either 7100 or 14 100 mg/m3 for 2 or 6 h, and hepatocytes were assessed for UDS immediately after exposure. In both studies, methylene chloride failed to induce UDS. Similar results were reported by Lefevre & Ashby (1989) following exposure of male B6C3F1 mice either by oral gavage in corn oil (1000 mg/kg) or by inhalation of an atmosphere containing 14 100 mg/m3 for 2 h. Slightly increased DNA damage, measured by alkaline elution of DNA from hepatocytes of rats orally exposed to methylene chloride (2.55 g/kg) in corn oil for 24 h, has been reported (Kitchin & Brown, 1989). DNA single strand breaks were found in mouse hepatocytes after in vivo exposure to 14 120 mg/m3 for 3 or 6 h but not in hepatocytes of similarly exposed rats. It was further observed in vitro that the lowest concentration of methylene chloride needed to induce DNA single strand breaks in mouse hepatocytes was 75 times below that in rat hepatocytes. This DNA damage was not accompanied by cytotoxicity. The relation of these findings to the mechanism for carcinogenic effects is discussed in section 8.8.1. 8.6.2.4 DNA binding Several studies have investigated the potential of methylene chloride to bind covalently to DNA after in vivo exposure. DNA has been isolated from the livers and lungs of mice and rats exposed to 14 100 mg/m3 (Green et al., 1987a,b,c). In both studies the DNA was hydrolysed and analysed by chromatography to distinguish between alkylation of DNA and incorporation of radioactivity though the C-1 pool. No evidence of alkylation was found, both studies having the power to detect one alkylation per 106 nucleotides. No alkylation of DNA was reported by Ottenwalder & Peter (1989), following a DNA binding assay of methylene chloride in rats and mice. Male mice and hamsters were exposed to 14 100 mg/m3, 6 h/day for 2 days, followed on the third day by a 6-h exposure to a decreasing concentration (15 900 to 8800 mg/m3) of 14C-labelled methylene chloride (Casanova et al., 1992). DNA-protein cross-links (DPX) were detected in mouse liver, but not in mouse lung or hamster liver or lung. The failure to detect DPX in mouse lung did not exclude possible formation in a sub-population of lung cells. These results demonstrate that formaldehyde derived from methylene chloride can form DNA-protein cross-links in the liver of B6C3F1 mice, the formation of DPX being dependent on the activity of the GST pathway. 8.6.2.5 Dominant lethal assay Groups of 20 Swiss-Webster male mice were injected subcutaneously 3 times/week for 4 weeks with 5 ml/kg of 5% v/v or 10% v/v methylene chloride in corn oil (Raje et al., 1988). Other groups of Swiss- Webster male mice were exposed to 350, 530 or 710 mg/m3 (2 h/day, 5 days/week for 6 weeks) (Basso et al., 1987; Raje et al., 1988). Mating was started 1 week later for the injection group and 2 days later for the inhalation group, each male mouse being mated with a virgin adult female. The mating continued for 2 weeks. The fetuses were examined on day 17 of gestation. No significant differences in any of the mutagenicity parameters were found between control and treated groups. No microscopic lesions were found in the testes (Basso et al., 1987; Raje et al., 1988) or brain (Basso et al., 1987) of the treated males. 8.6.2.6 Replicative DNA synthesis A number of studies have evaluated the ability of methylene chloride to induce replicative DNA synthesis (S-phase) in the livers and lungs of B6C3F1 mice and in the livers of Sprague-Dawley rats. A small, but statistically significant, increase in DNA synthesis was observed in the livers of mice exposed to 13 800 mg/m3 for 2 h, but not following a single oral dose of 1000 mg/kg (Lefevre & Ashby, 1989). The biological significance of these increases is unclear due to similar increases being seen in some control groups. There were no sustained increases in DNA synthesis in the livers of female mice exposed by inhalation to 3.53, 7.06, 14.12 or 28.24 g/m3 (1000, 2000, 4000 or 8000 ppm) for up to 4 weeks, nor in female mice exposed to 7.06 g/m3 for up to 2 years (Foley et al., 1993). Increases in DNA synthesis were not seen in rats exposed to 1770 mg/m3 for 6 or 12 months (Nitschke et al., 1988a,b). Replicative DNA synthesis was measured in the lungs of female mice exposed to 7.06 or 28.24 g/m3 for 1, 2, 3 or 4 weeks and in mice exposed to 7.06 g/m3 for 13 and 26 weeks. Small decreases in the labelling indices were reported at all these points (Kanno et al., 1993). In contrast, Foster et al. (1992) reported significant increases in the number of cells in S-phase in both the bronchiolar and alveolar epithelium of male mice exposed to 14.12 g/m3 for 13 weeks. 8.6.3 Appraisal Under appropriate exposure conditions, methylene chloride is mutagenic in prokaryotic microorganisms with or without metabolic activation (S. typhimurium or E. coli). In eukaryotic systems it gives either negative or, in one case, weakly positive results. In vitro gene mutation assays and tests for UDS in mammalian cells were uniformly negative. In vitro assays for chromosomal aberrations using different cell types gave positive results, whereas negative or equivocal results were obtained in tests for SCE induction. The majority of the in vivo studies reported have provided no evidence of mutagenicity of methylene chloride (e.g., chromosome aberration assay,micronucleus test or UDS assay). Where positive responses have been seen, they are restricted to tests using B6C3F1 mice. Marginal increases in the frequencies of SCEs, chromosomal aberrations and micronuclei in mice have been reported following inhalation of high concentrations of methylene chloride. Increases in hepatic DNA single strand breaks and DNA-protein crosslinks were seen in mice, but not in rats, exposed to 14 100 mg/m3. Within the limitations of the short-term tests currently available, in vivo genetic activity has only been detected in tests using B6C3F1 mice. 8.7 Chronic toxicity and carcinogenicity 8.7.1 Inhalation exposure 8.7.1.1 Rat Groups of 95 male and 95 female Sprague-Dawley rats (8 weeks old) were exposed by inhalation to 0, 1770, 5300 or 12 400 mg/m3 methylene chloride (99% pure) 6 h/day, 5 days per week for 2 years (Table 20). Overall survival in the study, including that of controls, was poor. Mortality among high-dose females was statistically increased from the 18th month when compared to controls. Increases in CO-Hb were found in treated groups from 6 months, but not in a dose- related manner. From 12 months, non-neoplastic pathological effects on the liver (increased hepatocellular vacuolization consistent with fatty change) were observed in both males and females at all exposure levels in a dose-related fashion. In males (5300 and 12 400 mg/m3) and females (12 400 mg/m3), a decrease in the incidence (females) and severity (males) of age-associated chronic progressive glomerulonephrotoxicity was observed. The only reported increases in tumour incidence occurred in benign mammary gland tumours in males and females, and tumours in the mid-cervical region close to the salivary gland in males. There was no significant increase in the proportion of animals with benign or malignant mammary tumours; however, the total number of benign mammary tumours showed a marginally significant dose- related increase in males (controls, 8/95; low-dose, 6/95; mid-dose 11/95; high-dose, 17/97; p = 0.046) and a dose-related increase in the total number of benign mammary tumours was observed in females (165/96; 218/95; 245/95; 287/97; p < 0.001). There was no indication of an increase in the number or incidence of malignant mammary tumours in either males or females. The background historical incidence for Sprague-Dawley rats in the laboratory normally exceeds 80% in females and is about 10% in males (Burek et al., 1984). Table 20. Carcinogenicity studies using the inhalation routes Species Strain/sex Route of Doses Observations Reference administration/protocol/ (mg/m3) group size Rat Sprague-Dawley, Inhalation, 6 h/day, 5 0, 1770, Increase in various types of sarcomas in the Burek et al. male and female days/week for 2 years 5300, mid-cervical area in the region of the (1984) 95 animals/sex/group 12 400 salivary gland in mid- and high-dose males (1/92, 0/95, 5/95, 11/97). Slight dose- related increase in total number of benign mammary tumours in males (8/95, 6/95, 11/95, 17/97; p =0.046); dose-related increase in total number of benign mammary tumours in females (165/96, 218/95, 245/95, 287/97; p<O.001) Rat Sprague-Dawley, Inhalation, 6 h/day, 5 O, 177, 710, No increase in incidence of benign Nitschke et al. male and female days/week for 20 1770 mammary tumours in males or in females (1988a) months (males), 24 exposed to 177 or 710 mg/m3; increased months (females); 90 incidence of benign mammary tumours in males/group females at 1770 mg/m3; no increase in the 198 females/group number of any malignant tumours; NOAEL 710 mg/m3 Rat Fischer-344/N, Inhalation, 6 h/day, 5 O, 3500, Dose-dependent increase in benign NTP (1986) male and female days/week for 102 7100, mammary tumours; male: 0/50, 0/50, 2/50, Mennear et al. weeks; 50 animals/sex 14 100 5/50; female: 5/50, 11/50, 13/50, 23/50 (1988) group Table 20 (Cont'd) Species Strain/sex Route of Doses Observations Reference administration/protocol/ (mg/m3) group size Rat Sprague-Dawley, Inhalation, 4 h/day, 5 350 No effect on percentage of animals bearing Maltoni et al. females, during days/week for 7 benign and/or malignant tumours, in (1988) and after weeks, then 7 h/day, 5 maternal or offspring animals; no pregnancy days per week for 97 statistically significant increase in total weeks; malignant tumours 54 females exposed 60 female controls Rat Sprague-Dawley, inhalation, protocol as 350 As above Maltoni et al. male and female above for 15 and 104 (1988) offspring from weeks; 60-70 males or 12th day in utero females/group, 158 male and 149 female controls Mouse B6C3F1 inhalation, 6 h/day, 5 7100 Mice exposed for more than one year Kari et al. (1992) days/week for various showed an excess of lung and liver tumours periods up to 104 weeks; killed at end of exposure; 364 females exposed, 364 female controls Table 20 (Cont'd) Species Strain/sex Route of Doses Observations Reference administration/protocol/ (mg/m3) group size Mouse B6C3F1 Inhalation to 6 h/day, 7100 All exposed groups showed excesses of Kari et al. (1992) 5 days/week for either lung and liver tumours 26, 52, 28 or 104 weeks; all mice maintained for 104 weeks; 68 females exposed, 68 female controls Mouse B6C3F1, male Inhalation, 6 h/day, 5 0, 7100, Dose-dependent increases in (1) NTP (1986); and female days/week for 102 14 100 alveolar/bronchiolar adenomas Mennear et al. weeks; 50 males: 3/50, 19/50, 24/50 (1988) animals/sex/group females: 2/50, 23/48, 28/48 (2) alveolar/bronchiolar carcinomas males: 2/50, 10/50, 28/50 females: 1/50, 13/48, 29/48 (3) hepatocellular adenoma and carcinoma - combined males: 22/50, 24/49, 33/49 females: 3/50, 16/48, 40/48 Hamster Syrian Golden, Inhalation, 6 h/day, 5 0, 1770, No significant increase in incidence of Burek et al. male and female days/week for 2 years; 5300, benign tumours (1984) 95 animals/sex/group 12 400 Males exposed to 5300 or 12 400 mg/ma showed an increase in the number of sarcomas in the mid-cervical area in the region of the salivary glands (1 subcutaneous sarcoma in controls, n = 92, 2 subcutaneous sarcomas and 3 salivary gland schwannoma at 5300 mg/m3, n = 95; and 5 subcutaneous fibrosarcomas and 2 subcutaneous mesofibrosarcomas as well as 2 salivary gland sarcomas at 12 400 mg/m3, n = 97). A number of uncertainties affect the toxicological significance of these observations. As stated by the investigators, all but two of these tumours were large and appeared to invade all adjacent tissues in the neck region. Histologically all were sarcomas. Some tumours morphologically resembled the overall type, fibrosarcoma while others resembled another, neurofibrosarcoma, and still others were undifferentiated or pleomorphic. Only two tumours were small enough to be localized in the salivary gland, appearing to arise in interstitial and capsular tissue. Based on these findings the author suggested that all probably arose from the salivary gland, although appearing to arise from mesenchymal tissue they represent a variety of tumour types. There is no explanation for the sex difference observed. Both male and female rats in the study had a viral disease (siabclacryoudenitis, which affects the salivary glands) during the first 2 months of the study. The infection did not increase mortality and all exposure groups appeared to be affected to the same degree (Burek et al., 1984). A later study in the same laboratory where the highest dose level was 1770 mg/m3 revealed salivary gland sarcomas in only two animals, a male at 1770 mg/m3 and a female at 177 mg/m3. The historical incidence in this laboratory was reported to range from 0 to 2% in control groups of Sprague-Dawley rats (Nitschke et al., 1988a). Groups of 90 male and 108 female Sprague-Dawley rats (6-8 weeks old) were exposed to 0, 177, 710 or 1770 mg/m3 6 h/day, 5 days/week for 20 and 24 months, respectively. Exposure-related increases in CO-Hb levels were found. Exposure-related histo-pathological changes were limited to the liver of male and female rats and mammary glands of female rats exposed to 1770 mg/m3. An increased incidence of hepatocellular vacuolization was found in males and females exposed to 1770 mg/m3. Females exposed to this dose level also had an increased incidence of multinucleated hepatocytes. The incidence of benign mammary tumours in female rats exposed to 177, 710 and 1770 mg/m3 was comparable to historical control values (79-82%), but the number (2.2) of benign mammary tumours per tumour-bearing female rat exposed to 1770 mg/m3 was greater than that of controls (1.8) (p < 0.05). There were no increases in the incidence of malignant tumours at any site in female rats, nor of benign or malignant tumours in male rats, exposed to methylene chloride. The no-observed-adverse-effect level for chronic inhalation exposure of Sprague-Dawley rats was judged to be 710 mg/m3 (Nitschke et al., 1988a). A group of 54 pregnant Sprague-Dawley rats was exposed to 350 mg/m3 4 h/day, 5 days/week for 7 weeks and subsequently for 7 h/day, 5 days/week for a further 97 weeks. A further group of 60 rats served as controls. In addition, groups of 60-70 males or females were exposed to 212 mg/m3 from the 12th day in utero for a total of either 15 or 104 weeks; there were 158 male and 149 female controls. In exposed maternal or offspring rats, methylene chloride did not affect the percentage of animals bearing benign and/or malignant tumours. Although there was a slight increase in total malignant rumours in rats exposed to 350 mg/m3 for 104 weeks, this was not deemed to be statistically significant (Maltoni et al., 1988). The Task Group noted the absence of relevant data on the design of the experiment such as the statistical methods, housing conditions and histopathological techniques. When groups of 50 male and 50 female F-344/N rats, 7-8 weeks old, were exposed by inhalation to 0, 3500, 7100 or 14 100 mg/m3 methylene chloride (99% pure) 6 h/day, 5 days per week, for 102 weeks (NTP, 1986), body weight gains for both exposed males and females were comparable to those of the control group. The survival of exposed male rats was also comparable to that of the controls, although the survival of all groups of males at the end of the study was low. Similarly, the survival of female rats was comparable with that of the controls, with the exception of the high-dose group. Non-neoplastic treatment-related increases in the incidence of renal tubular cell degeneration, splenic fibrosis and (females only) nasal cavity squamous metaplasia were observed. Increased incidences of benign tumours of the mammary gland (all fibroadenomas, except for one adenoma in the high-dose group) were observed in treated females (control, 5/50; low-dose, 11/50; mid-dose, 13/50; high-dose, 23/50; p < 0.001). There was a positive trend in the incidence of benign tumours of the mammary gland in males (0/50, 0/50, 2/50, 5/50; p < 0.01). The range of the historical control incidence of mammary gland fibroadenomas in this laboratory for the male rat was 0/50 to 6/49 and for the female rat was 5/50 to 24/49. There were no other exposure-related increases in tumour incidence (NTP, 1986; Mennear et al., 1988). 8.7.1.2 Mouse Groups of 50 male and 50 female B6C3F1 mice, 8-9 weeks old, were exposed to 0, 7100 or 14 100 mg/m3 (> 99% pure) 6 h/day, 5 days/week for 102 weeks and killed after 104 weeks of study. Survival to the end of the study period in males was: control, 39/50; low-dose, 24/50; and high-dose, 11/50; that in females was: 25/50, 25/49 and 8/49. This reduced survival may have been due to the chemically induced development of liver and lung neoplasia in both sexes, significant dose-related increases in lung and liver tumours having been found. The incidences of alveolar/bronchiolar adenomas were: males - 3/50, 19/50 and 24/50 (p < 0.001); and females - 2/50, 23/48 and 28/48 (p < 0.001). Those for alveolar/bronchiolar carcinomas were: males - 2/50, 10/50 and 28/50 (p < 0.001); and females - 1/50, 13/48 and 29/48 (p < 0.001). Incidence of hepatocellular adenomas or carcinomas (combined) were increased in high-dose males and dosed females (males: 22/50, 24/49 and 33/49; females: 3/50, 16/48 and 40/48). Dose-related increases were observed in the incidences of testicular atrophy in male mice and uterine and ovarian atrophy in female mice; these effects were considered to be secondary responses to neoplasia (NTP, 1986; Mennear et al., 1988). Studies have been conducted on the B6C3F1 mouse to study the time-dependency of exposure to methylene chloride leading to tumour formation. Groups of 364 female B6C3F1 mice were exposed to either air or 7100 mg/m3 methylene chloride for 6 h/day, 5 days/week for 104 weeks. Following exposure for either 462, 494, 515, 529, 571, 597, 618, 639 or 660 days, 10 exposed and 5 control mice were randomly selected and sacrificed for the purposes of following the progression of neoplasia in the lung and the liver. Increased incidences of alveolar/bronchiolar adenomas and carcinomas and of hepatocellular adenomas and carcinomas in mice exposed for 104 weeks were confirmed. In mice sacrificed following less than one year's exposure (368 days), there were no lung lesions observed and only minimal indications of proliferative lesions in the liver. Mice exposed for longer than one year showed a progressive increase in the incidence of both alveolar/bronchiolar adenomas and carcinomas. The incidence of liver tumours in mice exposed for longer than one year was relatively constant (40-60%), although a time-dependant increase in the total liver tumour burden per animal was reported (Kari et al., 1993). Groups of 68 female B6C3F1 mice were exposed to 100 mg/m3 6 h/day, 5 days/week for either 26, 52 or 78 weeks. All groups of mice were maintained for 104 weeks, at which time they were sacrificed. The exposures were split into two phases, i.e. exposure to either air or to methylene chloride for the specified period at the beginning or the end of the total exposure period. Further groups of 68 female mice were exposed either to air or to 7100 mg/m3 methylene chloride for the full 104 weeks. The percentage incidences of lung adenomas or adenocarcinomas or liver adenomas or adenocarcinomas are given in Table 21. The percentage incidence of both lung and liver tumours in mice exposed to methylene chloride during the first phase increased with the duration of exposure. In mice exposed to methylene chloride during the second phase, significant increases in the percentage incidence of lung and liver rumours were only observed following 78 weeks of exposure to methylene chloride. The study also showed that lung rumours appeared earlier in methylene chloride-exposed animals than did liver tumours. Comparison of the results of this study with those presented in the previous paragraph suggested that the percentage incidence of lung tumours but not liver tumours increased following withdrawal from exposure to methylene chloride, particularly when the mutagenicity of tumours was taken into account (Kari et al., 1993). Table 21. The percentage incidence of lung and liver tumours in female mice exposed to methylene chloride for different time periods Exposurea % incidence of tumours (adenomas or carcinomas) Phase I Phase II Lung Liver air 104 weeks 7.5 27 methylene chloride 26 weeks air 78 weeks 31b 40 methylene chloride 52 weeks air 52 weeks 63b 44b methylene chloride 78 weeks air 26 weeks 56b 62b methylene chloride 104 weeks 63b 69b air 26 weeks methylene chloride 78 weeks 19b 48b air 52 weeks methylene chloride 52 weeks 15 31 air 78 weeks methylene chloride 26 weeks 4 34 a exposure to methylene chloride was 7100 mg/m3, 6 h/day, 5 days/week b statistically significant p < 0.05 8.7.1.3 Hamster When groups of 95 male and 95 female Syrian golden hamsters (8 weeks old) were exposed by inhalation to 0, 1770, 5300 or 12 400 mg/m3 6 h/day, 5 days/week for 2 years, no exposure-related changes were observed in mean body weight and mean organ weights. CO-Hb levels were increased but this effect was not dose-related. The numbers of animals surviving to the end of the study were 16, 20, 11 and 14 for males and 0, 4, 10 and 9 for females, respectively. The incidence of lymphosarcomas was slightly higher in treated females than in controls (control, 1/91; low-dose, 6/92; mid-dose, 3/91; high- dose, 7/91; p = 0.032). The increased incidence of benign tumours was considered to be related to the higher survival of the exposed hamsters and not a direct result of exposure to methylene chloride (Burek et al., 1984). 8.7.2 Oral administration 8.7.2.1 Rat Groups of 85 male and 85 female F-344 rats (7 weeks old) were administered methylene chloride in the drinking-water at concentrations of 0, 5, 50, 125 and 250 mg/kg per day for 104 weeks (Table 22). Interim sacrifices were carried out at 26, 52 and 78 weeks, such that 50 males and 50 females per group received the treatment for 104 weeks. Additional groups of 50 male and 50 female F-344 rats received methylene chloride at a concentration of 250 mg/kg per day for 78 weeks, with further groups of 50 male or female rats serving as controls. Small changes in mean body weight and food/water consumption were seen in rats receiving either 125 or 250 mg/kg per day. Dose-related increases were noted in mean haematocrit, haemoglobin levels and red blood cell counts at the three highest doses. Decreases in serum alkaline phosphatase activity and in creatinine, blood urea nitrogen, serum protein and cholesterol levels in both sexes were found at 250 mg/kg per day. Treatment-related histopathological changes were seen in the liver of rats receiving 250 mg/kg per day. There were no increases in the incidence of any tumour in treated rats when compared to controls (Serota et al., 1986a). When groups of 50 male and 50 female Sprague-Dawley rats were given 0, 100 or 500 mg/kg methylene chloride (purity 99.97%) by gavage in olive oil, 4-5 days/week for 64 weeks, excess mortality was observed in rats of each sex receiving 500 mg/kg. A slightly higher incidence of adenocarcinomas of the mammary gland was observed in females receiving 500 mg/kg (4/50, 3/50, and 9/50 in the controls, low-dose and high-dose, respectively). There was no effect on the total tumour incidence in the exposed groups (Maltoni et al., 1988). The Task Group noted the short length of the exposure period as well as the absence of relevant data on the design of the experiment, such as the statistical methods applied, housing conditions, histopathological techniques and pathology schedule. Table 22. Carcinogenicity studies by oral route Species Strain/type Route of Doses Observations Reference administration/protocol/ group/size Rat Fischer-344, Drinking-water ad libitum for 0, 5, 50, 125 No increase in incidence of neoplasms; Serota et al. mate and female 104 weeks; 85 animals/sex or 250 mg/kg survival and other findings not affected (1986a) per dose; scheduled kills: 5 at per day by methylene chloride; significant 26 weeks, 10 at 52 weeks, 20 decreases in body weight gain at 125 at 78 weeks; also additional and 250 mg/kg per day and evidence of groups of controls and liver damage at doses above 50 mg/kg 250 mg/kg (50/sex) which per day received methylene chloride for 78 weeks only Rat Sprague-Dawley, Gavage in olive oil for 64 0, 100 or 500 No effect on total tumour incidence in Maltoni et male and female weeks; 50 animals/sex/dose; mg/kg per exposed rats. Higher incidence, not al. (1988) additional control group (not day statistically significant, of malignant dosed) of 20 males and 26 mammary tumours in high-dose females females; survival decreased in high dose males and females Mouse B6C3F1, male Drinking water ad libitum for 0, 60, 125, 185 No increase in incidence of neoplasms; Serota et al. and female 104 weeks; group size; or 250 mg/kg evidence of slight liver damage at 250 (1986b) 125 m, 100 f (controls) per day mg/kg per day 200 m, 100 f (60 mg/kg) 100 m, 50 f (125 mg/kg) 100 m, 50 f (185 mg/kg) 125 m, 50 f (250 mg/kg) Table 22 (Cont'd) Species Strain/type Route of Doses Observations Reference administration/protocol/ group/size Mouse Swiss, male and Gavage in olive oil for 64 0, 100 or Decrease in body weight in exposed Maltoni et female weeks 500 mg/kg males and females after 36-40 weeks; al, (1988) 50 animals/sex/dose per day dose-related increase in pulmonary 60 animals/sex/controls tumours in males - not significant without considering mortality rate; significant (p<0.05) taking into account the mortality rate; no treatment-related increase in the percentage of animals bearing benign and malignant tumours, or of animals bearing malignant tumours, or of the number of total malignant tumours per 100 animals 8.7.2.2 Mouse Groups of male and female B6C3F1 mice (7 weeks old) received methylene chloride in the drinking-water for 104 weeks at levels of 0 (control groups 60/65 males, 50/50 females), 60 (200 males, 100 females), 125 (100 males, 50 females), 185 (100 males, 50 females) or 250 (125 males, 50 females) mg/kg per day. Histopathological changes were observed in the liver of mice receiving 250 mg/kg per day. There was no increase in the incidence of tumours in any of the exposed groups, when compared to controls (Serota et al., 1986b). Groups of 50 male and 50 female Swiss mice received either 100 or 500 mg/kg methylene chloride (purity 99.97%) in olive oil by gavage on 4-5 days/week for 64 weeks. Groups of 60 males and 60 females served as controls and received only olive oil. In male mice dying between 52 and 78 weeks, an increase in the incidence of pulmonary adenomas was observed (0/27 in controls, 4/41 low dose level, 7/33 high dose level) although the effect was not statistically significant in exposed male mice when mortality was not taken into account. There was no increase in the total tumour burden in exposed mice. A decrease in body weight was observed in exposed males and females, compared to controls after weeks 36-40. No other exposure-related findings were reported (Maltoni et al., 1988) 8.7.3 Appraisal Methylene chloride is carcinogenic in the mouse, causing both lung and liver tumours, following exposure to high concentrations (7100 and 14 100 mg/m3 ). The incidence of both lung and liver tumours was increased in mice exposed to 7100 mg/m3 for 26 weeks and maintained for a further 78 weeks. Associated toxicity or hyperplasia in the target organs was not observed. Hamsters exposed to methylene chloride by inhalation at concentrations up to 12 400 mg/m3 for 2 years showed no evidence of a carcinogenic effect related to exposure to methylene chloride. Rats exposed to methylene chloride via various routes have shown increased incidences of tumours at certain sites. An excess of tumours in the region of the salivary gland was reported in male rats exposed to either 5300 or 12 400 mg/m3 for 2 years. This excess was only evident when the tumours, which were all of mesenchymal origin, were grouped together for statistical analysis. As the tumours arose from a variety of different tissues, the statistical approach of combining tumours was inappropriate. The response was not seen in a second study in which rats were exposed to either 3500, 7100 or 14 100 mg/m3 throughout their lifetime. A further inhalation study in rats exposed to methylene chloride at concentrations up to 1770 mg/m3 throughout their lifetime showed no evidence of carcinogenicity. These studies, taken together with the absence of effect on the salivary gland in all other inhalation studies, raise doubts regarding their biological and toxicological significance. Rats exposed to methylene chloride via the drinking-water or by gavage similarly showed no substantive evidence of carcinogenicity. An increase in either the incidence or the multiplicity of benign mammary tumours (fibroadenomas) in rats exposed to methylene chloride via inhalation has been reported in three studies. Increases in multiplicity were dose-related in male and female Sprague-Dawley rats (historical incidence 10% males and 80% females). The increase in multiplicity was observed only in females of the highest dose (1770 mg/m3 ) group. No effects were observed in males or the other dose groups of females. A dose-related increased incidence in benign mammary tumours was observed in female F-344/N rats, although the incidences were in the range of historical incidence (10 to 25%). An increased incidence in high dose (14 100 mg/m3 ) males was also within the historical incidence range of 0 to 11%. In one study, a slight increase in the incidence of adenocarcinomas in the mammary gland was observed in female Sprague- Dawley rats receiving 500 mg/kg by the oral route. A study in Fischer-344 rats with dose levels up to 250 mg/kg by the oral route (drinking-water) showed no carcinogenic effects. No increases in mammary tumours were observed in the mouse or hamster by inhalation or oral administration. 