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 Progra mme, the International Labour Organisation, or the World Health Organi zation.

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 Saf ety (IPCS) is a joint venture of the United Nations Environment Progr amme, the International Labour Organisation, and the World Health Organ ization. The main objective of the IPCS is to carry out and disse minate evaluations of the effects of chemicals on human health and th e quality of the environment. Supporting activities include the development of epidemiological, experimental laboratory, and r isk-assessment methods that could produce internationally comparable r esults, and the development of manpower in the field of toxicol ogy. Other activities carried out by the IPCS include the development of know-how for coping with chemical accidents, coordination of labora tory testing and epidemiological studies, and promotion of resea rch 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 Classificatio n: QV 633) ISSN 0250-863X

The World Health Organization welcomes requ ests for permission to reproduce or translate its publications, in par t 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 Organizati on enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Convention. All rights rese rved.
The designations employed and the presentat ion of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Hea lth Organization concerning the legal status of any country, ter ritory, city or area or of its authorities, or concerning the delimitat ion of its frontiers or boundaries.

The mention of specific companies or of cer tain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organization in preference to othe rs of a similar nature that are not mentioned. Errors and omissions ex cepted, the names of proprietary products are distinguished by initi al capital letters.

CONTENTS

ENVIRONMENTAL HEALTH CRITERIA FOR METHYLENE CHL ORIDE

1. SUMMARY

1.1. Identity, physical and chemical properties, an d analytical methods 

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1 of 102 3/23/2016 4:29 PM1.2. Sources of human and environmental exposure 1.3. Environmental transport, distribution and tran sformation 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 TRANSF ORMATION
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 b iological 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 andrelated activities 5.2.2.3 Aerosol production and use 5.2.2.4 Use as a process so lvent 5.2.2.5 Cleaning and degrea sing

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 

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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 an d 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 mammali an 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 ass ay 8.6.2.6 Replicative DNA syn thesis

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 extrapolat ion 

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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 effect s 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 ONTHE 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 infor mation in the criteria monographs as accurately as possible without un duly delaying their publication. In the interest of all users of th e Environmental Health Criteria monographs, readers are requested to c ommunicate any errors that may have occurred to the Director of the I nternational 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 ca n be obtained from the International Register of Potentially Toxic Che micals, Case postale 356, 1219 Chatelaine, Geneva, Switzerland (Tele phone 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 fina ncial support from the European Commission.

Environmental Health Criteria

PREAMBLE
Objectives

In 1973 the WHO Environmental Health Criter ia Programme was initiated with the following objectives:

(i) to assess information on the relationshi p between exposure to environmental pollutants and human healt h, and to provide guidelines for setting exposure limits;

(ii) to identify new or potential pollutants;

(iii) to identify gaps in knowledge concerningthe health effects of pollutants;
(iv) to promote the harmonization of toxicolo gical and epidemiological methods in order to have internationally comparable results.

The first Environmental Health Criteria (EH C) monograph, on mercury, was published in 1976 and since that t ime an ever-increasing number of assessments of chemicals and of physi cal 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 publ ications have been concerned with epidemiological guidelines, eval uation of short-term tests for carcinogens, biomarkers, effects on t he elderly and so forth.
Since its inauguration the EHC Programme ha s widened its scope, and the importance of environmental effects, in addition to health effects, has been increasingly emphasized in th e 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 impo rtance of occupational health and environmental effects was fully reco gnized. The EHC monographs have become widely established, usedand recognized throughout the world. 

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Development and the subsequent establishment of the Intergovernmental Forum on Chemical Safety with the priorities fo r action in the six programme areas of Chapter 19, Agenda 21, all l end further weight to the need for EHC assessments of the risks of ch emicals.

Scope
The criteria monographs are intended to pro vide critical reviews on the effect on human health and the environme nt of chemicals and of combinations of chemicals and physical and biol ogical agents. As such, they include and review studies that are of dir ect relevance for the evaluation. However, they do not describe every study carried out. Worldwide data are used and are quoted from ori ginal studies, not from abstracts or reviews. Both published and unpubl ished reports are considered and it is incumbent on the authors t o 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 des cribes the procedures used for unpublished proprietary data so that t his information can be used in the evaluation without compromising its confidential nature (WHO (1990) Revised Guidelines for the Preparat ion of Environmental Health Criteria Monographs. PCS/90.69, Geneva, World Health Organization).

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

The EHC monographs are intended to assist n ational and international authorities in making risk assess ments and subsequent risk management decisions. They represent a tho rough evaluation of risks and are not, in any sense, recommendation s for regulation or standard setting. These latter are the exclusiv e purview of national and regional governments.

Content
The layout of EHC monographs for chemicals is outlined below.

* Summary - a review of the salient facts andthe risk evaluation of the chemical

* Identity - physical and chemical properties , analytical methods

* Sources of exposure
* Environmental transport, distribution and t ransformation

* Environmental levels and human exposure

* Kinetics and metabolism in laboratory anima ls and humans

* Effects on laboratory mammals and in vitro test systems

* Effects on humans
* Effects on other organisms in the laborator y and field

* Evaluation of human health risks and effect s on the environment
* Conclusions and recommendations for protect ion of human health and the environment

* Further research
* Previous evaluations by international bodie s, e.g., IARC, JECFA, JMPR

Selection of chemicals

Since the inception of the EHC Programme, t he IPCS has organized meetings of scientists to establish lists of pr iority chemicals for subsequent evaluation. Such meetings have been held in: Ispra, Italy, 1980; Oxford, United Kingdom, 1984; Berlin, Ger many, 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 h uman or environmental exposure; the size and nature of populations at risk (both human and other species) and risks for environment; inter national concern, i.e. the substance is of major interest to several c ountries; adequate data on the hazards are available.
If an EHC monograph is proposed for a chemi cal not on the priority list, the IPCS Secretariat consults with the Co operating Organizations and all the Participating Institutions before e mbarking 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 designa ted staff member of IPCS, responsible for the scientific quality of the document, serves 

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and language. The first draft, prepared by cons ultants or, more usually, staff from an IPCS Participating Insti tution, is based initially on data provided from the Internation al 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 d etermine its scientific quality and objectivity. Once the RO finds the document acceptable as a first draft, it is distributed, in its unedit ed form, to well over 150 EHC contact points throughout the world who are asked to comment on its completeness and accuracy and, where nec essary, provide additional material. The contact points, usuall y designated by governments, may be Participating Institutions, IPCS Focal Points, or individual scientists known for their particula r expertise. Generally some four months are allowed before the comment s are considered by the RO and author(s). A second draft incorporating comments received and approved by the Director, IPCS, is then distrib uted to Task Group members, who carry out the peer review, at leas t 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, significa nce and relevance of the information in the document and to assess t he health and environmental risks from exposure to the chemic al. A summary and recommendations for further research and improv ed safety aspects are also required. The composition of the Task Grou p 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 organi zations. Representatives from relevant national and inte rnational associations may be invited to join the Task Group as observ ers. While observers may provide a valuable contribution to the proc ess, they can only speak at the invitation of the Chairperson. Obs ervers do not participate in the final evaluation of the chem ical; 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 monog raph must, in addition to serving in their personal capacity as scient ists, inform the RO if at any time a conflict of interest, whether act ual or potential, could be perceived in their work. They are required t o sign a conflict of interest statement. Such a procedure ensures th e transparency and probity of the process.

When the Task Group has completed its revie w and the RO is satisfied as to the scientific correctness and completeness of the document, it then goes for language editing, re ference checking, and preparation of camera-ready copy. After approva l by the Director, IPCS, the monograph is submitted to the WHO Off ice of Publications for printing. At this time a copy of the final draf t 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 avai lable that would substantially change the evaluation; there is p ublic concern for health or environmental effects of the agent be cause of greater exposure; an appreciable time period has elapse d since the last evaluation.
All Participating Institutions are informed , through the EHC progress report, of the authors and institution s proposed for the drafting of the documents. A comprehensive file of all comments received on drafts of each EHC monograph is mai ntained and is available on request. The Chairpersons of Task Groups are briefed before each meeting on their role and responsib ility in ensuring that these rules are followed. 

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Members

Dr L.A. Albert, Consultores Ambientales Associa dos, Xalapa, Veracruz, Mexico
Mr D. Farrar, ICI Chemicals and Polymers, Runco rn, Cheshire, United Kingdom (Rapporteur)

Dr R. Fransson-Steen, Institute of Environmenta l Medicine, Karolinska Institute, Stockholm, Sweden

Dr S. Henry, US Food and Drug Administration, W ashington, DC, USA
Mr P. Howe, Institute of Terrestrial Ecology, M onks Wood Experimental Station, Huntingdon, United Kingdom

Dr P. Standring, Health and Safety Executive, B ootle, Merseyside, United Kingdom

Dr L. Stayner, Division of Standards Developmen t and Technology Transfer, National Institute for Occupation al Safety and Health, Cincinnati, Ohio, USA
Dr T. G. Vermeire, Toxicology Advisory Centre, National Institute of Public Health and Environmental Hygiene, Bi lthoven, The Netherlands (Chairman)

Dr Ruqiu Ye, National Environmental Protection Agency, Beijing, China

Observers
Dr C. De Rooij, Solvay & Cie S.A., Brussels, Be lgium

Dr T. Green, ICI Chemicals & Polymers Ltd., Run corn, Cheshire, United Kingdom

Secretariat
Dr M. Gilbert, International Programme on Chemi cal 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 CHL ORIDE
A WHO Task Group on Environmental Health Cr iteria for Methylene Chloride met at the Institute of Terrestrial Ec ology, Monks Wood, 

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participants on behalf of the host institution, and Dr M. Gilbert opened the meeting on behalf of the three coope rating organizations of the IPCS (ILO/UNEP/WHO). The Task Group reviewe d and revised the draft monograph and made an evaluation of the risks f or human health and the environment from exposure to methylene chloride

. The first draft of this monograph was prepa red by Mr D. Farrar, ICI Chemicals and Polymers, Runcorn, United Kin gdom.
Dr M. Gilbert, IPCS, was responsible for th e overall scientific content of this monograph. After his death in J uly 1994, this responsibility was transferred to Dr P.G. Jenki ns, IPCS, who also dealt with the technical editing.
The efforts of all who helped in the prepar ation 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 concentrati on

NADPH reduced nicotinamide adenine dinucleoti de phosphate
NIOSH National Institute for Occupational Saf ety 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, an d analytical methods

Methylene chloride (dichloromethane) is a c lear, highly volatile, non-flammable liquid with a penetrating ether-l ike odour. The pure dry compound is very stable. Methylene chloride hyd rolyses slowly in the presence of moisture, producing small quantitie s of hydrogen chloride. Commercial methylene chloride normally contains small quantities of stabilizers to prevent decomposition.

Analytical methods are available for the de termination of methylene chloride in biological media and envi ronmental samples. All methods involve gas chromatography in combinati on with a suitable detector. In this way, very low detection limit s have been reached (e.g., in food: 7 ng/sample; water: 0.01 μg/lit re; air: 1.76 μg/m 3 (0.5 ppb); blood: 0.022 mg/litre).

1.2 Sources of human and environmental exposure

World production of methylene chloride is e stimated to be 570 000 tonnes/year. Most applications are base d on its solvent capacity for grease, plastics and paint bindingagents, in combination with its volatility and stability. The worldwid e 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 met al cleaning (10-15%). The usage of methylene chloride is tending to d ecrease, at least in western Europe.
More than 99% of the atmospheric releases o f methylene chloride result from its use as an end-product by variou s industries and the use of paint removers and aerosol products at h ome.
1.3 Environmental transport, distribution and tran sformation

Due to its high volatility, most of the met hylene chloride released to the environment will partition to t he atmosphere, where it will degrade by reaction with photochemically p roduced hydroxyl radicals with a lifetime of 6 months.

Abiotic degradation in water is slow compar ed to evaporation. Methylene chloride has been shown to disappear rapidly from soil and ground water.
The aerobic and anaerobic degradation of me thylene chloride has been established by a variety of different test systems. Complete biodegradation, especially by acclimated bacter ial cultures under

aerobic conditions, is rapid (e.g., 49-66% mine ralization in 50 h with 

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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/m 3 . In suburban areas, the average concentration is < 2 μg/m 3 and in urban areas < 15 μg/m 3 . In the vicinity of hazardous waste sites up to 43 μg/m 3 has been found. Precipitation may also contain met hylene chloride.

Methylene chloride enters the aquatic envir onment through waste water discharge from various industries, and me thylene chloride has been found in surface water, ground water and s ediment.

Exposure of members of the general public t o methylene chloride will occur from its use in consumer products su ch as paint removers, which can result in relatively high levels bein g found in indoor air. Occupational exposure during production arises primarily during filling and packaging (manufacturing is in clos ed systems). Because of its use in paint strippers, occupational exposu re to methylene chloride occurs during formulation of paint-rem over, original equipment manufacture, and in commercial furnit ure refinishing. Methylene chloride is widely used as a process solvent in the manufacture of a variety of products, in partic ular in the industries mentioned in section 1.2.

Biological monitoring of methylene chloride exposure can be based on measurement of the solvent itself in exhaledair 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 i s 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 exposu re 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 /m 3 in non-smokers.

1.5 Kinetics and metabolism

Methylene chloride is rapidly absorbed thou gh the alveoli of the lungs into the systemic circulation. It is also absorbed from the gastrointestinal tract, and dermal exposure res ults in absorption but at a slower rate than via the other routes of e xposure.
Methylene chloride is quite rapidly excrete d, mostly via the lungs in the exhaled air. It can cross the blood-brai n barrier and be transferred across the placenta, and small amou nts can be excreted in urine or in milk.

At high concentrations, most of the absorbe d methylene chloride is exhaled unchanged. The remainder is metabolizedto carbon monoxide, carbon dioxide and inorganic chloride. Metaboli sm occurs by either or both of two pathways, whose relative contributi on to the total metabolism is markedly dependent on the dose an d on the animal species concerned. One pathway involves oxidative metab olism mediated by cytochrome P-450 and leads to both carbon monox ide and carbon dioxide. This pathway appears to operate similarly in al l rodents studied and in man. Whilst this is the predominant metaboli c route at lower doses, saturation occurs at a relatively low dose (aro und 1800 mg/m 3 ). Increasing the dose above the saturation level does not lead to extra metabolism by this route.

The other pathway involves a glutathione tr ansferase (GST), and leads via formaldehyde and formate to carbon di oxide. This route seems only to become important at doses above the sat uration level of the "preferred" oxidative pathway. In some species (e.g., the mouse) it becomes the major metabolic pathway at sufficie ntly 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 correl ates 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 metab olic behaviour of methylene chloride in various species.

1.6 Effects on organisms in the environment

Algae and aerobic bacteria show no inhibiti on of growth below 500 mg/litre. Bacteria have been identified tha t are able to grow in the presence of methylene chloride at much high er concentrations including a saturated solution in water (sectio n 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 strongl y decreased the ATP content of the biomass including fungi and aero bic bacteria, and induced transient inhibition of enzyme activity . The no-observed- effect level was 0.1 mg/kg. In earthworms methy lene chloride is moderately toxic (100-1000 μg/cm 2 ) in the filter-paper contact toxicity test. In sediment no toxic effects wer e observed even at very high levels. 

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to 100 mg/m 3

. Adult fish seem to be relatively insensitiv e to methylene chloride even after prolonged exposure (14-day LC 50 > 200 mg per litre). The effect of methylene chloride on Daphnia is difficult to assess given the large variation in the outcome of the studi es performed. The lowest reported EC 50 was 12.5 mg/litre.

In the aquatic environment, fish and amphib ian 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 tes t systems
1.7.1 Single exposures

The acute toxicity of methylene chloride by inhalation and oral administration is low. The inhalation 6-h LC 50 values for all species are between 40 200 and 55 870 mg/m 3 . Oral LD 50 values of 1410-3000 mg/kg were recorded. Acute effects af ter methylene chloride administration by various routes of exposure ar e primarily associated with the central nervous system (CNS) and the l iver, and these occurred at high doses. CNS disturbances were f ound at concentrations of 14 100 mg/m 3 or more, with slight changes in EEG at 1770 mg/m 3

. Slight histological changes in the liver were f ound at 17 700 mg/m 3 or more. Occasionally other organs were affecte d such as the kidney or respiratory system. In mice, effects on the lun gs were restricted to the Clara cells after exposure to 7100 mg/m 3 . Cardiac sensitization to adrenaline-induced arrhythmia has been repor ted. Cardiovascular effects have been seen but the effects were inc onsistent.

