IPCS INCHEM Home


    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. Unpublished data are only used when relevant published
    data are absent or when they are pivotal to the risk assessment. A
    detailed policy statement is available that describes the procedures
    used for unpublished proprietary data so that this information can be
    used in the evaluation without compromising its confidential nature
    (WHO (1990) Revised Guidelines for the Preparation of Environmental
    Health Criteria Monographs. PCS/90.69, Geneva, World Health
    Organization).

        In the evaluation of human health risks, sound human data,
    whenever available, are preferred to animal data. Animal and  in vitro
    studies provide support and are used mainly to supply evidence missing
    from human studies. It is mandatory that research on human subjects is
    conducted in full accord with ethical principles, including the
    provisions of the Helsinki Declaration.

        The EHC monographs are intended to assist national and
    international authorities in making risk assessments and subsequent
    risk management decisions. They represent a thorough evaluation of
    risks and are not, in any sense, recommendations for regulation or
    standard setting. These latter are the exclusive purview of national
    and regional governments.

    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.

    Procedures

        The order of procedures that result in the publication of an EHC
    monograph is shown in the flow chart. A designated staff member of
    IPCS, responsible for the scientific quality of the document, serves

    as Responsible Officer (RO). The IPCS Editor is responsible for layout
    and language. The first draft, prepared by consultants or, more
    usually, staff from an IPCS Participating Institution, is based
    initially on data provided from the International Register of
    Potentially Toxic Chemicals, and reference data bases such as Medline
    and Toxline.

        The draft document, when received by the RO, may require an
    initial review by a small panel of experts to determine its scientific
    quality and objectivity. Once the RO finds the document acceptable as
    a first draft, it is distributed, in its unedited form, to well over
    150 EHC contact points throughout the world who are asked to comment
    on its completeness and accuracy and, where necessary, provide
    additional material. The contact points, usually designated by
    governments, may be Participating Institutions, IPCS Focal Points, or
    individual scientists known for their particular expertise. Generally
    some four months are allowed before the comments are considered by the
    RO and author(s). A second draft incorporating comments received and
    approved by the Director, IPCS, is then distributed to Task Group
    members, who carry out the peer review, at least six weeks before
    their meeting.

        The Task Group members serve as individual scientists, not as
    representatives of any organization, government or industry. Their
    function is to evaluate the accuracy, significance and relevance of
    the information in the document and to assess the health and
    environmental risks from exposure to the chemical. A summary and
    recommendations for further research and improved safety aspects are
    also required. The composition of the Task Group is dictated by the
    range of expertise required for the subject of the meeting and by the
    need for a balanced geographical distribution.

        The three cooperating organizations of the IPCS recognize the
    important role played by nongovernmental organizations.
    Representatives from relevant national and international associations
    may be invited to join the Task Group as observers. While observers
    may provide a valuable contribution to the process, they can only
    speak at the invitation of the Chairperson. Observers do not
    participate in the final evaluation of the chemical; this is the sole
    responsibility of the Task Group members. When the Task Group
    considers it to be appropriate, it may meet  in camera.

        All individuals who as authors, consultants or advisers
    participate in the preparation of the EHC monograph must, in addition
    to serving in their personal capacity as scientists, inform the RO if
    at any time a conflict of interest, whether actual or potential, could
    be perceived in their work. They are required to sign a conflict of
    interest statement. Such a procedure ensures the transparency and
    probity of the process.

        When the Task Group has completed its review and the RO is
    satisfied as to the scientific correctness and completeness of the
    document, it then goes for language editing, reference checking, and
    preparation of camera-ready copy. After approval by the Director,
    IPCS, the monograph is submitted to the WHO Office of Publications for
    printing. At this time a copy of the final draft is sent to the
    Chairperson and Rapporteur of the Task Group to check for any errors.

        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.

    FIGURE 1

    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)