8.8 Mechanistic studies 8.8.1 In vitro metabolic studies There are three transient reactive intermediates in the metabolism of methylene chloride. Two of them, formyl chloride and S-chloromethyl-glutathione, are assumed to be present on the basis of knowledge of the metabolic pathways; the third, formaldehyde, has been identified in vitro. All three have the reactivity necessary to bind covalently to macromolecules. Of these S-chloromethyl- glutathione is potentially the most potent alkylating agent, a conclusion based on the known reactivity of the halothioethers (Bohme et al., 1949), structural similarities to the mutagenic glutathione conjugates of the 1,2-dihaloethanes, and on the outcome of several studies using different liver fractions in the Salmonella mutation assay (Jongen et al., 1982; Green, 1983). Formyl chloride is highly unstable, existing chemically only at low temperatures (-80°C) in inert solvents (Staab & Datta, 1964). Formaldehyde is a common metabolic product in vivo which is efficiently metabolized in the liver to formic acid. The endogenous formation and metabolism of formaldehyde occurs at a high rate, and the additional formaldehyde derived from methylene would be metabolized by the same efficient pathways. Comparisons of the rates of metabolism of methylene chloride by each pathway in liver fractions from rats, mice, hamsters and man have been carried out (Green et al., 1986b,c; Reitz et al., 1989). These experiments demonstrated that the rates of metabolism in these pathways in vitro had similar differences to those seen in vivo in rats and mice, and enabled a comparison to be made with those species (hamster and man) where in vivo data was not available. Rates of metabolism in human liver have been measured for both glutathione- S-transferase (Green et al., 1987a,b,c; Reitz et al., 1989; Bogaards et al., 1993) and cytochrome P-450 (Green et al., 1987a,b,c; Reitz et al., 1989) pathways of methylene chloride metabolism. A total of 33 human liver samples have been assayed for glutathione- S-transferase activity and a range of activities reported. In the work by Bogaards et al. (1993), 3 samples had no activity, a further group of 11 had activity in the range of 0.20-0.41 (mean 0.31 ± 0.08) nmol/min per mg protein, and 8 samples had activity in the range 0.82-1.23 (1.03 ± 0.14) nmol/min per mg protein. The rates measured by Green et al. (1987a,b,c) were within the range found by Bogaards et al., (1993) (0.05-0.93 nmol/min per mg protein; mean 0.42 ± 0.32; n = 7) and those found by Reitz et al. (1989) were slightly higher (range 0-3.03 nmol/min per mg; mean 2.09 ± 1.40). The activity in all the samples assayed was at least 1.4 lower than that in rat liver cytosol. Two human lung samples were assayed and found to be lacking in glutathione- S-transferase activity (Reitz et al., 1989). However, enzyme activities of this type are significantly lower in the lung than the liver, and any such activity may not be detected by the currently available assays. The 10-fold difference in glutathione- S-transferase activity measured in vivo in mice and rats was also found in vitro. There is an excellent correlation between glutathione- S-transferase metabolism and the outcome of the 2-year cancer studies in the three animal species. More support for this correlation was obtained in DNA damage tests (section 8.6.2.3.) No such correlation exists for the cytochrome P-450 pathway where, for example, the metabolic rate in the hamster is very similar to that in the mouse. Cytochrome P-450- catalysed metabolism of methylene chloride could be detected in lung tissue from all three animal species, the relative activities being similar to those in the livers. Glutathione- S-transferase activity was detectable only in mouse lung fractions. The low rates of metabolism of methylene chloride by the glutathione- S-transferase pathway in human liver samples has been attributed to a deficiency in the transferase isoenzyme responsible. The same liver samples had similar activity to rat liver when assayed with an alternative substrate for these enzymes (Green et al., 1986b,c). 8.8.2 In vivo metabolic studies A comparative kinetic profile of methylene chloride and its metabolites was determined in B6C3F1 mice and F-344 rats both during and after a 6-h exposure to atmospheres containing various concentrations from 350 to 14 100 mg/m3 (Green et al., 1986b, 1987a,b,c, 1988). Blood levels of methylene chloride and CO-Hb and the rates of elimination of methylene chloride, carbon monoxide and carbon dioxide in exhaled air were measured. Stable isotopes were used to quantify the amount of carbon dioxide from each pathway at dose levels of 350, 1770 and 14 100 mg/m3, but only in the mouse. The steady- state blood levels of methylene chloride during exposure were up to 5 times higher in rats than in mice at the higher dose levels. A comparison of the CO-Hb levels in blood and carbon monoxide levels in expired air showed that rate of metabolism by the cytochrome P-450 pathway was similar in both rats and mice. The pathway was saturated in both species at exposures of less that 1770 mg/m3, resulting in maximal CO-Hb levels of 16% (Green et al., 1987a,b,c). Saturation of the cytochrome P-450 pathway in mice was also clearly shown by a 5-10 fold increase in the blood levels of methylene chloride when the inhaled concentration was doubled from 1770 to 3500 mg/m3 (Green et al., 1987a,b,c). The stable isotope studies demonstrated that the cytochrome P-450 pathway was the major source of carbon dioxide at low exposure levels (350 mg/m3) whereas at high levels ( 14 100 mg/m3) the glutathione- S-transferase pathway was the principal source of carbon dioxide. A comparison of the rate of elimination of the carbon dioxide by rats and mice at the top dose level showed the glutathione- S- transferase pathway to be 10-12 times more active in mice than rats. The higher rate of metabolic conversion of methylene chloride by mice when compared to rats largely accounts for the low blood levels of the parent chemical in this species. In summary, the in vitro and in vivo studies have provided evidence for the following: 1. The cytochrome P-450 pathway is saturated at 1770 mg/m3 and is quantitatively similar in rats and mice in vivo and in rat, mouse, hamster and human livers in vitro. 2. The glutathione- S-transferase pathway is a major pathway only in mice, its activity at the 14 100 mg/m3 dose level being an order of magnitude greater than in rats. 3. In all, 33 human liver samples have been assayed for glutathione- S-transferase activity. In all cases the levels of activity were lower than those measured in rat liver. 4. Methylene chloride metabolism is dose-dependent. The utilization of the two pathways is significantly different at the dose levels used in the carcinogenicity studies than at low dose levels. 5. These studies provided the metabolic rate constants used in the physiologically based pharmacokinetic models described in section 8.9. 8.8.3 Pulmonary effects A number of studies have examined the effects of methylene chloride on the mouse and rat lung (Eisenbrandt & Reitz, 1986; Hext et al., 1986; Green et al., 1987a, Foster et al., 1992; Kanno et al., 1993). Following a single exposure at concentrations of 7100 mg/m3 or more, a specific lesion characterized by marked vacuolization of Clara cells were seen in the mouse, but not the rat. No other cell types were affected in the mouse (Green et al., 1987a; Foster et al., 1992). The morphological damage in Clara cells recovered after 5 days of repeated 6-h exposures (Foster et al., 1992). The damage to Clara cells was accompanied by a change in the ability to metabolize methylene chloride by the two pathways. Cytochrome P-450 metabolism was suppressed while glutathione- S-transferase remained unchanged. In a thirteen week study (Foster et al., 1992), damage to the Clara cells was seen following the first exposure of each week of the study. Between days 2 and 9 of this study, a significant increase in the number of cells in S-phase was observed in both bronchiolar and alveolar epithelium. A similar study (Kanno et al., 1993) failed to detect an increase in the number of cells in S-phase. Significant pulmonary lesions were observed in male B6C3F1 mice 1 day after a single 6-h exposure to 14 100 mg/m3 (Eisenbrandt & Reitz, 1986). Necrosis of the epithelial cells in the bronchi and bronchioles were observed. Non-ciliated (Clara) cells were swollen and vacuolated. 8.8.4 Studies on oncogene activation Further studies have been conducted on the B6C3F1 mouse into the role of oncogene activation as a potential mechanism of action of the carcinogenic effect of methylene chloride. A group of 145 female B6C3F1 mice was exposed to 7100 mg/m3 for 6 h/day, 5 days/week for up to 27 months. Another group of 235 females acted as controls. These mice were used for the purpose of providing spontaneous and methylene- chloride-induced tumour tissue from both the liver and the lung for the analysis of proto-oncogene activation and tumour suppressor gene inactivation. The DNA recovered from 54 methylene-chloride-induced B6C3F1 lung tumours and from 7 spontaneous B6C3F1 lung tumours was analysed by the direct sequencing of PCR (polymerase chain reaction) amplified DNA fragments of the K-ras gene for first and second exon mutations. Twelve mutations were identified in the tumours from exposed mice, 5 in exon one and 7 in exon two. There was no difference in the frequency of K-ras activation in tumour tissue derived from both exposed mice when compared to controls. DNA was isolated from 49 spontaneous and 50 methylene-chloride-induced liver tumours and screened by the oligonucleotide hybridization of PCR (polymerase chain reaction) amplified H-ras gene fragments for codon 61 mutations. The mutation profile of the H-ras gene was similar in the tumour tissue derived from both control and treated mice (Devereux et al., 1993). Mutations of the p53 rumour suppresser gene were examined in lung tumours from female mice exposed to 7100 mg/m3 (2000 ppm) methylene chloride for 2 years (Hegi et al., 1993). The limited number of p53 mutations identified in this study and the small number of spontaneous tumours precluded any conclusions concerning the mutagenic spectrum or possible genotoxicity of methylene chloride. 8.8.5 The use of mechanistic studies in extrapolation Studies using liver fractions from rats, mice, hamsters and humans have confirmed the existence of two pathways for the metabolism of methylene chloride (the cytochrome P-450 pathway and the glutathione- S-transferase pathway) and have established substantial differences between species in the utilization of these pathways. The rates of metabolism of methylene chloride by the two enzymes in the liver fractions from a few species have been established. The activities of the cytochrome P-450 pathway in the mouse and hamster were similar, whereas those in the rat and human were lower. In marked contrast, the activity of the glutathione- S-transferase in the mouse was very high when compared with the other species; there being a 10-fold difference in activity between the mouse and the rat. Rates of metabolism by this pathway in hamsters and humans were even lower than in rats. The results of a full pharmacokinetic analysis of the behaviour of methylene chloride and its metabolites in vivo were consistent with the species differences observed in vitro. Saturation of the cytochrome P-450 pathway occurred in rats and mice at dose levels of less than 1770 mg/m3 and resulted in maximal CO-Hb levels of 16% in both species. Comparisons of the glutathion- S-transferase pathway based on expired carbon dioxide levels at high exposure concentrations found the same 10- to 12-fold difference between mice and rats that had been observed in vitro. The higher metabolic rates in mice accounted for the lower blood levels seen in this species compared to the rat. Exposure of mice to atmospheric concentrations of 7100 mg/m3 or more led to recurrent cytotoxicity, increases in DNA synthesis ( S-phase) and changes in the metabolic complement of mouse lung Clara cells. All the effects were specific to the mouse and to the Clara cells. Several of the changes (cytotoxicity and S-phase) were frequently associated with the development of tumours. However the significance of these findings with respect to the development of lung tumours in mice exposed to methylene chloride remains to be established. Effects on chromosomes have also been reported in the mouse lung. Studies on the potential role of activation of Ras oncogenes in the development of methylene-chloride-induced lung and liver tumours have been unable to distinguish between the tumours seen in methylene- chloride-treated animals and those occurring spontaneously in control animals. Studies in which mice were exposed for different intervals of a 2-year carcinogenicity study established that lung tumours developed quicker and after shorter exposures than liver tumours. Whether this indicates a different mechanism in the lung from the liver or reflects the cytotoxicity and cell division seen in the lungs of mice is unknown at the present time. The more recent studies in bacteria using glutathione-deficient strains and strains in which mammalian transferase enzymes have been expressed have established the role of this pathway in mutagenesis. Consistent with this are findings of DNA single strand breaks and DNA- protein cross-links in the livers of mice, but not rats or hamsters, exposed in vivo to 14 000 mg/m3. Both the single strand beaks and cross-links have been shown to be derived from metabolites of the glutathione- S-transferase pathway. At the present time, these effects have not been demonstrated in mouse lung. There is a consistency between the bacterial mutagenicity assays, the pharmacokinetic data and the studies of DNA single strand breaks and DNA protein cross-links which leads to the conclusion that the liver tumours seen in mice are derived from an interaction between metabolites of the glutathione- S-transferase pathway and DNA. The same level of detail is not available for the mouse lung. However the responses seen in the Clara cells and in mouse lung chromosomes are not inconsistent with this mechanism. The studies of the comparative metabolism and pharmaco-kinetics of methylene chloride in the rat, mouse and hamster also provided a plausible explanation for the species differences in the carcino- genicity of this chemical. The differing metabolic rates by the glutathione- S-transferase pathway are consistent with the outcome of the cancer studies whereas the blood levels of the parent chemical and the metabolic rates by the cytochrome P-450 pathway are not. These results are also consistent with the different responses seen in the three mouse cancer bioassays (NTP, 1986; Serota et al. 1986b; Maltoni et al., 1986). At the high dose levels used in the NTP study, the glutathione- S-transferase pathway would have been the major metabolic pathway and high tumour incidences were observed. At the lower dose levels used by Serota et al. (1986b) and Maltoni et al. (1986), methylene chloride would have been metabolized mainly by cytochrome P-450, and glutathione- S-transferase metabolism would have been minimal. Consequently there were no significant increases in either lung or liver tumours in these studies. 8.8.6 Mammary tumour promotion The dependence of mammary tumours upon pituitary hormones in both male and female rats has been established unequivocally (Welsch & Nagasawa, 1977; Welsch 1985). In the rat, prolactin acts as a promoter of mammary carcinogenesis. There is good evidence that increased prolactin levels increase the incidence of mammary tumours (Welsch et al., 1970), and there is a positive correlation between elevated blood prolactin levels and mammary rumours in aged R-Amsterdam female rats (Kwa et al., 1974). The mechanism by which methylene chloride induces mammary adenomas in the rat is important for human hazard assessment. Female Sprague- Dawley rats receiving methylene chloride have a high blood level of prolactin (Breslin & Landry, 1986). When male and female Sprague- Dawley rats were exposed to 10 600 mg/m3 (3000 ppm) methylene chloride for 15 to 19 consecutive days, a significant increase (2.3 x) in basal serum prolactin levels was observed in female rats. No significant effect was observed in male rats (Breslin & Landry, 1986). In humans, there is conflicting evidence on whether or not mammary rumours are as responsive to prolactin as in the case of rats (Sinha, 1981). The rat has elevated levels of prolactin when fed ad libitum in comparison to a restricted dietary regimen and this may explain why the mammary tumour incidence is so easily responsive to a variety of environmental and other effects. In the rat, however, prolactin is luteotrophic. An increase in the circulating levels of prolactin will lead to an increase in progesterone and exogenous oestrogen levels. The presence of all three factors that causes tubular-alveolar growth of the mammary glands may ultimately lead to tumour development. Prolactin is not luteotrophic in primates (Neumann, 1991). The mechanism of production of mammary tumours in the rat involving hyperprolactinaemia will probably occur only at doses of methylene chloride which affect prolactin levels. There is no direct information on prolactin levels in rats receiving low doses of methylene chloride, but no increase in mammary adenomas has been observed following the administration of low doses in either inhalation or drinking-water studies (i.e., below 250 mg/kg body weight or 1770 mg/m3). 8.8.7 Appraisal In vitro and in vivo metabolism and biochemical studies and mutagenicity assays in bacteria and B6C3F1 mice have provided a plausible explanation for the mechanism of action and the species differences in the carcinogenicity of methylene chloride to the lung and liver. This explanation is based on the existence of an isoenzyme of glutathione-S- transferase which specifically metabolizes methylene chloride to the reactive intermediates responsible for tumour induction in the mouse. Markedly lower levels of this enzyme in rats and hamsters are consistent with the fact that these tumours do not appear in these species. The levels of the enzyme are lower in human liver than those of the rat or hamster. Mutagenicity studies on methylene chloride in bacteria and in the B6C3F1 mouse, which shows a very high level of activity of the isoenzyme, reveal positive effects, whereas mutagenicity has not been demonstrated in standard in vivo mutagenicity assays using other systems. These observations are consistent with the above hypothesis and provide a mechanistic basis for the induction of tumours in the mouse. The role of the glutathione-S- transferase isoenzyme in the mediation of the demonstrated mutagenic effects, and the correlation between the activity of this pathway and the species differences in carcinogenic response in lung and liver, has led to its use as the dose surrogate in physiologically based pharmacokinetic models used for human health risk assessment. The pharmacokinetics of methylene chloride and the response seen in B6C3F1 mice suggest that this species is a poor model on which to base human hazard assessment to methylene chloride. The mechanism of mammary tumour formation in the rat is probably related to the effect of methylene chloride on prolactin levels in this species. 8.9 Interspecies and dose extrapolations by kinetic modelling Two physiologically based pharmacokinetic (PB-PK) models (Andersen et al., 1987; ECETOC, 1988) have been developed and provide quantitative estimates of the levels of methylene chloride metabolites in four mammalian species (mice, rats, hamsters and humans) following inhalation exposure. The models use information on various physiological parameters and metabolic constants for the cytochrome P-450 and glutathione- S-transferase pathways in the lung and liver, measured in vitro for four species and measured in vivo for the mouse, with values for rats, hamsters and humans scaled from the mouse data. The metabolic constants for these models were obtained from the data described in section 8.8 and from gas uptake studies described by Andersen et al. (1987). The models were validated against other human experimental data such as the elimination of carbon monoxide and blood levels of methylene chloride. Time-course concentration data from the model were compared to experimental results in F-344 rats, Syrian Golden hamsters, B6C3F1 mice and human volunteers. The predicted values for each of the four species were in agreement with the experimental data. The models were also shown to predict the appearance and elimination of methylene chloride metabolites. A similar model was used by Andersen et al. (1991) to predict the time course of the disappearance of CO-Hb after exposure to methylene chloride. This model was also shown to predict CO-Hb levels in rats and humans exposed to methylene chloride. Several authors have discussed the assumptions, range and variability of the data used to construct these models. While these kinetic models have been extremely useful in improving the characterization of human exposure and potential risk, it should be recognized that they are based on a set of assumptions with varying degrees of certainty. Portier & Kaplan (1989) investigated the impact of varying the intra-population values of the biological parameters used in the model developed by Andersen et al. (1987) using Monte Carlo and resampling statistical methods. The results from this analysis indicated that the estimates of "effective" doses in humans may vary widely if variability of the parameters is taken into account in the PBPK model. Dankovic & Baiter (1993) investigated the impact of exercise and human inter-subject variability on the estimates of dose derived from the PBPK model. The model developed by Andersen et al. (1987) and Reitz et al. (1989) assumed resting values for the parameters governing cardiac output, alveolar ventilation and blood flow to the tissues. The authors modified these parameters to reflect light working conditions. The metabolic parameters for humans used in the Andersen et al. (1987) model were based on the average of four individual liver samples. The authors examined the impact of using the individual values rather than the average value in the PBPK model. Modifying the physiological parameters to reflect light work conditions increased the glutathion- S-transferase pathway metabolic contribution by a factor of 2.9 for the liver and 2.4 for the lung. When the model was also modified to reflect metabolic inter-individual (n = 4) variability in humans, the glutathione- S-transferase pathway contribution estimates were increased by as much as 5.4-fold for the liver and 3.6-fold for the lung. More recent data have become available on human metabolic parameters, and are summarized in section 8.8.1. Based on 33 individual liver samples, they suggest that the Portier & Kaplan estimates which assume 200% variation for metabolic variability may be exaggerated. Since Dankovik & Bailer's calculations were based on the four actual values, their estimates would not change; however, three of their values represent the three highest values observed for human glutathione- S-transferase activity. 9. EFFECTS ON HUMANS 9.1 General population exposure 9.1.1 Environmental exposure Bell et al. (1991) conducted a study to examine the relationship between birth weight in Monroe County and exposure to emissions of methylene chloride from manufacturing processes of the Eastman Kodak Company in Rochester, New York, USA. County census tracts were categorized as high, moderate, low or no methylene chloride exposure, based on the Kodak Air Monitoring Program. Birth weight and information on variables known to influence birth weight were obtained from 91 302 birth certificates of white, single births to Monroe County residents from 1976 to 1987. At the level of methylene chloride exposure (highest predicted average concentration, 50 µg/m3), no significant adverse effect of exposure on birth weight was found, although several problems in the method of estimation of exposure were identified. 9.1.2 Oral exposure A 56-year-old woman was found deeply unconscious and cyanosed after ingesting approximately 300 ml of a paint remover containing mainly methylene chloride and methanol. Approximately one hour after ingestion the CO-Hb level measured was found to be 9%. This level varied between 2.5-12% over the following 2 days and dropped below 1% thereafter. The woman regained consciousness after 14 h, but over the following 3 weeks her condition was complicated by progressive renal failure, pneumonia, pancreatitis, on-going gastrointestinal haemorrhage and sepsis, which eventually led to death some 25 days following ingestion. It was considered that the corrosive properties of the formulation rather than the formation of CO-Hb were responsible for the lethal outcome (Hughes & Tracey, 1993). In an earlier poisoning case with the same paint remover formulation, there was recovery after ingestion of 0.5-1 litre (Roberts & Marshall, 1976). 