1.7.2 Short- and long-term exposure
Prolonged exposure to high concentrations o f methylene chloride ( > 17 700 mg/m 3 ) caused reversible CNS effects, slight eye irritation and mortality in several laboratory species. Body weight reduction was observed in rats at 3500 mg/m 3 and in mice from 17 700 mg/m 3 . Slight effects on the liver were noted in dogs continuously exposed to 3500 mg/m 3 for up to 100 days. After intermittent exposure, effects on the liver wer e observed in rats at 3500 mg/m 3 and in mice at 14 100 mg/m 3

. Other target organs are the lungs and the k idneys.
No evidence of irreversible neurological da mage was seen in rats exposed by inhalation to concentrations up to 7 100 mg/m 3 for 13 weeks.
Oral administration of methylene chloride t o 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 t o the skin and eyes of experimental animals.

1.7.4 Developmental and reproductive toxicity
Methylene chloride is not teratogenic in ra ts or mice at concentrations up to 16 250 mg/m 3 . No evidence of an effect on the incidence of skeletal malformations or other de velopmental effects were observed in three animal studies. Small ef fects on either fetal or maternal body weight were reported at 4400 m g/m 3 , 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/m 3 , 6 h/day, 5 days/week for 17 weeks did not show evidence of an adverse effect on any reproductive parameter, neonatal survival o r neonatal growth in either the F 0 or F 1 generation.

1.7.5 Mutagenicity and related end-points

Under appropriate exposure conditions, meth ylene 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, weakl y positive results. In vitro gene mutation assays and tests for unscheduled DNA synthesis (UDS) in mammalian cells were uniform ly negative. In vitro assays for chromosomal aberrations using differ ent cell types gave positive results, whereas negative or equivocal results were obtained in tests for sister chromatid exchange (SCE) in duction.

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 assa y). Marginal increase in frequencies of SCEs and micronuclei in mice has been reported following inhalation exposure to high concentra tions of methylene chloride.

There was no evidence of binding of methyle ne chloride to DNA or DNA damage in rats or mice given high doses of methylene chloride. These studies are potentially the most sensitiv e in vivo studies, the best of which are capable of detecting one alkylation in 10 6 nucleotides.

Within the limitations of the short-term te sts currently 

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genotoxic in vivo.

1.7.6 Chronic toxicity and carcinogenicity

Methylene chloride is carcinogenic in the m ouse, causing both lung and liver tumours, following exposure to high c oncentrations (7100 and 14 100 mg/m 3 ) of methylene chloride. The incidence of both lungand liver tumours was increased in mice exposed to 7100 mg/m 3 for 26 weeks and maintained for a further 78 weeks. Th ere was no substantial evidence of associated toxicity or hyperplasia in the target organs.
Syrian hamsters exposed to methylene chlori de by inhalation at concentrations up to 12 400 mg/m 3 for 2 years showed no evidence of a carcinogenic effect related to exposure to me thylene chloride.

Rats exposed to methylene chloride via vari ous routes have shown increased incidences of tumours at certain site s. An excess of tumours in the region of the salivary gland was reporte d in female rats exposed to either 5300 or 12 400 mg/m 3 for 2 years. This excess was only evident when the tumours, which were all o f mesenchymal origin, were grouped together for statistical analysis. As the tumours arose from a variety of different cells, the statisti cal approach adopted was inappropriate. Furthermore, it was reportedthat the rats in the study had been infected with a common viral dis ease (sialoda- cryoadenitis) early in the study, an infection that affects primarily the salivary gland. It is likely that these tum ours were not causally related to exposure to methylene chloride but t hat the exposure had exacerbated the response of the infection in th e 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 10 0 mg/m 3 for their lifetime. A further inhalation study on rats ex posed to methylene chloride at concentrations up to 1770 mg/m 3 for their lifetime showed no evidence of carcinogenicity. Rats exp osed to methylene chloride via their drinking-water or by gavage similarly showed no substantive evidence of carcinogenicity.

An increased incidence of benign mammary tu mours in rats exposed to methylene chloride has been reported in thre e studies, two following exposure by inhalation and the third by gavage. There are no reports of increases in mammary tumour incidenc e in hamsters or in mice receiving methylene chloride at comparable dose levels. The dependence of mammary tumours upon pituitary ho rmones in both male and female rats has been established unequivocally. In the rat, prolactin acts as both an initiator and promoter of mamma ry carcinogenesis. There is good evidence that increased prolactin levels increase the incidence of mammary tumours (e.g., the graftin g of multiple pituitary glands into Sprague-Dawley rats increases the i ncidence of mammary tumours and there is a positive correlation bet ween elevated blood prolactin levels and mammary tumours in aged R- Amsterdam female rats).
Treatments that induce hyperprolactinaemia in f emale rats that have received carcinogens produce a dramatic increas e in tumour incidence. These treatments include adrenalectomy, pituita ry homografts and high dietary fat.
The mechanism by which methylene chloride i nduces mammary adenomas in the rat is important for human hazard assess ment. 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-ind uced 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 wh en 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 th e 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 t o a variety of environmental and other effects. In the rat, ho wever, prolactin is luteotrophic. An increase in the circulating le vels of prolactin will lead to an increase in progesterone and exogeno us oestrogen levels. It is the presence of all three factors that cause s tubular-alveolar growth of the mammary glands, which ultimately leads to tumour development. Prolactin is not luteotrophic in p rimates. It is unlikely, therefore, that this mechanism of tum our development is of relevance to man.
The mechanism of production of mammary tumo urs in the rat involving hyperprolactinaemia will occur only a t doses of methylene chloride which affect prolactin levels. There i s no direct information on prolactin levels in rats receiving low doses of methylene chloride, but no increase in mammary adenomas has been ob served following the administration of low doses in either inhalatio n or drinking-water studies (i.e. below 250 mg/kg body weight).

1.8 Effects on humans

Methylene chloride irritates the skin and e yes especially when evaporation is prevented. In these circumstance s, prolonged contact may cause chemical burns. A case of serious pul monary oedema has been reported after excessive inhalation. Fatalities due to accidental inhalation and skin contamination have been rep orted. The main toxic effects of methylene chloride are reversible CN S depression and CO-Hb formation. Liver and renal dysfunctions and eff ects on haematological 

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chloride.

Neurophysiological and neurobehavioural dis turbances have been observed in human volunteers exposed to methyle ne chloride at concentrations of 694 mg/m 3 for 1.5-3.0 h. No evidence of neurological effects was seen in men with expos ure for several years to methylene chloride at concentrations rangingfrom 260 to 347 mg/m 3 . 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 neuro physiological 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 i n employees in Finnish pharmaceutical industries has been attributed t o exposure to methylene chloride. A causal relationship was not establi shed because of insufficiencies in the design of the study.
Several mortality studies in relevant cohor ts show an inconsistent pattern in the causes of death. Excesses in mor tality from specific diseases (e.g., pancreatic cancer, ischaemic he art disease) were not consistently increased, but confined to single studies. These effects cannot be attributed to exposure to methylene c hloride.

2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS
2.1 Identity

Formula: CH 2 CI 2

CI ' Structure: CI - C - H ' H

Relative molecular mass: 84.93

Common name: Methylene ch loride
Synonyms: DCM; dichlor omethane; methane dichloride; methylene bichloride; methylene di chloride; methylenum chloratum

Tradenames: Aerothene MM ; Freon 30; Narkotil; Solaestin; S olmethine

CAS name (9 CI): Methane, dic hloro-
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% (analy tical grade) product:
Impurities of technical Mostly C 1 - and C 2 -chlorinated product hydrocarbons (up to 200 mg/kg) (ECETOC, 198 4)
Stabilizer: Typically 0. 005-0.2% (w/w) methanol, et hanol, amylene (2-methyl-bu t-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 an d is non-corrosive. In the presence of water, it undergoes very slow h ydrolysis to produce small quantities of hydrogen chloride, which ca n lead to corrosion, e.g., to mild steel. This reaction is accelerat ed by elevated temperatures and the presence of alkalis or met als. In the vapour phase under abnormal conditions (elevated tempe ratures, high UV light exposure, flame, sparks, red hot surfaces), met hylene chloride may be decomposed to give small amounts of hydrogen ch loride, carbon monoxide and phosgene (ECSA, 1989). Other physical and c hemical properties are given in Table 1.
Commercial methylene chloride is normally s tabilized (section 2.1) to prevent decomposition. Applications in aggre ssive conditions, such as special metal cleaning operations may requir e more sophisticated stabilizer technology. Poorly stabilized methyl ene chloride can react violently with aluminium or other light metals.

2.3 Conversion factors
Conversion factor for methylene chloride co ncentrations in air, calculated at 20°C and 1.013 hPa are:

1 mg/m 3 = 0.28 ppm 

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and for carbon monoxide:
1 mg/m 3 = 0.86 ppm 1 ppm = 1.16 mg/m 3

2.4 Analytical methods

Details of sampling and methods of analysis used in biological media and environmental samples are given in Ta bles 2 and 3.
Table 1. Physical and chemical properties

Parameter, units Va lue Reference
Boiling temperature (°C at 1.013 hPa) 40 Weast et al. (1988)
Melting temperature (°C at 1.013 hPa) -9 5.1 Weast et al. (1988)
Relative density of liquid D ( 20 ) 1.3266 Weast et al. (1988) (water at 4°C = 1 kg/m 3 ) 4

Vapour pressure (hPa at 20°C) 47 0 ECSA (1989)

Saturation concentration in air 1. 7 Calculated (kg/m 3 at 20°C)
Vapour density at 20°C (air = 1) 2. 93 IPCS (1984)

Threshold odour concentration 74 3 Leonardos et al. (mg/m 3 ) (1969) (odour: ether-like) 70 0-1060 DFG (1983) 88 0 Amoore & Hautala (1983) 54 0-2160 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 P ow(octanol/water) 1.3 Hansch & Leo (1979) log K oc0.89 calculated from K ow (Karickhoff, 1981) Henry's Law constant, Pa.m 3 /mol at 380 20°C Smith (1989)
Flash point, closed cup (°C) No ne ECSA (1989)

Explosion limits in air a(%) 13-22 ECSA (1989)
Auto-flammability, ignition temp. (°C) 60 5 ECSA (1989)

a This is with a high energy source; these conditio ns are unlikely to arise in normal operations.

Table 2. Analytical methods for determinin g methylene chloride in biological monitoring (ATSD R, 1991)

Sample matrix Preparation method Analytical Sample detection Percentag e Reference method alimit 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 ga s GC/FID 0.706 ± 0.353 No data Di Vincenzo et al. sample loop mg/m 3 (1971) (0.2 ± 0.1 ppm)

Adipose tissue Hydrolyse with acid, heat GC/FID 1.6 mg/kg bNo 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 therma lly

a FID = flame ionisation detector; GC = gas chromat ography; MS = mass spectrometry bLowest reported concentration

Table 3. Analytical methods for determining me thylene chloride in environmental samples (ATSDR, 1 991)

Sample Preparation method Analytical Sample detection Percentage Reference matrix method alimit recovery 

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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/m 3 No data Woodrow et al. benzyl alcohol (approx. 0.5 ppb) (1988)
Water Purge with inert gas, trap on so rbent GC/HSD No data 85 US EPA (1989c) trap, desorb thermally
Water Purge with inert gas, trap on so rbent GC/ELCD 0.01 μg/litre 97-100 US EPA (1989) trap, desorb thermally
Water Purge with inert gas, trap on so rbent GC/MS 1.0 μg/litre 99 US EPA (1989b) trap, desorb thermally
Water Purge with inert gas, trap on so rbent HRGC/MS 0.03-0.09 μg/litre 95-97 US EPA (1989a) trap, desorb thermally
Water Purge with inert gas, trap on so rbent 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 method alimit recovery

Water Purge with inert gas, trap on so rbent HRGC/MS 0.02-0.2 μg/litre 95±5 APHA (1989b) trap, desorb thermally
Water Purge with helium, trap on sorbe nt GC/MS No data 99-105 Michael et al. trap, desorb thermally (1988)

Waste Purge with inert gas, trap on so rbent GC/HSD 0.25 μg/litre 97.9±2.6 US EPA (1982a) water trap, desorb thermally
Waste Purge with inert gas, trap on so rbent GC/MS 2.8 μg/litre 89±28 US EPA (1982b) water trap, desorb thermally
Soil/solid Purge with inert gas, trap on so rbent GC/MS 5 μg/kg D-221 US EPA (1986a) waste trap, desorb thermally
Soil/solid Purge with inert gas, trap on so rbent GC/HSD No data 25-162 US EPA (1986b) waste trap, desorb thermally; or injec t directly into GC

Food Equilibrate in heated sodium sul fate GC/ELCD 0.05 ppm No data Page & Charbonneau solution, collect headspace vapo ur (1984)

Food Isolate solvent by closed systemGC/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 method alimit recovery

Food Isolate solvent by closed systemGC/ECD 7 ng 100 Page & Charbonneau vacuum distillation with toluene as (1977) carrier solvent

Food Purge with nitrogen, trap on sor bent GC/ELCD 1.2 mg/kg d84-96 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 = electroly tic conductivity detector; FID = flame ionisation d etector; GC = gas chromatography; HRGC = high resolution gas chromatography; H SD = halogen-specific detector; MS = mass spectrome try bLowest value for various compounds reported durin g collaborative testing of this method

c Estimated accuracy of the method when the persona l sampling pump is calibrated with a charcoal tube in the line dLowest reported concentration

3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

3.1 Natural occurrence

Methylene chloride is not known to occur na turally in the environment.

3.2 Anthropogenic sources

3.2.1 Production

Methylene chloride is produced almost exclu sively by the Stauffer process. Methyl chloride is first produced by t he reaction of methanol 

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and, to a lesser extent, carbon tetrachloride a re also produced. Historically the direct route to methylene chlo ride by chlorination of methane was also used; this also produced the o ther three chloromethanes in varying proportions dependingon the conditions used (CEC, 1986; ICI, personal communication to the IPCS).
World production of methylene chloride in 1 980 was estimated to be 570 000 tonnes (Edwards et al., 1982); a simila r figure is considered to apply currently (ECSA, 1992). USA production was 229 000 tonnes in 1988, the demand being 207 000 tonnes. The tota l amount produced in western Europe ranged from 331 500 tonnes in 19 86 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, 1 986) to 175 000 tonnes/year in 1989 and to 150 000 tonn es/year in 1992 (CEFIC, 1993).

Most of the applications of methylene chlor ide are based on its considerable solvent capacity, especially for g rease, plastics and various paint-binding agents. Other important p roperties are its volatility and stability; it is also non-flamma ble. Among its uses are (CEFIC, 1983):

- a component of paint and varnish stripp ers, and adhesive formulations

- a solvent in aerosol formulations
- an extractant in food and pharmaceutica l industries

- a process solvent in cellulose ester pr oduction and fibre and film forming

- a process solvent in polycarbonate prod uction
- a blowing agent in flexible polyurethan e foams
- the extraction of fats and paraffins

- plastics processing, and metal and text ile treatment

- a vapour degreasing solvent in metal-wo rking industries

An estimated breakdown of usage worldwide b efore 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 changedsince. Reliable reports on present trends are not available.

3.2.3 Consumer applications

The main use in consumer products is in pai nt strippers, where methylene chloride is the main constituent (70- 75%). The second important use is in hairspray aerosols, where i t acts as a solvent and vapour pressure modifier. In the European Commu nity (EC) it may be used in such products at concentrations of up t o 35% w/w (European Council, 1982). The US Food and Drug Administra tion has banned the use of methylene chloride in cosmetic products. It is also used in aerosol paints. Other types of methylene chloride-conta ining products are household cleaning products and lubricating, de greasing 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 accordi ng to the Swedish Code of Statutes. Furthermore, it may not be used fo r working purposes after 1st January 1996 (National Chemical Inspe ctorate, 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 indus tries, 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 

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Methylene chloride is released to the atmos phere during its production, storage and transport, but more tha n 99% of the atmospheric releases result from industrial andconsumer uses (US EPA, 1985). It has been estimated that 85% of the to tal amount of methylene chloride produced in the USA is lost to the env ironment, of which 86% is released to the atmosphere (US EPA, 1985). D ata reported to the US EPA for the 1988 Toxic Chemical Release Invento ry indicate that approximately 170 000 tonnes of the USA product ion volume for 1988 (230 000 tonnes) was lost to the atmosphere; of this, 60 000 tonnes resulted from industrial methylene chloride emi ssions and 110 000 tonnes from the use of consumer products and fr om other sources such as hazardous waste sites.
Estimates of annual global emissions of 500 000 tonnes have been reported for methylene chloride (WMO, 1991). Th e short atmospheric lifetime of methylene chloride (see section 4.2 .1) implies that emissions quantities given on a seasonal as wel l as on a regional basis are more relevant for comparison with atm ospheric measurements. The total emission into the air in western Euro pe was estimated to be 173 000 tonnes for 1989 and 180 000 tonnes in 1 991.

4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANS FORMATION
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 p roduced hydroxyl radicals with a lifetime of 6 months. Given an int ra-hemispheric mixing time of approximately 1 month, transport ca n occur to regions far removed from the emission source. The atmosphe ric lifetime is fairly short relative to the inter-hemispheric tra nsport time of 1 to 1.5 years, resulting in higher concentrations of m ethylene chloride in the northern hemisphere, where most of the emis sions occur at present.
Methylene chloride is expected to have no signific ant impact on stratospheric ozone depletion. It will not contrib ute significantly to photochemical smog formation.

Hydrolysis and photolytically induced degradation in water are slow compared to evaporation. Methylene chloride h as been shown to disappear rapidly from soil and ground water due t o bio- transformation.

The aerobic and anaerobic degradation of methylenechloride has been proven by a variety of different test systems . Complete biodegradation by acclimated bacterial cultures un der aerobic conditions is rapid. There is no evidence that significant bioaccumulation or biomagnification of methylene c hloride along the food chain will occur.

4.1 Transport and distribution between media

4.1.1 Water/air

Methylene chloride enters the hydrosphere e ither directly, via aqueous effluents, or indirectly from the atmos phere by dissolution in sea water and in rain water. Due to its high vo latility (Henry's Law constant 380 Pa.m 3 /mol at 20°C) and low liquid-film transfer coefficient (Kp = 0.005 m/h), methylene chlorid e is rapidly transferred from the hydrosphere to the atmosph ere.
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 t he 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 t he surface of the water caused an increase of 17% in volatilization ove r a period of 20 min compared to the presence of still conditions (D illing et al., 1975). A decrease in the water temperature decreased the rate of volatilization. For example, over a period of 3 0 min, a 28% decrease in rate was seen at 1-2°C compared to that at 2 5°C (Dilling et al., 1975).

When measured under field conditions in exp erimental ponds, half- lives for methylene chloride of 26-28 h have be en reported (Merlin et al., 1992). Its half-life for evaporation from the river Rhine has been estimated to be 33-38 clays (Zoeteman et a l., 1980). Further estimates of the half-life for its evaporation are between 3 and 48 h depending on wind and mixing conditions (Halbar tschlager et al., 1984). In a further study, methylene chloride w as 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 poin t in a river basin (De Walle & Chain, 1978).
Rain-out is considered to be a limited proc ess for removal of methylene chloride from the troposphere. If it is assumed that its aqueous-phase concentration is in equilibrium w ith the background 

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(35-38 ppt) (Cox et al., 1976; WMO, 1991), the total amount of methylene chloride rained out in the northern h emisphere will be 700 tonnes/year (assuming a rain fall of 2.5x10 14 tonnes/year containing 9.9 ng/m 3 (2.8 ppt) at 10°C). The same calculation performed at 20°C (Henry's constant is 1.57 tim es higher) would lead to a value of 445 tonnes methylene chloride rai ned out annually in the northern hemisphere. For the southern hemispher e, 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 rele ases at ground level may reach the stratosphere (Derwent & Eggleton, 1978).

4.1.2 Soil/air

Methylene chloride present in the soil is p redicted 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 f or methylene chloride is 8-10 (log K oc= 0.89-1.05). Methylene chloride has a low tendenc y to adsorb to soil (adsorption coefficient 0.25 for a soil containing 1% organic carbon, Giger et al., 1983). Therefo re there is a potential for it to leach to ground water.

The amount of adsorption of methylene chlor ide 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 absor bed 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 chlo ride over water, soil and air compartments may be estimated by means of the fugacity model developed by MacKay (Slooff & Ros, 1988). Appli cation of this model suggests that over 98% of the total emissions o f the chemical will be found in air, 1 to 2% in water and far less tha n 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 ch loride is scavenged from the atmosphere is the reaction with hydrox yl rate of methylene chloride can be calculated from the rate consta nt for the initiating breakdown reaction with HO. and the varying con centration of these radicals in the troposphere. Determination of t he rate constant for the reaction of methylene chloride with hydroxy l radicals has been the subject of various investigations. WMO (1991) r ecommends the following value:

k OH= 5.8 × 10 -12 exp(-1100/T) cm 3 molecule -1 s -1

Other reactive species (e.g., ozone, oxygen atoms, chlorine atoms and nitrate radicals) are not thought to contri bute 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) G lobal average [X] Lifetime with: (cm 3 molecule -1 s -1 ) (molecule cm -3 ) (years)

.Cl 4.1 × 10 -13 10 3 77 (IUPAC, 1992) (estimated) (estimated)

.NO 3 <3.2 × 10 -17 1.2 × 10 8 > 8.3

.O( 3 p) 6.44 × 10 -16 2.5 × 10 4 approx. 2000 (Barassin & Cambourieu, 1973)

.O( 1 D) < 5 × 10 -10 0.5 > 120 (estimated)

Carbon dioxide and hydrogen chloride are th e major breakdown products and minor quantities of carbon monoxid e and phosgene are formed (Sanhueza & Heicklen, 1975; Rayez et al. , 1987). The breakdown reaction can be described as follows:

CH 2 Cl 2 + HO. --> .CHCl 2 + H 2 O .CHCl 2 + O 2 --> .CHCl 2 O 2 .CHCl 2 O 2 + NO --> .CHCl 2 O + NO 2 .CHCl 2 O --> .Cl + HCOCl or .CHCl 2 O + O2 --> COCl 2 + HO 2 (minor reaction)

Formyl chloride may be taken up by cloud dr oplets, hydrolysed to 

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or land surfaces and then hydrolysed. The overa ll lifetime for wet or dry deposition is unlikely to exceed a few mont hs 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 (C O, CO 2 ) and HCl (Libuda et al., 1990).

Phosgene is known to hydrolyse slowly in th e gas phase, but rapidly once dissolved in liquid water, to give CO 2 and HCl.

HCl is removed from the troposphere by wet deposition (dissolution in atmospheric water droplets and subsequent ra in-out) or dry deposition (direct uptake by the oceans, land s urfaces, vegetation etc.) with an average lifetime of about 1 week. The amount of chloride
deposited in this manner is completely negligib le compared to the natural atmospheric chloride flux of around 10 10 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 aqare 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 h ydroxyl radicals being dominant. The total rate constant for the sunli ght-induced transformation in surface water (with a depth o f 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 es timated 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 irridia tion for 5 days at 22°C (Chodola et al., 1989).
The half-life of a 1 mg/litre aqueous solut ion of methylene chloride was found to be about 1.5 years when m easured 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 pH4 or 9.2 after 7 days in the dark (Chodola et al., 1989). Under acidi c and basic conditions in the temperature range of 80-150°C, the hydro lysis of methylene chloride results in the formation of formaldehy de and HCl (Fells & Moelwyn-Hughes, 1958). Extrapolation of these d ata 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 hyd rolysis of methylene chloride varies with temperature, the extrapola tion of rate data from 80-150°C may not be valid.

No reductive dehalogenation of methylene ch loride 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, hydrolys is is probably not an important process in the removal of methylene c hloride from soil (see section 4.2.2).

In a lysimeter experiment, a 90% decrease o ver 2.5 m soil column was obtained (Nellor et al., 1985).

In the report of a spillage, the concentrat ions of methylene chloride were up to 802 mg/m 3 and 26 900 mg/m 3 near the point of leakage. In both cases, methylene chloride coul d not be detected some hundred metres away from the points of contamin ation even in the direction of the groundwater flow (ECSA, 1989). In the neighbourhood of polluted areas, an increase of bacterial act ivity has been found. In well-documented cases of accidental spills t o 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 deg radation test following the Japanese MITI standards (Kawasaki, 1980). H owever, complete degradation occurred during a static-culture fl ask test (Tabak et al., 1981).

In laboratory studies methylene chloride wa s almost completely transformed within days by bacteria enriched fr om a primary sewage sludge, municipal activated sludge (with or wit hout acclimitization) and industrial waste water (Rittmann & McCarty, 1980; Davis et al., 1981; Klecka, 1982; Stover & Kincannon, 1983; H albartschlager et al., 1984).

In field studies it has been shown that met hylene chloride is efficiently removed from water treatment works (Namkung & Rittmann, 1987).

Certain strictly aerobic, facultative methy lotrophic bacteria, 

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from contaminated soil and waste-water treatmen t plants, are capable of using methylene chloride as a sole carbon so urce for growth (Brunner et al., 1980; Stucki et al., 1981).

Secondary substrate utilisation of methylen e 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 ch loride. It converted methylene chloride to formaldehyde via the nucl eophilic displacement of chloride and the formation of S- chloromethyl glutathione and

S-hydroxymethyl glutathione. This enzymic dehalogenat ion in extracts of methylene-chloride-grown cells amounted to 1 160 mg/g protein per h under alkaline (pH 8-9) conditions (Stucki et a l., 1981; Leisinger, 1983).

Eight other bacteria (mainly Pseudomonads ), capable of growing on methylene chloride as their sole carbon sour ce, were isolated from enriched cultures. Maximum degradation rates fo r methylene chloride (up to 860 mg/litre per h) were found for an in itial saturated solution of 14.5 g/litre in a pH-controlled fer menter (flow rate 10 ml/h). Further increases in degradation rate were limited by the high salt concentration resulting from the neut ralization of the degradation products. In a fluidized bed reacto r with bacteria immobilized on silica, a degradation rate of me thylene chloride of up to 1600 mg/litre per h was observed (Gälli and Leisinger, 1985; Stucki, 1990).

Ubiquitous soil- and water-dwelling nitrify ing bacteria such as Nitrosomonas europaea, which depends for growth on the oxidation of ammonia, were able to degrade 1 mg methylene ch loride/litre completely within 24 h in the presence of ammonia and by 6 7% in the absence of ammonia (Vannelli et al., 1990).
The removal of methylene chloride from aero bic soil was significantly increased following exposure to m ethane (Henson et al., 1988).

Flathman et al. (1992) described the remedi ation of ground water contaminated with dichloromethane after a leak. Air stripping was used initially on water pumped out from the contamin ated site, and 97% of the contamination was removed in this way. This was followed by the first phase of bioremediation, in which contami nated water was withdrawn from the site and added to a bioreact or containing bacteria acclimated to DCM. The treated water was reinje cted on the site together with the bacteria. This phase decrease d the concentration by 97% over a period of 40 days. A second phase of bioremediation followed some 3 years later, dealing with a sub section of the original site. In this case, the indigenous bacteria wer e used and nutrients were added to the site. Concentrations before t reatment were up to 5200 mg/litre; after 10 months these had reduce d to < 2 mg/litre. At this point active treatment ceased, but the lev els 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 ethylben zene (Scholz-Muramatsu et al., 1988).

Aerobic biodegradation of methylene chlorid e was observed in a variety of surface soils including sand, a sand y loam and a sandy clay loam, as well as in subsurface clay soil. Degra dation occurred over concentrations ranging from approximately 0.1 t o 5 mg/litre. The time required for 50% disappearance of the parent co mpound varied between 1.3 and 191.4 days.

4.3.2 Anaerobic

Details of studies on the anaerobic biodegr adation of methylene chloride are given in Table 6.

Methylene chloride was degraded at a concen tration of 200 μg/litre in the aqueous phase of natural sediment. Degra dation was observed to proceed via methyl chloride, although accumulat ion was not observed (Wood et al., 1981). After a varying acclimatio n period using anaerobic digestion in waste water, 86-92% conv ersion to CO 2 will occur (Gossett, 1985). The half-life of methyle ne chloride in an anaerobic water/sludge system is 11 days (Bayar d et al., 1985).
Methylene chloride degradation was observedunder anaerobic conditions in sandy loam soil (Davis & Madsen, 1991).

4.3.3 Bioaccumulation

The n- octanol/water partition coefficient for methylene c hloride is 18 (log P ow= 1.25-1.3). As a consequence, its bioaccumulation is not expected to be significant. Moreover, its h igh depuration and degradation rate will reduce the probability of bioaccumulation.

No experimental bioconcentration factor (BC F) for methylene chloride is available. Its theoretical BCF rang es between 0.91 and 7.9 (Veith et al., 1980; Lyman et al., 1982; Veith and Kosian, 1983; 

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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 b iological factors
The ozone-depletion potential (ODP) of meth ylene chloride, as compared to the standard ODP of CFC11, can be e stimated from the numbers of chlorine atoms (2 as compared to 3 f or 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 tha t of CFC11.

Table 6. Aerobic biodegradation of methyle ne chloride

Test system Condition Duration Degradation Initia l 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 volati lization 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 volati lization 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 264-13 00 mg/litre Halbartschlager et al. (6 weeks per hour (1984) acclimatization) transformation

Table 6 (Cont'd)

Test system Condition Duration Degradation Initia l 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% Namkun g & Rittmann activated sludge transformation (1987) plant

At the current estimated total emission rat e of 500 000 tonnes per year, the calculated tropospheric chlorine load ing due to methylene chloride is 35 ppt, i.e. approximately 1% of th e total chlorine loading of 3600 ppt (WMO, 1991).

As methylene chloride has a low photochemic al ozone creation potential in the troposphere (0.9), when compar ed 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 ai r. 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 lo wer environmental levels of methylene chloride.

For the general population, air is the major sourc e of exposure to methylene chloride. In indoor air, higher level s may result from the use of consumer products which contain methyle ne chloride. High levels of methylene chloride may occur for short p eriods of time when paint strippers and aerosols are used.

Exposure to methylene chloride can occur during it s production and use as a paint stripper. cleaner, degreaser, p rocess solvent and as an aerosol. 