9.2 Occupational exposure 9.2.1 Short-term exposure 9.2.1.1 Case studies A number of case-reports have been published regarding short-term exposure to methylene chloride in the occupational environment. Hall & Rumack (1990) described four cases of serious methylene chloride poisoning, including two fatalities, in small-scale furniture-stripping shops in Denver, Colorado, USA. In the three patients discovered while still alive, cardiac irregularities were recorded. Corneal burns with first- and/or second-degree burns were reported in areas having direct contact with the methylene chloride- based paint-stripping compound, and measured CO-Hb levels did not exceed 8.6%. In each case, no respiratory protection was worn and ventilation was inadequate, but exposure levels were not known. The authors concluded that the toxic effects were due to the anaesthetic properties of methylene chloride. A 67-year-old male who had been using a paint stripper in a poorly ventilated location was brought to a hospital emergency room complaining of headache and chest pain; he was also confused, disorientated, had a progressive loss of mental alertness, increased fatigue and lethargy, slurred speech, little recall of either recent or past events, and was disorientated to time (ATSDR, 1993). These and other case studies of methylene chloride poisoning during paint-stripping operations have demonstrated that inhalation can be fatal to humans (Hall & Rumack, 1990; Novak & Hain, 1990; Leikin et al., 1990; Manno et al., 1992). In the majority of cases reported, quantitative estimates of exposure levels have not been reported, although methylene chloride was detected in various tissues. In one case (Manno et al., 1989), air samples collected a few hours later from a well in which two men were found dead, were analysed by gas chromatography/mass spectrometry and were found to contain up to 583 mg methylene chloride/litre and much lower or trace amounts of other solvents; blood levels collected at necropsy contained 571 and 601 mg methylene chloride/litre and only trace to a few mg/litre of other solvents. The CO-Hb levels were about 30% in blood taken 24 h after death. In most cases, the cause of death was not clarified. However, in the report of five victims, including two deaths, described by Leikin et al. (1990), the authors concluded that the cause of death was due to solvent-induced narcosis and not carbon monoxide poisoning. Signs of CNS depression, narcosis, irritation of the eyes and respiratory tract, lung oedema and sometimes death were found after accidental exposures to methylene chloride or paint remover containing this compound (Moskowitz & Shapiro, 1952; Hughes, 1954; Bonventre et al., 1977; Fagin et al., 1980). Three myocardial infarctions in one subject were reported to have followed three exposures to a paint remover containing methylene chloride over a period of approximately 8 months. The subject was exposed in a poorly ventilated room, and concentrations were up to 4511 mg/m3 in the breathing zones (Stewart et al., 1976). Electrocardiographic changes resembling those after carbon monoxide poisoning were found in an exposed man with a history suggesting ischaemic heart disease (Benzon et al., 1978). Three probable cases of phosgene poisoning occurred after the use of methylene-chloride-based paint remover near a source of heat (Gerritsen & Buschmann, 1960; English, 1964). A serious case of pulmonary oedema with bilateral exudative pleural effusions was reported in a 34-year-old man who presented with respiratory distress. Buie et al. (1986) speculated that hydrochloric acid, a product of dichloromethane under warm, moist conditions, may have played a role in this patient's parenchymal abnormalities. Recovery was complete with the exception of neuropsychiatric abnormalities thought to be related to exposure to methylene chloride. Miller et al. (1985) reported the case of a 19-year-old man using a tile remover, again in a poorly ventilated room. This patient presented with an array of signs and symptoms ranging from liver enzyme elevations to poorly localized abdominal pain. Renal studies and biopsy confirmed the diagnosis of acute tubular necrosis. Histological studies demonstrated plasma membrane changes in addition to mitochondrial effects suggestive of anoxic damage. Serum enzyme changes noted during the patient's stay in hospital suggested that hepatocellular injury accompanied the nephrotoxic sequelae. Another case of chemically induced hepatitis resulting from accidental exposure to methylene chloride alone has been described by Cordes et al. (1988). The liver was palpable but not enlarged or tender. The results of initial tests were normal except for a leukocyte count of 4900 µl with a left shift, and elevated serum enzyme levels of alkaline phosphatase 142, lactic dehydrogenase and serum aspartate aminotransferase (ALAT). Five days after admission, the patient was discharged from hospital. Laboratory tests for hepatitis A and B antibodies were negative. Further evidence for hepatic effects of methylene chloride were reported by Puurunen & Sotaniemi (1985). One week after a brief but extensive body exposure to methylene chloride, serum ALAT was elevated three-fold in a 24-year-old male chemical factory worker. The serum ALAT returned to normal within 2 weeks. 9.2.1.2 Skin and eye effects The irritating action of methylene chloride on the eyes and skin has been shown in several cases (see section 9.2.1.1). Slight erythema was found when methylene-chloride-containing aerosol-spray deodorants were used twice a day for 12 weeks by 75 men and women (Meltzer et al., 1977). On direct contact, methylene chloride caused a burning sensation and pain (Stewart & Dodd, 1964). Weber et al. (1990) reported a case of an individual who fell into a vat containing methylene chloride and methanol. After being immersed for about 15 min, the subject suffered extensive lesions, including skin burns of superficial and deep severe epidermal damage and a severe kerato-conjunctivitis. Wells & Waldron (1984) briefly reported on a young employee who climbed into a small open vessel with a bucket of about two litres of methylene chloride in order to clean the walls. The concentration of methylene chloride vapour within the vessel built up and he became unconscious, overturning the bucket as he slumped into the bottom of the vessel. After about 30 min, the man was rescued. During the time that he was in the vessel, the man sustained second and third degree burns to both legs, the areas affected being those which were bearing the weight of his body while he was unconscious. On his discharge from hospital, these areas were dry and required no skin grafting (Wells & Waldron, 1984). 9.2.1.3 Laboratory studies Neurobehavioural changes were observed at low exposure level after volunteers were exposed to 694 mg/m3 for 1.5-3 h. Vigilance disturbance and impaired combined tracking monitoring performance were found (Putz et al., 1976). The critical flicker frequency, one of the measures for visual function, was reduced after 95 min of exposure to 1040 mg/m3 (Fodor & Winneke, 1971). Visually evoked responses (one of the surrogate methods of measuring visual functions) were altered after 1 h of exposure to 2400 mg/m3, while exposed subjects experienced lightheadedness. Blood and urine variables, except CO-Hb levels, were normal in this study after 1-2 h of exposure to levels of methylene chloride between 739 and 3420 mg/m3. No eye, nose, or throat irritation was observed (Stewart et al., 1972). Most neurobehavioural effects observed were less pronounced or absent, with carbon monoxide exposures resulting in comparable CO-Hb levels (Putz et al., 1976). In a double-blind laboratory experiment, a short inhalation exposure to 2.5 mg methylene chloride/litre did not impair vigilance performance in human volunteers (time of exposure and number of subjects not stated) (Kozena et al., 1990). A clinical laboratory evaluation of 266 exposed volunteer workers and 251 reference volunteer workers from two cellulose di- and tri-acetate plants in the USA, which took into account smoking habits, race, sex, age, intensity of exposure, and time of venepuncture, revealed increases in red cell counts, haemoglobin levels and haematocrit among white women exposed to a methylene chloride level of approximately 1650 mg/m3. CO-Hb levels were elevated in all exposed groups at all exposure levels (section 5.3). A dose-related increase was observed in serum bilirubin for exposed subjects of both sexes. A group of 24 exposed male volunteers and 26 reference male volunteers from the above two industries was also selected for 24-h electro- cardiographic monitoring. Three exposed and 8 reference workers had reported a history of heart disease. Neither increased ventricular or supraventricular ectopic activity nor increased episodic ST-segment depression was found to be associated with methylene chloride exposure (Ott et al., 1983). 9.2.2 Long-term exposure 9.2.2.1 Case studies Irreversible damage to the central nervous system with acoustic and optical illusions and hallucinations was diagnosed in one man who had been exposed for 5 years to methylene chloride at levels ranging from 2290 to 12 500 mg/m3 (Weiss, 1967). Another man, exposed for 3 years to levels of methylene chloride ranging from 1735 to 3470 mg/m3 showed a bilateral temporal lobe degeneration (Barrowcliff & Knell, 1979). A case of delirium and seizures was reported in a man who was exposed to methylene chloride for 4 years. The man reported a 12-month history of intermittent headache, nausea, blurred vision, shortness of breath, and transient memory disturbances. Neuropsychological and EEG examinations revealed a dysfunction of the right hemisphere. All symptoms and signs cleared with removal from the workplace (Tariot, 1983). Between December 1984 and June 1986, 34 men with occupational exposure to methylene chloride were evaluated at the Greater Cincinnati Occupational Health Centre. The mean value of the exposure was reported to be 240 mg/m3, ranging from 11 to 544 mg/m3. Although the primary complaint of these employees involved problems associated with central nervous dysfunction, 8 of the 34 complained of testicular, epididymal or lower abdominal pain, and had clinical histories relating to infertility. Low sperm counts were reported in workers who used methylene chloride in bonding operations which also resulted in possible skin exposure. It is uncertain whether the effect was due to methylene chloride since the workers were also exposed to other chemicals (Kelly, 1988). a) Morbidity studies The few reports available deal with small groups of occupationally exposed subjects. Workers exposed occupationally to a time-weighted average of 114 mg/m3 had CO-Hb levels of between 0.8 and 2.5%. No effects were found on clinical chemistry, haematology or electrocardio-grams (Di Vincenzo & Kaplan, 1981a). Cherry et al. (1981) did not find any exposure-related, long-term damage in 29 subjects as shown by subjective symptoms, neurobehavioural tests, motor nerve conduction velocity, electrocardiograms and clinical examinations. The men had been exposed for several years to levels of methylene chloride ranging from 260 to 347 mg/m3. Age-matched controls were used. In a study without a control group, neurasthenic disorders and irritation of the eyes and respiratory passages were experienced by half of the 33 workers exposed to methylene chloride for an average of 2 years. Digestive disorders were reported by one-third of these workers. Formic acid was found in the urine. No other deviations were found during the internal, nervous system, eye and laboratory examinations. The methylene chloride concentrations measured varied between 100 and 17 000 mg/m3 (Kuzelova & Vlasak, 1966). A group of 1758 retired airline maintenance workers was surveyed by mail and telephone to identify a cohort of workers with more than 22 years of methylene chloride exposure following the stripping of paint from airplanes. A cohort of 25 exposed and 21 non-exposed retirees met the criteria and were tested extensively (Becker & Lash, 1990; Lash et al., 1991). Following a specially prepared battery of neuropsychological and neurophysio-logical tests performed by professionals without prior knowledge of exposure status of the employees, exposed and control outcome measures were all within the "normal" range. No statistically significant difference was found between exposed and control groups, although subtle differences in attention and memory were detected. In 46 subjects exposed to methylene chloride concentrations of 6-34 mg/m3 for several years, an excess (not significant) of digestive disorders and hypotonia was found over controls, while symptoms of gall bladder pathology and swollen liver were frequent. No details were given concerning drinking or smoking habits (Kashin et al., 1980). A case-control study on 44 women who had a spontaneous abortion was performed within a cohort of female workers employed in Finnish pharmaceutical factories during 1973 or 1975 to 1980. Three controls matched for age at conception within 2.5 years were chosen for every case except two. Information about pregnancy outcome was collected from hospital data, and data on exposures from health personnel at the factories. The odds ratio for methylene chloride exposure, based on 11 exposed cases, was of borderline significance (2.3 with a 95% confidence interval, 1.0-5.7; p = 0.06). Odds ratios were also increased for exposures to many other solvents. For those exposed to methylene chloride less than once a week the odds ratio was 2.0 (95% CI = 0.6-6.6); whereas for those exposed more than once a week the odds ratio was 2.8 (95% CI = 0.8-9.5) (Taskinen et al., 1986). A group of active workers (n = 150) who had worked for at least 10 years in an area where average exposures were 1677 mg/m3 were compared to an unexposed group of workers (n = 260) with regards to symptoms and blood chemistry. The methylene chloride workers were also exposed to acetone and methanol (900 ppm and 100 ppm 8-h TWAs, respectively). Health history and blood samples had been collected as part of a company-sponsored health monitoring programme in which both exposed and unexposed workers were participants. No remarkable or statistically significant differences were observed in the selected symptoms (including irregular heartbeat, dizziness or loss of memory) or in SGPT, bilirubin or haematocrit. The only noticeable difference was in SGOT, where the non-exposed group had higher levels than the exposed group (means of 28.2 versus 25.1, p = 0.06). A limitation to this study is that both groups consisted of active healthy workers. The age and sex distribution of the two groups was reported to be similar, but was not given Soden, 1993). b) Mortality studies Several studies have evaluated the effects of long-term exposure to methylene chloride on mortality of workers. The first study was by Friedlander et al. (1978), who performed both a retrospective cohort mortality, i.e. standardized mortality ratio (SMR) study, and a proportionate mortality ratio (PMR) study of men exposed to methylene chloride at a Kodak photographic film production facility in Rochester, New York. The PMR study included 334 deaths that occurred between 1956 and 1976 among former workers who were exposed to methylene chloride at the facility. The retrospective cohort mortality study included 751 workers employed during 1964 and involved follow-up of this cohort up to 1976. Hearne & Friedlander (1981) extended the follow-up of this cohort to 1980 and subsequently expanded the study population to include all workers (n = 1013) who were exposed for at least one year between 1964 and 1970 (Hearne et al., 1987). In a more recent publication Hearne et al. (1990) extended follow-up of this expanded cohort to 1988. The Friedlander et al. (1978) study was initially conducted to test the hypothesis that exposure to methylene chloride increases the risk of ischaemic heart disease. This hypothesis was based on the fact that methylene chloride is metabolized to carbon monoxide and induces the formation of CO-Hb in humans (Stewart et al., 1972). Increases in CO-Hb as low as 2% (Allred et al., 1989) have been shown to induce electrocardiographic changes in exercising patients with pre-existing coronary artery disease. An excess of ischaemic heart disease mortality has also been reported in a cohort of tunnel workers exposed to carbon monoxide (Stern et al., 1988). Liver and lung cancer were also considered a priori hypotheses in the subsequent articles by Hearne et al., based on the results from the animal bioassay data described in chapter 8. Comparisons in both studies (Friedlander et al., 1978 and Hearne et al., 1990) were made with mortality rates (or proportions) from New York State and from an internal unexposed cohort from the Kodak facility. Extensive industrial hygiene measurements were available for this cohort from after 1980, which indicated that 8-h TWA exposure concentrations for different occupational classifications ranged from approximately 35.3-402 mg/m3 (10-114 ppm) and the mean exposure was 91.8 mg/m3 (26 ppm) (Hearne et al., 1987). An exposure-response analysis, which was presented by Hearne et al. (1987), failed to demonstrate an increasing risk for these causes of death with increasing methylene chloride exposure. Hearne et al. (1987) observed an excess of pancreatic cancer mortality (8 observed, SMR = 2.58, 95% confidence interval (CI) = 1.11-5.08). Mirer et al. (1988) published a letter suggesting that the excess of pancreatic cancer mortality increased with time since first exposure (latency) and was greatest among workers in the highest exposure (750 ppm-years) and latency (> 30 years) categories (4 observed, SMR = 4.49, 95% CI = 1.22-11.49). No new pancreatic cancer cases were identified with additional follow-up, and with the additional data the excess was not statistically significant (SMR = 1.90, 95% CI = 0.82-3.75) (Hearne et al., 1990). The study by Friedlander et al. (1978) and the subsequent studies by Hearne et al. (1987,1990) failed to detect a significantly increased risk of ischaemic heart disease, lung cancer, liver cancer or other cancers among methylene-chloride-exposed workers. It also important to recognize that workers at the Kodak facility (T. Hearne, personal communication to the IPCS) were not permitted to smoke at their workstations and that this fact may have induced a negative bias in these studies, particularly with respect to lung cancer or cardiovascular disease. Unfortunately detailed information on cigarette smoking was not available for this cohort and thus adjustments for this potential bias could not be made. Ott et al. (1983) evaluated a cohort of workers exposed to methylene chloride in the production of triacetate fibre at a manufacturing plant in Rock Hill, South Carolina, USA. This cohort included 1271 males and female workers who were employed for at least 3 months, sometime between 1954 and 1977, Workers from another textile facility that were not exposed to methylene chloride, but met the same inclusion criteria as the exposed cohort, were also included for comparison purposes. All workers (both exposed and unexposed) were followed for vital status ascertainment up to June 1977. Eight-hour TWA methylene chloride exposures in this cohort were estimated to range from 494 to 1677 mg/m3 (140 to 475 ppm) from a survey conducted in 1977 and 1978. Workers in this study were also reported to have been exposed to methanol and acetone. The mortality experience of the exposed cohort was compared with the mortality of the USA population using a modified life-table approach (SMRs). Direct comparisons were also made between the mortality of the exposed and unexposed cohorts. Mortality from cardiovascular disease or any other cause was not found to be significantly increased relative to the USA population. However, the authors did observe a significant increase in the risk of ischaemic heart disease (RR = 3.1, p < 0.05) among white men in the analysis when the mortality rates of the exposed and unexposed cohorts were compared. It was also noted that 8 of the 14 ischaemic heart disease deaths among exposed white men occurred among workers who were actively employed. Although Ott et al. (1983) did not report any increase in cancer mortality, this study was not designed to evaluate cancer and only included seven malignant neoplasms. The follow-up of the exposed cohort (but not the unexposed one) studied by Ott et al. (1983) was subsequently extended to 1986 by Lanes et al. (1990). The analyses presented in this paper were solely based upon comparisons with the USA population and did not include any direct comparisons with the unexposed cohort as did the study by Ott et al. (1983). This study failed to detect an excess of cardiovascular or ischaemic heart disease. A significant excess of cancers of the biliary passages and liver (SMR = 5.75, 95% CI = 1.82-13.78) was observed. Three of the cancers were cholangiocarcinomas of various biliary sites while the fourth was a liver adenocarcinoma. The SMR for biliary cancer was estimated using mortality rates from 1973 to 1977. The SMR for biliary cancer alone was 20 (95% CI = 5.2-56). Three of the four liver and biliary cancer deaths observed in this study were thought to have occurred among workers with 10 or more years of employment and at least 20 years since first employment (0.35 expected, SMR = 11.43), a pattern consistent with a potential occupational etiology. One of the cases had only been exposed to methylene chloride for one year. Lanes et al. (1993) recently extended the follow-up of the cohort for an additional 4 years. Although no additional cases of liver or biliary cancer were observed, an excess from the previous study persisted (SMR = 2.98, 95% CI = 0.81-7.63]. This latest report did not include an analysis for biliary cancer alone. Another retrospective cohort mortality study of workers from a Hoechst-Celanese cellulose acetate fibre plant in Cumberland, Maryland, USA was reported by Gibbs (1992). This study included 3211 cellulose fibre workers employed in or after 1970 and followed until 1989. The cohort was divided into three groups: high (> 1235 mg/m3, > 350 ppm) methylene chloride exposure, low exposure (176-350 mg/m3, 50-100 ppm), and no exposure. Comparisons were made with USA, Maryland and county mortality rates. Estimates of exposure levels for this population were based on industrial hygiene measurements from the plant studied by Ott et al. (1983), which used similar production methods. Cancer of the prostate was significantly elevated among men, and particularly among those with long latency and with high levels of methylene chloride exposure. A significant excess of cervical cancer was observed among women in the low exposure group, based on Maryland rates (SMR = 4.75 based upon 5 observed), but there was no evidence of a dose-response relationship. Two cases (exp = 1.40) of biliary cancer were observed among the combined high and low exposure groups. An excess of ischaemic heart disease mortality was observed among workers in all three groups when comparisons were based upon Maryland rates, but not when local county rates were used. Mortality from lung, pancreatic, liver/biliary and other cancers was not observed to be significantly elevated in this study. As with the Kodak study, workers at the Hoechst-Celanese facilities were not permitted to smoke at their workstations. Again this fact may have induced a negative bias in these studies, particularly with respect to lung cancer or cardiovascular disease. A cohort study of chemical workers included a sub-cohort of 226 men employed for at least one year in chlorinated methanes production (Ott et al., 1983). Methylene chloride is principally produced by a method involving the hydrochlorination of methanol which also results in the production of chloroform and carbon tetrachloride (IARC, 1986). The men had been employed between 1940 and 1969 and were followed for mortality until 1979. In all, 42 deaths were observed and no excesses of respiratory cancer (SMR = 0.70, based on 3 observed) or circulatory disease (SMR = 0.68, based on 18 observed) were seen. The results for liver and biliary cancer were not reported, but three cases of pancreatic cancer were observed (0.9 expected). All three persons had worked in chlorinated methanes production between 1942 and 1946; two had been employed for less than 5 years, the third for 6 years. No further information on exposure for the individuals or the sub-cohort was given and the mixed exposure to methylene chloride, chloroform, and carbon tetrachloride limits the interpretation of the results with respect to methylene chloride. Finally, Heineman et al. (1994) reported the results of a case- control study of astrocytic brain cancer and occupational exposure to chlorinated aliphatic hydrocarbons. The study included 300 cases with a hospital diagnosis of astrocytic brain cancer and 320 controls matched on age, year of death and geographical area. A job-exposure matrix was used to classify cases and controls in terms of potential exposure to chlorinated aliphatic hydrocarbons including methylene chloride (Gomez et al., 1994); 119 cases and 108 controls were classified as being potentially exposed to methylene chloride in this study. The risk was reported to increase with the probability of exposure (Odds Ratio (OR) - 2.4 for high probability, 95% CI = 0.9-6.4) and duration of employment (OR = 1.9 for > 20 years, 95% CI = 0.7-5.2) in jobs considered to be exposed to methylene chloride after adjustment for other solvent exposures. The exposure information used in this study is weaker than that generally used in the retrospective cohort mortality studies described above and the results should therefore be viewed more cautiously. 9.3 Appraisal of human effects The main toxic effects of methylene chloride are reversible CNS depression and CO-Hb formation. Liver and renal dysfunctions and effects on haematological parameters have also been reported following exposure to methylene chloride. Methylene chloride will irritate the skin and eyes especially when evaporation is prevented. Prolonged contact may cause chemical burns. Neurophysiological and neurobehavioural disturbances have been observed in human volunteers exposed to methylene chloride at concentrations of 694 mg/m3 for 1.5-3.0 h. No evidence of neurological effects was seen in men exposed to methylene chloride for several years at concentrations ranging from 260 to 347 mg/m3. Similarly, the performance of a group of retired airplane strippers, with a long history of exposure to methylene chloride (22 years) at high but unspecified levels, in a battery of neurophysiological and psychological tests was within the "normal" range when compared with a control group who had a history of either no or only low exposure to methylene chloride. Fatalities due to excessive oral exposure to methylene chloride have been reported. A case of serious pulmonary oedema has been reported after excessive inhalation. An increased rate of spontaneous abortion in employees in Finnish pharmaceutical industries has been attributed to exposure to methylene chloride. This isolated finding from a limited study makes it difficult to interpret the significance of the observations. Five mortality studies on methylene chloride have been conducted and evaluated specifically with regard to cancer and cardiovascular disease. None of the studies demonstrated a relationship between exposure to methylene chloride and lung or liver cancer mortality. With regard to the lung cancers, the lack of smoking histories hampers the interpretability of the results. An excess of mortality from biliary cancer was reported in one study, but this was not corroborated by other studies. Two studies showed an excess in mortality from pancreatic cancer. In one of the studies no new pancreatic cancer cases were identified with additional follow-up; with the additional data the excess was not statistically significant. It should be noted that the size of these studies resulted in very low statistical power to detect an excess particularly for rare cancers such as liver and biliary tract tumours. Associations between exposure to methylene chloride and prostate and cervical cancers have been reported in studies, each of which had its limitations. An association between the potential for exposure to methylene chloride and other organic solvents and brain cancer was found in a case-control study which classified exposure to methylene chloride using a job exposure matrix. This finding should be viewed with caution. The results from these studies have been contradictory with respect to mortality from ischaemic heart disease. A role for methylene chloride in the induction of ischaemic heart disease is plausible based on the fact that methylene chloride is metabolized to carbon monoxide and induces the formation of carboxyhaemoglobin in humans. An excess of cardiovascular disease was reported in one of the mortality studies. The fact that further studies did not provide any compelling evidence of an increased risk of cardiovascular disease might be attributable to their reliance on comparisons with the general population as the referent group. The use of general mortality rates in occupational cohort mortality studies may bias the results towards the null (i.e. no effect) due to the "healthy worker effect" which is particularly strong for cardiovascular diseases. This bias may have been further exacerbated by the fact that workers were not permitted to smoke at their workstations. The currently available epidemiological studies are inadequate for drawing any firm conclusions with regard to either cancer or cardiovascular disease risk. 10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT 10.1 Evaluation of human health risks Human exposure to methylene chloride is mainly by inhalation of the vapour. Exposure of the general population to methylene chloride depends strongly on the indoor air concentration. Owing to the use of products containing methylene chloride, peak concentrations of up to 4000 µg/m3 have been reported. However, 24-h average exposures are in general below 50 µg/m3. Methylene chloride is rapidly absorbed through the lung and also from the gastrointestinal tract. It is absorbed via the skin, but at a much slower rate than by the other routes. Once absorbed, methylene chloride is distributed throughout the body and will cross both the placenta and the blood-brain barrier. It is rapidly excreted, the majority being exhaled unchanged via the lungs. The remainder is metabolized to carbon monoxide, carbon dioxide and inorganic chloride. Two metabolic pathways have been identified, one involving cytochrome P-450 and the second involving glutathione- S-transferase. Clear species differences exist in the relative contributions of these two pathways. These differences have been used as a basis for a physiologically based pharmaco-kinetic (PB-PK) model for methylene