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Environmental levels measured before 1980 w ere summarized in EHC 32: Methylene Chloride (IPCS, 1984). This m onograph 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 area s, mean background levels of methylene chloride are 0.07-0.29 μg/m 3 (Table 7). The average concentrations in suburban and urban ar eas, respectively, are reported to be < 2 μg/m 3 and < 15 μg/m 3 . In the vicinity of hazardous waste sites, up to 43 μg/m 3 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 (He llmann, 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 ambi ent air
Country/ Location Year of Concentration Referenc e region measurement (μg/m 3 )

Germany urban area: Frankfurt 1980 2.1-4.2 Arendt e t al. (1982)

Italy northern part 1983-1984 < 14 De Borto li et al. (1986)

Netherlands Delft, Vlaardingen (urban 1980-1981 14.1 (max. annual mean) Guicheri t & Schulting (1985) area) Isle of Terschelling (rural, 1980-1981 1.4 (max. annual mean) Guicheri t & Schulting (1985) suburban area) mean concentration in 1980-1981 9 Guicheri t & Schulting (1985) the country

USA rural, suburban areas - 0.18-2.1 Shah & H eyerdahl (1988) San Francisco Bay area 1984 3.2-9.1 Levaggi et al. (1988) urban areas 1980 0.8-6.7 Shah & H eyerdahl (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 e t al. (1985)

Arctic Spitzbergen July 1982 0.26±0.04 Hov et a l, (1984) March 1983 0.29±0.06 Hov et a l. (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 Ref erence measurement (μg/litre)

Ground water

Italy Milan 1983 4.5 CEF IC (1986)

USA Iowa 128 wells 1984-1985 1-5 (4 wells) Kel ley (1985)

Surface water

Germany Mosel 1983 1.5-2.0 Hel lmann (1984)

Neckar 1983 0.6-1.0 Hel lmann (1984)

Elbe 1983 0.7-2.1 Hel lmann (1984)

Elbe 1988 11 (max) LWA (1990)

Weser 1982-1983 < 0.5 Hel lmann (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) Hel lmann (1984)

Rhine at the Wesel 1983 < 2.0 Hel lmann (1984)

Rhine at Duisburg 1984 1.5 (max) LWA (1984) 

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Table 8 (Cont'd)

Country Location Year of Concentration Ref erence 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 Vande 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 Ref erence measurement (μg/litre)

USA Susquehanna river, 1987 10 (mean) Smi th (1989) Columbia

Lancaster 1987 4.7 (mean) Smi th (1989)

Ohio river basin (11 1980-1981 > 1 (238 samples) How ard et al. (1990) stations, 4972 samples) > 10 (19 samples)

Sea and estuarine East Pacific Ocean 1981 0.002 (mean) Sin gh et al. (1983) water (30 samples)

East Sea (German Coast) 1983 1.3-2.6 Hel lmann (1984)

North Sea (German 1983 0.06-0.20 Hel lmann (1984) Coast)

In surface water, levels of methylene chlor ide have been reported to vary from not detectable to 10 μg/litre. Acc ording to data recorded in the US EPA STORET database, 30% of the sampl es showed methylene chloride levels above the detection limits. A m edian concentration of 0.1 μg/litre was estimated (Staples et al., 198 5).
Limited information concerning the contamin ation of sea water and estuaries by methylene chloride is available. I t 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 ch loride is generally not detected in open oceans. A mean concentration o f 2.2 ng/litre has been reported in the South Pacific Ocean.

Methylene chloride enters the aquatic envir onment primarily through waste water discharge. An estimated amo unt of 0.2% of the total methylene chloride production is releasedin waste water (Dequinze et al., 1984). The input from air rai n-out has been estimated for the northern and southern hemisph ere (section 4.1.1).

Waste water from certain industries has bee n reported to contain methylene chloride at average concentrations in excess of 1000 μg/litre, these being coal mining, alumini um forming, photographic equipment and supplies, pharmaceut ical manufacture, organic chemical/plastics manufacture, paint an d ink formulation, rubber processing, foundries and laundries. The maximum concentration measured was 210 mg/litre in waste water from t he paint and ink industry and the aluminium-forming industry (US EPA, 1981).

In the US EPA STORET database on industrial effluents, 38.8% of 

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concentration of 10 μg/litre (Staples et al., 1 985).

Samples from the outfalls of four municipal treatment plants in Southern California, USA, with both primary andsecondary treatment, contained < 10 to 400 μg methylene chloride/lit re (Young et al., 1983). In 30 Canadian water-treatment facilitie s, 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 l andfills, 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 contaminati on by methylene chloride are limited. It is the sixth most freq uently detected organic contaminant in ground water at hazardous waste disposal sites in the CERCLA database (178 sites), the detection freq uency being 19% (Plumb, 1987). In contaminated ground water in Minnesot a, USA, up to
250 μg/litre has been detected (Sabel & Clark, 1984). Levels of up to 110 μg/litre were found in percolation water fr om a waste-disposal site in Germany. However, methylene chloride wa s not found (< 1 μg/litre) in the ground water below the si te (Heil et al., 1989).

5.1.3 Aquatic organisms

Concentrations of methylene chloride in fre shwater organisms have been reported for oyster and clams from Lake Po nchartrain, Louisiana, USA. Levels ranging from 4.5 to 27 μg/kg (wet w eight) 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/k g wet weight were found in marine bottom fish taken from Commencement B ay in the state of Washington, USA (Nicola et al., 1987).

Data on biota collected in the US EPA STORE T data base show an average level of 660 μg/kg in the 28% of the sa mples in which methylene chloride was detected (Staples et al. , 1985).

5.1.4 Soil and sediment

No data are available on the levels of meth ylene chloride in soil.

The levels of methylene chloride found in s ediment from Lake Pontchartrain, Louisiana ranged from not detect able 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 sedimen t sampling data (Staples et al., 1985).

The levels of methylene chloride found in s ediments from the river Rhine in 1987-1988 varied from non-detectable t o 30-40 μg/kg. At one site maximum concentrations of 220-2200 μg/kg w ere 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 meth ylene chloride are used, air levels of methylene chloride much hig her than outdoor levels (< 15 μg/m 3 , see section 5.1.1.1) may be found (Table 7).
Relatively high levels (mean 670 μg/m 3 , peak level 5000 μg/m 3 ) have been found in the indoor air of residentia l houses (De Bortoli et al., 1986).

5.2.1.2 Drinking-water
Methylene chloride has been detected in dri nking-water supplies (estimations made before 1980) in numerous citi es in the USA (Dowty et al., 1975; Coleman et al., 1976; Kopfler et al. , 1977; Kool et al., 1982), the mean concentrations reported being g enerally less than 1 μg/litre. An average of 3-10 μg/litre and a m aximum of 50 μg/litre were observed in a Canadian study of 30 drinkab le water treatment facilities (Otson et al., 1982).
Samples from 128 drinking-water wells in th e 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 dr inking-water of Santiago de Compostela, Spain, in 1987. Methyle ne chloride was found in 98.4% of the samples; the average concentrat ion was 14.1 μg/litre, with a range of 1.2-93.2 μg/litre. Other halome thanes were also found and measured in the samples at average concentr ations ranging from 9 to 25 μg/litre.

A wide sampling exercise involving 630 publ ic community water supplies (serving 6.9 million people in New Jer sey, USA) was carried 

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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/li tre.

5.2.1.3 Foodstuffs
Although methylene chloride is used in foodprocessing (solvent extraction of coffee, spices, hops), there is l ittle information on its residual levels in food. In the USA, residu es of methylene chloride were found in decaffeinated coffee bea ns (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-c ream and yoghurt (BUA, 1986).

In seven types of decaffeinated ground coff ee 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 (P age & Charbonneau, 1984).

Heikes & Hopper (1986) analysed samples of grains and intermediate grain-based foods for a range of fumigants usin g 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 wa s found in some of the intermediate foods such as bleached flour (30 μ g/kg), yellow corn meal (4.7 μg/kg), lasagne noodles (5.4 μg/kg) and ye llow cake mix (4.6 μg/kg).

One of the authors (Heikes, 1987) investiga ted levels of methylene chloride in table-ready foods, taken from the U S Food and Drug Administration's Total Diet Study. Of the 19 fo ods examined, eight contained methylene chloride above the quantifi cation limit (not given). Detailed results for six of the foods a re given in Table 9.

Table 9. Dichloromethane content of table read y foods (Heikes, 1987)

Food Number of Numbe r Range of samples positi ve 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 foods a12 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 and66% of aerosol spray paints sold as household products contained met hylene chloride (US EPA, 1987). Over 100 consumer products in Swede n contain methylene chloride (National Chemical Inspectorate, Swede n, personal communication to the IPCS). In Norway the numbe r is around 140, including 45 paint removers (AKZO, personal communication to the IPCS).
Methylene chloride does not appear to be su bject to widespread volatile substance abuse. Statistics on deaths resulting from substance abuse in the United Kingdom were coll ected 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 p roducts, 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 b asis of USA investigations of household solvent products. T he estimated levels ranged from less than 35 mg/m 3 to a few short-term exposures of 14 100 to 21 200 mg/m 3 . The majority of the concentration estimates were below 1770 mg/m 3 (US EPA, 1990).

Methylene chloride exposure was estimated w hile 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, inv olving furniture stripping in a room with through ventilation, t he operator exposure was found to be 289 mg/m 3 on a 2-h TWA. Peaks of exposure were observed during application (460 mg/m 3 ) and during scraping-off (710-1410 mg/m 3 ) (ICl, 1988, personal communication to the IPCS).

A series of paint-stripping exercises were performed in a small room. Various ventilation conditions were evalu ated while using a number of formulations of paint stripper. A wor st possible case was 

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chloride in the room rose to 14.1-17.6 g/m 3 (4000-5000 ppm), although it is questionable whether anyone coul d work in such conditions without breathing apparatus. Further exercises with the door and windows open (as recommended by suppli ers) reduced atmospheric concentrations by more than a facto r of 10. Exposure to methylene choride resulted in an 8-h TWA of 187 -226 mg/m 3 (53-64 ppm). The actual stripping operations to ok between 1 and 1.5 h. Maximum exposures occurred during initial appli cation and scraping- off. These exposures were of a few minutes dura tion and concentrations never exceeded 3.53 g/m 3 (1000 ppm) (ICI, 1990, personal communication to the IPCS).

The effect of variation in the formulation was also investigated. During paint stripping, background concentratio ns in the room varied from 710-1410 mg/m 3 to less than 350 mg/m 3 depending on the formulation. However, within 5-6 min of applica tion the level of methylene chloride fell to an equilibrium conce ntration of around 71 mg/m 3 , irrespective of the formulation used. The shortes t time before reaching equilibrium was about 2-2.5 min (ICI, 1990, personal communication to the IPCS).

Studies in the Netherlands have measured pe ak concentrations in salons of 21-106 mg/m 3 (6-30 ppm), with an 8-h TWA of 3.53-17.65 mg/m 3 (1-5 ppm) (CEC Scientific committee of

Cosmetology). The same study measured a peak co ncentration of 265 mg/m 3 (75 ppm) arising from home use of a hairdressing a erosol containing 35% methylene chloride. This equates to a TWA of 2.65 mg/m 3 (0.75 ppm).

Studies in the United Kingdom simulating co nsumer exposure during salon use yielded values well within the nation al Maxium Exposure Limit (353 mg/m 3 or 100 ppm for an 8-h TWA). The hairdresser received an exposure of 77.7 mg/m 3 (22 ppm, on an 8-h TWA) during what is considered to be exceptionally heavy us e (i.e. a 10-second spray every 15 min for an 8-h period). The cust omer exposure was found to be 106-265 mg/m 3 (30-75 ppm) (10-min TWA) (ICI, 1990, personal communication to the IPCS).
The same study simulated home use of person al-care aerosols containing methylene chloride. Even adverse con ditions (small room, no ventilation) resulted in an exposure of 353 mg/ m 3 (100 ppm) (10-min TWA), equating to 7.06 mg/m 3 (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 du ring its production and use as a paint stripper, cleaner, degreaser, pr ocess 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 ind ustries.

5.2.2.1 Production

Production of methylene chloride is normall y carried out in a closed system, with a relatively small number o f people being involved. Exposure arises primarily during fill ing and packing operations. Occupational exposures are listed i n Table 10. Some measured ranges, e.g., 35-81 mg/m 3 and 85-244 mg/m 3 (HSE, 1992), indicate that engineering control techniques ca n bring 8-h TWA exposures below 350 mg/m 3

. 5.2.2.2 Paint stripping and related activities

Workers in the formulation of paint remover s are exposed while transferring methylene chloride from storage ta nks, during mixing (blending) operations and while packaging. The extent of exposure will depend on the control measures and work practic es in force. Exposure levels (8-h TWA) range from a low of 0-18 mg/m 3 to over 1770 mg/m 3 (US EPA, 1990).

Table 10. Occupational exposure to methyle ne chloride

Industry Activity E xposure range Comments aReference (8 -h TWA) (mg/m 3 )

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) 

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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 E xposure range Comments aReference (8 -h TWA) (mg/m 3 )

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 E xposure range Comments aReference (8 -h TWA) (mg/m 3 )

Quarry Laboratory work, 71-1370 High exposures due to inadequat e 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 = loc al exhaust ventilation

Paint strippers are widely used in a number of industries: automotive, rubber products, furniture and fixt ures, plastic, and electronic industries. Exposure to methylene ch loride takes place during application, removal of the substrate so aked in methylene chloride, and the disposal of the spent paint r emover. Typical exposures (8-h TWA) range from 18 mg/m 3 to about 1770 mg/m 3 (US EPA, 1990).
Exposure of commercial furniture refinisher s to methylene chloride occurs when stripping involves either the dippi ng of furniture into a tank containing a mixture of solvents includingmethylene chloride (typically 65%) or coating it manually with a b rush. Exposure levels are highly variable and greatly influenced by t he size of the organization, engineering controls in place andwork practices. Some refinishers may operate on a part-time basis an d from their homes. In some instances where the worker was leaning ove r the tank or using a brush to scrub the surface coating, concentrati ons of > 7100 mg/m 3 have been recorded (McCammon et al., 1991; HSE, 1992). Better work stations and work practices have helped to redu ce 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 genera lly below 180 mg/m 3 (ECSA, 1989). Potential occupational exposure t o methylene chloride as a result of aerosol products varies according t o the use and the work undertaken. 

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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 proc ess solvent in the manufacture of a variety of products. Most of t he processes are carried out in closed systems, with the excepti on of triacetate fibre and film manufacture. Normally, exposure levels are low, but occasionally high exposures (> 350 mg/m 3 ; 10-min TWA) may occur in such operations as filter changing, charging an d discharging. Some industrial processes involve somewhat higher ex posure levels; in particular, the manufacture of cellulose triace tate fibres and films can involve exposure up to 350 mg/m 3 (8-h TWA) even when good engineering controls are installed (Zahm et al. , 1987; ECSA, 1989). As part of an epidemiological study of cellulose f ibre production workers, Ott et al. (1983) reported exposure le vels ranging from 177 to 2436 mg/m 3 in the processing area, and exposure ranging from 18 to 1341 mg/m 3 in the preparation area based on sampling performe d in 1978. The range for the entire plant was later reported to be from below detectable limits to 6000 mg/m 3 (Lanes et al., 1990). As part

of an epidemiological study of photographic fil m workers, Friedlander et al. (1978) reported exposures ranging from 0 to 1236 mg/m 3 based on sampling carried out between 1959 and 1975. The highest mean exposures at this plant were reported for group leaders (402 mg/m 3 ) (Hearne et al., 1987).

In the pharmaceutical industry, methylene c hloride 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/m 3 (Zahm et al., 1987; HSE, 1992).

Methylene chloride is also used as an extra ction medium in the nutrition industry, where the exposure levels a re generally low when the processes are adequately controlled (Cohen et al., 1980).
Methylene chloride is used in the foam indu stry for cleaning process equipment, purging spray guns, and as a n auxiliary blowing agent. It is also used as a releasing agent in the moulding of polyurethane products. Exposure levels ranging from a few mg/m 3 to short-term exposures of over 1770 mg/m 3 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 applicatio n of the adhesive, to short-term levels in excess of 350 mg/m 3 (Fleeger & Lee, 1988; HSE, 1992). Processes involving the formulation of a dhesives are likely to be well controlled.
Methylene chloride is also used as a solven t in the analysis of bitumen samples. This work is normally carried out in small laboratories and exposure levels will be high u nless adequate control measures are used.
5.2.2.5 Cleaning and degreasing

In the manufacture of metal products, clean ing (degreasing) is required before painting, plating, plastic coat ing, etc. The degree of exposure to methylene chloride will be influenc ed by many factors, including the age of the equipment, type of eng ineering controls available, their maintenance, handling, and dry ing methods. In general, it is possible to reduce exposure leve ls to below 124 mg/m 3 (Swedish National Board of Occupational Safety & Health, personal communication to the IPCS).

5.2.3 Occupational exposure limits

A listing of some national occupational exp osure limits is given in Table 11.