chloride, which allows interspecies comparison of the concentrations of active metabolites at the target tissues, thus enhancing the value of results from animal studies in human health risk assessment. This approach has been used in assessing the human cancer risk associated with exposure to methylene chloride. The acute toxicity of methylene chloride is low. The predominant effects in human beings are CNS depression and elevated blood carboxyhaemoglobin (CO-Hb) levels. These effects are reversible. Other target organs can be the liver and, occasionally, the kidney. The odour threshold concentration of methylene chloride is reported to be 540 mg/m3 or more. Mild CNS effects have been reported following exposure to concentrations as low as 694 mg/m3 for 1.5-3 h (behavioural disturbances). More significant effects occur at concentrations in excess of 2000 mg/m3. Narcosis has been reported to occur following a 0.5-h exposure to 69 000 mg/m3. Metabolism to carbon monoxide leads to increases in blood CO-Hb levels following acute exposure to the vapour, a process which becomes saturable following exposures to high levels of methylene chloride. Exposure to either 100 or 530 mg/m3 for 7.5 h leads to CO-Hb levels of 3.4% and 5.3%, respectively, in human volunteers. This effect forms the basis of most, if not all, published occupational exposure limits, where a level of 5.0% is judged to be acceptable. The predominant effects following repeated or long-term exposure to methylene chloride are the same as for acute exposure. Reversible symptoms of CNS depression are seen in several species, including humans. The lowest-observed-effect level (LOEL) for this effect in all animal species is 7100 mg/m3 by inhalation. No evidence of irreversible neurological damage was seen in rats exposed to methylene chloride by inhalation at concentrations up to 7100 mg/m3 for 13 weeks. Additional target organs reported in various species chronically exposed to methylene chloride include the liver and, occasionally, the kidney. The no-observed-adverse-effect level (NOAEL) for chronic intermittent inhalation exposure was judged to be 710 mg/m3 in rats. After continuous exposure, slight cytoplasmic vacuolization in the liver of both mice and rats was observed at 88-350 mg/m3. A single study has reported the presence of methylene chloride in the placenta, fetus and breast milk of women following occupational exposure. The teratogenic potential of methylene chloride has been assessed in three animal studies. Small effects on either fetal or maternal body weights were reported, but no evidence of an effect on the incidence of skeletal malformations or other developmental effects was seen. A well-conducted two-generation reproductive toxicity study in rats exposed to methylene chloride by inhalation at concentrations up to 5300 mg/m3, 6 h/day, for 5 days/week showed no evidence of an adverse effect on any reproductive parameter, neonatal survival or neonatal growth in either the F0 or F1 generations. Under appropriate exposure conditions, methylene chloride is mutagenic in prokaryotic microorganisms (Salmonella or E. coli) with or without metabolic activation. In eukaryotic systems it gave either negative or, in one case, weakly positive results. In vitro gene mutation assays and tests for unscheduled DNA synthesis (UDS) in mammalian cells were uniformly negative. In vitro assays for chromosomal aberrations using different cell types gave positive results, whereas negative or equivocal results were obtained in tests for sister chromatid exchange (SCE) induction. The majority of the in vivo studies reported provided no evidence of mutagenicity of methylene chloride (e.g., chromosome aberration assay, micronucleus test or UDS assay). A very marginal increase in frequencies of SCEs, chromosomal aberrations and micronuclei in mice has been reported following inhalation exposure to high concentrations of methylene chloride. The significance of these results is questionable due to methodological deficiencies in the statistical analysis. There was no evidence of binding of methylene chloride to DNA or DNA damage in rats or mice given high doses. Within the limitations of the short-term tests currently available, there is no conclusive evidence that methylene chloride is genotoxic in vivo. Methylene chloride is carcinogenic in the mouse, causing both lung and liver tumours, following lifetime exposure to high concentrations (7100 and 14 100 mg/m3). These tumours were not seen in the rat or the hamster. Increased incidence of benign mammary tumours in female rats was observed in one study, and increased incidence and multiplicity were observed in two other rat studies. The increased incidence of these tumours was within the historical control range; nevertheless there was a dose-response relationship within one study. It is considered that an increase in a tumour type, which occurs with high and variable incidences in control animals, which does not progress to malignancy, and which may be related to changes in prolactin levels, is of little importance in human hazard assessment. In vitro and in vivo metabolism and biochemical studies, and mutagenicity assays in bacteria and B6C3F1 mice have provided a plausible explanation for the mechanism of action and the species differences in the carcinogenicity of methylene chloride to the lung and liver. This explanation is based on the existence of an isoenzyme of glutathione- S-transferase which specifically metabolizes methylene chloride to the reactive intermediates responsible for tumour induction in the mouse. Markedly lower levels of this enzyme in rats and hamsters are consistent with the fact that these tumours do not appear in these species. The levels of the enzyme in the liver are lower in humans than in rats or hamsters. The variability of the current estimates of this enzyme activity in human liver is low, but the possibility of wider variation existing in subpopulations cannot be discounted. Although the currently available information on enzyme activity in human lung is limited, it is expected to be lower than in human liver. The carcinogenic potency of methylene chloride in man is expected to be low. Mutagenicity studies on methylene chloride in bacteria and in the B6C3F1 mouse, which shows a very high level of activity of the isoenzyme, reveal positive effects, whereas mutagenicity has not been demonstrated in standard in vivo mutagenicity assays using other systems. These observations are consistent with the above hypothesis and provide a mechanistic basis for the induction of tumours in the mouse. The role of the glutathione- S-transferase isoenzyme in the mediation of the demonstrated mutagenic effects, together with the correlation between the activity of this pathway and the species differences in carcinogenic response, has led to its use as the dose surrogate in physiologically based pharmacokinetic models used for human health risk assessment. Overall, animal inhalation studies have shown effects on the liver from 710 mg/m3 and on other organs from 1700 mg/m3. However, these effects have not been observed in epidemiological studies. Effects on the CNS have been observed in both animals and humans and a threshold in humans has been defined, based on the level of the metabolite carbon monoxide in the blood, leading to exposure limits of the order of 177 mg/m3. 10.2 Evaluation of effects on the environment Due to its high volatility methylene chloride released to the environment will end up in the atmosphere where it can be transported to regions far removed from the emission source. Methylene chloride is degraded in the troposphere by reaction with hydroxyl radicals giving carbon dioxide and hydrogen chloride as major breakdown products. Based on a lifetime in the troposphere of about 6 months it may be assumed that only a few percent, if any, of methylene chloride will reach the stratosphere. No significant impact on stratospheric ozone depletion is expected. Methylene chloride will also not contribute significantly to photochemical smog formation. In ambient air in rural and remote areas, background levels of 0.07-0.29 µg/m3 have been measured. In suburban and urban areas levels up to 2 and 15 µg/m3 have been found. Concentrations of methylene chloride in the surface water of rivers in industrialized areas stay generally below 10 µg/litre. In industrial effluents, outfalls of municipal water treatment plants and leachates of landfills, concentrations of methylene chloride of up to 200 mg/litre have been measured. In the aquatic environment, fish and amphibian embryos have been shown to be the most sensitive to methylene chloride, with effects on hatching from 5.5 mg/litre; adult aquatic organisms are relatively insensitive even after prolonged exposure. There is no evidence to suggest that methylene chloride and/or its metabolites bioaccumulate in the environment. Given the concentrations observed in surface water (< 10 µg/litre) and those in contaminated effluents (< 200 mg/litre), no significant impact on the aquatic environment is expected. Localized contamination of soils will not significantly disperse despite the mobility of methylene chloride; in groundwaters and soils, biological degradation processes have been identified capable of mineralizing methylene chloride in a few days. From the limited information on soil organisms, it may be assumed that contamination of soil has only a local and transient effect. Apart from accidental spills, it is concluded that the present use of methylene chloride has no significant impact on the environment. REFERENCES Abernethy S, Bobra AM, Shiu WY, Wells PG, & MacKay D (1986) Acute lethal toxicity of hydrocarbons and chlorinated hydrocarbons to two planktonic crustaceans: the key role of organism- water partitioning. Aquatic Toxicology 8:163-174. Abrahamson S & Valencia R (1980) Evaluation of substances of interest for genetic damage using Drosophila melanogaster. In: Mutagenicity of methylene chloride. Oakridge, Tennessee, National Toxicology Programme. ACGIH (1992) Documentation of the threshold limit values and biological exposure indices, 6th ed. Cincinnati, Ohio, US American Conference of Governmental Industrial Hygienists. Adams JD & Erickson HH (1976) The effects of repeated exposure to methylene chloride vapour. Preprint from the Annual Scientific Meeting of the Aerospace Medical Association. Washington, DC, Aerospace Medical Association, pp 61-62. AFS (1990) [Labour Protection Board Statute Book. Hygiene threshold values.] Stockholm, 13 pp (in Swedish). Agency for Toxic Substances and Disease Registry (1993) Methylene chloride toxicity. Am Fam Phys, 47(5): 1159-1166. Ahmed AE & Anders MW (1978) Metabolism of dihalomethanes to formaldehyde and inorganic chloride II. Studies on the mechanism of the reaction. Blochem Pharmacol, 27: 2021-2025. Ahmed AE & Anders MW (1976) Metabolism of dihalomethanes to formaldehyde and inorganic chloride. Drug Metab Dispos, 4: 357-361. Alexander HC, McCarty WM, & Bartlett EA (1978) Toxicity of perchloroethylene and methylene chloride to fathead minnows. Bull Environ Contam Toxicol, 20: 344-352. Alexeef G & Kiglore W (1983) Learning impairment in mice following acute exposure to dichloromethane and carbon tetrachlorlde. J Toxicol Environ Health, 11: 569-581. Allen J, Kligerman A, Campbell J, Westbrook-Collins B, Erexson G, Kari F, & Zeiger E (1990) Cytogenetic analysis of mice exposed to dichloromethane. Environ Mol Mutagen, 15: 221-228. Allred EN, Bleecker ER, Chaitman BR, Dalims TE, Gotlieb SO, Hackney JD, Hayes D, Pagano M, & Selvester RH (1989) Acute effects of carbon monoxide exposure on individuals with coronary artery disease. Health Effects Institute, 98 pp (Research Report No. 25). Amoore JE & Hautala E (1983) Odor as an aid to chemical safety: odor thresholds compared with threshold limit values and volatilities for 214 industrial chemicals in air and water dilution. J Appl Toxicol, 3: 272-290. Anders MW & Sunram JM (1982) Transplacental passage of dichloromethane and carbon monoxide. Toxicol Lett, 12: 231-244. Anders MW, Kubic VL, & Ahmed ME (1977) Metabolism of halogenated methanes and macromolecular binding. J Environ Pathol Toxicol, 1: 117-121. Andersen ME, Clewell III H J, Gargas ML, Smith FA, & Reitz RH (1987) Physiologically based pharmacokinetics and the risk assessment process for methylene chloride. Toxicol Appl Pharmacol, 87: 185-205. Andersen CHJ, Gargas ML, MacNaughton MG, Reitz RH, Nolen RJ, & McKenna MJ (1991) Physiologically based pharmacokinetic modelling with dichloromethane, its metabolite, carbon monoxide, and blood carboxyhaemoglobin in rats and humans. Toxicol Appl Pharmacol, 108: 14-27. Anderson BE, Zeiger E, Shelby MD, Resnick MA, Gulati DK, Ivett JL, & Loveday KS (1990) Chromosome aberration and sister chromatid exchange test results with 42 chemicals. Environ Mol Mutagen, 16: 55-137. Andrae U & Wolff T (1983) Dichloromethane is not genotoxic in isolated rat hepatocytes. Arch Toxicol, 52: 287-290. Angelo MJ, Pritchard AB, Hawkins DR, Waller AR, & Roberts A (1986a) The pharmacokinetics of dichloromethane, I. Disposition in B6C3F1 mice following intravenous and oral administration. Food Chem Toxicol, 24: 965-974. Angelo MJ, Pritchard AB, Hawkins DR, Waller AR, & Roberts A (1986b) The pharmacokinetics of dichloromethane, II. Disposition in Fischer 344 rats following intravenous and oral administration. Food Chem Toxicol, 24: 975-980. Antoine SR, de Leon IR, & O'Dell-Smith RM (1986) Environmentally significant volatile organic pollutants in human blood. Bull Environ Contam Toxicol, 36(3): 364-371. APHA (1977) Methods of sampling and analysis. 2nd ed. Washington, DC: American Public Health Association, pp 894-902. APHA (1989a) Purge and trap capillary-column gas chromatographic method. In: Standard methods for the examination of water and wastewater, 17th ed. Washington, DC, American Public Health Association. APHA (1989b) Purge and trap capillary-column gas chromatographic/mass spectrometric method In: Standard methods for the examination of water and wastewater, 17th ed. Washington, DC, American Public Health Association. Arbeidsinspectie (1991) Labour Inspectorate (1991) [The national MAC list 1991.] Directorate of Labour, Ministry for Social Affairs and Employment (in Dutch). Arbeidstilsynet (1990) Labour Inspectorate (1991) [Administrative standards for atmospheric pollution in the workplace 1990.] Directorate for the Supervision of Labour (in Norwegian). Arendt G, Haag F, & Puggmayer D (1982) [Determination of air pollution with organic compounds in conurbations. Research report of the Federal Office of the Environment.] (UFOPLAN 104 02 510). Frankfurt/Main, Batelle Institut (in German). Åstrand I, Övrum P, & Carlsson A (1975) Exposure to methylene chloride. I. Its concentration in alveolar air and blood during rest and exercise and its metabolism. Scand. J Work Environ Health, 1: 78-94. ATSDR (1992) Toxicological Profile for Methylene Chloride. Atlanta, Georgia, Agency for Toxic Substances and Disease Registry (TP-SZ-13). Aviado DM (1978) Effects of fluorocarbons, chlorinated solvents and inosine on the cardiopulmonary system. Environ Health Perspect, 26: 207-215. Aviado DM & Belej MA (1974) Toxicity of aerosol propellants on the respiratory and circulatory systems. 1. Cardiac arrythmia in the mouse. Toxicology, 2: 31-42. Aviado DM, Zakhari S, & Watanabe T (1977a) Methylene chloride. In: Goldberg L ed. Non-fluorinated propellants and solvents for aerosols. Cleveland, Ohio, CRC Press, pp 19-36. Aviado DM, Zakhari S, & Watanabe T (1977b) Interactions among hydrocarbon propellants, methylene chloride and ethanol. In: Goldberg L ed. Non-fluorinated propellants and solvents for aerosols. Cleveland, Ohio, CRC Press, pp 83-89. Baldanf G (1981) The case of Grenzach - example of groundwater pollution by environmentally relevant substances. DVGW Schr.reihe Wasset, 29: 53-69. Ballantyne B, Gazzard MF, & Swanston DW (1976) The ophthalmic toxicology of dichloromethane. Toxicology, 6: 173-187. Balmer MF, Smith FA, Leach LJ, & Yuile CL (1976) Effects in the liver of methylene chloride inhaled alone and with ethyl alcohol. Am Ind Hyg Assoc J, 37: 345-352. Barassin J & Combourieu J (1973) Etude cinétique des reactions entre l'oxygène atomique et les derivés chlorés du méthane. 1. Réaction CH2Cl2+O. Bull Soc Chim France, 7-8: 2173-2177. Barber ED, Donish WH, & Mueller KR (1980) Quantitative measurement of the mutagenicity of volatile liquids in the Ames Salmonella/microsome assay. Environ Mutagen, 2(2): 307 (Abstract P39). Barrowcliff DF & Knell AJ (1979) Cerebral damage due to endogenous chronic carbon monoxide poisoning caused by exposure to methylene chloride. J Soc Occup Med, 29: 12-14. Barsoum GS & Saad K (1934) Relative toxicity of certain chlorine derivatives of the aliphatic series. Q J Pharm Pharmacol, 7: 205-214. Basso M, Raje R, & Greening M (1987) Evaluation of in vivo mutagenicity of methylene chloride following inhalation exposure in mice by dominant lethal test [abstract]. Toxicologist, 7: 1034. Bauchop T (1967) Inhibition of rumen methanogenesis by methane analogues. J Bacteriol, 94: 171-175. Bayard SP, Bayliss DL, Davidson IWF, Fowle III Jr, Greenberg M, Haberman BH, Kotchmar D, Benignus V, Parker JC, & Singh D (1985) Health assessment document for dichloromethane (methylene chloride). Final report. Washington, DC, US Environmental Protection Agency (EPA-600/8-82-004B; NTIS PB85 191559). Becker CE & Lash A (1990) Study of neurological effects of chronic methylene chloride exposure in airline maintenance mechanics [abstract 7]. Vet Hum Toxicol, 32: 342. Belej MA, Smith GA, & Aviado DM (1974) Toxicity of aerosol propellants in the respiratory and circulatory system. IV. Cardiotoxicity in the monkey. Toxicology, 2: 381-395. Bell BP, Franks P, Hildreth N, & Melius J (1991) Methylene chloride exposure and birthweight in Monroe County, New York. Environ Res, 55: 31-39. Benzon HT, Claybon L, & Brunner EA (1978) Elevated carbon monoxide levels from exposure to methylene chloride. J Am Med Assoc, 239: 2341. Berger M & Fodor GG (1968) CNS disorders under the influence of air mixtures containing dichloromethane. Zent.bl Bakteriol, 215: 517. Bergman K (1979) Whole body radiography and allied tracer techniques in distribution and elimination studies of some organic solvents: benzene, toluene, xylene, styrene, methylene chloride. Scand J Work Environ Health 5(1): 1-263. Binnemann PH, Sandmeyer U, & Schmuk E (1983) [Contents of heavy metals, organochlorine pesticides, PCB and volatile organohalogen compounds in fish in the Upper Rhine and Lake Constance.] Z Lebensm.unters Forsch, 176: 253-261 (in German). Birge WJ, Black JA, & Kuehne RA (1980) Effects of organic compounds on amphibian reproduction. Lexington. Kentucky, Kentucky University, Water Resources Research Institute (Research Report No. 121; NTIS PB80-147523). Black JA, Birge WJ, McDonnell WE, Westerman AG, Ramey BA, & Bruser DM (1982) The aquatic toxicity of organic compounds to embryo-larval stages of fish and amphibians. Lexington, Kentucky, Kentucky University, Water Resources Research Institute (Research Report No. 133: NTIS PB82-224601). Blum DJW & Speece RE (1991) Quantitative structure-activity relationships for chemicals toxicity to environmental bacteria. Ecotoxicol Environ Saf, 22: 198-224. Boeniger MF (1991) Nonisocyanate exposures in 3 flexible polyurethane manufacturing facilities. Appl Occup Hyg, 6: 945-952. Bogaards JJP, Van Ommen B, & Bladeten PJ (1993) Individual differences in the in vitro conjugation of methylene chloride with glutathione by cytosolic glutathione S-transferase in 22 human liver samples. Blochem Pharmacol, 45(10): 2166-2169. Bohme H, Fischer H, & Frank R (1949) Preparation and properties of the x-halogenated thioethers. Ann Chem, 563: 54-72. Bonnet P, Francin JM, Gradiski D, Raoult G, & Zissu D (1980) Détermination de la concentration léthale50 des principaux hydrocarbures aliphatiques chlorés chez le rat. Arch Mal Prof Med, 41: 317-321. Bonventre J, Brennan O, Jason D, Henderson A, & Bastos ML (1977) Two deaths following accidental inhalation of dichloromethane and 1,1,1-trichloroethane. J Anal Toxicol, 1: 158-160. Bornmann G & Loeser A (1967) [The question of the chronic toxic action of dichloromethane.] Z Lebensm. unters Forsch, 136: 14-18 (in German). Bornschein RL, Hastings L, & Manson JM (1980) Behavioral toxicity in the offspring of rats following maternal exposure to dichloromethane. Toxicol Appl Pharmacol, 52: 29-37. Breslin WJ & Landry TD (1986) Methylene chloride: Effects on estrous cycling and serum prolactin in Sprague-Dawley rats. Midland, Michigan, Dow Chemical USA (Internal report). Bringmann G & Kühn R (1977a) [Findings on the harmful effects of water pollutants on Daphnia magna.] Z Wasser Abwasser Forsch, 10: 161-166 (in German). Bringmann G & Kühn R (1977b) Limiting values for damaging action of water pollutants to bacteria (Pseudomonas putida) and green algae (Scenedesmus quadricauda) in the cell multiplication inhibition test. Z Wasset Abwasser Forsch, 10: 87-98. Bringmann G & Kühn R (1978) [Threshold values for the harmful effects of water pollutants on blue algae (Microcystis aeruginosa) and green algae (Scenedemus quadricauda) in the cell reproduction inhibition test.] Vom Wasser, 50: 45-60 (in German). Bringmann G & Kühn R (1980) Determination of the harmful biological effect of water pollutants on protozoa. II. Bacteriovorous ciliates. Z Wasser Abwasser Forsch, 13(1): 26-31. Bringmann G & Kühn R (1982) [Findings on the harmful effect of water pollutants on Daphnia magna in a further developed standardized test procedure.] Z Wasser Abwasser Forsch, 15: 1-6 (in German). Bringmann G & Meinck F (1964) [Water toxicological assessment of industrial waste waters.] Gesundheits-Ingenieur, 85: 229-236 (in German). Briving C, Hamberger A, Kjellstrand P, Rosengren L, Karlsson JE, & Haglid KG (1986) Chronic effects of dichloromethane on amino acids, glutathione and phosphoethanolamine in gerbil brain. Scand J Work Environ Health, 12: 216-220. Brown KW & Donnelly KC (1988) An estimation of the risk associated with the organic constituents of hazardous and municipal waste landfill leachates. Hazard Waste Hazard Mater, 5: 1-30. Brunner W, Staub D, & Leisinger TH (1980) Bacterial degradation of dichloromethane. Appl Environ Microbiol, 40: 950-958. BUA (Advisory body on wastes with environmental implications) (1986) [Dichlormethane.] BUA-Substance Report 6. Weinheim, VCH (BUA Report No. 6) (in German). Buccafusco RJ, Ells SJ, & Leblanc GA (1981) Acute toxicity of priority pollutants to bluegill (Lepomis macrochirus). Bull Environ Contam Toxicol, 26: 446-452. Buie SE, Pratt DS, & May JJ (1986) Diffuse pulmonary injury following paint remover exposure. Am J Med, 81: 702-704. Burek JD, Nitschke KD, Bell TJ, Wackerle DL, Childs RC, Beyer JE, Dittenber DA, Rampy LW, & McKenna MJ (1980) Methylene chloride: A two- year inhalation toxicity and oncogenicity study in rats and hamsters. Final report, Toxicology Research Laboratory, Health and Environmental Sciences, Midland, Michigan, Dow Chemical USA. Burek JD, Nitschke KD, Bell TJ, Wackerle DL, Childs RC, Beyer JE, Dittenber DA, Rampy LW, & McKenna MJ (1984) Methylene chloride: A two- year inhalation toxicity and oncogenicity study in rats and hamsters. Fundam Appl Toxicol, 4: 30-47. Burton DT & Fischer DJ (1990) Acute toxicity of cadmium, copper, zinc, ammonia, 3,3'-dichlorobenzidine, 2,6-dichloro-4-nitroaniline, methylene chloride, and 2,4,6-trichlorophenol to juvenile grass shrimp and killifish. Bull Environ Contam Toxicol, 44: 776-783. Callen DF, Wolf CR, & Philpot RM (1980) Cytochrome P-450 mediated genetic activity and cytotoxicity of seven halogenated aliphatic hydrocarbons in Saccharomyces cerevisiae. Mutat Res, 77: 55-63. Carlsson A & Hultengren M (1975) Exposure to methylene chloride, III. Metabolism of 14C-labelled methylene chloride in rat. Scand J Work Environ Health, 1: 104-108. Carreon R (1981) Methylene chloride: Acute oral toxicity. Midland, Michigan, Dow Chemical Company (Internal report). Casanova M, Deyo DF, & Heck Hd'A (1992) Dichloromethane (methylene chloride): Metabolism to formaldehyde and formation of DNA-protein cross-links in B6C3F1 mice and Syrian golden hamsters. Toxicol Appl Pharmacol, 114: 162-165. CEC (1980) Carcinogenicity volume II: Summary reviews of scientific evidence. Luxembourg, European Commission, Health and Safety Directorate, Directorate-General for Employment, Industrial Relations and Social Affairs, pp 67-74. CEC (1982) Multiannual programme of the Joint Research Centre 1980-1983, 1982 Annual Status Report - Protection of the environment. Luxembourg, European Commission (Report EUR 8527-EN). CEC (1986) Organochlorine solvents, health risks to workers. Luxembourg, European Commission (Report EUR-10531-EN). CEFIC (1986) The occurrence of chlorinated solvents in the environment. European Chemical Industry Council. Chem Ind, 15: 861-869. CEFIC (1993) Methylene chloride: Use in industrial applications. Brussels, European Chemical Industry Council. Cherry N, Venables H, Waldron HA, & Wells GG (1981) Some observations on workers exposed to methylene chloride. Br J Ind Med, 38: 351-355. Chodola GR, Biswas N, Bewtra JK, St Pierre CC, & Zytner RG (1989) Fate of selected volatile organic substances in aqueous environment. Water Pollut Res J Can, 24: 119-142. Chrostek WJ & Levine MS (1981) Health Hazard Evaluation Report No. HHE-80-154-1207: Bechtel Powder Corporation, Betwick, Pennsylvania, 24 pp. Clark DG & Tinston DJ (1982) Acute inhalation toxicity of some halogenated and non-halogenated hydrocarbons. Hum Toxicol, 1: 239-247. Cohen JM, Dawson R, & Koketsu M (1980) Extent-of-exposure survey of methylene chloride. Cincinnati, Ohio, National Institute of Occupational Safety and Health, 53 pp (Document No. 80-131). Coleman WE, Lingg RD, Melton RG, & Kopfler FC (1976) The occurrence of volatile organics in five drinking water supplies using gas chromatography/mass spectrometry. In: Keith LH, ed. Identification and analysis of organic pollutants in water. Ann Arbor, Michigan, Ann Arbor Science, pp 305-327. Condie LW, Smallwood CL, & Laurie RD (1983) Comparative renal and hepatotoxicity of halomethanes: bromodichloromethane, bromoform, chloroform, dibromochloromethane and methylene chloride. Drug Chem Toxicol, 6: 563-578. Cordes DH, Brown WD, & Quinn KM (1988) Chemically induced hepatitis after inhaling organic solvents. West J Med, 148: 458-460. Cornish HH, Ling BP, & Barth ML (1973) Phenobarbital and organic solvent toxicity. Am Ind Hyg Assoc J, 34: 487-492. Corsi GC, Valentini F, & Bertazzon A (1983) Effects of subtoxic amounts of furan, acetylfuran and methylene chloride on some serum enzymes of rat. Boll Soc Ital Biol Sper, 59: 1049-1052. Cox RA, Derwent RG, & Eggleton AE (1976) Photochemical oxidation of halocarbons in the troposphere. Atmos Environ, 10: 305-308. Crebelli R, Andreoli C, Carere A, Conti G, Conti L, Cotta Ramusino M, & Benigni R (1992) The induction of mitotic chromosome malsegregation in Aspergillus nidulans. Quantitative structure activity relationship (QSAR) analysis with chlorinated aliphatic hydrocarbons. Mutat Res, 266: 117-134. Crebelli R, Benigni R, Franekic J, Conti G, Conti L, & Carere A (1988) Induction of chromosome malsegregation by halogenated organic solvents in Aspergillus nidulans: unspecific or specific mechanism? Murat Res, 201: 401-411. Cunningham ML, Gandolfi AJ, Brendel K, & Sipes IG (1981) Covalent binding of halogenated volatile solvents to subcellular macromolecules in hepatocytes. Life Sci, 29: 1207-1212. Cuppit LT (1980) Fate of toxic and hazardous materials in the air environment. Washington, DC, US Environmental Protection (NTIS PB80-221948). Daft JL (1987) Determining multifumigants in whole grain and legumes, milled and low-fat grain products, spices, citrus fruit, and beverages. J Assoc Off Anal Chem, 70: 734-739. Daft JL (1989) Determination of fumigants and related chemicals in fatty and nonfatty foods. J Agric Food Chem, 37: 560-564. Daniels SL, Hoerger FD, & Moolenaer RJ (1985) Environmental exposure assessment: experience under the toxic substances control act. Environ Toxicol Chem, 4: 107-117. Dankovic DA & Baiter AJ (1994) The impact of exercise and intersubject variability on dose estimates for dichloromethane derived from a physiologically based pharmacokinetic model. Fundam Appl Toxicol, 22: 20-25. Danni O, Brossa O, Burdino E, Milillo P, & Ugazio G (1981) Toxicity of halogenated hydrocarbons in pretreated rats - an experimental model for the study of integrated permissible limits of environmental poisons. Int Arch Occup Health, 49: 164-176. Davis JW & Madsen SS (1991) The biodegradation of methylene chloride in soils. Environ Toxicol Chem, 10: 463-474. Davis EM, Murray HE, Liehr JG, & Powers EL (1981) Basic microbial rates and chemical byproducts of selected organic compounds. Water Res, 15: 1125-1127. De Walle FB & Chain ESK (1978) Presence of trace organics in the Delaware river and their discharge by municipal and industrial sources. Proc Ind Waste Conf, 32: 908-919. De Bortoli M, Knöppel H, Pecchio E, Peil A, Rogora L, Schauenburg H, Schlitt H, & Vissers H (1986) Concentrations of selected organic pollutants in indoor and outdoor air in Northern Italy. Environ Int, 12: 343-350. Dequinze J, Scimar C, & Edeline F (1984) Identification of the substances and their derived products on the list of 129 substances (list 1 of the Directive 76/464/EEC), present in the refuse of chlorine derived organic chemistry industry CEBEDEAU (Final report No. 83/205). Derwent RG & Eggleton AEJ (1978) Halocarbon lifetimes and concentration distributions calculated using a two-dimensional tropospheric model. Atmos Environ, 12: 1261-1269. Derwent RG & Jenkin ME (1991) Hydrocarbons and the long-range transport of ozone and PAN across Europe. Atmos Environ, 25A: 1661-1678. Devereux TR, Foley JF, Maronpot RR, Kari F, & Anderson MW (1993) RAS proto-oncogene activation in liver and lung tumors from B6C3F1 mice exposed chronically to methylene chloride. Carcinogenesis, 14(5): 795-801. DFG (Deutsche Forschungsgemeinschaft) (1983) In: Henschler D ed. [Maximum workplace concentration and biological tolerance values for working materials.] Verlage Chemie, Weinheim, Germany (in German). DFG (Deutsche Forschungsgemeinschaft) (1991), Senate Committee for the Testing of Working Materials Endangering Health (1991) Maximum workplace concentrations and biological tolerance values for working materials. Weinheim, Germany, VCH Publishers (in German). Di Renzo AB, Gandolt AJ, & Sipes IG (1982) Microsomal and covalent binding of aliphatic halides to DNA. Toxicol Lett, 11: 243-252. Di Vincenzo GD & Kaplan CJ (1981a) Uptake, metabolism and elimination of methylene chloride vapor by humans. Toxicol Appl Pharmacol, 59: 130-140. Di Vincenzo GD & Kaplan CJ (1981b) Effect of exercise or smoking on the uptake, metabolism, and excretion of methylene chloride vapor. Toxicol Appl Pharmacol, 59: 141-148. Di Vincenzo GD & Hamilton ML (1975) Fate and disposition of 14C- methylene chloride in the rat. Toxicol Appl Pharmacol, 32: 385-393. Di Vincenzo GD, Yanno FJ, & Astill BD (1971) The gas chromatographic analysis of methylene chloride in breath, blood and urine. Am Ind Hyg Assoc J, 32: 387-391. Di Vincenzo GD, Yanno F J, & Astill BD (1972) Human and canine exposure to methylene chloride vapor. Am Ind Hyg Assoo J, 33: 125-135. Dill DC, Watanabe PG, & Norris JM (1978) Effect of methylene chloride on the oxyhemoglobin dissociation curve of rat and human blood. Toxicol Appl Pharmacol, 46(1): 125-129. Dill DC, Murphy PG, & Mayes MA (1987) Toxicity of methylene chloride to life stages of fathead minnow, Pimephales promelas Rafinesque. Bull Environ Toxicol, 39: 869-876. Dilling WL, Tefertiller NB, & Kallos GJ (1975) Evaporation rates of methyl chloride, chloroform, 1,1,1-trichloroethane, trichloroethylene, tetrachloroethylene, and other chlorinated compounds in dilute aqueous solutions. Environ Sci Technol, 9: 833-838. Dilling WL (1977) Interphase transfer processes. II. Evaporation rates of chloromethanes, ethanes, ethylenes, propanes, and propylenes from dilute aqueous solutions; Comparisons with theoretical predictions. Environ Sci Technol, 4: 405-409. Dillon DM, Combos RD, McConville M, & Zeiger E (1990) The role of metabolism and glutathione in the mutagenicity of vapour phase dichloromethane in bacteria. Environ Mol Mutagen, 15 (Suppl 17): Abstr 52. Dillon DM, Edwards IR, Combos RD, McConville M, & Zeiger E (1992) The role of glutathione in the bacterial mutagenicity of vapor phase dichloromethane. Environ Mol Mutagen, 20: 211-217. Dowty BJ, Carlisle DR, & Laseter JL (1975) New Orleans drinking water sources tested by gas chromatography-mass spectrometry. Environ Sci Technol, 9: 762-765. Duprat P, Delsaut L, & Gradiski D (1976) Pouvoir irritant des principaux solvents chlorés aliphatiques sur la peau et les muqueuses oculaires du lapin. J Eur J Toxicol, 9(3): 171-177. ECETOC (1984) Joint assessment of commodity chemicals No. 4, Methylene chloride. Brussels, European Centre for Ecotoxicology and Toxicology of Chemicals. ECETOC (1988) Methylene chloride (dichloromethane): human risk assessment using experimental animal data. Brussels, European Centre for Ecotoxicology and Toxicology of Chemicals (Technical Report No. 32). ECETOC (1989) Methylene chloride (dichloromethane): an overview of experimental work investigating species, differences in carcinogenicity and their relevance to man. Brussels, European Centre for Ecotoxicology and Toxicology of Chemicals (Technical Report No. 34). ECSA (European Chlorinated Solvents Association) (1989) Methylene chloride, its properties, uses, occurrence in the environment, toxicology and safe handling. Brussels, European Chemical Industry Council. ECSA (European Chlorinated Solvents Association) (1992) ECSA production figures for methylene chloride. Brussels, European Chemical Industry Council. Edwards PR, Campbell I, & Milne GS (1982) The impact of chloromethanes on the environment. Part 2. Methyl chloride and methylene chloride. Chem Ind, 619-622. Eisenbrandt DL & Reitz RH (1986) Acute toxicity of methylene chloride: Tumorigenic implications for B6C3F1 mice. Toxicologist, 6: 662. English JM (1964) A case of probable phosgene poisoning. Br Med J, 1: 38. Engström J & Bjurström R (1977) Exposure to methylene chloride: Content in subcutaneous adipose tissue. Scand J Work Environ Health, 3: 215-224. European Council (1982) Council Directive of 17 May 1982 amending for the second time Directive 76/768/EEC on the approximation of the laws of the Member States relating to cosmetic products (82/368/EEC). Off J Eur Communities, L167: 1-8. Fagin J, Bradley J, & Williams D (1980) Carbon monoxide poisoning secondary to inhaling methylene chloride. Br Med J, 281: 1461. Fells I & Moelwyn-Hughes EA (1958) The kinetics of the hydrolysis of metylene chloride. J Chem Soc, 1326-1333. Ferguson J & Pirie H (1948) The toxicity of vapours to the grain weevil. Ann Appl Biol, 35: 532-550. Ferrario JB, Lawler GC, DeLeon IR, & Laseter JL (1985) Volatile organic pollutants in biota and sediments of Lake Pontchartrain. Bull Environ Contam Toxicol, 34: 246-255. Flanagan R J, Ruprah M, Meredith T J, & Ramsey JD (1990) An introduction to the clinical toxicology of volatiles substances. Drug Saf, 5(5): 359-383. Flathman PA, Jerger DE, & Woodhall PM (1992) Remediation of dichloromethane (DCM)-contaminated ground water. Environ Prog, 11(3): 202-209. Fleeger AK & Lee JS (1988) Characterization of worker exposures to methylene chloride resulting from application of aerosol glue in the asbestos abatement industry. Appl Ind Hyg, 3: 245-250. Flury F & Zernik F (1931) [Harmful gases, yapours, mists, smokes, and dusts]. Berlin, Julius Springer, pp 311-312 (in German). Foder GG, Prajsnar D, & Schlipkoter KW (1973) Endogenous conformation by incorporated halogenated hydrocarbons of the methane series. Staubreinhalt Luft, 33: 260-261. Fodor GR & Winneke (1971) Nervous system disturbances in men and animals experimentally exposed to industrial solvent vapors. In: England HM ed. Proceedings of the 2nd International Clean Air Congress. New York, Academic Press, pp 238-243. Fodor GG, Schlipköter HW, & Zimmermann M (1973) The objective study of sleeping behaviour in animals as a test of behavioural toxicity. In: Horvath M (ed.) Adverse effects of environmental chemicals and psychoretapic drugs: quantitative interpretation of functional tests. London, Elsevier, pp 115-123. Foley JF, Tuck PD, Ton TVT, Frost M, Kari F, & Anderson MW (1993) Inhalation to a hepatocarcinogenic concentration of methylene chloride does not induce sustained replicative DNA synthesis in hepatocytes of female B6C3F1 mice. Carcinogenesis, 14(5): 811-817. Foster JR, Green T, Smith LL, Lewis RW, Hext PM, & Wyatt I (1992) Methylene chloride - an inhalation study to investigate pathological and biochemical events occurring in the lungs of mice over an exposure period of 90 days. Fundam Appl Toxicol, 18: 376-388. Friedlander BR, Hearne T, & Hall S (1978) Epidemiologic investigation of employees chronically exposed to methylene chloride. J Occup Med, 20: 657-666. Fuxe K, Andersson K, Hansson T, Agnati LF, Eneroth P, & Gustafsson JA (1984) Central catecholamine neurons and exposure to dichloromethane. Selective changes in amine levels and turnover in tel- and diencephalic DA and NE nerve terminal systems and in secretion of anterior pituitary hormones in the male rat. Toxicology, 29: 293-305. Gälli R & Leisinger T (1985) Specialized bacterial strains for the removal of dichloromethane from industrial waste. Conserv Recycl, 8: 91-100. Gargas ML, Clewell III HJ, & Anderson ME (1986) Metabolism of inhaled dihalomethanes in vivo: differentiation of kinetic constants for two independent pathways. Toxicol Appl Pharmacol, 82: 211-223. Gerritsen WB & Buschmann CH (1960) Phosgene poisoning caused by the use of chemical paint removers containing methylene chloride in ill- ventilated rooms heated by kerosene stoves. Br J Ind Med, 17: 187-189. Ghittori S, Marraccini P, Franco G, & Imbirani M (1993) Methylene chloride exposure in industrial workers. Am Ind Hyg Assoc J, 54(1): 27-31. Gibbs GW (1992) The mortality of workers employed at a cellulose acetate and triacetate fibers plant in Cumberland Maryland, a "1970" cohort followed 1970-1989. Final report by Safety Health Environmental International Consultants, Winterburn, Alberta (TO). Somerville, New Jersey, Hoechst Celanese. Giger W, Schwarzenbach RP, Hoehn E, Schellenberg K, Schneider JK, Wasmer HR, Westall J, & Zobrist J (1983) [Das Verhalten organischer Wasser-inhaltstoffe bei der Grundwasserbildung und im Grundwasser.] Gaz-Eaux-usées, 63: 517-531 (in German). Gocke E, King M-T, Eckhardt K, & Wild D (1981) Mutagenicity of cosmetic ingredients licensed by the European Communities. Murat Res, 90: 91-109. Gomez MR, Cocco P, Dfosemeci M, & Stewart PA (1994) Occupational exposure to chlorinated aliphatic hydrocarbons: Job exposure matrix. Am J Ind Med, 26(2): 171-183. Gossett JM (1985) Anaerobic degradation of C1 and C2 chlorinated hydrocarbons. Air Force Engineering Service Centre, Engineering Service Laboratory, 153 p (ESL-TR-85-38). Gradiski D, Bonnet P, Raoult G, Magadur JL, & Francin JM (1978) Toxicité aiguë comparée par inhalation de principaux solvants alphatiques chlorés. Arch Mal Prof Méd Trav Sécur Soc, 39: 249-257. Green T (1983) The metabolic activation of dichloromethane in a bacterial assay using Salmonella typhimurium. Mutat Res, 118: 277-288. Green T, Provan WM, Collinge DC, & Guest AE (1986a) Methylene chloride (dichloromethane): Interaction with rat and mouse liver and lung DNA in vivo. Alderley Park, Macclesfield (Cheshire), ICI (Technical Report CTL/R/851). Green T, Provan WM, Nash JA, & Gowans N (1986b) Methylene chloride (dichloromethane): In vivo inhalation pharmacokinetics and metabolism in F344 rats and B6C3F1 mice. Alderley Park, Macclesfield (Cheshire), ICI (Technical Report CTL/R/880). Green T, Nash JA, & Mainwaring G (1986c) Methylene chloride (dichloromethane): In vitro metabolism in rat, mouse and hamster liver and lung fractions and in human liver fractions. Alderley Park, Macclesfield (Cheshire), ICI (Technical Report CTL/R/879). Green T, Provan WM, & Gowans N (1987a) Methylene chloride (dichloromethane): In vivo inhalation pharmacokinetics in B6C3F1 mice (using stable isotopes) and F344 rats. AlderIcy Park, Macclesfield (Cheshire), ICI (Technical Report CTL/R/931). Green T, Nash JA, & Hill SJ (1987b) Methylene chloride (dichloromethane): Glutathione- S-transferase metabolism in vitro in rat, mouse, hamster, and human liver cytosol fractions. Alderley Park, Macclesfield (Cheshire), ICI (Technical Report CTL/R/934). Green T, Nash JA, Hill SJ, & Foster JR (1987c) Methylene chloride (Dichloromethane): The effects of exposure to 4000 ppm on mouse lung enzymes. Alderley Park, Macclesfield (Cheshire), ICI (Report CTL/R/935). Green T, Provan WM, Collinge DC, & Guest AE (1988) Molecular interactions of inhaled methylene chloride in rats and mice. Toxicol Appl Pharmacol, 93(1): 1-10. Gu Z & Wang Y (1988) [Evaluation of genotoxic effect of 16 chemicals using the micronucleus assay in vitro]. Weisheng Dulixue Zazhi, 2: 1-4 (in Chinese). Guengerich FP, Shimada T, Raney KD, Yun CH, Meyer DJ, Ketterer B, Harris TM, Groopman JD, & Kadlubar FF (1992) Elucidation of catalytic specificites of human cytochrome P450 and glutathione-S-transferase enzymes and relevance to molecular epidemiology. Environ Health Perspect, 98: 75-80. Guicherit R & Schulting FL (1985) The occurrence of organic chemicals in the atmosphere of The Netherlands. Sci Total Environ, 43: 193-219. Guidelines on the Evaluation and Treatment of Groundwater Pollution with Readily Volatile chlorohydrocarbons (1983) Published by the Ministry for Nutrition, Agriculture and Environment, Baden- Württemberg, Heft, 13 August. Hake CL, Stewart RD, Wu A, & Graff SA (1975) Carboxyhaemoglobin levels of humans exposed to methylene chloride. 14th Annual Meeting of the Society of Toxicity, Williamsbergh, Virginia. Toxicol Appl Pharmacol, 33(1): 145 (Abstract 59). Halbartschlager J, Kohler H, Swerinski H, & Bardtke D (1984) [Studies on the biological decomposition of chlorohydrocarbons, using the example of dichlormethane (methylene chloride).] GWF-Wasser/Abwasser, 125: 380-386 (in German). Hall AH & Rumack BH (1990) Methylene chloride exposure in furniture- stripping shops: Ventilation and respirator use practices. J Occup Med, 32: 33-37. Hallier E, Laughof T, Dannappel D, Leutbecher M, Schroeder K, Goergeus HW, Müller A, & Bolt HM (1993) Polymorphism of glutathione conjugation of methylbromide, ethylene oxide and dichloromethane in human blood: Influence of the induction of sister chromatid exchanges (SCE) in lymphocytes. Arch Toxicol 67: 173-178. Hansch C & Leo A (1979) Substituent Constants for Correlation Analysis in Chemistry and Biology. New York, Chichester, Brisbane, Toronto, John Wiley and Sons. Hardin BD & Manson JM (1980) Absence of dichloromethane teratogenicity with inhalation exposure in rats. Toxicol Appl Pharmacol, 52: 22-28. Harkov R (1984) Comparison of selected volatile organic compounds during the summer and winter at urban sites in New Jersey. Sci Total Environ, 38: 259-274. Harkov R, Giante SJ, Bozelli JW, & LaRegina JE (1985) Monitoring volatile organic compounds at hazardous and sanitary landfills in New Jersey. J Environ Sci Health, A20: 491-501. Hatch GG, Mamay PD, Ayer ML, Casto BC, & Nesnoro S (1983) Chemical enhancement of viral transformation in Syrian hamster embryo cells by gaseous volatile chlorinated methanes and ethanes. Cancer Res, 43: 1945-1950. Haun CC, Vernot EH, Darmer KI, & Diamond SS (1972) Continuous animal exposure to low levels of dichloromethane. In: Proceedings of the 3rd Annual Conference on Environmental Toxicology. Dayton, Ohio, Wright- Patterson Air Force Base, Aerospace Medical Research Laboratory, pp. 199-208 (Paper No. 12; AMRL-TR-130). Hayes WC & Bailey RE (1977) The inhibition of anaerobic sludge gas production by 1,1,1-trichloroethane, methylene chloride, trichloroethylene and perchloroethylene. Report dated 27 Jan 1977. Midland, Michigan, USA, Dow Chemical. Hearne FT, Grose F, Pifer WJ, Friedlander BR, & Raleigh RL (1987) Methylene chloride mortality study: Dose-response characterization and animal model comparison. J Occup Med, 29: 217-228. Hearne FT & Friedlander BR (1981) Follow-up of methylene chloride study. J Occup Med, 23: 660. Hearne FT, Pifer JW, & Grose F (1990) Absence of adverse mortality effects in workers exposed to methylene chloride. An update. J Occup Med, 32(3): 234-240. Hegi ME, Soderkvist P, Foley JE, Schronhoven R, Swenberg JA, Kari F, Maronpot RR, Anderson MW, & Wiseman RW (1993) Characterization of p53 mutations in methylene chloride-induced lung tumors from B6C3F1 mice. Carcinogenesis, 14(5): 803-810. Heikes DL & Hopper ML (1986) Purge and trap method for determination of fumigants in whole grains, milled grain products and intermediate grain based foods. J Assoc Off Anal Chem, 69(6): 990-998. Heikes DL (1987) Purge and trap method for determination of volatile hydrocarbons and carbon disulphide in table ready foods. J Assoc Off Anal Chem, 70(2): 215-226. Heil H, Eikman T, Einbrodt HJ, König H, Lahl U, & Zeschmar-Lahl B (1989) [Consequences of the Bielefeld-brake Atlas case.] Vom Wasser, 72: 321-348 (in German). Heineman EF, Cocco P, Gomez MR, Dosemeci M, Stewart PA, Hayes RB, Zahim SH, Thomas TL, & Blair A (1994) Occupational exposure to chlorinated aliphatic hydrocarbons and risk of astrocytic brain cancer. Am J Ind Med, 26(2): 155-169. Heitmuller PT, Hollister TA, & Parrish PR (1981) Acute toxicity to 54 industrial chemicals to sheepshead minnows (Cyprinodon variegatus). Bull Environ Contam Toxicol, 27: 596-604. Hellmann H (1984) [Readily volatile chlorohydrocarbons in the inland waters of the Federal Republic of Germany - occurrence and quantities.] Gesundheits-Ingenieur, 105: 269-278 (in German). Helz GR & Hsu RY (1978) Volatile chloro- and bromocarbons in coastal waters. Limnol Oceanogr, 23: 858-869. Henson JM, Yates MV, Cochran JW, & Shackleford DL (1988) Microbial removal of halogenated methanes, ethanes, and ethylenes in an aerobic soil exposed to methane. FEMS Microbiol Ecol, 53: 193-201. Heppel LA, Neal PA, Perrin TL, Orr ML, & Porterfield VT (1944) Toxicology of dichloromethane (methylene chloride). I. Studies on effects of daily inhalation. J Ind Hyg Toxicol, 26: 8-16. Heppel LA & Neal PA (1944) Toxicology of dichloromethane (methylene chloride). II. Its effect upon running activity in the male rat. J Ind Hyg Toxicol, 26: 17-21. Hermens J, Busser F, Leeuwangh P, & Musch A (1985) Quantitative structure-activity relationships and mixture toxicity of organic chemicals in Photobacterium phosphoreum: the microtox test. Ecotoxicol Environ Saf, 9: 17-25. Hext PM, Foster J, & Millward SW (1986) Methylene chloride (Dichloromethane): 10-day inhalation toxicity study to investigate the effects on rat and mouse liver and lungs. ICI Report No. CTL/P/1432. Horvath AL (1982) Halogenated hydrocarbons: solubility-miscibility with water. New York, Marcel Dekker, Inc. Hov O, Penkett SA, lsaksen ISA, & Semb A (1984) Organic gases in the Norwegian Arctic. Geophys Res Left, 11: 425-428. Howard PH (1990) Dichloromethane. In: Howard PH ed. Handbook of environmental fate and exposure data for organic chemicals. Chelsea, Michigan, Lewis Publishers, Inc., pp 176-183. Howard PH, Sage GW, & Jarvis WF ed. (1990) Handbook on Environmental Fate and Exposure Data for Organic Chemicals. Chelsea, Michigan, Lewis Publishers Inc., pp 176-183. HSE (UK Health and Safety Executive) (1987) ACTS review: dichloromethane. London, Her Majesty's Stationery Office. HSE (UK Health and Safety Executive) (1992) National exposure data base. Bootle (Merseyside), United Kingdom. Health and Safety Executive. Hughes JP (1954) Hazardous exposure to some so-called safe solvents. J Am Med AssPc, 156: 234-237. Hughes NJ & Tracey JA (1993) A case of methylene chloride (nitremans) poisoning, effects on carboxyhaemoglobin levels. Hum Exp Toxicol, 12(2) 159-160. Hutchinson TC, Hellebust JA, Tam D, MacKay D, Mascarenhas RA, & Shiu WY (1978) The correlation of the toxicity to algae of hydrocarbons and halogenated hydrocarbons with their physical-chemical properties. Environ Sci Res, 16: 577-586. IARC (1986) Some halogenated hydrocarbons and pesticide exposure. Lyon, International Agency for Research on Cancer, 43-85 (IARC Monograph on the Evaluation of Carcinogenic Risk of Chemicals to Humans, Volume 41). ILO (1991) Occupational exposure limits for airborne toxic substances, 3rd ed. Geneva, International Labour Office (Occupational Safety and Health Series No. 37). IPCS (International Programme on Chemical Safety) (1984) Environmental health criteria 32: methylene chloride. Geneva, World Health Organization. Ito A, Kawata F, Takeshita T, & Ito M (1990) [Experimental studies of effects of methylene chloride on living body (1)]. Hochudoku, 8: 64-65 (in Japanese). Janssen PJM & Pot TE (1988a) Acute oral toxicity study with dichloromethane in rats. Weesp, The Netherlands, Solvay Duphar (Unpublished document 56645/33/88). Janssen PJM & Pot TE (1988b) Acute dermal toxicity study with dichloromethane in rats. Weesp, The Netherlands, Solvay Duphar (Unpublished document 55645/24/88). Jensen AA (1983) Chemical contaminants in human milk. Residue Rev, 89: 1-108. Jenson RA (1978) A simplified bioassay using finfish for estimating potential spill damage. In: Proceedings of the Meeting on Control of Hazardous Material Spills, Rochville, Maryland, pp 104-108. Jernelov M & Antonsson AB (1987) [Exposure to solvents when pouring polyurethane into moulds.] Oslo, IVL (Report No. B869) (in Norwegian). Jongen WMF, Alink GM, & Koeman JH (1978) Mutagenic effect of dichloromethane on Salmonella typhimurium. Mutat Res, 56: 245-248. Jongen WMF, Lohman PHM, Kottenhagen M J, Alink GM, Betends F, & Koeman JH (1981) Mutagenicity testing of dichloroethane in short-term mammalian test systems. Mutat Res, 81(2): 203-213. Jongen WMF, Harmsen EGM, Alink GM, & Koeman JH (1982) The effect of glutathione conjugation and microsomal oxidation on the mutagenicity of dichloromethane in Salmonella typhimurium. Mutat Res, 95: 183-189. Jongen WMF (1984) Relationship between exposure time and metabolic activation of dichloromethane in Salmonella typhimurium. Mutat Res, 136(2): 107-108. Juhnke I & Ltidemann D (1978) [Results of the testing of 200 chemical compounds for acute toxicity for fish in the golden orfe test.] Z Wasser Abwasser Forsch, 11: 161-164 (in German). Kanazawa S & Filip Z (1987) Effects of trichloroethylene and dichloromethane on soil biomass and microbial counts. Zent.bl Bakteriol Hyg, 184; 24-33. Kanazawa S & Filip Z (1986) Effect of trichloroethylene, tetrachloroethylene and dichloromethane on enzymatic activities in soil. Appl Microbiol Biotechnol, 25: 76-81. Kanno J, Foley JF, Kari F, Anderson MW, & Maronport RR (1993) Effect of methylene chloride inhalation on replicative DNA synthesis in the lungs of female B6C3F1 mice. Environ Health Perspect, 101 (Suppl 5): 271-286. Kari FW, Maronpot RR, & Anderson MW (1992) Testimony for the OSHA hearing on the proposed occupational standard for methylene chloride. Research Triangle Park, North Carolina, National Institute of Environmental Health Sciences. Kari FW, Foley JF, Seilkop SK, Maronpot RR, & Anderson MW (1993) Effect of varying exposure regimens on methylene chloride induced lung and liver tumors in female B6C3F1 mice. Carcinogenesis, 14(5): 819-826. Karickhoff SW (1981) Semi-empirical estimation of sorption of hydrophobic pollutants on natural sediments and soils. Chemosphere, 10(8): 833-847. Karlsson J-E, Rosengren LE, Kjellstrand P, & Haglid KG (1987) Effects of low-dose inhalation of three chlorinated aliphatic organic solvents on deoxyribonucleic acid in gerbil brain. Scand J Work Environ Health, 13: 453-458. Kashin LM, Makotchenko VM, Malinina-Putsenko VP, Mikhailovskaja LF, & Shmuter LM (1980) [Experimental and clinico-hygienic investigations of methylene chloride toxicity.] Vrach Delo, 1: 100-103 (in Russian). Kawasaki M (1980) Experiences with the test scheme under the chemical control law of Japan: An approach to structure-activity correlations. Ecotoxicol Environ Saf, 4: 444-454. Kelley RD (1985) Synthetic organic compound sampling survey of public water supplies. Washington, DC, Environmental Protection Agency (NTIS PB85-214427). Kelly M (1988) Case reports of individuals with oligospermia and methylene chloride exposures. Reprod Toxicol, 2: 13-17. Kim YC & Carlson GP (1986) The effect of an unusual workshift on chemical toxicity, I. Studies on the exposure of rats and mice to dichloromethane. Fundam Appl Toxicol, 6: 162-171. Kimura ET, Ebert DM, & Dodge PW (1971) Acute toxicity and limits of solvent residue for sixteen organic solvents. Toxicol Appl Pharmacol, 19: 699-704. Kirschman JC, Brown NM, Coots RH, & Morgareidge K (1986) Review of investigations of dichloromethane metabolism and subchronic oral toxicity study as the basis for the design of chronic oral studies in rats and mice. Food Chem Toxicol, 24: 943-949. Kirwin CJ, Thomas WC, & Simmon VF (1980) In vitro microbiological mutagenicity studies of hydrocarbon propellants. J Soc Cosmet Chem, 31(7): 367-370. Kitchin KT & Brown JL (1989) Biochemical effects of three carcinogenic chlorinated methanes in rat livers. Teratogen Carcinogen Mutagen, 9: 61-69. Kjellstrand P, Bjerkemp M, Adler-Maihofer M, & Holmquist B (1986) Effects of methylene chloride on body and organ weight and plasma butyrylcholinesterase activity in mice. Acta Pharmacol Toxicol, 59: 73-79. Kjellstrand P, Mansson L, Holmquist B, & Jonsson I (1990) Tolerance during inhalation of organic solvents. Pharmacol Toxicol, 66: 409-414. Klaassen CD & Plaa GL (1966) Relative effects of various chlorinated hydrocarbons on liver and kidney function in mice. Toxicol Appl Pharmacol, 9: 139-151. Klaassen CD & Plaa GL (1967) Relative effects of various chlorinated hydrocarbons on liver and kidney function in dogs. Toxicol Appl Pharmacol, 10: 119-131. Klecka GM & Gonsior SJ (1984) Nonenzymatic reductive dechlorination of chlorinated methanes and ethanes in aqueous solution. Chemosphere, 13: 391-402. Klecka GM (1982) Fate and effects of methylene chloride in activated sludge. Appl Environ Microbiol, 44: 701-707. Klimmer OR (1968) Working paper on dichloromethane. Presented to the Committee on Foreign Substances in Food of the German Research Council, Bad Godesberg, 5 July 1968. Bonn, German Research Council (in German). Kluwe WM, Harrington FW, & Cooper SE (1982) Toxic effects of organohalide compounds on renal tubular cells in vivo and in vitro. J Pharmacol Exp Ther, 220: 597-603. KNIE (1988) The dynamic daphnia test - practical experience in the monitoring of inland waters. Gewasserschutz-Wasser-Abwasser, 102: 341-357 (in German). Koch M, Dolfing J, Whurmann K, & Zehnder AJB (1983) Pathways of propionate degradation by enriched methanogenic cultures. Appl Environ Microbiol, 45: 1411-1414. Könemann H (1981) Quantitative structure-activity relationships in fish toxicity studies, part 1: Relationship for 50 industrial pollutants. Toxicology, 19: 209-211. Kool HJ, Van Kreijl CF, & Zoeteman BCJ (1982) Toxicology assessment of organic compounds in drinking water. CRC Crit Rev Environ Control, 12: 307-350. Kopfler FC, Melton RG, Lingg RD, & Coleman WE (1977) Human exposure to water pollutants. Adv Environ Sci Technol, 8: 419-433. Kozena L, Frantik E, & Vodickova A (1990) Methylene chloride does not impair vigilance performance at blood levels simulating limit exposure. Acta Nerv Super, 32: 35-37. Kramers PGN, Mout HCA, Bissumbhar B, & Mulder CR (1991) Inhalation exposure in Drosophila mutagenesis assays: experiments with aliphatic halogenated hydrocarbons, with emphasis on the genetic activity profile of 1,2-dichloroethane. Mutat Res, 252:17-33. Kubic VL & Andera MW (1978) Metabolism of the dichloromethane to carbon monoxide III. Studies on the mechanism of the reaction. Blochem Pharmacol, 27: 2349-2355. Kubic VL, Anders MW, Engel RR, Bertow CH, & Caughey WS (1974) Metabolism of dichloromethanes to carbon monoxide I. In vivo studies. Drug Metab Dispos, 2: 53-57. Kubic VL & Anders MW (1975) Metabolism of dihalomethanes to carbon monoxide. II. In vitro studies. Drug Metab Dispos, 3: 104-112. Kühn R, Pattard M, Pernak KD, & Winter A (1989) Results of the harmful effects of selected water pollutants (anilines, aliphatic compounds) to Daphnia magna. Water Res, 23: 495-499. Kühn R (1979) Results of ecotoxicological testing of about 200 selected compounds. Paris, Organisation for Economic Co-operation and Development, Chemicals Testing Programme (ECO 22). Kurppa K, Kivisto H, & Vainio H (1981) Dichloromethane and carbon monoxide inhalation: carboxyhaemoglobin addition and drug metabolising enzymes in rat. Int Arch Occup Environ Health, 49: 83-87. Kurppa K & Vainio H (1981) Effects of intermittent dichloromethane inhalation on blood carboxyhaemoglobin concentration and drug metabolizing enzymes in rat. Res Commun Chem Pathol Pharmacol, 32: 535-544. Kutob SD & Plaa GL (1962) A procedure for estimating the hepatoxic potential of certain industrial solvents. Toxicol Appl Pharmacol, 4: 354-361. Kuzelova M & Vlasak R (1966) [The effect of methylene chloride on the health of workers in production of film-foils and investigation of formic acid as a methylene-dichloride metabolite]. Prac Lek, 18: 167-170 (in Czech). Kwa HG, Van der Gugten AA, & Verhofstad F (1974) Radioimmunoassay of rat prolactin. Prolactin