Table 11. Occupational exposure limit valu es a

Country TWA STEL TWA STEL Remarks Reference (mg/m 3 , 20°C) b(ppm) (ppm) (ppm)

Australia 350 - 100 - Suspected carcinogen ILO (1991)

Austria 360 1800 c 100 500 Suspected of carcinoge nic 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 1800 c 100 500 Suspected of carcinoge nic DFG (1991) 

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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/m 3 , 20°C) b(ppm) (ppm) (ppm)

Portugal 174 - 50 - ILO (1991)

Sweden 120 250 d35 70 Classified as a low p otent AFS (1990) carcinogen

Switzerland 360 1800 100 500 Classification for teratogen ic ILO (1991) effects not possible; biolog ical 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 wo rking period); STEL = short-term exposure limit (15 min, unless specified) bOfficial values; some countries use different con version factors and/or other ambient temperature

c 30 min d15 min

5.3 Human monitoring data

5.3.1 Body burden

Methylene chloride was detected in all eigh t samples of human milk from four urban areas (Pellizzari et al., 1982) . Hardin & Manson (1980) could still find methylene chloride in m other's milk 17 h after the end of exposure.
In 12 male volunteers exposed to 2600 mg/m 3 , biopsies showed adipose tissue levels of 10.2, 8.4 and 1.6 mg/k g at 1, 4, and 22 h, respectively, after a 1-h exposure (Engström & Bjurström, 1977). When Antoine et al. (1986) analysed whole blood from250 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 fro m people living in industrialized areas of Beckum, Germany. They r eported that no methylene chloride was detected.

5.3.2 Occupational exposure studies

In a cohort of 14 furniture strippers expos ed to methylene chloride at concentrations of 53 to 1290 mg/m 3 , post-exposure breath concentrations of methylene chloride ranged fro m 8.1 to 590 mg/m 3 (McCammon et al., 1991).

Mother's milk in Soviet women manufacturingrubber articles contained a mean of 74 μg/kg in 17 out of 28 sa mples approximately 5 h after start of work, the level declining after termination of work (Jensen, 1983).

A group of seven non-smoking workers, who h ad previously been exposed to methylene chloride for several years , were exposed to a mean concentration of 635 mg/m 3 (in addition, there was exposure to 154 mg/m 3 (mean) chloroform). The pre-exposure average carbo n monoxide level in alveolar air was 34 mg/m 3 , increasing to 58 mg/m 3 during exposure; before the next exposure, the car bon monoxide level was 27 mg/m 3 : this corresponded to carboxyhaemoglobin (CO-Hb) levels of 4.9%, 8.3% and 3.9%, respecti vely. The biological half-life of CO-Hb was 13 h (Rathey et al., 197 4). Although methylene chloride does not accumulate following repeatedexposure, these data clearly indicate that CO-Hb levels will be cumu lative if the periods between exposure are insufficient to allow the CO-Hb levels to return to normal.
CO-Hb levels in a worker accidentally overc ome 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-H b level of 6% on the day following the exposure (Benzon et al., 1978 ).
Methylene chloride levels in alveolar air a nd blood were measured in 14 shoe-sole factory workers. The alveolar c oncentration, mean blood levels, and CO-Hb levels, following expos ure to 74±28 mg/m 3 methylene chloride, were: 49 mg/m 3 , 0.41 mg/litre, and 4.0%, respectively; upon exposure to 124+42 mg methyl ene chloride/m 3 : 71 mg/m 3 , 0.99 mg/litre, and 5.2%, respectively; and upon e xposure 

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6.5%, respectively. In this factory, the methyl ene chloride exposure was highly variable; the data are too limited t o allow valid extrapolation (Perbellini et al., 1977).
The relationship between low environmental levels of methylene chloride, carbon monoxide in alveolar air and u rinary 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 excret ion 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 refe rence values intended for guidelines for the evaluation of potential health hazards in industrial hygiene practice. The BEIs for methy lene chloride at the end of a working shift have been given as: CO-H b 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 exhaledair 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 analysi s of carbon monoxide in exhaled air or of CO-Hb in blood is to be prefe rred. However, this can only be applied in non-smoking subjects. Sampli ng should be carried out either about 0-2 h after exposure or 16 h l ater, i.e. on the following morning.

In the case of an 8-h exposure to less than 350 mg methylene chloride/m 3 in non-smokers, CO-Hb levels 2-h after exposure ce ases are not expected to exceed 2-3%, and after 16 hto 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 ab sorbed from the gastrointestinal tract and dermal exposure resultsin absorption but at a slower rate than that of the other exposure r outes.

Distribution studies indicate that, via inhalationor dermal exposure, methylene chloride distributes to all ti ssues. It can cross the blood-brain barrier and it can be transferred across the placenta. Concentrations of methylene chloride ris e more slowly in adipose tissue and longer exposures are required b efore these tissue levels equal those of the blood. Data indicate tha t methylene chloride and/or its metabolites do not accumulate in tissues.

Methylene chloride is metabolized to carbon monoxi de, carbon dioxide and inorganic chloride. Methylene chlorideis eliminated from the body primarily via the lungs in expired air. Urinary excretion plays a minor role in its elimination. As exposurelevels increase, a large proportion of methylene chloride is exhaled unchanged. Metabolism occurs by either or both of two pathway s; their relative contribution to the total metabolism is markedly d ependent 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 p athway appears to operate similarly in a qualitative and quantitative sense in all rodents studied and in humans. This is the predomi nant metabolic route, but saturation occurs at around 1800 mg/m 3 . Increasing the dose above the saturation level does not lead to e xtra metabolism by this route.

The other pathway involves a glutathione transfera se, and leads via formaldehyde and formate to carbon dioxide. Th is route seems only to become important at doses above the saturation level of the "preferred" oxidative pathway. There are marked sp ecies and dose- dependent differences in the contribution that thi s 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 me thylene chloride is inhalation. Evaluation of pulmonary uptake indi cated that 70-75% of inhaled vapour was absorbed in human subjects e xposed to 180, 350, 530
and 710 mg/m 3 . Initial absorption was rapid, as indicated by lev els of methylene chloride in the blood of approxima tely 0.6 mg/litre in the first hour of exposure to levels of 350-710 mg/m 3 . At a level of 180 mg/m 3 , the increase in blood methylene chloride concentr ation was 0.2 mg/litre for the first hour. There was a direct correlation between the steady-state blood methylene chlori de values and the exposure concentration, both during the exposur e 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 u ntil the end of exposure. Once exposure ceased, methylene chlor ide was rapidly cleared from the blood. Seven hours after exposure, les s than 0.1 mg/litre was detected following exposure to 180, 350 or 530 mg/m 3 . Only 1 mg/litre was detected in the highest group (7 10 mg/m 3 ) 16 h after 

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baseline concentrations (Di Vincenzo & Kaplan, 1981a,b).

In common with other lipophilic organic vap ours, 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 methy lene chloride absorbed by the body, due to an increase in ventilation rate and cardiac output, since these factors increase blood flowthrough the lungs and promote absorption (Di Vincenzo et al., 1972; A strand et al., 1975).

Uptake also increases with the body fat per centage, since methylene chloride dissolves in fat to a greate r extent than it dissolves in aqueous media. Therefore, obese su bjects will absorb and retain more methylene chloride than lean subjec ts exposed to the same vapour concentration. Under controlled conditio ns, there was a 30% greater absorption and retention of methylene c hloride by obese subjects exposed to 2650 mg/m 3 for 1 h as compared to lean subjects (Engström & Bjurström, 1977). Åstrand et al. (1975) reported that the amo unt of methylene chloride taken up increases with physical workl oad, whereas the retention decreases. With a 50-watt workload, t he 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 pos t-exposure period compared with exposure under rest conditions.

6.1.1.2 Animal studies
Studies of the relationship between inhalat ion exposures of animals and their blood methylene chloride conc entrations indicate that absorption is proportional to the magnitud e and duration of the exposure over a methylene chloride concentratio n range of 350 to 28 200 mg/m 3 . This conclusion is based on the monitoring of blo od methylene chloride concentrations following inh alation exposure in
dogs and rats (Di Vincenzo et al., 1972; MacEwe n et al., 1972; McKenna et al., 1982). As is the case with humans, bloo d methylene chloride levels reach a steady-state value which does no t increase further as the duration of exposure increases (McKenna et al., 1982).

The data from studies of blood methylene ch loride values during a 6-h exposure of rats to between 180 and 5300 mg /m 3 suggest that the steady-state blood/air concentration ratio incr eases as the exposure concentration increases. The ratio of the stead y-state methylene chloride concentrations in plasma to the exposu re concentration was 0.001, 0.005 and 0.006 at levels of 180, 1800 a nd 5300 mg/m 3 , respectively (McKenna et al., 1982). It is post ulated that the increased ratio at steady state results from sa turation of metabolic pathways as exposure increases, rather than fro m an increased absorption coefficient.

Kim & Carlson (1986)conducted experiments t o 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 inh alation. Rats and mice were exposed to 710, 1800 or 3500 mg/m 3 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 chlo ride level was found to be dependent upon the methylene chloride exposu re concentration, but the half-life was independent of the duration o f 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 c hloride in water or in corn oil suggests that methylene chloride is ea sily absorbed from the gastrointestinal tract. Methylene chloride leve ls were measured in gut segments up to 40 min after rats were administe red 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 th e administered dose up to the 40-min test interval (Angelo et al., 198 6b).

In mice administered oral doses of non-radi oactive methylene chloride at 10 or 50 mg/kg in water, approximat ely 25% of the administered dose was detected in the upper gas trointestinal tract within < 20 min. Similarly, after treatment wit h methylene chloride at 10, 50, or 1000 mg/kg in corn oil, approxima tely 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 abs orbed through human skin (Stewart & Dodd, 1964). In this study, a v olunteer immersed a thumb in methylene chloride for 30 min under co nditions which precluded the inhalation of vapour. The subsequ ent alveolar air concentration was 3.1 ml/m 3 ; 2 h later it had fallen to 0.69 ml/m 3

. Various studies on the rate of absorption t hrough animal skin and subsequent pharmacokinetics have been reported. Tissue concentrations of methylene chloride were measured in various organs (lung, liver, 

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chromatography, following immersion of two-thir ds 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 methyle ne chloride concentrations in fatty tissues increased marke dly after 3 h of exposure. After 4 h of exposure, methylene chlo ride concentrations remained elevated in fatty tissues and were inc reased 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 cm 2 (Tsuruta, 1975).

The dermal permeability constant for the ab sorption of methylene chloride vapour through rat skin in vivo has been measured following exposure to 106, 212 and 353 g/m 3 for 4 h. Blood levels of methylene chloride were shown to reach steady state level s after 1 h of exposure to the two lower concentrations and after 3 h o f exposure to 353 g/m 3 . The mean dermal permeability constant was calcula ted to be 0.28 mg/h per cm 2 (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/m 3 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 amo unt absorbed. The amount or methylene chloride absorbed was highl y 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 ob ese 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 chlorid e in certain tissues from pregnant or nursing women has been reporte d. The study was conducted following observations of disturbance s 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 expose d to several chemicals during their work for at least 3 years. The che micals included gasoline, ethylene dichloride and methylene chl oride. An estimate of the average workplace concentration of methylen e chloride was reported to be 85.6 mg/m 3 . A control group (number unspecified) was constituted from women working in the same faci lity but who had had no direct contact with the chemicals. The tissues examined were the blood, the fetal membranes and the fetus, all t issue samples being obtained at the time of abortion of the fetus. The mean tissue concentrations of methylene chloride (54 observ ations) 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, compar ed to 0.12 ± 0.07, 0.013 ± 0.01 and 0.016 ± 0.001 mg/kg in the con trols. Methylene chloride was also detected in 17 out of 28 spec imens of breast milk taken from exposed nursing women. An average co ncentration 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 insignifica nt quantity of methylene chloride was reported 17 h after cess ation of exposure (Vosovaja et al., 1974).

6.2.1.2 Animal studies

Distribution studies in rats demonstrate th at methylene chloride (and/or its metabolites) is present in the live r, kidney, lungs, brain, muscle and adipose tissues after inhalat ion exposures (Carlsson & Hultengren, 1975; McKenna et al., 1982). One hour after exposure, the highest concentration of radioactive materi al was found in the white adipose tissue, followed by the liver. Th e concentration in the kidney, adrenal and brain were less than half t hat in the liver. Radioactivity in the fat deposits declined rapi dly during the first 2 h after exposure (Carlsson & Hultengren, 1975 ). Concentrations in the other tissues declined more slowly. Whole b ody autoradiography in mice at one hour after inhalation of 10 μl 14 C-methylene chloride for 10 min showed a rapid and even distribution immediately after exposure. A high uptake was noted in brain, bod y fat, blood, liver and kidney. Evaporated sections showed a high reten tion of non-volatile radioactivity, presumably representing metaboli tes, in the liver, bronchi and kidneys. Thirty minutes after inhal ation, radioactivity started to appear in tissues with a high cell t urnover such as bone marrow, thymus and gastrointestinal mucosa, andin tissues with a high rate of protein synthesis such as the spleen, e xocrine pancreas and salivary glands (Bergman, 1979).

On the other hand, after 5 days of exposure to 710 mg/m 3 for 6 h/day, the concentration of methylene chlorid e in the perirenal fat was 6-7 times greater than that in the blood an d liver (Savolainen et al., 1977). It has been suggested that methylen e 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 chlo ride 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 chlo ride leads to exposure 

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Sunram, 1982).

6.2.2 Oral exposure

No studies are available regarding distribu tion of methylene chloride in humans following oral exposure.
In animals, radioactivity from labelled met hylene chloride was detected in the liver, kidney, lung, brain, epi didymal fat, muscle, and testes after exposure of rats to a single g avage dose of 1 or 50 mg/kg. The tissue samples were taken 48 h af ter dosing. At that time, the lowest concentration of radioactivity was found in the fat. The highest concentrations were in the liver an d kidney. This was true for both doses (McKenna & Zempel, 1981).

Similar results were observed in rats admin istered methylene chloride doses of 50-1000 mg/kg for 14 days. At each dose tested, and in each tissue, the label was rapidly cleared d uring the 240 min following each exposure (Angelo et al., 1986b).

6.2.3 Dermal exposure
No information is available regarding distr ibution 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, sli ces and hepatocytes, mainly from the rat, confirmedthe presence of the two metabolic pathways. The primary reaction, f irst described by Kubic & Anders (1975), appears to be an oxidative deh alogenation giving

carbon monoxide and chloride ion. The reaction is catalysed by rat liver microsomal fractions and is dependent upo n NADPH and molecular oxygen. The presence of a binding spectrum and inducers confirmed the involvement of the cytochrome P-450 mixed funct ion oxidase system. More recent studies have identified the cytochr ome P-450 isoenzyme as cytochrome P-450 IIEl (Pankow et al., 1991; Gue ngerich et al., 1992; Pankow & Jagielki, 1993). The highest activity was found in liver microsomes, which were five times more active t han lung microsomes and thirty times more active than kidney microsomes . The proposed metabolic route involves rearrangement of the p rimary hydroxylation product to formyl chloride followed by decompos ition to carbon monoxide (Kubic & Anders, 1978). Although the t ransient intermediates have not been isolated or identified, their for mation is consistent with the enzyme involved and the products forme d.
The effect of pyrazole on methylene chlorid e 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 in jection. The metabolism of methylene chloride to carbon monoxide can be stimulated or inhibited by pyrazole; the effect depends on th e 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 inhi bition was observed following pre-treatment with high pyrazole dose s or following simultaneous administration of pyrazole and met hylene chloride. The inhibition may reflect the competition between pyrazole and methylene chloride for oxidation by CYP2El as long as pyr azole is present in the blood, or may also reflect the hepatotoxic effe ct of pyrazole.
Hepatic cytochrome P-450 levels were not in creased in rats exposed by inhalation to methylene chloride (5.29-10.59 g/m 3 (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/m 3 (500 or 1000 ppm) 6 h/day for 2 weeks (Kurppa & Vainio, 1981). Marginal increases wer e seen in a third study (Norpoth et al., 1974) in which rats were expos ed to 0.176-17.6 g/m 3 (50-5000 ppm), 5 h/day for 10 days. In the stud y by Kurppa & Vainio (1981) an increase in renal ethoxycoumarin de-e thylase activity was reported.
The second metabolic pathway occurring in r at liver is localized in the soluble (cytosolic) fraction (Ahmed & An ders, 1976, 1978). It does not require oxygen but is dependent upon g lutathione and a glutathione- S-transferase enzyme, the products in vitro being formaldehyde and chloride ion. The rapid and al most quantitative conversion of formaldehyde to formic acid and t hen carbon dioxide known to occur in vivo (Neely, 1964) is consistent with this pathway being the source of carbon dioxide exhaled afte r exposure to methylene chloride. The intermediates involved in the met abolism of methylene

chloride to formaldehyde are unknown, but the n ature of the enzyme involved and the dependence upon glutathione su ggest that S- chloromethyl-glutathione is formed and rapidly hydr olysed and degraded to glutathione and formaldehyde (Ahmed& Anders, 1978). The isoenzyme involved in the metabolism of methyle ne 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 

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Formaldehyde, in addition to its metabolism to carbon dioxide, becomes incorporated into the C-1 metabolic pool via fo rmic acid. Therefore, exposure to radiolabelled methylene chloride re sults in the incorporation of radioactivity into macromolecu les 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 erythy rocytes (Thier et al., 1991) and to be due to the presence of a glutat hione- S- transferase enzyme (Schroeder et. al., 1992). The work by S chroeder describes the detection of enzyme activity in erythrocytes us ing methyl bromide as a substrate, not methylene chloride. Furthermore, in experiments investigating the influence of cofactors on enz yme activity, glutathione could be replaced by L-cysteine, su ggesting 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 var ious animal species and in humans has been studied extensively (e.g., F odor et al., 1973; Kubic et al., 1974; Roth et al., 1975; Lee Rodk ey & Collison, 1977; Peterson, 1978; McKenna & Zempel, 1981; McKenna et al., 1982; Angelo et al., 1986a,b).

Methylene chloride and the other dihalometh anes are unique in being the only class of industrial chemicals kn own to be metabolized to carbon monoxide. This metabolic pathway (dep endent on cytochrome P-450), first discovered in humans (Stewart et al., 1972), results in elevated levels of CO-Hb and in increased level s of carbon monoxide in expired air. Subsequent studies in experimental animals and in humans established that this pathway is rate-limited b y enzyme saturation, so that at high doses the levels of CO-Hb become c onstant and independent of dose (Lee Rodkey & Collison, 1977). Later ex periments in animals using radiolabelled methylene chloride identifi ed carbon dioxide as the other major metabolite (Di Vincenzo & Hamil ton, 1975). Although

carbon dioxide is a known metabolite of carbon monoxide, the amount of carbon dioxide formed from the monoxide was tho ught 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 p retreatment or co- administration of other organic solvents on the metabolism of methylene chloride to carbon monoxide. Pankow e t al. (1991) described increases in CO-Hb levels in rats pretreated wi th 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 in creased 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 pretrea ted with a single garage dose of methanol (Pankow & Jagiel ki, 1993). In both studies, the levels of CO-Hb were reduced when the solvents were co- administered with methylene chloride. The resul ts of both studies were considered to be consistent with the metabolismof methylene chloride by cytochrome P-450 IIE1.

At first sight it might appear that the rel ative 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 (Garga s et al., 1986; Reitz et al., 1986). Similar studies in mice using me tabolic inhibitors have confirmed these findings, leading to the conclu sion 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 wi th glutathione to form S- formyl glutathione. The rapid enzymatic and chemica l breakdown of this conjugate (Uotila & Koivusalo, 1974a,b) wo uld yield formic acid and hence carbon dioxide. Thus, a quantitative correlation between the amount of carbon monoxide and carbon dioxide ex haled and the activity of the two pathways no longer appears to be val id.

Levels of CO-Hb in the blood, following exp osure to methylene chloride, are both dose- and time-dependent. Hu man subjects exposed to concentrations of 1770 mg/m 3 or less for 1 h have CO-Hb levels of 1-4%. These levels rose to an average of 10% sa turation within 1 h after exposure to 3500 mg/m 3 for 2 h (Stewart et al., 1972). Hake et al. (1975) reported CO-Hb levels in excess of 8 % following exposure to 880 mg/m 3 for 7.5 h.

Human volunteers were exposed to 350 or 124 0 mg/m 3 for 6 h and levels of methylene chloride in blood and exhal ed air, CO-Hb and exhaled CO were measured. At the end of the 6-hexposure, the CO-Hb concentration of the group exposed to 1240 mg/m 3 was 1.4 times
higher than that of the group exposed to the lo wer dose. Likewise, the concentration of exhaled CO in the high-dose gr oup was 2.1 times higher than that of the low-dose group. The aut hors concluded that their finding of non-linearity between administ ered 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 

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workers (Åstrand et al., 1975; Di Vincenzo & Ka plan, 1981b). Under a moderate workload, an exposure to 350 mg/m 3 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, inclu ding 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 me thylene chloride (350 mg/m 3 ) for 2 h, an average of 22.6 μg methylene chloride was excreted in the urine within 24 h after the exp osure. In seven subjects exposed to 710 mg/m 3 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. Methyle ne chloride excretion in expired air was most evident during the firs t 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/m 3 and fell to about 18 mg/m 3 at the end of 30 min. Small amounts of methylene chloride remained in the e xpired air at 2.5 h.
A detailed study of the relationship betwee n the measurements of methylene chloride in expired air or blood, car bon monoxide in expired air and CO-Hb in blood was undertaken by Di Vin cenzo & Kaplan (1981a,b). At the end of exposure of non-smokin g, sedentary volunteers for 7.5 h to methylene chloride vapour concentr ations of 180-710 mg/m 3 , 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/m 3 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 unsuitabl e for use as a marker of occupational exposure.

Table 12. Methylene chloride in expired ai r and blood, and carboxyhaemoglobin (CO-Hb) levels of human volunteers f ollowing 7.5 h exposure (from Di Vincenzo & Kaplan, 1981a)

Methylene Methylene choride M ethylene chloride CO-Hb chloride exposure in expired air in blood levels (mg/m 3 ) (mg/m 3 ) (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/m 3 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/m 3 is equivalent to an 8-h exposure to 124 mg carbon monoxide/m 3 , 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 investiga ted the effects of exercise and cigarette smoking on the uptake, m etabolism and excretion of methylene chloride. The effects of smoking a nd methylene chloride exposure on CO-Hb saturation levels were found to be additive. Exercise was found to increase the absorption o f methylene chloride and CO-Hb levels. However, the effects of exerc ise on CO-Hb were not observed to increase with heavy workloads beyon d the level achieved with moderate work-loads, suggesting a saturati on of this effect (see also section 5.3.2).

Engström & Bjurström (1977) found that, dur ing the first 2 h after exposure, the concentration in alveolar air ten ded to be lower and declined more rapidly in obese subjects than in slim ones. After this, the concentration dropped more slowly in the ob ese group. During the late phase of elimination, the obese subjects t ended 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 exposu re to 180, 1800 or 53 000 mg methylene chloride/m 3 (McKenna et al., 1982). At 180 mg/m 3 , only 5% of the exhaled label was found as methyle ne

chloride. The remainder was exhaled as CO and C O 2 . As the exposures increased, so did the exhalation of non-metabol ized methylene chloride. Methylene chloride accounted for 30% of the label from the 1800 mg/m 3 dose and 55% of the label for the 53 000 mg/m 3 dose. A combination of exhaled methylene chloride, CO 2 and CO accounted for 

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1800 and 53 000 mg/m 3 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 exc reted 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). T he radiolabel was present in the exhaled air as CO and CO 2 , as well as in expired methylene chloride. The amount of methylene chl oride in the expired air increased from 12% to 72% as the dose was i ncreased 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 lun gs 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 excre tion 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 t he 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 obs erved at 3 mg/litre in anaerobic biological sludge.

In the aquatic environment, fish and amphibian emb ryos are the most sensitive with effects on hatching from 5.5 m g/litre.

Adult fish are relatively insensitive to methylenechloride. even after prolonged exposure (14-day LC 50> 200 mg/litre). The effect of methylene chloride on Daphnia is variable; the lowest reported EC50was 135 mg/litre in a closed system.

In soil, 10 mg/kg strongly decreased the ATP conte nt of the biomass, adversely affected the growth of fungi an d aerobic bacteria. and induced transient inhibition of enzyme activit y. The no-observed- effect level was 0.1 mg/kg. In earthworms, LC50values were in the range of 300 to more than 1000 μg/cm 2). In sediment, no toxic effects were observed even at very high levels.

In higher plants, no effects were found after expo sure for 14 days to 100 mg/m 3

. 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 w ith Pseudomonas putida, a threshold of 500 mg/litre for methylene chloride was determined (Bringmann & Kühn, 1977b). The glyco lysis of Pseudomonas putida was inhibited after a 16-h exposure to 1000 mg/litr e (Bringmann & Meinck, 1964).

Nenzda & Seydel (1988) report minimum inhib itory 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., 198 1).

The IC 50 for inhibition of multiplication of Escherichia coli was 37.2 mg/litre (Nendza & Seydel, 1988).

In the OECD activated sludge respiration-in hibition test (method 209) using sealed vessels, the EC 50 value for methylene chloride was more than 1000 mg/litre after 30 min (Volskay & Grady, 1988).
Concentrations up to 1000 mg/litre had no e ffect 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 grow th (Stucki et al., 1981). 

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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 in hibited from 136 mg/litre (Bauchop, 1967). At 93 mg/litre, t he growth of a mixed bacterial population from an anaerobic digester was inhibited by 50% (Thiel, 1969). Addition of methylene chloride t o anaerobic sludge from an operating municipal digester showed, after 4 8 h, a 20% inhibition of gas production at 3 mg/litre and a 50% inhib ition at 50 mg/litre (Hayes & Bailey, 1977). Addition of methylene c hloride to the feed of a mixed anaerobic culture, developed in the lab oratory from seed from a sewage treatment plant, decreased the gas pro duction to such an extent that at 3.3 mg/litre it had virtually ce ased after 5 days, compared with 15 days in controls (Vargas & Ahl ert, 1987).

Blum & Speece (1991) determined the toxicit y of methylene chloride to methanogenic bacteria and found an IC 50 for inhibition of gas production of 7.2 mg/litre.

Stuckey et al. (1980) used a batch system i n 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 conce ntration 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 (EC 5 inhibition cell proliferation) (Bringmann & Küh n, 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, CO 2 uptake) and cell number were only affected by meth ylene chloride from 1450 mg/litre (Bringmann & Kühn, 1978; Hut chinson et al., 1978; US EPA, 1980). The threshold (7-day EC 3 ) 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 presen ts difficulties in aquatic toxicity testing. Therefore, care shoul d be taken when interpreting results based on nominal concentra tions and open static systems. Flow-through systems or closed static systems are necessary to conduct adequate toxicity studies on volatil e substances. However, these systems were not always used (Tables 14 a nd 15).

7.2.1 Plants

The EC 50 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 inse cts was investigated in adult Tribolium confusum and grain weevil (Calandra granaria). The LC 50 for a 5-h exposure in fumigation vessels was 82 an d 380 mg/litre, respectively (Ferguson & Pirie, 1 948; Negherbon, 1959).

Table 13. Acute aquatic toxicity of methyl ene chloride to algae

Organism Description Me thod Parameter aConcentration Reference (mg/litre)

Diatom Skeletonema costatum Ch lorophyll a content, 96-h EC 50 > 662 US EPA (1980) (salt water) ce ll number

Green alga Selenastrum Ch lorophyll a content, 96-h EC 50 > 662 US EPA (1980) capricornutum ce ll number

Green alga Scenedesmus Ce ll number 8-day TT 1450 Bringmann & Kühn quadricauda (1978)

Green alga Chlorella vulgaris CO 2 uptake 3-h EC 50 2292 Hutchinson et al. (1978)

Green alga Chlamydomonas CO 2 uptake 3-h EC 50 1477 Hutchinson et al. angulosa (1978) 

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(blue-green alga) (1978)

a TT = toxicity threshold (i.e. the concentration a t which cell multiplication was inhibited by more t han 3%)

Table 14. Acute aquatic toxicity of methylene chloride in fresh water

pH/dissolved Hardness Organism Temperature oxygen (mg CaCO 3 Flow/ Parameter Concentratio n References (°C) (mg/litre) per litre) Static (mg/litre) and remarks

Crustacean

Water flea 22 7.4-9.4/ 173 Static 48-h LC 50 220 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 EC 50 2100-2270 Bringmann & Kühn (Daphnia magna) 24-h NOEC 1550-1707 (1977a, 1982) (nominal concentration)

Water flea Unknown Unknown Unknown 48-h LC 50 1250 Bringmann & Meinck (Daphnia magna) (1964)

Water flea Unknown Unknown Unknown Static 24-h EC 50 1959 Kühn et al, (1989) (Daphnia magna) 48-h EC 50 1682 (closed system)

Water flea Unknown Unknown Unknown Static 48-h EC 50 135 Abernethy et al. (1986) (Daphnia magna) (closed system)

Water flea 18-20 8/8.7-8.8 11.7 Unknown 48-h LC 50 480 RIVM (1986) (Daphnia magna) 48-h NOEC 100 (nominal concentration closed system) Fish

Goldfish Unknown Unknown Unknown Static 24-h LC 50 420 Jenson (1978) (Carrassius (nominal concentration) auratus)

Table 14 (Cont'd)
pH/dissolved Hardness Organism Temperature oxygen (mg CaCO 3 Flow/ Parameter Concentratio n References (°C) (mg/litre) per litre) Static (mg/litre) and remarks

Fathead minnow 12 7.8-8.0/ 67 Static 98-h LC 50 310 Alexander et al, (1978) (Pimephales > 5 Flow 96-h LC 50 193 (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 LC 50 502 Dill et al. (1987) (Pimephales (analysed concentration) promelas) (juvenile)
Bluegill, 21-23 6.5-7.9/ 32-48 Static 96-h LC 50 220 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/dissolve d Hardness Flow/ References Organism (°C) oxygen (mg CaCO 3 Stat Parameter Concentration and rema rks (mg/litre) per litre) (mg/litr e)

Crustacean
Mysid shrimp Unknown Unknown Unknown Static 96-h LC 50 260 US EPA (1980) (nominal (Mysidopsis bahia) concentration)

Grass shrimp 20±2 6.1-8.0 8-12 Static 48-h LC 50 108.5 Burton & Fischer (1990) (Palaemonetes pugia) > 4

Fish

Golden orfe Unknown Unknown Unknown Static 48-h LC 50 521-528 Juhnke & Lüdemann (1978) (Leuciscus idus)
Killifish (juvenile) 20±2 6.1-8.0/ Unknown Static 48-h LC 50 97.0 Burton & Fischer (1990) (Fundulus > 4 heteroclitus) a

Sheepshead minnow 25-31 Unknown 10-31 Static 98-h LC 50 330 Heitmuller et al. (1981) (Cyprinodon 98-h NOEC 130 (nominal concentration) variegatus) 

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7.2.2.2 Crustaceans

Data on the toxicity of methylene chloride to crustaceans are presented in Table 14. Daniels et al. (1985) an d Knie (1988) reported a 48-h LC 50 of 27 mg/litre and a 24-h LC 50 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 a ssessed.

7.2.2.3 Molluscs
In seawater, metamorphosis was induced in u p 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 chl oride 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 eq uilibrium, melanization, narcosis and swollen, haemorrhagi ng gills) were reversible at a sublethal level (Alexander et a l., 1978).
7.2.3.2 Chronic toxicity and reproduction

Data on the chronic and embryo-larval toxic ity of methylene chloride to fish are summarized in Table 16.