levels of rats with spontaneous pituitary tumours, primary oestrogen-induced pituitary tumours or pituitary transplants. Eur J Cancer, 5: 571-579. Laham S (1978) Toxicological studies on dichloromethane, a solvent simulating carbon monomide poisoning. Toxicol Eur Res, 1: 63-73. Landry TD, Burek JD, Bell TJ, & Wolfe EL (1981) Methylene chloride: An acute inhalation toxicity study in rats. Midland, Michigan, Dow Chemical Company (Internal report). Lanes SF, Cohen A, Rothman KJ, Dreyer NA, & Soden KJ (1990) Mortality of cellulose fiber production workers. Scand J Work Environ Health, 16: 247-251. Lanes SF, Rothman KG, Dreyer NA, & Soden KJ (1993) Mortality update of cellulose fiber production workers. Scand J Work Environ Health, 19(6): 426-428. Lapat-Polasko LT, McCarty PL, & Zehnder AJB (1984) Secondary substrate utilisation of methylene chloride by an isolated strain of Pseudonomas sp. Appl Environ Microbiol, 47: 825-830. Lash AA, Becker CE, So Y, & Shore M (1991) Neurotoxic effects of methylene chloride: Are they long lasting in humans? Br J Ind Med, 48: 418-426. Lazarew NW (1929) The narcotic effect of vpours from the chlorine derivatives of methane, ethane and ethylene. Arch Exp Pathol Pharmakol, 141: 19-24. Le Blanc GA (1984) Interspecies relationships of priority pollutants to water flea (Daphnia magna). Bull Environ Contam Toxicol, 24: 684-691. Le Blanc GA (1980) Acute toxicity of priority pollutants to water flea (Daphnia magna). Bull Environ Contam Toxicol, 24: 684-691. Lee Rodkey F & Collison HH (1977) Biological oxidation of [14C] methylene chloride to carbon monoxide and carbon chloride by the rat. Toxicol Appl Pharmacol, 40: 33-38. Lefevre PA & Ashby J (1989) Evaluation of dichloromethane as an indicator of DNA synthesis in the B6C3F1 mouse liver. Carcinogenesis, 10: 1067-1072. Lehman J & Paech C (1972) [Einfluss einiger lipophiler Lösungsmittel in gasförmigem Zustand auf die CO2-Fixierung durch Luzerne.] Experientia (Basel), 28: 1415-1416. Leikin JB, Kaufman D, Lipscomb JW, Burda AM, & Hryhorczuk DO (1990) Methylene chloride: Report of five exposures and two deaths. Am J Emerg Med, 8: 534-537. Leisinger T (1983) Microorganisms and xenobiotic compounds. Experientia (Basel), 39: 1183-1191. Leonardos G, Kendall D, & Barnard N (1969) Odor threshold determination of 53 odorant chemicals. J Air Pollut Control Assoc, 19: 91-95. Leuschner F, Neumann BW, & Hubscher F (1984) Report on subacute toxicological studies with dichloromethane in rats and dogs by inhalation. Arzneimittel forschung, 34: 1772-1774. Levaggi DA, Siu W, Zerrudo RV, & La Voie JW (1988) Experiences in gaseous toxic monitoring in the San Francisco Bay area. Proc APCA Annual Meet, 81: 88-95A. Libuda HG, Zabel F, Fink EH, & Becker KH (1990) Formyl chloride: UV absorption cross sections and rate constants for the reactions of Cl and OH. J Phys Chem, 94: 5860-5865. Longstaff E, Robinson M, Bradbrook C, Styles JA, & Purchase IFH (1984) Genotoxicity and carcinogenicity of fluorocarbons assessment by short- term in vitro tests and chronic exposure in rats. Toxicol Appl Pharmacol, 72(1): 15-31. Loyke HF (1973) Methylene chloride and chronic renal hypertension. Arch Pathol, 95(2): 130-131. LWA (1980) [Water quality report.] Düsseldorf, Germany, State Board for Water and Waste of North Rhein-Westphalia (in German). LWA (1981) [Water quality report.] Düsseldorf, Germany, State Board for Water and Waste of North Rhein-Westphalia (in German). LWA (1982) [Water quality report.] Düsseldorf, Germany, State Board for Water and Waste of North Rhein-Westphalia (in German). LWA (1983) [Water quality report.] Düsseldorf, Germany, State Board for Water and Waste of North Rhein-Westphalia (in German). LWA (1984) [Water quality report.] Düsseldorf, Germany, State Board for Water and Waste of North Rhein-Westphalia (in German). LWA (1989) [Water quality report.] Düsseldorf, Germany, State Board for Water and Waste of North Rhein-Westphalia (in German). LWA (1990) [Water quality report.] Düsseldorf, Germany, State Board for Water and Waste of North Rhein-Westphalia (in German). LWA (1991) [Water quality report.] Düsseldorf, Germany, State Board for Water and Waste of North Rhein-Westphalia (in German). LWA (1992) [Water quality report.] Düsseldorf, Germany, State Board for Water and Waste of North Rhein-Westphalia (in German). Lyman WJ, Reehl WF, & Rosenblatt DH ed. (1982) Handbook of Chemical Property Estimation Methods: Environmental Behavior of Organic Compounds. New York, McGraw-Hill Book Co. MacEwen JD & Vernot EM (1972) Toxic hazards research unit annual technical report. Dayton, Ohio, Wright Patterson Air Force Base. Aerospace Medical Research Laboratory (AMRL-TR-72-62). MacEwen JD, Vernot EH, & Haun CC (1972) Continuous animal exposure to dichloromethane. Dayton, Ohio, Wright Patterson Air Force Base. Aerospace Medical Research Laboratory (AMRL-TR-72-28). Makisimov GG & Mamleyeva NK (1977) [An assessment of the hazard presented by methylene chloride entering the organism percutaneously.] Absorbtion of industrial poisons through the skin, and prevention thereof.] 83-88 (in Russian). Maltoni C, Cotti G, & Perino G (1986) Experimental research on methylene chloride carcinogenesis. In: Maltoni C & Mehlman MA (eds.) Archives Research Industrial Carcinogenesis, Volume 4, Princeton, New Jersey, Princeton Scientific Publishing Co. Maltoni C, Cotti G, & Perino G (1988) Long-term carcinogenicity bioassays administered by ingestion to Sprague-Dawley rats and Swiss mice and by inhalation to Sprague-Dawley rats. Ann NY Acad Sci, 534: 352-366. Manno M, Chirillo R, Danlotti G, Cocheo V, & Albrizio F (1989) Carboxyhaemoglobin and fatal methylene chloride poisoning. Lancet, 2(8657): 274. Manno M, Rugge M, & Cockeo V (1992) Double fatal inhalation of dichloromethane. Hum Exp Toxicol, 11(6): 540-545. Masuda Y, Yano I, & Murano T (1980) Comparative studies on the hepatoxic actions of chloroform and related halogenomethanes in normal and phenobarbital-pretreated animals. J Pharm Dyn, 3: 53-64. Mattsson JL, Albee RR, & Streeter CM (1988) Evaluation of the acute neuropharmacologic effects of dichloromethane in rats. Midland, Michigan, Dow Chemical Company (Internal Report). Mattsson JL, Albee RR, & Eisenbrandt DL (1990) Neurotoxicologic evaluation of rats after 13 weeks of inhalation exposure to dichloromethane or carbon monoxide. Pharmacol Blochem Behav, 36: 671-681. McCammon CS, Glaser RA, Wells VE, Plupps FC, & Halperin WE (1991) Exposure of workers engaged in furniture stripping to methylene chloride as determined by environmental biological monitoring. Appl Occup Environ Hyg, 6: 371-379. McCarroll NE, Cortina TA, Zito MJ, & Farrow MG (1983) Evaluation of methylene chloride and vinylidene chloride in mutational assays. Environ Mutagen, 5(3): 426-427. McCarty WM (1979) Toxicity of methylene chloride to Daphnids. Midland, Michigan, Dow Chemical (DR 001-5849-099-005). McCarty LP, Flannagan DC, Randall SA, & Johnson KA (1992) Acute toxicity in rats of chlorinated hydrocarbons given via the intracheal route. Hum Exp Toxicol, 11: 173-177. McDougal JN, Jepsono GW, Clewell III HJ, MacNaughton MG, & Andersen ME (1986) A physiological pharmacokinetic model for dermal absorption of vapors in the rat. Toxicol Appl Pharmacol, 85: 286-294. McGeorge L, Krietzman S, Bukowski G, & Hamill B (1987) Implementation and results of a mandatory state-wide program for organic contaminant analysis of delivered water. In: Proceedings of the Water Quality Technology Conference, Vol 15, pp 71-102. McGregor DB (1979) Practical experience in testing unknowns in vitro. In: Pagel GE (ed) Mutagenesis in Submammalian Systems, Status and Significance. MTP Press, pp 53-71. McKenna MJ, Saunders JH, & Boeckler WH (1980) The pharmacokinetics of inhaled methylene chloride in human volunteers. Toxicol Appl Pharmacol, Abstr 59. McKenna MJ & Zempel JA (1981) The dose-dependent metabolism of 14C-methylene chloride following oral administration to rat. Food Cosmet Toxlcol, 19: 73-78. McKenna MJ, Zempel JA, & Braun WH (1982) The pharmacokinetics of inhaled methylene chloride in rats. Toxicol Appl pharmacol, 65: 1-10. Meltzer N, Rampy L, Bielinski P, Garofalo M, & Sayad R (1977) Skin irritation, inhalation toxicity studies of aerosols using methylene chloride. Drug Cosmet Ind, 120: 38-40. Mennear JH, McConnell EE, Huff JE, Renne RA, & Giddens E (1988) Inhalation toxicology and carcinogenesis studies of methylene chloride (dichloromethane) in F344/N rats and B6C3F1 mice. Ann NY Acad Sci, 534: 343-351. Merlin G, Thiebaud H, Blake G, Sembiring S, & Alary J (1992) Mesocosms and microcosms utilization for ecotoxicity evaluation of dichloromethane, a chlorinated solvent. Chemosphere, 24: 37-50. Meyer DJ, Coles B, Pemble SE, Gilmore KS, Fraser GM, & Kitterer B (1991) Theta, a new class of glutathione transferases purified from rat and man. Biochem J, 274: 409-414. Michael LC, Pellizzari ED, & Wiseman RW (1988) Development and evaluation of a procedure for determining volatile organics in water. Environ Sci Technol 22: 565-570. Miller L, Pateras V, Friederici H, & Engel G (1985) Acute tubular necrosis after inhalation exposure to methylene chloride. Report of a case. Arch Intern Med, 145: 145-146. Mirer FE, Silverstein M, & Park R (1988) Methylene chloride and cancer of the pancreas [letter]. J Occup Med, 30: 475-476. Moody DE, James JL, Clawson GA, & Smuckler EA (1981) Correlations among the changes in hepatic microsomal components after intoxication with alkyl halides and other hepatoxins. Mol Pharmacol, 20: 685-693. Morris JB, Smith FA, & Garman RH (1979) Studies on methylene chloride- induced fatty liver. Exp Mol Pathol, 30: 386-393. Moskowitz S & Shapiro H (1952) Fatal exposure to methylene chloride vapor. Am J Ind Hyg Occup Med, 5: 116-123. Mueller S, Weise M, Krug T, & Hoffmann P (1991) Adrenergic cardiovascular actions in rats as affected by dichloromethane exposure. Biomed Blochim Acta, 50: 307-311. Myhr B, McGregor D, Bowers L, Riach C, Brown AG, Edwards I, McBride D, Martini R, & Caspary WJ (1990) L5178Y mouse lymphoma cell mutation assay results with 41 compounds. Environ Mol Mutagen, 16 (Suppl 18): 138-167. Namkung E & Rittmann BE (1987) Estimating volatile organic compound emissions from publicly owned treatment works. J WPCF, 59: 670-678. Narotsky MG, Hamby BT, Mitchell DS, & Kavlock RJ (1992) Full-litter resorptions caused by low-molecular weight hydrocarbons in F-344 rats [abstract 67]. Teratology, 45: 472. Neely WB (1964) Metabolic fate of formaldehyde-14C intraperitoneally administered to the rat. Biochem Pharmacol, 13: 1137-1142. Negherbon WO (1959) Methylene chloride. In: Negherborn WO (ed.) Handbook of toxicology III: insecticides, a compendium. London, Saunders, pp 485-486. Nellor MH, Baird RD, & Smyth JR (1985) Health effects of indirect potable water reuse. J Am Water Works Assoc, 77(7): 88-96. Nendza M & Seydel JK (1988) Multivariate data analysis of various biological test systems used for the quantification of ecotoxic compounds. Quant Struct-Act Relatsh, 7: 165-174. Nestmann ER, Otson R, Williams DT, & Kowbel DJ (1981) Mutagenicity of paint removers containing dichloromethane. Cancer Lett, 11: 295-302. Nestmann ER, Lee EGH, Matula TI, Douglas GR, & Mueller JC (1980) Mutagenicity of constituents identified in pulp and paper mill effluents using the Salmonella/mammalian-microsome assay. Mutat Res, 79: 203-212. Neuhauser EF, Loehr RC, Malecki MR, Milligan DR, & Durkin PR (1985) The toxicity of selected organic chemicals to the earthworm Eisenia fetida. J Environ Qual, 14: 383-388. Neumann F (1991 ) Early indicators for carcinogenesis in sex-hormone- sensitive organs. Mutat Res, 248: 341-356. Nicola RM, Branchflower R, & Pierce D (1987) Chemical contaminants in bottomfish. J Environ Health, 49: 342-347. NIOSH (1976) Criteria for a Recommended Standard ... Occupational Exposure to Methylene Chloride. Cincinnati, Ohio, National Institute of Occupational Safety and Health (DHEW Publication No. 76-138). NIOSH (1987) Method No. 1005, Revision 1. NIOSH Manual of Analytical Methods, 3rd ed. Cincinnati, Ohio, National Institute for Occupational Safety and Health. Nishio A, Yajema S, Yahogi M, Sasaki Y, Sawano Y, & Miyao N (1984) [Studies on the teratogenicity of dichloromethane in rats]. Gakujutsu Hikoku-Kagoshima Daigaku Nogakubu, 34: 95-103 (in Japanese). Nitschke KD, Stevens GA, Kociba RJ, Keyes DG, & Rampy LW (1981) Methylene chloride: a four week inhalation toxicity study in rats, hamsters and mice. Midland, Michigan, Dow Chemical Company (Internal report). Nitschke KD, Burek JD, Bell TJ, Kociba RJ, Rampy LW, & McKenna MJ (1988a) Methylene chloride: A 2-year inhalation toxicity and oncogenicity study in rats. Fundam Appl Toxicol, 11: 48-59. Nitschke KD, Eisenbrandt DL, Lomax LG, & Rao KS (1988b) Methylene chloride: Two-generation inhalation reproductive study in rats. Fundam Appl Toxicol, 11: 60-67. Norpoth K, Witting U, & Springorum M (1974) Induction of microsomal enzymes in the rat liver by inhalation of hydrocarbon solvents. Int Arch Arbeitsmed, 33: 315-321. Novak JJ & Hain JR (1990) Furniture stripping vapor inhalation fatalities: two case studies. Appl Occup Environ Hyg, 5: 843-847. Novakova V, Musil J, Buckiova D, Taborsky O, Sollova H, & Vyborny P (1981) Effect of tetrachloromethane and other chlorinated hydrocarbons on the hepatic metabolism in the isolated perfused rat liver. J Hyg Epidemiol Microbiol Immunol, 25: 369-383. NTP (US National Toxicology Program) (1986) Toxicology and carcinogenesis studies of dichloromethane (methylene chloride) (CAS No.75-09-2 ) in F344/N rats and B6C3F1 mice (inhalation studies). Research Triangle Park, North Carolina, National Toxicology Programme (Technical Report No. 306; NIH Publication No. 86-2562). Otson R, Williams DT, & Bothwell PD (1982) Volatile organic compounds in water at thirty Canadian potable water treatment facilities. J Assoc Off Anal Chem, 65: 1370-1375. Ott MG, Skory LK, Holder BB, Bronson JM, & Williams PR (1983) Health evaluation of employees occupationally exposed to methylene chloride. Scand J Work Environ Health, 9 (Suppl 1): 1-38. Ottenwalder H & Peter H (1989) DNA binding assay of methylene chloride in rats and mice [letter]. Arch Toxicol, 63: 162-163. Ottenwalder H, Jager R, Thier R, & Bolt HM (1989) Influence of cytochrome P-450 inhibitors on the inhalative uptake of methyl chloride and methylene chloride in male B6C3F1 mice. Arch Toxicol, 13 (Suppl): 258-261. Page BD & Charbonneau CF (1977) Gas chromatographic determination of residual methylene chloride and trichloroethylene in decaffeinated instant and ground coffee with electrolytic conductivity and electron capture detection. J Assoc Off Anal Chem, 60: 710-715. Page BD & Charbonneau CF (1984) Headspace gas chromatographic determination of residual methylene chloride in decaffeinated tea and coffee with electronic conductivity detection. J Assoc Off Anal Chem, 67: 757-761. Pankow D, Gutewort R, Glatzel W, & Tieze K (1979) Effect of dichloromethane on the sciatic motor conduction velocity of rats. Experientia (Basel), 35: 373-374. Pankow D, Dretschmer S, & Weise M (1991) Effect of pyrazole on dichloromethane metabolism to carbon monoxide. Recent Developments in Toxicology: Trends, Methods and Problems. Arch Toxicol, 14 (Suppl): 246-248. Pankow D & Jagielki S (1993) Effect of methanol or modifications of the hepatic glutathione concentration on the metabolism of dichloromethane to carbon monoxide in rats. Hum Exp Toxicol, 12(3): 227-231. Pellizzari ED, Hartwell TD, Harris BS, Waddell RD, Whitaker DA, & Erickson MD (1982) Purgeable organic compounds in mother's milk. Bull Environ Contam Toxicol, 28: 322-328. Pennington JT & Hadfield MG (1989) Larvae of nudibranch mollusc (Phestilla sibogae) metamorphose when exposed to common organic solvents. Biol Bull, 177: 350-355. Penverne Y & Montiel A (1985) Etude des organohalogénés volatils dans les eaux souterraines du departement du Val-de-Marne (France). Trib Cebedeau, 505: 23-30. Perbellini L, Brugnone F, Grigolini L, Cunegatti P, & Tacconi A (1977) Alveolar air and blood dichloromethane concentration in shoe sole factory workers. Int Arch Occup Environ Health, 40(4): 241-247. Perocco P & Prodi G (1981) DNA damage by haloalkanes in human lymphocytes cultured in vitro. Cancer Lett, 13: 213-218. Peterson JE (1978) Modeling the uptake, metabolism, and excretion of dichloromethane by man. Am Ind Hyg Assoc J, 39: 41-47. Plaa GL & Larson RE (1965) Relative nephrotoxic properties of chlorinated methane, ethane, and ethylene derivatives in mice. Toxicol Appl Pharmacol, 7: 37-44. Pleil JD & McClenny WA (1990) Canister-based sampling and subsequent GC/MS analysis for measurement of trace-level volatile organohalogen compounds. In: Proceedings of the 10th International Meeting Dioxin 90-Organohalogen compounds, Vol 2, pp 411-414. Plumb RH Jr (1987) A comparison of ground water monitoring from CERCLA and RCRA sites. Ground Water Monit Rev, Fall 1987: 94-100. Poplawski-Tabarelli S & Uehleke H (1982) Inhibition of microsomal drug oxidations by aliphatic hydrocarbons: correlation with vapour pressure. Xenobiotica, 12: 55-61. Portier CJ & Kaplan NL (1989) Variability of safe dose estimates when using complicated models of the carcinogenic process. Fundam Appl Toxicol, 13: 533-544. Post W, Kromhout H, Heederik D, Noy D, & Duijzentkunst RS (1991) Semiquantitative estimates of exposure to methylene chloride and styrene: The influence of quantitative exposure data. Appl Occup Environ Hyg, 6(3): 197-204. Price P J, Hassett CM, & Mansield JI (1978) Transforming activities of trichloroethylene and proposed industrial alternatives. In Vitro, 14: 290. Putz VR, Johnson BL, & Setzer JV (1976) A comparative study of the effects of carbon monoxide and methylene chloride on human performance. J Environ Pathol Toxicol, 2: 97-112. Puurunen J & Sotaniemi E (1985) Usefulness of follow-up liver-function tests after dichloromethane exposure. Lancet, 1: 822. Radding SB, Liv DH, Johnson HL, & Mill T (1977) Review of the environmental fate of selected chemicals. Washington, DC, US Environmental Protection Agency (EPA-560/5-77-003). Raje R, Basso M, Tolen T, & Greening M (1988) Evaluation of in vivo mutagenicity of low-dose methylene chloride in mice. J Am Coll Toxicol, 7: 699-703. Ranna R, Rugge R, & Cocheo V (1992) Double fatal inhalation of dichloromethane. Hum Exp Toxicol, 11: 540-545. Rapson WH, Nazar MA, & Butsky VV (1980) Mutagenicity produced by aqueous chlorination of organic compounds. Bull Environ Contam Toxicol, 24(4): 590-596. Ratney RS, Wegman DH, & Elkins HB (1974) In vivo conversion of methylene chloride to carbon monoxide. Arch Environ Health, 28: 223-226. Rayez JC, Rayez MT, Halvick P, Duguay B, & Lesclaux R (1987) A theorical study of the decomposition of halogenated alkoxy radicals. I. Hydrogen and chlorine extrusions. Chem Phys, 116: 203-213. Rebert CS, Matteuci M J, & Pryor GT(1989) Acute effects of inhaled dichloromethane on the EEG and sensory-evoked potentials of Fischer- 344 rats. Pharmacol Biochem Behav, 34: 619-629. Reitz RH, Smith FA, & Andersen ME (1986) In vivo metabolism of 14C-methylene chloride [abstract]. Toxicologist 6, A 1048. Reitz RH, Mendrala AL, & Guengerich FP (1989) In vitro metabolism of methylene chloride in human and animal tissues: use of physiologically based pharmacokinetic models. Toxicol Appl Pharmacol, 97: 230-246. Reynolds ES & Yee AG (1967) Liver parenchymal cell injury. V. Relationships between patterns of chloromethane-C14 incorporation into constituents of liver in vivo and cellular injury. Lab Invest, 16: 591-603. Rittmann BE & McCarty PL (1980) Utilization of dichloromethane by suspended and fixed-film bacteria. Appl Environ Microbiol, 39: 1225-1226. RIVM (Rijksinstituut voor Volksgezondheid en Milieuhygiene) (1986) Methylene chloride; 48 hour IC50/EC50 Daphnia magna (86/HO63) and embryotoxicity for Oryzia latipes (86/HO65) (Project No. 840820) Bilthoven, The Netherlands, National Institute of Public Health and Environmental Protection. Roberts BL & Dorough HW (1984) Relative toxicity of chemicals to the earthworm Eisenia foetida. Environ Toxicol Chem, 3: 67-78. Roberts CYC & Marshall FPP (1976) Recovery after "lethal" quantity of paint remover. Br Med J, 1: 20-21. Rodruigez Rojo A, Freiria Gandara M J, Alvarez Devesa A, Lorenzo Ferreira RA, & Bermejo Martinez F (1989) Determination of halogenated hydrocarbons in the water supply of Santiago de Compostela (Spain). Environ Technol Lett, 10(8): 717-724. Rosengren LE, Kjellstrand P, Aurell A, & Haglid KG (1986) Irreversible effects of dichloromethane on the brain after long term exposure: A quantitative study of DNA and the glial cell marker proteins S-100 and GFA. Br J Ind Med, 43: 291-299. Rossman TG, Molina M, Meyer L, Boone P, Klein CB, Wang Z, Li F, Lin WC, & Kinney PL (1991) Performance of 133 compounds in the lambda prophage induction endpoint of the Microscreen assay and a comparison with S. typhimurium mutagenicity and rodent carcinogenicity assays. Murat Res, 260: 349-367. Roth RP, Drew RT, Lo RJ, & Fouts JR (1975) Dichloromethane inhalation, carboxyhaemoglobin concentrations and drug metabolizing enzymes in rabbits. Toxicol Appl Pharmacol, 33: 427-437. Ruhe RL, Watanabe A, & Stein G (1981) Health Hazard Evaluation Report No. HHE-80-49-808: Superior Tube Company, Collegeville, PA, Cincinnati, Ohio, National Institute of Occupational Safety and Health. Ruhe RL, Singal M, & Hervin RL (1982) Health Hazard Evaluation Report No. HETA-80-79-1189.: Rexall Drug Company, St Louis, MO, Cincinnati, Ohio, National Institute of Occupational Safety and Health. Ruth H (1986) Odor thresholds and initiation levels of several chemical substances. A review. Am Ind Hyg Asspc J, 47: A141-A151. Sabel GV & Clark TP (1984) Volatile organic compounds as indicators of municipal solid waste leachate contamination. Waste Manage Res, 2: 119-130. Sahn SC & Lowther DK (1981) Pulmonary reactions to inhalation of methylene chloride: Effects on lipid peroxidation in rats. Toxicol Lett, 8(4-5): 253-256. Sanhueza E & Heicklen J (1975) Chlorine-atom sensitized oxidation of dichloromethane and chloromethane. J Phys Chem, 79: 7-11. Savolainen H, Pfäffli P, Tengen M, & Vainio H (1977) Biochemical and behavioral effects of inhalation exposure to tetrachloroethylene and dichloromethane. J Neuropathol Exp Neurol, 36: 941-949. Sawhney BL (1989) Movement of organic chemicals through landfill and hazardous waste disposal sites. Soil Science Society of America and American Society of Agronomy, pp 447-474. Scholz-Muramatsu H, Schneider V, Gaiser S, & Bardtke D (1988) Biological elimination of dichloromethane from contaminated groundwater-interference by components of the groundwater. Wat Sci Tech, 20: 393-397. Schubert R (1979) Toxicity of organohalogen compounds towards bacteria and their degradability. Spez Ber Kernforschungsaulage, 1979: 211-218. Schumacher H & Grandjean E (1960) Comparative investigations on the anaesthetic effect and acute toxicity of nine solvents. Arch Gewerbepathol Gewerbehyg, 18: 109-119. Schutz E (1960) Effects of organic liquids on the skin. Arzeimittelforschung, 10: 1027-1029. Schroeder KR, Hallier E, Peter N, & Bolt HM (1992) Dissociation of a new glutathione-S-transferase activity in human erythrocytes. Blochem Pharmacol, 43: 1671-1674. Schwetz BA, Leong BKJ, & Gehring PJ (1975) The effect of maternally inhaled trichloroethylene, perchloroethylene, methyl chloroform, and methylene chloride on embryonal and fetal development in mice and rats. Toxicol Appl Pharmacol, 32: 84-96. Scott JB, Smith FA, & Garman RH (1979) Exposure of mice to CH2Cl2 and CH3OH alone and in combination [abstract]. Toxicol Appl Pharmacol, 48: A 105. Selan FM & Evans MA (1982) Role of hepatic microtubule system in chlorinated hydrocarbon induced hepatic steatosis. Toxicologist, 2: 134 [abstract]. Selenka F & Bauer U (1978) [Survey of organochlorine compounds in water.] Forsch Ber, A27: 187-188 (in German). Serota DG, Thakur AK, Ulland BM, Kirschman JC, Brown NM, Cotts RG, & Morgareidge K (1986a) A two-year drinking-water study of dichloromethane in rodents I. Rats. Food Chem Toxicol, 24: 951-958. Serota DG, Thakur AK, Ulland BM, Kirschman JC, Brown NM, Cotts RG, & Morgareidge K (1986b) A two-year drinking-water study of dichloromethane in rodents. II. Mice. Food Chem Toxicol, 24: 959-963. Shah JJ & Heyerdahl EK (1988) National ambient volatile organic compounds (VOCs): data base update. Rep. by Nero and Associated, Portland (OR), EPA/600/3-88/010a. Research Triangle Park, North Carolina, Atmospheric Sciences Research Laboratory. Sheldon T, Richardson CR, & Elliott BM (1987) Inactivity of methylene chloride in the mouse bone marrow micronucleus assay. Mutagenesis, 2: 57-59. Shikiya J, Tsou G, Kowalski J, & Leh F (1984) Ambient monitoring of selected halogenated hydrocarbons and benzene in the California South Coast Air Basin. Proceeding of 77th Annual Meeting of the Air Pollution Control Association 24-29 June. Shmuter LM & Kashin LM (1978) Experimental study of the sensitising effect of some chlorinated aliphatic hydrocarbons. Gig Tr Prof Zabol, 3: 57-59 (in Russian). Simmon VF, Kauhanen K, & Tardill RG (1977) Mutagenic activity of chemicals identified in drinking water. Dev Toxicol Environ Sci, 2: 249-258. Singh HB, Salas LJ, & Stiles RE (1983) Selected man-made halogenated chemicals in the air and oceanic environment. J Geophys Res, 88: 3675-3683. Sinha YN (1981) Plasma prolactin analysis as a potential predictor of murine mammary tumorigenesis. In: McPike PK, Sitteri, & Welsch CW ed. Hormones and breast cancer. Cold Spring Harbor, New York, Cold Spring Laboratory (Banbury Report No. 8). Slooff W & Ros JPM (1988) Integrated criteria document: Dichloromethane. Bilthoven, The Netherlands, National Institute of Public Health and Environmental Protection (Report No. 758473009). Smith RL (1989) A computer assisted, risk-based screening of a mixture of drinking water chemicals. Trace Subst Environ Health, 22: 215-232. Soden KJ (1993) An evaluation of chronic metylene chloride exposure. J Occup Med, 35(3): 282-286. Staab HA & Datta AP (1964) Formyl chloride. Angew Chem Int ed, 3: 132. Staples CA, Frances Werner A, & Hoogheem TJ (1985) Assessment of priority pollutant concentrations in the United States using STORET database. Environ Toxicol Chem, 4: 131-142. Stern FB, Halperin WE, Hornung RW, Ringenburg VL, & McCammon CS (1988) Heart disease mortality among bridge and tunnel officers exposed to carbon monoxide. Am J Epidemiol, 128(6): 1276-1288. Stevenson MF, Chemoweth MB, & Cooper GL (1978) Effect on carboxyheamoglobin of exposure to aerosol spray paints with methylene chloride. Clin Toxicol, 12: 551-561. Stewart RD & Dodd HC (1964) Absorption of carbon tetrachloride, trichloroethylene, tetrachloroethylene, methylene chloride, and 1,1,1-trichloroethane through the human skin. Am Ind Hyg Assoc J, 25: 439-446. Stewart RD, Hake CL, & Wu A (1976) Use of breath analysis to monitor methylene chloride exposure. Scand J Work Environ Health, 2: 57-70. Stewart RD, Fisher TN, Hosko MJ, Peterson JE, Baretta ED, & Dodd HC (1972) Experimental human exposure to methylene chloride. Arch Environ Health, 25: 342-348. Stover EL & Kincannon DF (1983) Biological treatability of specific organic compounds found in chemical industry wastewaters. J Water Pollut Control Fed, 55: 97-109. Stuckey DC, Owen WF, & McCarty PL (1980) Anaerobic toxicity evaluation by batch and semi-continuous assays. J Water Pollu Control Fed, 52(4): 720-729. Stucki G, Gälli R, Ebersold H-R, & Leisinger TH (1981) Dehalogenation of dichloromethane by cell extracts of Hyphomicrobium DM2. Arch Microbiol, 130: 366-371. Stucki G (1990) Biological decomposition of dichloromethane from a chemical process effluent. Biodegradation, 1: 221-228. Svirbely JL, Highman B, Alford we, & Von Oettingen WF (1947) The toxicity and narcotic action of mono-chloro-mono-bromo-methane with special reference to inorganic and volatile bromide in blood, urine and brain. J Ind Hyg Toxlcol, 29: 382-389. Tabak HH, Quave SA, Mashni CI, & Barth EF (1981) Biodegradability studies with organic priority pollutant compounds. J Water Pollut Control Fed, 53: 1503-1518. Takashita T, lto A, Kawata F, Ito M, & lto K (1991) [Experimental studies of effects of methylene chloride on living bodies (2)]. Hochudoku, 9: 100-101 (in Japanese). Tariot PM (1983) Delirium resulting from methylene chloride exposure: Case report. J Clin Psychiatry, 44: 340-342. Taskinen H, Lindbohm M-L, & Hemminki K (1986) Spontaneous abortions among women working in the pharmaceutical industry. Br J Ind Med, 43: 199-205. Taylor G J, Drew R J, Lores EM, & Clemmer TA (1976) Cardiac depression by haloalkane propellants, solvents, and inhalation anesthetics in rabbits. Toxicol Appl Pharmacol, 38: 379-387. Tham R, Bunnfors I, Erikkson B, Larsby B, Lindgren S, & Odkvist LM (1984) Vestibulo-ocular disturbances in rats exposed to organic solvents. Acta Pharmacol Toxicol, 54: 58-63. Thiel PG (1969) The effect of methane analogues on methanogenesis in anaerobic digestion. Water Res, 3: 215-223. Thier R, Forst U, Deutschmann S, Schroeder KR, Westphal G, Hallier E, & Peter H (1991) Distribution of CH2Cl2 in human blood. Recent developments in toxicology: trends, methods and problems. Arch Toxicol, 14(Suppl): 254-258. Thief R, Pemble SM, Taylor JB, Humphreys WG, & Ketterer B (1993) Glutathione S-transf erases 5-5 expression in Salmonella typhimurium increases mutation rate caused by methylene dihalides. Pharmacol Toxicol, 73(Suppl 11): 42 (Abstract FCZ/12). Thilagar AK, Back AM, Kirby PE, Kumaroo PV, Pant KJ, Clarke JJ, Knight R, & Haworth SR (1984a) Evaluation of dichloromethane in short-term in vitro genetic toxicity assays. Environ Mutagen, 6: 418-419. Thilagar AK, Kumaroo PV, Clarke JJ, Kott S, Back AM, & Kirby PE (1984b) Induction of chromosome damage by dichloromethane in cultured human peripheral lymphocytes, CHO cells and mouse lymphoma L5178Y cells. Environ Mutagen, 6: 422. Thilagar AK & Kumaroo PV (1983) Induction of chromosome damage by methylene chloride in CHO cells. Mutat Res, 116: 361-367. Toftgard R, Nilsen OG, & Gustafsson J-A (1982) Dose dependent induction of rat liver microsomal P-450 and microsomal enzymatic activities after inhalation of toluene and dichloromethane. Acta Pharmacol Toxicol, 51: 108-114. Trevors JT (1985) Effect on methylene chloride on respiration and electron transport system (ETS) activity in freshwater sediment. Bull Environ Contam Toxicol, 34: 239-245. Trueman RW & Ashby J (1987) Lack of UDS activity in the livers of mice and rats exposed to dichloromethane. Environ Mol Mutagen, 10: 189-195. Trueman RW, Burlinson B, Lefevre PA, & Ashby J (1987) Inactivity of methylene chloride as a UDS-initiating agent in mouse and rat hepatocytes exposed in vivo and in vitro. Mutat Res, 181: 346 [abstract]. Truhaut R, Boudene C, Jounany JM, & Bouant A (1972) The application of the physiogram to the investigation of the acute toxicity of chlorinated solvents. Eur J Toxicol, 5(5): 284-292. Tsuruta H (1975) Percutaneous absorption of organic solvents. 