In a 32-day embryo-larval test with fatheadminnow (Pimephales promelas), the larval survival and weight was affected from 20 9 and 142 mg/litre, respectively. The maximum accepta ble toxicant concentration (MATC) based on body weight was c alculated to be 108 mg/litre. The ratio between the acute 8-day LC 50 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 t o 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 analytical ly determined post- hatching LC 50 s were > 32 mg/litre for the pickerel frog

Table 16. Chronic and embryo-larval toxici ty of methylene chloride to fish

Dissolved Hardness Flow/ References and Description Temperature pH oxygen (mg CaCO 3 Static Parameter Concentration rem arks (°C) ( mg/litre) per litre) (mg/litre)

Chronic toxicity

Guppy 22±1 ? > 5 25 daily LC 50 , 14 days 295 Könemann (Poecilia reticulata) renewal (1981) (covered with glass, nominal)

Fathead minnow 25±1 6.8-8.6 > 9 73-82 flow LC 50 , 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 LC 50 a34 Black et al. (Pimephales promelas) (1982) (embryo-larva)

Fathead minnow 25±1 6.8-8.6 > 9 73-82 flow LOEC, 32 day s 209 Dill et al. (Pimephales promelas) (survival) (1987) (embryo-larva) LOEC, 32 day s 142 (analytical (weight) concentration)

Rainbow trout 13.3±0.3 7.8 9.4 106 flow LC 50 a,b13.1 Black et al. (Salmo gairdneri) (1982) (embryo)

Table 16 (Cont'd)

Dissolved Hardness Flow/ References and Description Temperature pH oxygen (mg CaCO 3 Static Parameter Concentration rem arks (°C) ( mg/litre) per litre) (mg/litre) 

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38 of 102 3/23/2016 4:29 PMRice fish 25±1 7.6-8.4 4.5-8.8 11.7 renewal LC 50 , 3 weeks 106 RIVM (1986)

(Oryzias latipes) 23±2 3 times/ NOEC, 3 week s 75 (analytical (egg-larva) week concentration)

a Eggs were exposed from 30 min after fertilization to 4 days post-hatch bTeratogenic 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 6.7 3 mg/litre. For the pickerel frog and Fowler's toad, hatching was d ecreased by 14 and 20% at 10 and 32 mg/litre, respectively. In the hat ched populations slightly higher incidences of teratogenic effec ts were observed (Birge et al., 1980).

Black et al. (1982) exposed several amphibi an species to methylene chloride from 30 min after fertilization to 4 d ays post-hatch. Post- hatching LC 50 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 m ost sensitive to methylene chloride.

7.3 Terrestrial organisms
The toxicity of methylene chloride to highe r plants (Phaseolus vulgaris, Raphanus sativus radicula, Lepidum sativ um, Trifolium pratense, Saintpaula ionatha, Petunia hybrida) was evaluated, using the LIS (Landesanstalt für Immissionsschutz, Es sen) test; no effect was observed at 100 mg/m 3 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 14 CO 2 was tested; photosynthesis appeared to be reduced f rom 388 000 mg/m 3 (Lehman & Paech, 1972).

In a 48-h filter-paper contact toxicity tes t on the earthworm Eisenia fetida, the LC 50 was 304 μg/cm 2 in one study and > 1000 μg/cm 2 in another. Therefore, methylene chloride was classified as moderately toxic (100-1000 μg/cm 2 ) (Roberts & Dorough, 1984; Neuhauser et al., 1985).
In embryos of White leghorn chicken, the LD 50 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 w eight), methylene chloride decreased the ATP content of the soil biomass by 80-85%, compared to controls, and adversely affected th e growth of soil fungi and aerobic bacteria after 3 days. A slight rec overy was observed by
the end of the 56-day experiment. Anaerobic bac teria were hardly influenced and, in the case of the obligate ana erobic Clostridium sp., the growth was even increased. The replacement of o xygen probably explained the stimulation of growth in the latter case (Kanazawa & Filip, 1987).

Incubation of soil for 2 months with 1-10 m g/kg (dry weight) methylene chloride reduced the activity of ß-gl ucosidase, ß-acetylglucosaminidase and proteinase during t he first 28 days, with recovery after 2 months; no effect was observedat 0.1 mg/kg (Kanazawa & Filip, 1986).

7.4.2 Sediment microorganisms

In sediment from a freshwater stream, methy lene chloride did not significantly affect the electron transport sys tem (ETS) activity during a 1-h enzymatic assay at 66 500 mg/kg. W hen assayed over an 11-day period, 1330-66 500 mg/kg caused a fluct uating stimulation of ETS activity, which may indicate a marked alter ation of the stability of the biological activity in the sediment. Mic robial respiration, measured by CO 2 evolution, was inhibited (EC 50 ) after 7 days at 15 500 mg/kg. However, when measured by oxyg en 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 day s. At 1000 mg/litre a decrease in oxygen content of the water was obs erved, together with mortality in snails and algae, as well as necro sis on fronds of Lemna minor. The photosynthesis of the plants was inhibited. The se effects were less at 500 mg/litre, but this concentrati on was still lethal to snails. In outdoor mesocosms, containing a larg e diversity of species, initial concentrations of 137-156 mg/litre did not induce any toxicity (Merlin et al., 1992). 

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39 of 102 3/23/2016 4:29 PM8. 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, hypoth ermia and haematuria were also noted (dose threshold not reported). The gastrointestinal tract was found to be congested with micro-haem orrhages or partial destruction from doses of 530 mg/kg or more. Bl ood CO-Hb increased in a dose-related manner (Laham, 1978).

Various effects have been reported followin g 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-phosp hatase activity in male rats (strain unspecified) receiving 2210 mg/kg (Reynolds & Yee, 1967), and decreased hepatic secretion of triglyceride s 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-Da wley 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 we re found in several studies. Decreased running activity was found i n rats exposed to 17 700 mg/m 3 for 1.5 h (Heppel & Neal, 1944); hypothermia, hypotension and convulsion in Sprague-Dawley ra ts from 28 200 mg/m 3 (6-h exposure) (Laham, 1978); CNS depression in Alderley-Park rats at 31 800 mg/m 3 (10-min EC 50 ) (Clark & Tinston, 1982) and in rats at 40 000 mg/m 3 (2-h exposure) (Ulanova & Yonovskayo, 1959); and dyspnoea and anaesthesia in rats from 53 000 mg /m 3 (30-min exposure) (Schumacher & Grandjean, 1960; Kashin et al., 1 980).

Table 17. Acute toxicity of methylene chlo ride

Species Route Ve hicle Parameter Concentration Reference

Rat (Wistar, male) oral n one LD 50 1710-2250 mg/kg Klimmer (1988)

Rat CDF (F-344) oral n one LD 50 1530-2524 mg/kg Carreon (1981)

Rat (Sprague-Dawley) oral n one LD 50 2120 mg/kg Kimura et al, (197 1) young male

Rat (Sprague-Dawley) - male oral n one LD 50 2280 mg/kg Laham (1978) - female oral n one LD 50 1410 mg/kg

Mouse (CF-1, male) oral un known LD 50 1987 mg/kg Aviado et al. (197 7a,b)

Dog oral mucil lage of LD 50 3000 mg/kg Barsoum & Saad

ac acia (1934)

Rat (Alderley Park) inhalation 15-min LC 50 197 790 mg/m 3 Clark & Tinston (1982)

Rat (Sprague-Dawley) inhalation 6-h LC 50 52 000 mg/m 3 Bonnet et al. (1980) male

Rat (Sprague-Dawley) inhalation 6-h LC 50 > 28 000 mg/m 3 Landry et al, (1981)

Mouse (CF-1, male) inhalation 20-min LC 50 92 680 mg/m 3 Aviado et al. (1977a,b)

Mouse (LF1, female) inhalation 6-h LC 50 49 100 mg/m 3 Gradiski et al, (1978)

Mouse (ICR, male) inhalation 6-h LC 50 55 870 mg/m 3 Scott et al. (1979)

Guinea-pig inhalation 6-h LC 50 40 200 mg/m 3 Balmer et al. (1976)

Table 17 (Cont'd)

Species Route Ve hicle Parameter Concentration Reference 

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40 of 102 3/23/2016 4:29 PMRat (Sprague-Dawley) intratracheal ALD a350 mg/kg McCarty et al. (19 92)

male
Mouse (CF-1, male) intraperitoneal un known LD 50 448 mg/kg Aviado et at. (197 7a,b)

Mouse intraperitoneal un known LD 50 500 mg/kg Schumacher & Grandjean (1960) Mouse (Swiss-Webster, intraperitoneal cor n oil LD 50 1990 mg/kg Klaassen & Plaa (1 966) male)
Dog intraperitoneal cor n oil LD 50 1260 mg/kg Klaassen & Plaa (1 967)

Mouse subcutaneous oli ve oil LD 50 6500 mg/kg Kutob & Plaa (1962 )

a "ALD = Approximate Lethal Dose, the lowest dose c ausing death within 3 days

Studies on sleeping patterns in Wistar rats by measuring electroencephalographic (EEG) and electromyogra phic (EMG) activity have shown a dose-related increase in total sle eping time and intervals between rapid eye movement (REM) slee p when the rats were exposed to 1770, 3500 or 10 600 mg/m 3 (500, 1000 or 3000 ppm) methylene chloride for 3 h. During exposure to 3500 or 10 600 mg/m 3 , the percentage of sleep defined as REM decrease d by nearly 20% (Fodor & Winneke, 1971; Fodor et al., 1973). Studies u sing similar techniques on the narcotic effects of methylene chloride i n rats (strain unspecified) have been reported by Berger & Fod or (1968). Rats were exposed to a range of concentrations of methyle ne chloride from 9900 to 99 000 mg/m 3 (2800 to 28000 ppm) for unspecified periods of tim e. There was an initial period of excitation follo wed 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/m 3 (25 000 or 28 000 ppm) and after a 6-h exposure to 54 000 or 64 000 mg/m 3 (16000 or 18000 ppm). Following exposure to 17 700 to 31 800 mg/m 3 (5000 to 9000 ppm) methylene chloride, long per iods of sleep occurred without desynchronization phases. Following exp osure to concentrations of methylene chloride below 17 700 mg/m 3 (5000 ppm), there were no measurable effects on either EEG or EMG activit y (Berger & Fodor, 1968).
Exposure of F-344 rats to 7100 mg/m 3 for 2.5 h caused statistically significant changes in somatosens ory evoked responses and EEG. The lack of effect of 157 mg carbon mo noxide/m 3 (which induces a CO-Hb level of 10%, comparable with t hat produced by exposure to 7100 mg methylene chloride/m 3 ) on evoked responses, indicated that the effects were probably due to methylene chloride itself and not to its principal metabolite carb on monoxide (Mattsson et al., 1988). Alterations in somatosensory evo ked potentials were also observed after a 1-h exposure of F-344 rat s to dose levels of 17 700 mg/m 3 or more (Rebert et al., 1989).

No macroscopic lesions were found in rats a t the 6-h LC 50 of 53 000 mg/m 3 (Bonnet et al., 1980). Congestion of various organ s, as well as oedema of the brain, heart, lungs and h ip region, was noted following exposure to 71 000 mg/m 3 (6-h exposure) in Sprague-Dawley rats (Laham, 1978).

Increased CO-Hb levels were found from 1770 mg/m 3 in various strains of rats (MacEwen et al, 1972; Laham, 19 78; Dill et al., 1978; Kurppa et al., 1981). Ascorbic acid content of the liver was increased in rats exposed to 40 000 mg/m 3 for 2 h (Ulanova & Yonovskayo, 1959), but no effect on cytochrome P-450 or spe cific liver enzymes was noted in Wistar rats after a 3-h exposure to 35 00 mg/m 3 or in Sprague-Dawley rats after a 5-min (repeated 5 t imes) exposure to 350 mg/m 3 , except for increased microsomal and decreased lys osomal ß-glucuronidase activity (Kurppa et al., 1981).

Intratracheal administration of methylene c hloride in Sprague- Dawley rats showed lethal levels at 350 mg/kg ( corresponding to 17.5% of the oral LD 50 ), death occurring in a few seconds. This result emphasises that aspiration of methylene chlorid e may present more of a hazard than oral ingestion (McCarty et al., 199 2).

8.1.3.2 Mouse
CNS depression resulting in reversible narc osis was reported in mice exposed to between 14 100 and 52 200 mg/m 3 methylene chloride for 2-6 h (Flury & Zernik, 1931) or to 47 700 m g/m 3 for 128 min (EC 50 ) (Kashin et al., 1980). This effect was also notedin CF-1 mice exposed to 35 300 mg/m 3 for 20 min (Aviado et al., 1977a,b) or in Swiss mice exposed to 45 900 mg/m 3 for 7 h (Svirbely et al., 1947). Exposure at 35 000 mg/m 3 for 2 h was the minimal CNS effective concentration found in the mouse (Laz arew, 1929).

Ability to learn a simple passive avoidance task, 1-4 days after exposure, was reduced in Swiss-Webster mice exp osed to 168 000 mg/m 3 for 20 seconds (Alexeef & Kiglore, 1983).
Fatty changes were noted in the liver and, less frequently, in the kidney from 56 500 mg/m 3 after a 7-h exposure (Svirbely et al., 1947). Microscopic lesions in the liver, kidney s, lungs and adrenal were found (no dose-response relationship) in m ice exposed to lethal doses for 6 h (Gradiski et al., 1978). 

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41 of 102 3/23/2016 4:29 PMCardiac sensitization to the effects of adr enaline was reported in

Swiss mice exposed to 710 000 mg/m 3 for 6 min) (Aviado & Belej, 1974).
Male NMRI mice were exposed to methylene ch loride by inhalation in a study of tolerance, i.e. decreased responsive ness to a chemical that arises as a result of previous exposure to the same chemical (Kjellstrand et al., 1990). Motor activity meas ured with Doppler radar units was used to monitor the behavioural react ions of the animals. Motor activity increased on exposure to methyle ne 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/m 3 , the concentration of triglycerides was increased in the liver of gui nea-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/m 3 (Morris et al., 1979). Slight to moderate vacuolation in t he liver of guinea-pigs

was seen after a 6-h exposure to 38 800 mg/m 3 . 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/m 3 for 2 h (Truhaut et al, 1972). Only serum AST was significantly increased.

In Beagle dogs, ECG changes as well as decr eased blood pressure, heart and respiratory rate were found at 141 20 0 mg/m 3 (7-h exposure). Behavioural effects were already not ed after a 1-h exposure to 53 000 mg/m 3 (reduced reflexes) (Von Oettingen et al., 1950).

Increased CO-Hb values, but no change in ha ematocrit, haemoglobin concentration or red cell count, were reported in dogs and rhesus monkeys exposed for 24 h to 17 700 mg/m 3 . Slight pathological changes in the liver (fatty changes, vacuolizat ion) could be attributed to the treatment (MacEwen et al., 19 72).

Cardiac effects such as arrhythmia, tachyca rdia and hypotension were found in monkeys, dogs and rabbits exposedfor 1-5 min to levels of methylene chloride exceeding 35 300 mg/m 3 (Belej et al., 1974; Adams & Erickson, 1976; Taylor et al., 1976; Av iado et al., 1977a,b; Aviado, 1978).

One study on rabbits showed allergic reacti ons after inhalation (Shmuter & Kashin, 1978). However, the experime ntal protocol of this study is questionable and the result has not be en confirmed.

8.1.4 Dermal administration

No effect was noted on rats receiving a der mal 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 (s welling) were found in Wistar rats receiving 2000 mg/kg under an occlu sive dressing for 24 h (Janssen & Pot, 1988b). Haemoglobinuria was obs erved in rats when abdominal skin was immersed in methylene chlori de for 2-20 min (Schutz, 1960).

8.1.5 Intraperitoneal administration

A single intraperitoneal injection of 510 m g methylene chloride/kg in rats slowed down the sciatic motor conductio n velocity by 11% and gave rise to a CO-Hb level of 6.8% (Pankow et a l., 1979).
Signs of CNS depression were found in Wista r rats and CFI mice receiving, respectively, 1060 mg/kg (with pheno barbital pretreatment) and 114 mg/kg (Aviado et al., 1977; Masuda et a l., 1980).