1. Comparative study of the in vivo percutaneous absorption of chlorinated solvents in mice. Ind Health, 13: 227-236. Ulanova IP & Yonovskayo BI (1959) Changes in the ascorbic acid content of the internal organs of white rats in response to the action of chlorinated hydrocarbons II. Effect of methylene chloride. Biull Eksp Biol Med, 48: 846. Uotila L & Koivusalo M (1974a) Formaldehyde dehydrogenase from human liver. J Biol Chem, 249: 7653-7663. Uotila L & Koivusalo M (1974b) Distribution and properties of S-formylglutathione hydrolase from human liver. J Biol Chem, 249: 7664-7672. US EPA (1980) Ambient water quality criteria for halomethanes. Washington, DC, US Environmental Protection Agency (EPA 440/5-80-051; NTIS PB81-117 624). US EPA (1981) Environmental risk assessment of dichloromethane. Washington, DC, US Environmental Protection Agency (Draft report). US EPA (1982a) Purgeable halocarbons-method 601. In: Methods for organic chemical analysis of municipal and industrial wastewater. Cincinnati, Ohio, US Environmental Protection Agency, Environmental Monitoring and Support Laboratory (EPA-600/4-82-057). US EPA (1982b) Purgeables method 624. in: Methods for organic chemical analysis of municipal and industrial wastewater. Cincinnati, Ohio, US Environmental Protection Agency, Environmental Monitoring and Support Laboratory (EPA-600/4-82-057). US EPA (1985) Health assessment document for dichloromethane (Methylene chloride). Washington, DC, US Environmental Protection Agency (EPA/600/8-82/OO4F). US EPA (1986a) Gas chromatography/mass spectrometry for volatile organics-method 8240. In: Test methods for evaluating solid waste, 3rd ed. Washington, DC, US Environmental Protection Agency, Office of Solid Waste and Emergency Response (SW-846). US EPA (1986b) Halogenated volatile organics-method 8010. In: Test methods for evaluating solid waste, 3rd ed. Washington, DC, US Environmental Protection Agency, Office of Solid Waste and Emergency Response (SW-846). US EPA (1987) Household solvent products: A national usage survey. Washington, DC, US Environmental Protection Agency. US EPA (1989a) Measurement of purgeable organic compounds in water by capillary column gas chromatography/mass spectrometry-Method 524.2. In: Methods for the determination of organic compounds in drinking water. Cincinnati, Ohio, US Environmental Protection Agency, Environmental Monitoring Systems Laboratory (EPA/600/4-88/039). US EPA (1989b) Measurement of purgeable organic compounds in water by packed column gas chromatography/mass spectrometry-Method 524.1. In: Methods for the determination of organic compounds in drinking water. Cincinnati, Ohio, US Environmental Protection Agency, Environmental Monitoring Systems Laboratory (EPA/600/4-88/039). US EPA (1989c) Volatile halogenated organic compounds in water by purge and trap gas chromatography-method 502.1. In: Methods for the determination of organic compounds in drinking water. Cincinnati, Ohio, US Environmental Protection Agency, Environmental Monitoring Systems Laboratory (EPA/600/4-88/039). US EPA (1989d) Volatile organic compounds in water by purge and trap capillary column gas chromatography with protoionization and electrolytic conductivity detectors in series-method 502.2. In: Methods for the determination of organic compounds in drinking water. Cincinnati, Ohio, US Environmental Protection Agency, Environmental Monitoring Systems Laboratory (EPA/600/4-88/039). US EPA (1990) Paint stripping, options selection paper. Washington, DC, US Environmental Protection Agency. Van Beck L (1990) Investigation of a possibility to reduce the use of rabbits in skin irritation tests; experiments with dichloromethane, trichloroethylene, tetrachloroethylene and 1,1,1-trichloroethane. Doc. 56645/34/90, rep. V 89.265. Zeist, The Netherlands, TNO-CIVO Institutes. Van Haut H & Prinz B (1979) [Evaluation of the relative harmfulness to plants of organic air pollutants in the LIS short-term test.] Staub- Reinhalt Luft, 39: 408-414 (in German). Van de Graaff S (1986) [Estimation of harmful effects of environmentally relevant compounds in river water.] Münch Beitr Abwasser-Fisch-Flussbiol, 40: 556-572 (in German). Vannelli T, Logan M, Arciero DM, & Hooper AB (1990) Degradation of halogenated aliphatic compounds by the ammonia oxidzing bacterium Nitrosomas europaea. Appl Environ Microbiol, 56: 1169-1171. Vargas C & Ahlert RC (1987) Anaerobic degradation of chlorinated solvents. J Water Pollut Control Fed, 59(11): 964-968. Veith GD, Macek KJ, Petrocelli SR, & Carroll J (1980) An evaluation of using partition coefficients and water solubility to estimate bioconcentration factors for organic chemicals in fish. Philadelphia, Pennsylvania, American Society for Testing and Materials, pp 116-129 (ASTM-STP 707). Veith GD & Kosian P (1983) Estimating bioconcentration potential from octanol/water partition coefficients. Physical behaviour of PCBs in Great Lakes. Ann Arbor, Michigan, Ann Arbor Science Publishers, pp 269-282. Verrett M J, Scott WF, Reynaldo EF, Alterman EK, & Thomas CA (1980) Toxicity and teratogenicity of food additive chemicals in the developing chicken embryo. Toxicol Appl Pharmacol, 56: 265-273. Verschueren K (1983) Handbook of Environmental data on Organic Chemicals, 2nd ed. New York, Van Nostrand Reinhold Publishers, pp 848-849. Verschuuren HG & Wilmer JW (1983) Neurotoxicity of 1,1,1- trichloroethane questioned. Scand J Work Environ Health, 16: 144-146. Volskay VT & Grady CPL (1988) Toxicity of selected RCRA compounds to activated sludge microorganisms. J Water Pollut Control Fed, 60: 1850-1856. Von Oettingen WF, Powell CC, Sharpless NE, Alford WC, & Pecora LJ (1950) Comparative studies on the toxicity and pharmacodynamic action of chlorinated methanes with special reference to their physical and chemical characteristics. Arch Int Pharmacodyn Ther, 81: 17-34. Vosovaja MA, Maljorowa LR, & Yenikeyera KM (1974) Levels of methylene chloride in biological fluids of pregnant or lactating workers in an industrial rubber products company. Gig Tr Prof Zabol, 4: 42-43. Weast RC, Astle M J, & Beyer WH (eds) (1988) CRC Handbook of chemistry and physics. 69th ed. (1988-1989). Boca Raton, Florida, CRC Press, pp C-161., D-212. Weber M, Martin A, Bollaert P-E, Bauer Ph, Leroy F, Meley M, Mur J-M, Carry C, & Lambert H (1990) Intoxication aiguä par chlorure de méthylène et méthanol par voie percutanée. Arch Mal Prof, 51: 103-106. Weinstein RS, Boyd D, & Back KC (1972) Effects of continuous inhalation of dichloromethane in the mouse: morphologic and functional observations. Toxicol Appl Pharmacol, 23: 660-679. Weinstein RS & Diamond SS (1972) Hepatotoxicity of dichloromethane (methylene chloride) with continuous exposure at a low dose level. In: Proceedings of the 3rd Annual Conference on Environmental Toxicology. Dayton, Ohio, Wright-Patterson Air Force Base, Aerospace Medical Research Laboratory (AMRL-TR-72-130, 209-220). Weiss G (1967) Toxic encephalosis in occupational contact with methylene chloride. Zent.bl Arbeitsmed Arbeitsschutz, 17(9): 282-285. Wells GG & Waldron HA (1984) Methylene chloride burns. Br J Ind Med, 41: 420. Welsch CW (1985) Host-factors affecting the growth of carcinogen- induced rat mammary carcinomas: a review and tribute to Charles Brenton Huggins. Cancer Res, 45: 3415-3443. Welsch CW, Jenkins JW, & Mertes J (1970) Increased incidence of mammary tumors in female rat grafted with multiple pituitaries. Cancer Res, 30: 1024-1029. Welsch CW & Nagasawa H (1977) Prolactin and murine mammary tumorigenesis: a review. Cancer Res, 37: 951-963. Westbrook-Collins B, Allen JW, Sharief Y, & Campbell J (1990) Further evidence that dichloromethane does not induce chromosome damage. J Appl Toxicol, 10: 79-81. Westbrook-Collins B, Allen JW, Kligerman A, Campbell JA, Erexson GL, Kari F, & Zeiger E (1989) Dichloromethane-induced cytogenetic damage in mice. Environ Mol Mutagen, 14(Suppl 15): [abstact 630]. Wesforook-Collins B, Campbell JA, Poorman PA, Sharief Y, & Allen JW (1988) SCE, chromosome aberration, and synaptonemal complex analyses in mice exposed to dichloromethane. Environ Mol Mutagen, 11(Suppl 11): 112 [abstract 2741. WMO (World Meteorological Organization) (1991) Scientific assessment of ozone depletion: 1991. Report No 25, 8.8. WMO, Geneva, p 8.8. Wood PR, Parsons FZ, DeMarco J, Harween HJ, Lang RF, Payan IL, & Ruiz MC (1981) Introductory study of the biodegradation of the chlorinated methane, ethane, and ethene compounds. Presented at the American Water Works Association Meeting, June 1981. Woodrow JE, McChesney MM, & Seiber JN (1988) Determination of methyl bromide in air samples by headspace gas chromatography. Anal Chem, 60: 509-512. Yagafarova AB, Ivanova TS, & Khabirova FY (1981) The toxic allergic action of hydrocarbons on the eyes. Kazanski Med Zh, 67: 72-74. Yesair DW, Jacques D, Schepspis P, & Liss RH (1977) Dose-related pharmacokinetics of 14C methylene chloride in mice. Fed Proc, 36: 998. Young DR, Gossett RW, Baird RB, Brown DA, Taylor PA, & Mille MJ (1983) Waste water inputs and marine bioaccumulation of priority pollutant organics off Southern California, USA. In: Jolley RL ed. Proceedings of the 4th Conference on Water Chlorination. Volume 4: Environmental Impact and Health Effect - Part 2: Environmental health and risk. Ann Arbor, Michigan, Ann Arbor Science Publishers. Zahm SH, Stewart ZP, & Blair A (1987) A study of mortality among workers exposed to methylene chloride. Feasibility report. Bethesda, Maryland, US National Cancer Institute. Zeiger E, Haseman JK, Shelby MD, Margolin BH, & Tennant RW (1990) Evaluation of four in vitro genetic toxicity tests for predicting rodent carcinogenicity: Confirmation of earlier results with 41 additional chemicals. Environ Mol Mutagen, 16: 1-14. Zielenska M, Ahmed A, Pienkowska M, Anderson M, & Glickman BW (1993) Mutational specificlties of environmental carcinogens in the LACL gene of Escherichia coli. VI. Analysis of methylene chloride induced mutational distribution in UVR + UVRB-strains. Carcinogenesis, 14(5): 789-794. Zoeteman BC, Harmsen K, Linders JB, Morra CF, & Slooff W (1980) Persistent organic pollutants in river water and ground water of the Netherlands. Chemosphere, 9: 231-249. RESUME 1. Identité, propriétés physiques et chimiques, et méthodes d'analyse Le chlorure de méthylène (dichlorométhane) est un liquide limpide, ininflammable et extrêmement volatil qui possède une puissante odeur éthérée. Lorsqu'il est pur et anhydre, ce composé est très stable. Le chlorure de méthylène s'hydrolyse lentement en présence d'humidité, pour donner une petite quantité de chlorure d'hydrogène. Le chlorure de méthylène du commerce est généralement additionné de petites quantités de stabilisants afin d'en éviter la décomposition. Il existe des méthodes d'analyse pour le dosage du chlorure de méthylène dans les milieux biologiques et les échantillons prélevés dans l'environnement. Dans tous les cas, on fait appel à la chromatographie en phase gazeuse avec un détecteur convenable. On obtient ainsi des limites de détection très basses (par exemple dans les denrées alimentaires 7 ng/échantillon; dans l'eau 0,01/µg/litre; dans l'air 1,76/µg/m3 (0,5 ppb); dans le sang 0,022 mg/litre). 2. Sources d'exposition humaine et environnementale On estime à 570 000 tonnes la production annuelle mondiale de chlorure de méthylène. On l'utilise la plupart du temps comme solvant des graisses, des matières plastiques et des liants pour peinture, en particulier à cause de sa volatilité et de sa stabilité. Dans l'ensemble du monde, il est utilisé à 20-25% dans des aérosols, à 25% comme décapant des peintures, à 35-40% comme solvant au cours des différentes opérations de l'industrie chimique et pharmaceutique, et enfin à 10-15% dans diverses applications allant de la fabrication de mousse de polyuréthane au décapage des métaux. Son utilisation tend à augmenter, tout au moins en Europe de l'Ouest. Les émissions atmosphériques de chlorure de méthylène proviennent à plus de 99% de son utilisation comme produit final par diverses industries ou encore comme décapant des surfaces peintes et comme constituant des bombes aérosols à usage domestique. 3. Transport, distribution et transformation dans l'environnement En raison de sa forte volatilité, la majeure partie du chlorure de méthylène libéré dans l'environnement se répartit dans l'atmosphère où il est décomposé en l'espace de six mois par réaction avec des radicaux hydroxyles d'origine photochimique. Dans l'eau, il est décomposé par voie abiotique beaucoup plus lentement qu'il ne s'évapore. On a montré que le chlorure de méthylène disparaissait rapidement du sol et des eaux souterraines. Grâce à divers systèmes d'épreuve on a pu établir les modalités de la décomposition aérobic et anaérobie du chlorure de méthylène. Sa biodécomposition complète est rapide, notamment en aérobiose, sous l'action de cultures bactériennes acclimatées (par exemple 49 à 66% de minéralisation en 50 heures en présence de boues d'effluents municipaux acclimatées). Dans les réacteurs biologiques, la décomposition peut atteindre 10% à l'heure. Rien n'indique qu'il y ait une bioaccumulation ou une bioamplification importante. 4. Concentrations dans l'environnement et exposition humaine On a décelé la présence de chlorure de méthylène dans l'air ambiant de régions rurales et de zones écartées, à la concentration de 0,07-0,29/µg/m3. Dans les zones de banlieue, la concentration moyenne est inférieure à 2 µ/m3 et dans les zones urbaines, elle est inférieure à 15 µg/m3. A proximité de décharges jugées dangereuses, on a trouvé des concentrations allant jusqu'à 43 µg/m3. Les précipitations peuvent également contenir du chlorure de méthylène. Le chlorure de méthylène pénètre dans l'environnement aquatique par suite de la décharge d'eaux résiduaires provenant des diverses industries et on en a retrouvé dans les eaux superficielles et souterraines ainsi que dans les sédiments. S'il y a exposition au chlorure de méthylène de personnes appartenant à la population générale, c'est par suite de son utilisation dans certains produits de consommation tels que les décapants pour peinture, dont l'emploi peut entraîner la présence de teneurs relativement importantes dans l'air intérieur. En ce qui concerne l'exposition professionnelle au cours de la production de chlorure de méthylène, elle se produit essentiellement au cours du remplissage et du conditionnement (la fabrication s'effectue en circuit fermé). Du fait de l'utilisation de ce composé comme décapant pour peinture, il peut également y avoir exposition professionnelle lors de la préparation de ces décapants, lors de la fabrication de certains équipements et également lorsqu'on procède au décapage du mobilier. Le chlorure de méthylène est largement utilisé comme solvant lors de la préparation de différents produits, en particulier dans les industries citées à la section 1.2. La surveillance biologique de l'exposition au chlorure de méthylène peut s'effectuer par dosage du solvant lui-même dans l'air expiré ou dans le sang. Toutefois, étant donné que la production de monoxyde de carbone constitue le facteur limitant du risque en cas d'exposition supérieure à 3 ou 4 heures par jour, il vaut mieux que la surveillance biologique s'effectue soit par dosage du monoxyde de carbone dans l'air expiré, soit par dosage de la carboxyhémoglobine (CO-Hb) dans le sang. Toutefois cette méthode ne vaut que pour les sujets non fumeurs. Les prélèvements doivent s'effectuer environ 0 à 2 heures après l'exposition, ou au bout de 16 heures, c'est-à-dire le matin suivant. Les taux de CO-Hb, 2 heures après cessation de l'exposition, ne devraient pas dépasser 2 à 3%, et au bout de 16 heures 1%, dans le cas d'une exposition de 8 heures à moins de 350 mg de chlorure de méthylène par m3 chez un non fumeur. 5. Cinétique et métabolisme chez les animaux de laboratoire et l'homme Le chlorure de méthylène est rapidement absorbé au niveau des alvéoles pulmonaires et pénètre dans le courant sanguin. Il est également absorbé dans les voies digestives et aussi par voie percutanée, mais cette voie est la plus lente de toutes. Le chlorure de méthylène est très rapidement excrété, en majeure partie dans l'air expiré. Il peut traverser la barrière hémato- encéphalique ainsi que le placenta et on peut le retrouver en petites quantités dans les urines ou le lait. A fortes concentrations, la majeure partie du chlorure de méthylène absorbée est expirée tel quel. Le reste est métabolisé en monoxyde, dioxyde de carbone et chlorures minéraux. La métabolisation s'effectue selon l'une ou l'autre de ces deux voies ou les deux à la fois, et la prédominance de l'une ou de l'autre dépend largement de la dose et de l'espèce animale en cause. Une de ces voies comporte un processus oxydatif, par l'intermédiaire du cytochrome P-450, et alle conduit à la production de monoxyde et de dioxyde de carbone. Il semble que cette voie soit identique chez tous les rongeurs étudiés et chez l'homme. Il s'agit de la voie prédominante aux faibles doses, mais il y a saturation à des doses relativement modérées (autour de 1800 mg/m3). Même si la dose dépasse la valeur de saturation, il n'y pas accroissement de la métabolisation par cette voie. Dans l'autre voie métabolique intervient une glutathion- transférase (GST) qui conduit à la formation de dioxyde de carbone par l'intermédiaire du formaldéhyde et du formiate. Il semble que cette voie ne prenne de l'importance que lorsque les doses dépassent la valeur de saturation de la voie oxydative "préférentielle". Chez certaines espèces (par exemple la souris), elle devient la voie principale lorsque la dose est suffisamment élevée. En revanche, chez d'autres espèces (par exemple le hamster et l'homme), elle semble n'être que peu utilisée, qu'elle que soit la dose. Les différentes interspécifiques touchant le métabolisme par la voie de la GST sont en bonne corrélation avec les différences observées selon les espèces, notamment en ce qui concerne la cancérogénicité du chlorure de méthylène. Le taux de métabolisation selon cette voie chez les espèces concernées est utilisé comme modèle cinétique pour la description du comportement métabolique du chlorure de méthylène chez diverses espèces. 6. Effets sur les êtres vivants dans leur milieu naturel Aux concentrations inférieures à 500 mg/litre, il n'y a aucune inhibition de la croissance des algues et des bactéries aérobies. Il existe des bactéries qui sont capables de croître en présence de chlorure de méthylène à des concentrations beaucoup plus élevées et notamment en solution aqueuse (section 4.2.4.1 ). Les bactéries anaérobies sont beaucoup plus sensibles; ainsi on a observé un blocage de la croissance à la dose de 1 mg/litre, dans des boues biologiques anaérobies. A la concentration de 10 mg/kg de terre, on a constaté une forte diminution de la teneur en ATP de la biomasse, notamment des champignons et des bactéries aérobies, et une inhibition transitoire de l'activité enzymatique. La dose sans effets observables était dans ce cas de 0,1 mg/kg. Le chlorure de méthylène est modérément toxique pour les lombrics (100 à 1000 µg/cm2) soumis au test de toxicité par contact sur papier filtre. Dans les sédiments, on n'a pas observé d'effets toxiques, même à des doses très élevées. Chez les plantes supérieures, on n'a pas constaté d'effets après une exposition de 14 jours à la dose de 100 mg/m3. Les poissons adultes semblent être relativement insensibles au chlorure de méthylène, même après une exposition prolongée (la CL50 à 14 jours est supérieure à 200 mg/litre). Il est difficile d'apprécier l'effet du chlorure de méthylène sur les daphnies en raison de la très grande dispersion des résultats fournis par les différentes études. La CE50 la plus basse qui ait été rapportée était égale à 12,5 mg/litre. En milieu aquatique, c'est chez les embryons de poissons et d'amphibiens que l'on a constaté la sensibilité la plus élevée avec des effets sur l'éclosion â partir de 5,5 mg/litre. 7. Effets sur les mammifères de laboratoire et les systèmes d'épreuve in vitro 7.1 Exposition unique Lorsqu'il est absorbé par inhalation ou par voie orale, le chlorure de méthylène présente une faible toxicité aiguë. Ainsi, les valeurs de la CL50 à 6 heures pour l'ensemble des espèces étudiées, se situent entre 40 200 et 55 870 mg/m3. En ce qui concerne la DL50 par voie orale, on a obtenu des valeurs allant de 1410-3000 mg/kg. Les effets aigus observés après administration de chlorure de méthylène par diverses voies d'exposition affectent essentiellement le système nerveux central (SNC) et le foie, et tous les effets observés l'ont été à des doses élevées. Des troubles neurologiques ont été observés à des concentrations supérieures ou égaies à 14 100 mg/m3, avec de légères altérations du tracé électro- encéphalographique à la dose de 1770 mg/m3. De légères modifications histologiques ont été également constatées dans le foie aux concentrations supérieures ou égaies à 17 700 mg/m3. Il est également arrivé que d'autres organes soient touchés, comme les reins ou le système respiratoire. Chez la souris, les effets au niveau pulmonaire étaient limités, après exposition à 7100 mg/m3, aux cellules de Clara. On a également fait état d'une sensibilisation cardiaque à l'arythmie induite par l'adrénaline. D'autres effets cardio-vasculaires ont également été observés, mais pas de façon systématique. 7.2 Exposition à court et à long terme Une exposition prolongée à de fortes concentrations de chlorure de méthylène (> 17 700 mg/m3) a causé des effets réversibles sur le SNC, une légère irritation oculaire et une certaine mortalité chez diverses espèces d'animaux de laboratoire. Chez le rat on a observé à 3500 mg/ma une réduction du poids corporel, cet effet étant également observé chez la souris à partir de 17 700 mg/m3. De légers effets hépatiques ont été également observés chez des chiens exposés de manière continue à des doses de 3500 mg/m3 pendant des périodes allant jusqu'à 100 jours. Après exposition intermittente, des effets ont également été observés au niveau du foie chez des rats à la dose de 3500 mg/m3 et chez des souris à 14 100 mg/m3. Les autres organes cibles sont les poumons et les reins. Chez des rats exposés à du chlorure de méthylène pendant 13 semaines par voie respiratoire à des concentrations allant jusqu'à 7100 mg/m3, on a observé aucun signe de lésion neurologique irréversible. L'administration de chlorure de méthylène par voie orale à des rats a entraîné des effets hépatiques à partir d'une dose journalière d'environ 200 mg/kg. 7.3 Irritation cutanée et oculaire Le chlorure de méthylène se révèle modérément irritant pour la peau et les yeux chez les animaux de laboratoire. 7.4 Effets toxiques sur le développement et la reproduction Le chlorure de méthylène n'est pas tératogène chez le rat ou la souris à des concentrations allant jusqu'à 16 250 mg/m3. Trois études effectuées sur ces animaux n'ont pas permis de relever d'effets se traduisant par l'apparition de malformations squelettiques ou autres anomalies du développement. Un léger effet, se manifestant par une modification du poids foetal ou maternel, a été relevé à la dose de 4400 mg/m3, et l'on a constaté qu'après la naissance, le gain de poids des rats mâles était également affecté à la dose de 0,04% dans la nourriture. Une étude toxicologique portant sur deux générations a été effectuée sur des rats qui ont été exposés par la voie respiratoire à du chlorure de méthylène, à des concentrations allant jusqu'à 5300 mg/m3, et cela pendant 17 semaines, 6 heures par jour et 5 jours par semaine. Aucun effet nocif, de quelque nature ce que soit, n'a été relevé sur la reproduction, ni sur la survie ou la croissance des ratons nouveaux-nés appartenant à la génération F0 ou F1 7.5 Mutagénicité et points d'aboutissement correspondants Dans certaines conditions d'exposition, le chlorure de méthylène se révèle mutagène pour les microorganismes procaryotes, avec ou sans activation métabolique (Salmonella ou Escherichia coli) Dans les systèmes eucaryotes, il est sans effet mais, dans un cas, il a donné des résultats légèrement positifs. On a également obtenu des résultats uniformément négatifs lors des tests de mutation génique in vitro et des tests de recherche d'une synthèse non programmée de l'ADN sur cellules mammaliennes. La recherche d'aberrations chromosomiques in vitro sur différents types de cellules a donné des résultats positif s; en revanche, des tests visant à mettre en évidence l'induction d'échanges entre chromatides soeurs ont été soit négatif s, soit ambigus. Dans leur majorité, les études in vivo publiées n'ont pas fourni d'éléments en faveur d'une mutagénicité du chlorure de méthylène (par exemple recherche d'aberrations chromosomiques, recherche de micronoyaux ou synthèse non programmée de l'ADN). Après avoir fait inhaler à des souris de fortes concentrations de chlorure de méthylène, on a observé un accroissement minime de la fréquence des échanges entre chromatides soeurs et du nombre de micronoyaux. Après administration à des rats et à des souris de fortes doses de chlorure de méthylène, on n'a pas constaté de liaison de ce composé à FADN ni de lésions de l'ADN. Ces méthodes pourraient être les plus sensibles in vivo et les meilleures d'entre elles sont capables de déceler un site d'alkylation sur 106 nucléotides. Dans les limites des épreuves à court terme actuelles, rien ne permet de conclure que le chlorure de méthylène soit génotoxique in vivo. 7.6 Toxicité chronique et cancérogénicité Le chlorure de méthylène est cancérogène pour la souris et il provoque l'apparition de rumeurs du poumon et du foie après exposition à des concentrations élevées (7100 et 14 100 mg/m3). Chez les souris qui avaient été exposées 26 semaines à la dose de 7100 mg/m3, on a constaté que l'incidence des tumeurs pulmonaires et hépatiques augmentait lorsqu'on poursuivait l'exposition pendant 78 semaines supplémentaires. Rien n'indique la présence d'effets toxiques concomitants ou d'une hyperplasie au niveau des organes cibles. Des hamsters dorés exposés pendant deux ans à du chlorure de méthylène, à des concentrations allant jusqu'à 12 400 mg/m3, n'ont présenté aucun signe d'effets cancérogènes qui soient attribuables à ce composé. Chez des rats exposés par différentes voies à du chlorure de méthylène, on a constaté un accroissement de l'incidence tumorale à certaines localisations. Ainsi, chez des rattes exposées pendant deux ans à des doses égaies soit à 5300, soit à 12 400 mg/m3, on a constaté un excès de la fréquence tumorale au niveau des glandes salivaires. Cet excès n'apparaissait que lorsque les rumeurs, toutes d'origine mésenchymateuse, étaient regroupées en vue de l'analyse statistique. Etant donné que ces rumeurs trouvent leur origine dans des cellules de types différents, l'analyse statistique utilisée s'est révélée inappropriée. En outre, il a été précisé que les rats utilisés pour cette étude avaient contracté une virose commune (sialodacryoadénite) au début de l'expérience, affection qui concerne essentiellement les glandes salivaires. Il est donc probable qu'il n'y a pas de lien causal entre ces tumeurs et l'exposition au chlorure de méthylène, mais que l'exposition à ce composé a exacerbé la réaction à l'infection au niveau de la glande salivaire. D'ailleurs cette réaction n'a pas été observée lors d'une deuxième étude au cours de laquelle des rats ont été exposés pendant toute la durée de leur vie à des doses respectivement égaies à 3500, 7100 et 14 100 mg/m3. Une autre étude au cours de laquelle des rats ont été également exposés à du chlorure de méthylène par la voie respiratoire et durant toute leur vie, à des concentrations allant jusqu'à 1770 mg/m3, n'a pas révélé de signe de cancérogénicité. Aucun signe notable de cancérogénicité n'a été non plus observé chez des rats à qui l'on avait administré du chlorure de méthylène, soit par gavage, soit par mélange à leur eau de boisson. Trois études font état d'une augmentation de l'incidence des tumeurs mammaires bénignes chez des rats exposés à du chlorure de méthylène; dans une des études le composé a été administré par garage tandis que dans les deux autres l'exposition a eu lieu par la voie respiratoire. Il n'y a aucune publication faisant état d'un accroissement des tumeurs mammaires chez des hamsters ou des souris qui avaient reçu du chlorure de méthylène à des doses comparables. On a établi sans aucun doute possible que les tumeurs mammaires étaient liées aux hormones hypophysaires tant chez les rats mâles que chez les femelles. Chez le rat, la prolactine se comporte à la fois comme un initiateur et comme un promoteur des cancers mammaires. On a de bonnes raisons de penser qu'un accroissement du taux de prolactine augmente l'incidence des tumeurs mamamaires (par exemple la greffe de plusieurs hypophyses à des rats Sprague-Dawley augmente l'incidence des tumeurs mammaires chez ces animaux et on a relevé l'existence d'une corrélation positive entre un taux sanguin élevé de prolactine et la présence de tumeurs mammaires chez des rattes âgées de souche R-Amsterdam). Après administration de composés cancérogènes à des rattes, une hyperprolactinémie provoquée chez ces animaux entraîne un accroissement spectaculaire de l'incidence tumorale. On peut notamment provoquer l'hyperprolactinémie par surrénalectomie, homogreffe d'hypophyse et régime alimentaire hyperlipidique. Il est important pour l'évaluation du risque chez l'homme, de connaître le mécanisme par lequel le chlorure de méthylène provoque l'apparition d'adénomes mammaires chez le rat. Les rattes Sprague- Dawley à qui l'on a administré du chlorure de méthylène présentent un taux sanguin élevé de prolactine. De même qu'avec les autres composés qui agissent par l'intermédiaire d'une hyperprolactinémie, la réaction au chlorure de méthylène se traduit uniquement par l'apparition de néoformations à caractère bénin. Rien n'indique que le chlorure de méthylène se lie à l'ADN d'autres tissus et par conséquent il paraît improbable qu'il se lie à l'ADN des tissus mammaires alors qu'il est principalement métabolisé dans le foie. Il paraît donc probable que l'accroissement de l'incidence des adénomes mammaires résulte d'un mécanisme indirect agissant par l'intermédiaire d'une hyperprolactinémie. Chez l'homme, les faits relatifs à la question de savoir si les tumeurs mammaires sont sous la dépendance de la prolactine comme chez le rat, apparaissent contradictoires. Le rat présente un taux élevé de prolactine lorsqu'on le laisse s'alimenter ad libitum plutôt que de le soumettre à un régime strict et cela pourrait expliquer pourquoi l'incidence des tumeurs mammaires est si clépendante des divers effets environnementaux ou autres. Chez le rat toutefois, la prolactine a un caractère lutéotrope. L'augmentation du taux de prolactine dans le sang circulant conduit à une augmentation du taux de progestérone et d'oestrogène exogènes. C'est la présence de l'ensemble de ces trois facteurs qui provoque la croissance tubulo-alvéolaire des glandes mammaires et qui finit par déboucher sur la formation de tumeurs. Ce mécanisme de formation tumorale n'a donc vraisemblablement pas à être pris en considération chez l'homme. Le mécanisme de formation de tumeurs mammaires chez le rat par l'intermédiaire d'une hyperprolactinémie n'intervient qu'à des doses où le chlorure de méthylène agit sur les taux de prolactine. On ne dispose pas de données de première main sur les taux de prolactine chez des rats soumis à de faibles doses de chlorure de méthylène, mais l'administration de faibles doses de ce composé, soit par inhalation, soit par mélange à l'eau de boisson (doses inférieures à 250 mg/kg de poids corporel) ne conduit pas, selon les études effectuées, à un accroissement des adénomes mammaires. 8. Effets sur l'homme Le chlorure de méthylène irrite la peau et les yeux, en particulier lorsqu'il ne peut pas s'évaporer. Dans ces conditions, un contact prolongé peut entraîner des brûlures chimiques. On a signalé un cas grave d'oedème pulmonaire consécutif à une inhalation excessive de chlorure de méthylène. On a également signalé des cas de décès par suite de l'inhalation accidentelle de chlorure de méthylène ou d'un contact cutané avec ce composé. Les principaux effets toxiques du chlorure de méthylène consistent dans une dépression réversible du système nerveux central et dans la formation de carboxyhémoglobine. On a également rapporté des cas d'insuffisance hépatique et rénale avec anomalies hématologiques, à la suite d'une exposition à du chlorure de méthylène. Chez des volontaires humains exposés pendant une heure et demie à trois heures à du chlorure de méthylène à la concentration de 694 mg/m3, on a observé des troubles neurophysiologiques et neuro- comportementaux. En revanche, aucun signe d'effet neurologique n'a été observé chez des hommes exposés plusieurs années à du chlorure de méthylène à des concentrations allant de 260 à 347 mg/m3. De même, on a soumis à une batterie de tests neurophysiologiques et psychologiques un groupe d'anciens décapeurs d'aéronefs qui avaient été longtemps exposés à du chlorure de méthylène (22 ans), à des doses élevées mais non précisées; comparés à ceux d'un groupe témoin qui n'avait jamais été exposé à du chlorure de méthylène ou du moins uniquement à de faibles doses, les résultats obtenus par les ouvriers se sont révélés "normaux". On a attribué à une exposition au chlorure de méthylène une augmentation du taux d'avortements spontanés constatée chez des employées des industries pharmaceutiques finlandaises. L'étude en question présentait cependant des défauts de conception qui n'ont pas permis d'établir une relation causale. Plusieurs éludes de mortalité sur des cohortes exposées au chlorure de méthylène font ressortir une absence d'uniformité dans les causes de décès. La surmortalité due à certaines maladies (par exemple cancer du pancréas, cardiopathies ischémiques) ne se manifeste pas de façon uniforme, mais seulement dans certaines éludes. Ces effets ne peuvent être attribués à l'exposition au chlorure de méthylène. RESUMEN 1. Identidad, propiedades físicas y químicas, y métodos analíticos El cloruro de metileno (diclorometano) es un liquido claro, altamente volátil y no inflamable, con un olor penetrante parecido al del éter. El compuesto puro en polvo es muy estable. El cloruro de metileno se hidroliza lentamente en presencia de humedad, dando lugar a pequeñas cantidades de ácido clorhídrico. Al compuesto comercial se agregan pequeñas cantidades de estabilizadores para prevenir su descomposición. Existen métodos analíticos para determinar el cloruro de metileno en medios biológicos y muestras ambientales; en todos ellos se utiliza cromatografía de gases y un detector apropiado. De esta manera se han alcanzado limites de detección muy bajos (p. ej., alimentos: 7 ng/muestra; agua: 0,01 µg/litro; aire: 1,76 µg/m3 (0,5 ppb); sangre: 0,022 mg/litro). 2. Fuentes de exposición humana y ambiental Se estima que la producción mundial de cloruro de metileno asciende a 570 000 toneladas/año. La mayoría sus aplicaciones se basan en su capacidad para disolver grasas, plásticos y agentes aglutinantes de pintura, así como en su volatilidad y estabilidad. Su uso a nivel mundial se reparte del siguiente modo: aerosoles (20%-25%), quitapinturas (25%), disolvente en la industria química y farmacéutica (35%-40%), usos varios (p. ej., la fabricación de espuma de poliuretano) y limpieza de metales (10%-15%). El uso de cloruro de metileno tiende a disminuir, al menos en Europa occidental. Más del 99% del cloruro de metileno liberado a la atmósfera procede de diversas industrias que lo emiten como producto fina], o es el resultado del uso doméstico de quitapinturas y aerosoles. 3. Transporte, distribución y transformación en el medio ambiente Debido a su alta volatilidad, la mayor parte del cloruro de metileno liberado al medio pasa a la atmósfera, donde se degrada reaccionando con radicales hidroxilo de origen fotoquímico; su tiempo de permanencia es de seis meses. La degradación abiótica del compuesto en agua es lenta en comparación con la evaporación. Se ha comprobado que el cloruro de metileno desaparece rápidamente del suelo y de las aguas subterráneas. Se han utilizado diversos sistemas de ensayo para determinar la degradación aerobia y anaerobia del cloruro de metileno. La biodegradación completa, sobre todo en cultivos bacterianos tratados y en condiciones aerobias, es rápida (p. ej., mineralización del 49%-66% en 50 horas en fangos urbanos tratados). En los biorreactores se puede alcanzar una degradación de hasta un 10% por hora. No hay indicios de una bioacumulación o biomagnificación importantes. 4. Niveles medioambientales y exposición humana Se ha detectado cloruro de metileno en el aire ambiente de zonas rurales y remotas a concentraciones comprendidas entre 0,07 y 0,29 µg/m3. En zonas suburbanas la concentración promedio es <2 µg/m3, y en zonas urbanas, <15 µg/m3. En las proximidades de vertederos de desechos peligrosos se han hallado hasta 43 µg/m3. Las precipitaciones también contienen a veces cloruro de metileno. El cloruro de metileno penetra en el medio acuático a través de las descargas de aguas residuales de diversas industrias, habiéndose detectado su presencia en aguas superficiales, aguas subterráneas y sedimentos. La población general se expone al cloruro de metileno cuando utiliza productos de consumo tales como los quitapinturas, cuyo empleo puede acompañarse de la presencia de niveles relativamente altos en el aire del interior de los domicilios. La exposición ocupacional durante la producción tiene lugar sobre todo durante el llenado y envasado (la fabricación se lleva a cabo en sistemas cerrados). Tratándose de un compuesto usado en los quitapinturas, la exposición laboral al cloruro de metileno se produce durante la elaboración de quitapinturas, la fabricación de material para ordenadores y el acabado comercial de muebles. El cloruro de metileno es ampliamente empleado como disolvente industrial en la elaboración de diversos productos, sobre todo en las industrias que se mencionan en la sección 1.2. La vigilancia biológica de la exposición al cloruro de metileno puede basarse en la medición del propio disolvente en el aire espirado o en la sangre. No obstante, dado que la producción de monóxido de carbono con una exposición de más de 3-4 horas/día parece ser el factor limitante en lo que respecta a los riesgos para la salud, es preferible basar la vigilancia biológica en el análisis bien del monóxido de carbono presente en el aire espirado, o bien de la carboxihemoglobina (CO-Hb) en sangre. Así y todo, esto sólo se puede aplicar a las personas no fumadoras. Deben tomarse muestras antes de transcurridas aproximadamente dos horas tras la exposición, o bien al cabo de 16 horas, esto es, a la mañana siguiente. Los niveles postexposición de CO-Hb a las dos horas de interrumpir la exposición no deben sobrepasar el 2%-3%, y a las 16 horas el 1%, en los no fumadores expuestos durante ocho horas a menos de 350 mg/m3 de cloruro de metileno. 5. Cinética y metabolismo El cloruro de metileno es absorbido rápidamente por los alvéolos pulmonares, a través de los cuales llega a la circulación sistémica. Es absorbido también por el tracto gastrointestinal, así como por vía cutánea, si bien en este último caso la velocidad de absorción es menor que por otras vías de exposición. El cloruro de metileno se excreta con considerable rapidez, fundamentalmente a través del aire espirado por los pulmones. Puede atravesar la barrera hematoencefálica, así como la placenta, y se excreta también en pequeñas cantidades por la orina y la leche. A altas concentraciones la mayoría del cloruro de metileno absorbido se espira inalterado. El resto es metabolizado en monóxido de carbono, dióxido de carbono y cloruro inorgánico. Hay dos vías posibles de metabolización, cuya contribución relativa al metabolismo total depende en gran medida de la dosis y de la especie animal considerada. Una vía consiste en un proceso de metabolismo oxidativo mediado por el citocromo P-450, que conduce a la producción tanto de monóxido de carbono como de dióxido de carbono. Esta vía funciona de manera parecida en todos los roedores estudiados y en el hombre. Si bien es la vía metabólica predominante a dosis bajas, se satura también a dosis relativamente bajas (en torno a 1800 mg/m3). Aumentar la dosis por encima de ese nivel de saturación no conlleva un mayor metabolismo a través de esa vía. La otra vía está mediada por una glutatión-transferasa (GTF) y conduce, previa producción de formaldehído y de formato, a la formación de dióxido de carbono. Al parecer esta vía sólo adquiere importancia a dosis superiores al nivel de saturación de la vía oxidativa «preferente». En algunas especies (p. ej., el ratón) constituye la principal vía metabólica a dosis suficientemente altas. Por el contrario, en otras especies (p. ej., el hámster o el hombre) esta vía apenas es utilizada, cualquiera que sea la dosis. Las diferencias interespecies del metabolismo mediado por la GTF guardan una clara relación con las diferencias interespecies observadas en lo que respecta a la carcinogenicidad. Analizando la intensidad del metabolismo mediado por esta vía en determinadas especies, se ha elaborado un modelo cinético del metabolismo del cloruro de metileno en diversas especies. 6. Efectos en organismos presentes en el medio ambiente Por debajo de 500 mg/litro no se observa inhibición del crecimiento de algas y de bacterias aerobias. Se han descubierto bacterias capaces de crecer en presencia de cloruro de metileno a concentraciones mucho mayores, incluida una solución saturada en agua (sección 4.2.4.1 ). Las bacterias anaerobias son más sensibles; se ha observado inhibición del crecimiento a una concentración de 1 mg/litro en fangos biológicos anaerobios. En el suelo, se observó que una concentración de 10 mg/kg reducía considerablemente el contenido de ATP de la biomasa, incluidos hongos y bacterias aerobias, e inducía una inhibición transitoria de la actividad enzimática. El nivel sin efectos observados fue de 0,1 mg/kg. En las lombrices de tierra el cloruro de metileno tiene un efecto moderadamente tóxico (100-1000 µg/cm2), como demuestra la prueba de toxicidad de contacto con papel de filtro. En sedimentos no se observaron efectos tóxicos ni siquiera a concentraciones muy altas. En plantas superiores no se observaron efectos al cabo de 14 días de exposición a 100 mg/m3. Los peces adultos parecen relativamente insensibles al cloruro de metileno, incluso después de una exposición prolongada (14 días, CL50 > 200 mg/litro). El efecto del cloruro de metileno en Daphnia resulta difícil de evaluar porque hay grandes diferencias entre los resultados de los estudios realizados. La CE50 más baja notificada es de 12,5 mg/litro. En cuanto al entorno acuático, se ha demostrado que los embriones de peces y anfibios son los más sensibles, observándose efectos sobre la incubación a partir de 5,5 mg/litro. 7. Efectos en mamíferos de laboratorio y en sistemas de prueba in vitro 7.1 Exposiciones aisladas La toxicidad aguda del cloruro de metileno por vía respiratoria y por vía oral es baja. La CL50-6h por inhalación está comprendida en todas las especies entre 40 200 y 55 870 mg/m3. Se han registrado DL50 orales de 1410-3000 mg/kg. Los efectos agudos de la administración de cloruro de metileno por diversas vías de exposición se manifiestan fundamentalmente en el sistema nervioso central (SNC) y en el hígado, y se producen a dosis altas. Se han observado trastornos del SNC a concentraciones de 14 100 mg/m3 o más, con ligeras variaciones del EEG a 1770 mg/m3. A concentraciones de 17 700 mg/m3 o más se observaron leves cambios histológicos en el hígado. Ocasionalmente se vieron afectados otros órganos, tales como el riñón o el sistema respiratorio. En el ratón, los efectos sobre los pulmones se limitaron a las células de Clara después de una exposición a 7100 mg/m3. Se ha notificado la aparición de sensibilización cardiaca a la arritmia inducida por adrenalina. Se han observado efectos cardiovasculares, si bien de manera irregular. 7.2 Exposición a corto y a largo plazo La exposición prolongada a concentraciones altas de cloruro de metileno (>17 700 mg/m3) causó efectos reversibles sobre el SNC, ligera irritación ocular y mortalidad en varias especies de laboratorio. Se observó una reducción del peso corporal en ratas a 3500 mg/m3, y en ratones a partir de 17 700 mg/m3. El hígado de perros expuestos continuamente a 3500 mg/m3 por espacio de hasta 100 días se vio ligeramente afectado. Se observaron asimismo efectos en el hígado tras la exposición intermitente a 3500 mg/m3 en la rata, y a 14 100 mg/m3 en el ratón. Otros órganos diana son los pulmones y los riñones. No se hallaron indicios de daño neurológico irreversible en ratas expuestas por inhalación a concentraciones de hasta 7100 mg/m3 durante 13 semanas. La administración oral de cloruro de metileno a ratas causó efectos hepáticos a partir de 200 mg/kg al día. 7.3 Irritación cutánea y ocular El cloruro de metileno es moderadamente irritante para la piel y los ojos de animales experimentales. 7.4 Toxicidad para el desarrollo y la reproducción El cloruro de metileno no es teratógeno en la rata o el ratón a concentraciones de hasta 16 250 mg/m3. En tres estudios realizados con animales no se observaron indicios de variación de la incidencia de malformaciones esqueléticas ni otros efectos sobre el desarrollo. Se notificaron efectos leves sobre el peso corporal fetal o materno a una concentración de 4400 mg/m3, así como sobre el aumento de peso postnatal de ratas macho a una concentración del 0,04% en la dieta. Un estudio de toxicidad reproductiva llevado a cabo en dos generaciones de ratas expuestas a cloruro de metileno por inhalación a concentraciones de hasta 5300 mg/m3, 6 h/día, 5 días/semana durante 17 semanas no puso de manifiesto ningún efecto adverso en lo tocante a los parámetros reproductivos, la supervivencia neonatal o el crecimiento neonatal en ninguna de las generaciones, F0 o F1. 7.5 Mutagenicidad y criterios de evaluación relacionados En condiciones de exposición adecuadas el cloruro de metileno tiene efectos mutágenos en microorganismos procariotas, con o sin activación metabólica (Salmonella o Escherichia coli). En los sistemas eucariotas los resultados son negativos, salvo en un caso en que fueron débilmente positivos. Los ensayos y pruebas de mutación genética in vitro basados en la síntesis no programada de ADN (UDS) en células de mamífero fueron siempre negativos. Los ensayos in vitro realizados para detectar aberraciones cromosómicas en diferentes tipos de células dieron resultados positivos, mientras que en las pruebas de inducción de intercambio de cromátides hermanas (SCE) se obtuvieron resultados negativos o ambiguos. La mayoría de los estudios in vivo publicados no han aportado ningún dato indicativo de mutagenicidad del cloruro de metileno (determinada por ejemplo, mediante la prueba de aberración cromosómica, la prueba de los micronúcleos o el ensayo UDS). Se ha notificado un aumento mínimo de la frecuencia de SCE y de micronúcleos en el ratón tras la exposición por inhalación a altas concentraciones de cloruro de metileno. En ratas o ratones a los que se administraron dosis altas de cloruro de metileno no se observaron indicios de unión del cloruro de metileno al ADN ni de lesiones de éste. Son éstos los estudios in vivo potencialmente más sensibles, el mejor de los cuales permite detectar una alquilación por cada 106 nucleótidos. Dentro de las limitaciones de las pruebas a corto plazo actualmente disponibles, no hay pruebas concluyentes de que el cloruro de metileno sea genotóxico in vivo. 7.6 Toxicidad crónica y carcinogenicidad El cloruro de metileno es carcinógeno en el ratón, en el que la exposición a altas concentraciones (7100 y 14 100 mg/m3) es causa de tumores tanto pulmonares como hepáticos. La incidencia de esos dos tipos de tumores aumentó en ratones expuestos a 7100 mg/m3 durante 26 semanas y estudiados durante 78 semanas más. No se observaron signos claros de toxicidad o hiperplasia asociadas en los órganos diana. La exposición de hámsters sirios a cloruro de metileno por inhalación a concentraciones de hasta 12 400 mg/m3 durante dos años no tuvo efectos carcinógenos. Se ha observado que las ratas expuestas al cloruro de metileno por diversas vías sufren una mayor incidencia de tumores en determinados lugares. Se ha notificado un exceso de tumores en la región de las glándulas salivales en ratas hembra expuestas a 5300 ó 12 400 mg/m3 durante dos años. Ese exceso sólo se hizo patente cuando se procedió a agrupar los tumores, todos ellos de origen mesenquimatoso, con fines estadísticos. El método estadístico utilizado era inapropiado dado que los tumores procedían de células de diverso tipo. Además, se señaló que las ratas utilizadas se habían visto infectadas al principio del estudio por un virus causante de una enfermedad común, la sialodacrioadenitis, que afecta sobre todo a la glándula salival. Probablemente los tumores no estaban relacionados causalmente con la exposición al cloruro de metileno, y la exposición se limitó a exacerbar la respuesta a la infección en la región de la glándula salival. El efecto no se reprodujo en un segundo estudio realizado con ratas expuestas a 3500, 7100 ó 14 100 mg/m3 durante su ciclo de vida. Un estudio ulterior realizado con ratas expuestas por inhalación a concentraciones de hasta 1770 mg/m3 de cloruro de metileno durante todo su ciclo de vida no reveló indicios de carcinogenicidad. En ratas expuestas al cloruro de metileno a través del agua que consumían o de alimentos administrados con sonda tampoco se observaron indicios significativos de carcinogenicidad. Tres estudios han puesto de manifiesto un aumento de la incidencia de tumores mamarios benignos en ratas expuestas a cloruro de metileno, en dos de los casos por inhalación y en el tercero por administración forzada. No se ha notificado ningún aumento de la incidencia de tumores mamarios en hámsters o en ratones sometidos a dosis comparables de cloruro de metileno. La dependencia de los tumores mamarios de las hormonas hipofisarias en la rata, tanto macho como hembra, es un dato incontrovertible. En la rata, la prolactina actúa como iniciador y como promotor de la carcinogénesis mamaria. Hay datos convincentes de que el aumento de los niveles de prolactina incrementa la incidencia de tumores mamarios (p. ej., el injerto de varias hipófisis en ratas Sprague-Dawley aumenta la incidencia de tumores mamarios, y se ha observado además una correlación positiva entre la existencia de niveles elevados de prolactina en sangre y la incidencia de tumores mamarios en ratas hembra R-Amsterdam viejas). En las ratas hembra que han recibido carcinógenos, los tratamientos inductores de hiperprolactinemia dan lugar a un aumento espectacular de la incidencia de tumores. Entre esos tratamientos cabe citar la adrenalectomía, los homoinjertos hipofisarios y el consumo de alimentos ricos en grasas. El conocimiento de los mecanismos de inducción de adenomas mamarios por el cloruro de metileno en la rata es importante para poder evaluar los riesgos para el hombre. Las ratas Sprague-Dawley hembras sometidas a cloruro de metileno presentan una elevada concentración de prolactina en sangre. Al igual que la respuesta a otros agentes cuya acción está mediada por una hiperprolactinemia, la respuesta inducida por el cloruro de metileno se limita a la aparición de neoplasias benignas. No hay datos indicativos de una unión del cloruro de metileno al ADN de otros tejidos, por lo que parece improbable que pueda unirse al tejido mamario, tanto más cuanto que su metabolismo se produce fundamentalmente en el hígado. Es más probable, por tanto, que el aumento de la incidencia de adenomas mamarios se deba a un mecanismo indirecto en el que intervenga la hiperprolac- tinemia. En cuanto al hombre, hay datos contradictorios respecto a si los tumores mamarios son tan sensibles a la prolactina como en la rata. Este animal presenta niveles elevados de prolactina cuando es alimentado ad libitum en lugar de sometido a una dieta restringida, lo cual explica quizá la gran sensibilidad de la incidencia de tumores mamarios a diversos efectos ambientales y de otro tipo. En la rata, no obstante, la prolactina es luteotrófica. Un aumento de la prolactina circulante da lugar a un aumento de los niveles de progesterona y de estrógenos exógenos. Es la coincidencia de estos tres factores lo que causa el crecimiento túbulo-alveolar de las glándulas mamarias y, finalmente, el desarrollo del tumor. La prolactina no es luteotrófica en los primates; es improbable, por tanto, que este mecanismo de desarrollo tumoral pueda tener importancia en el hombre. En la rata, el mecanismo de desarrollo de tumores mamarios mediado por la hiperprolactinemia sólo entra en juego a las dosis de cloruro de metileno que alteran los niveles de prolactina. No se dispone de información directa sobre los niveles de prolactina en ratas sometidas a dosis bajas de cloruro de metileno, pero no se ha observado ningún aumento de la incidencia de adenomas mamarios tras la administración de dosis bajas por inhalación o a través del agua de bebida (p. ej., dosis inferiores a 250 mg/kg peso corporal). 8. Efectos en el hombre El cloruro de metileno es irritante para la piel y para los ojos, sobre todo cuando se impide su evaporación. En estas condiciones, el contacto prolongado puede causar quemaduras químicas. Se ha notificado un caso de edema pulmonar grave por inhalación excesiva. Se han producido también defunciones en casos de inhalación o contaminación cutánea accidentales. Los principales efectos tóxicos del cloruro de metileno son la depresión reversible del SNC y la formación de CO-Hb. Se ha señalado también la aparición de disfunciones hepáticas y renales y de trastornos hematológicos tras la exposición al producto. Se han observado problemas neurofisiológicos y neurocomporta- mentales en voluntarios humanos expuestos a concentraciones de cloruro de metileno de 694 mg/m3 durante 1,5-3,0 horas. No se han observado efectos neurológicos en hombres expuestos durante varios años a concentraciones del producto comprendidas entre 260 y 347 mg/m3. De forma parecida, un grupo de raspadores de pintura de aviones ya jubilados con antecedentes de una larga (22 años) exposición a concentraciones altas, si bien no especificadas, de cloruro de metileno obtuvieron resultados "normales" en una batería de pruebas neurofisiológicas y psicológicas en comparación con un grupo testigo sin antecedentes de exposición, o en todo caso con antecedentes de una baja exposición al compuesto. Un aumento de la tasa de abortos espontáneos entre empleadas de la industria farmacéutica finlandesa se ha atribuido a la exposición a cloruro de metileno. Sin embargo, el diseño incorrecto del estudio ha impedido establecer una relación causal. Varios estudios de mortalidad realizados en cohortes pertinentes muestran resultados dispares en cuanto a las causas de defunción. Se ha observado un aumento de la mortalidad por enfermedades especificas (como por ejemplo el cáncer pancreático o la cardiopatía isquémica), pero de forma irregular y sólo en determinados estudios. Estos efectos no se pueden atribuir a la exposición al cloruro de metileno.
See Also: Toxicological Abbreviations Methylene chloride (EHC 32, 1984, 1st edition) Methylene chloride (HSG 6, 1987) Methylene chloride (PIM 343)