Various biological investigations in rats a nd mice showed a dose- related increase in serum AST and/or ALT activi ty 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/k g in corn oil (Klaassen & Plaa, 1967). No histological changes in the l iver could be found in rats exposed to < 1300 mg/kg (Cornish et al., 1973; Corsi et al., 1983). However, mild hepatic inflammation was n oted in Swiss Webster mice receiving lethal doses of methylene chlori de (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 r enal histological change could be found in Swiss mice receiving 1 300 mg/kg in corn oil (Plaa & Larson, 1965). Slight histological chan ges were noted in the liver and kidneys of mongrel dogs receiving 130 0 mg/kg (Klaassen & Plaa, 1967).

8.1.6 Intravenous administration

The minimum lethal concentration in anaesth etized dogs was found to be 200 mg/kg in olive oil after intravenous administration (Barsoum & Saad, 1934).

Methylene chloride shortened the duration o f nystagmus induced by 

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42 of 102 3/23/2016 4:29 PMrotation in Sprague-Dawley rats during intraven ous 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 s ensitize the myocardium of rats to arrhythmia development in response t o catecholamines. The release by methylene chloride of endogenous cat echolamines is possibly a cause of these modified cardiovascular action s (Mueller et al., 1991).

8.1.7 Subcutaneous administration

The minimum lethal concentration in the rab bit 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 inhala tion and oral administration is low. The inhalation 6-h LC 50values for all species lie between 40 200 and 55 870 mg/m 3 . Oral LD 50values of 1410-3000 mg/kg have been recorded. Acute effects after methylene

chloride administration by various routes of expos ure are primarily associated with the central nervous system (CNS) a nd the liver. CNS disturbances were found at 14 100 mg/m 3or more and slight changes in EEG at 1770 mg/m 3 . Slight histological changes in the liver were found at concentrations of 17 700 mg/m 3or more. In the mouse, but not in the rat, effects on the lungs restricte d to the Clara cells were observed after exposure to 7100 mg/m 3 . Occasionally the kidney is affected. Cardiac sensitization to adren aline-induced arrhythmias has been reported, and cardiovascular effects were seen at concentrations above 35 000 mg/m 3 . However, the effects were inconsistent.

8.2 Short-term exposure

8.2.1 Oral administration

CD 1 mice given doses of up to 665 mg/kg per day of met hylene chloride in corn oil by gavage for 14 days did not show any effect on liver enzymes. Microscopic examination revealedslight vacuolation in the liver from 333 mg/kg. No damage to the kidn eys 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 exp osure to 2000 mg/kg twice a week for 17 weeks. No effect was found in normotensive rats. Very slight histopathological changes in the li ver 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 reporte d after exposure to methylene chloride (3500 mg/m 3 ) 2 h/day for 15 days; decreased body weight, increased hepatic lipid peroxidation, a nd high concentrations of methylene chloride in the brain, kidney and blood immediately after inhalation were observed (Ito et al., 1990). Si milar hepatic effects (hypertrophic hepatocytes, and increased lipid peroxidation and glutathione peroxidase activity) were reported in male Wistar rats exposed to 3500 mg/m 3 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 we re exposed to 250, 1100 or 3500 mg/m 3 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 ne rve terminal systems, was reported. A dose-dependent increase in nora drenaline turnover in the anterior periventricular hypothalamic area and dose-dependent
decreased noradrenaline concentration in the po sterior 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 r ats were exposed to methylene chloride at concentrations of 5740, 1 1 500, 22 900, 45 900 or 56 500 mg/m 3 , 6 h/day for 19 days. Intermittent scratching, ataxia and hyperactivity were seen in all rats exposed to 22 900 mg/m 3 or more. Dyspnoea and anaesthesia were observed in animals exposed to 45 900 mg/m 3 or more. Some deaths were also observed at these concentrations (NTP, 1986).

No, or limited, lesions were found in the l iver and lungs of F-344 rats, exposed to 7100 or 14 100 mg/m 3 , 6 h/day for 10 days, after light and electron microscopic examination (Hex t et al., 1986). No effect on the liver was found in rats (strain u nspecified) after inhalation of 880 mg/m 3 , 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/m 3 (5 h/day, 5 days/week) for 4 weeks revealed an inflammatory response and cell dama ge in the lungs as demonstrated by the increase biochemical respon se (enzymatic and non

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43 of 102 3/23/2016 4:29 PMenzymatic) observed in cell-free lavage effluen ts from the lungs (Sahn

& Lowther, 1981).

8.2.3.2 Other animals

Increased liver weight and increased mitoti c activity in hepatocytes were observed in male B6C3F 1 mice after 2 weeks of repeated exposure to 14 100 mg/m 3 6 h/day for up to 3 weeks (Eisenbrandt & Reitz, 1986). Necrosis of occasi onal epithelial cells in the bronchi and bronchioles, together with r eactive hyperplasia of adjacent lymphoid tissue, was observed in a fewanimals.

Groups of five male and five female B6C3F 1 mice were exposed to methylene chloride at concentrations of 5740, 1 1 500, 22 900, 45 900 or 56 500 mg/m 3 , 6 h/day for 19 days. Hyperactivity (dose-related) was seen in exposed mice, but no exposure-relat ed pathological findings were observed. Some deaths occurred in mice exposed to 45 900 mg/m 3 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/m 3 , 6 h/day, 5 days/week, for a total of 19, 18, 19 or 7 exp osures, respectively, over a period of 21 to 28 days. Animals exposedto 28 000 mg/m 3 showed anaesthetic effects and a decrease in th e body weight of rats. At 18 000 mg/m 3 there was slight anaesthesia, decreased body weigh t in male rats, increased aspartate aminotransfer ase (ASAT) in female mice and Sprague-Dawley rats, and increased liv er weights in female

mice, hamsters and rats. At 8800 mg/m 3 , the animals exhibited more scratching activity than the controls, but show ed no other effects attributable to exposure (Nitschke et al., 1981 )
Following exposure to 17 700 mg/m 3 , a reduction in body weight of mice was observed, and relative liver weight s were increased up to the end of the 7-days of continuous exposure. F atty infiltration, an increase in the triglyceride concentration and hydropic degeneration of the endoplasmic reticulum gradually disappea red. Protein synthesis was depressed. Necrosis was observed in a few h epatocytes (Weinstein et al., 1972).

Carboxyhaemoglobin levels were raised after continuous exposure of monkeys to 88.25 mg/m 3 (25 ppm) for 28 days (MacEwen & Vernot, 1972; Haun et al., 1972).

A group of NMRI mice was continuously expos ed to 130-1059 mg/m 3 (37-300 ppm) for 30 days, while another was int ermittently exposed for 1-12 h/day to 2118-25 416 mg/m 3 (600-7200 ppm) for 30 days, corresponding to an average exposure (24-h mean value) of 1059 mg/m 3 (300 ppm). In addition, groups of mice were con tinuously exposed to 1059 mg/m 3 for 4, 8, 15 and 90 days. The blood level of butyrylcholinesterase was significantly increas ed from 265 mg/m 3 (75 ppm) in continuously exposed male mice and after intermittent exposure in male rats. Moreover, liver weight w as significantly increased in a dose-related manner from 265 mg/ m 3 (75 ppm) and 330 mg/m 3 (150 ppm) in male and female mice, respectively. F inally, fatty accumulation was found in both sexes at 2 65 mg/m 3 (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/m 3 (25 ppm) for 28 days (MacEwen et al., 1972, Haun e t 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 mal e and 20 female Sprague-Dawley rats were exposed to 35 g/m 3 , 6 h/day for 90 days. Haematological, clinical chemistry and urinary parameters were measured and histological examinations were per formed. A slight redness of the conjunctiva lasting for 1-10 h w as observed after each exposure. No other treatment-related signs of t oxicity were reported.

Groups of 10 male and 10 female F-344 rats were exposed to methylene chloride at concentrations of 1850, 3 700, 7400, 14 800 and 29 700 mg/m 3 for 6 h/day, 5 days/week for 13 weeks. One male an d one female rat exposed to 29 700 mg/m 3 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/m 3 . The mean body weight in males and females exposed to 29 700 m g/m 3 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/m 3 and in females exposed to 14 800 mg/m 3 when compared to controls (NTP, 1986).

Male and female F-344 rats were exposed to 177, 710 or 7100 mg/m 3 6 h/day, 5 days/week for 13 weeks. No treatment-re lated alterations in sensory evoked potentials (flash , auditory brainstem, somatosensory or caudal nerve) or neuropatholog y were observed at any of the exposure levels (Mattsson et al., 1990).

CNS depression was found in rats during eac h daily session of repeated exposure to 35 000 mg/m 3 7 h/day, 5 days/week for 6 months (Heppel et al., 1944). 

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44 of 102 3/23/2016 4:29 PMRats (sex and strain unspecified) were cont inuously exposed to

either 88 or 350 mg/m 3 for 100 days. Slight cytoplasmatic vacuolization with positive fat stains in the l iver and tubular degeneration in the kidney were observed (Haun et al., 1972).

8.3.1.2 Oral exposure

Rats receiving methylene chloride in the dr inking-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 female s. Slightly decreased body weights were observed in mid-dose males an d high-dose females throughout the study. There were no differences between treated and control animals with regard to mortality, physi cal observations, food consumption or gross necropsy results. There we re no exposure-related effects observed following the histopathologica l evaluation of rat tissues from the 1 month interim necropsies. Ho wever, hepatocellular changes were observed following treatment for 3 months; central lobular necrosis, granulomatous foci, ceroid or lipofuscin accumulation, and cytoplasmic eosinophilic bodi es were observed in high-dose males and females and in some mid-dos e females. A dose- dependent increased incidence of hepatocyte vac uolation was also observed, many of the vacuoles containing lipidwhich 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, 3 700, 7400, 14 800 or 29 700 mg/m 3 for 6 h/day, 5 days/week for 13 weeks. Exposure-re lated deaths were seen in some mice exposed to 29 700 mg/m 3 . Hepatic centrilobular hydropic degeneration was observe d in males and females exposed to 29 700 mg/m 3 and in females exposed to 14 800 mg/m 3

. Both mean body weights and liver lipid to liver weight ratios were reduced in males and females exposed to 29 700 mg/m 3 when compared to controls (NTP, 1986).

Mice (strain and sex unspecified) were cont inuously exposed to either 88 or 350 mg/m 3 for 100 days. Slight cytoplasmatic vacuolization was found at both dose levels, an d a decrease in the microsomal cytochrome P-450 content was found i n the liver of mice exposed to methylene chloride at 350 mg/m 3 (Haun et al., 1972).

Female ICR mice were continuously exposed t o 350 mg/m 3 for 10 weeks. Fatty infiltration, vacuolization and en larged hepatocyte nuclei persisted up to the end of the exposure period. A reversible increase in plasma triglycerides was also obser ved (Weinstein & Diamond, 1972).

8.3.2.2 Oral exposure

Groups of 20 male and 20 female B6C3F 1 mice were given 0.15, 0.45 and 1.50% methylene chloride in drinking-w ater 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 an d control animals did not differ with respect to physical and ophthal mological observations or food consumption. There were no exposure-rel ated effects observed following the histopathological evaluation of m ice 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 les ions related to exposure at 17 700 mg/m 3 (7 h/day, 5 days/week for 6 months) in studies on dogs, monkeys, rats, rabbits and gui nea-pigs, with the exception of moderate centrilobular fatty degen eration of the liver and pneumonia in 3 out of 14 guinea-pigs. CNS d epression was found in

all species following exposure to 35 000 mg/m 3 ; all animals became inactive, sometimes after initial excitement. A t 35 000 mg/m 3 , dogs also showed fatty degeneration of the liver.
Hepatic changes (slight cytoplasmatic vacuo lation) and vacuolar changes in the renal tubules were found in dogs exposed continuously to 3500 mg/m 3 for up to 100 days. Abnormal haematology and incre ased activity of serum enzymes were reported after 4 weeks. Oedema of the brain was observed at a concentration of 17 350 mg/m 3 (Haun et al., 1972).

Three male and three female beagle dogs wer e exposed to 17 700 mg/m 3 , 6 h/day for 90 days. Haematology, clinical chemis try and urinary parameters were measured and ECG an d circulatory functions were examined. At the end of the study, histolo gical examinations were 

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and all dogs had slight erythema, lasting up to 10 h after exposure. No deaths and no other signs of toxicity were o bserved (Leuschner et al., 1984).

Decreased levels of neurotransmitter amino acids were observed in gerbil brains after continuous inhalation expos ure to methylene chloride (340 mg/m 3 ) for 3 months (Briving et al., 1986). In gerbils exposed by inhalation to 1240 mg/m 3 for 3 months, followed by a 4- month solvent-free period, increased brain conc entrations of two astroglial proteins and decreased levels of DNAin the hippocampus and cerebellum were observed (Rosengren et al., 198 6). Decreased hippocampal DNA levels were also observed in ge rbils exposed to 740 mg/m 3 (Rosengren et al., 1986; Karlsson et al., 1987). I t was suggested by the authors that this effect may h ave been the result of the loss of nerve cells.
8.3.4 Appraisal

Prolonged exposure to high concentrations of methylene chloride ( > 17 700 mg/m 3) caused reversible CNS effects, slight eye irritation and mortality in several laboratory spe cies. Body weight reduction was observed in rats at 3500 mg/m 3and in mice at > 17 700 mg/m 3. After intermittent exposure, effects on the live r were observed in rats at 3500 mg/m 3and in mice at 14 100 mg/nz 3

. After continuous exposure, slight cytoplasmatic va cuolization in the liver of both rats and mice were found at 88 and 3 50 mg/m 3

. No evidence of irreversible neurological damage wa s seen in rats exposed by inhalation to concentrations of < 7100 mg/m 3for 13 weeks.

Oral administration of methylene chloride to rats caused effects on the liver with a no-observed-effect level of 12 5 mg/m 3

. 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 inta ct skin, caused severe erythema and oedema with necrosis and acanthosi s (Duprat et al., 1976). Rabbits exposed to 0.5 ml methylene chlo ride 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, 1 990).

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 ins tillation. Moderate to severe changes were seen in the conjunctiva, to gether with increased corneal thickness and intra-ocular tension. All effects were reversible. Vapour exposure of the eyes to 17 7 00 mg/m 3 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 t o 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 Swis s-Webster mice were exposed to methylene chloride at a concentratio n of 4400 mg/m 3 on days 6-15 of pregnancy for 7 h/day, maternal bo dy weight in the mice was increased and the dams of both rats and mic e 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 a l., 1975). No significant teratogenic or fetotoxic effects we re observed in either species.
Groups of 18 rats were exposed before and/o r during 17 days of pregnancy to a methylene chloride concentration of 16 250 mg/m 3 for 6 h/day. The exposed dams exhibited increased b lood CO-Hb levels, ranging from 7.1 to 10.1%, and increased relati ve and absolute liver weights. Fetal body weight was decreased, but n o increase in the incidences of dead fetuses and/or resorptions n or any skeletal and/or

visceral malformations were observed (Hardin & Manson, 1980). After exposure to methylene chloride using the same e xperimental conditions, litters from four groups of 10 rats were used f or behavioural testing. Body weight gain, food and water consumption, w heel running activity and avoidance learning were all unaffected by t he exposure. However, changes in the general activity of pups were fo und 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 c annot be definitely and directly attributed to methylene chloride, howe ver, since elevated maternal CO-Hb- or methylene chloride-induced c hanges in maternal- litter interactions could have been contributin g factors (Bornschein et al., 1980). 

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46 of 102 3/23/2016 4:29 PMGroups of seven female Wistar rats were fedmethylene chloride at

levels of 0.04, 0.4 and 4.0% in their diet fromdays 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. Althou gh a reduction in the fetal weight of the females in the 0.4% group w as observed, there were no differences in any group in the number of implantations and resorptions. No external malformations were obs erved by fetal, skeletal and visceral examination, and no diffe rences were observed in any group in the frequency of delayed ossificat ions or in the dilation of the renal pelvis. A decrease in postnatal we ight gain and in absolute liver weight was found in the 0.04% gr oup 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) i n corn oil on gestation days 6- 15. The compound was tested with at lea st two dose levels plus a concurrent control group, the high dose (not specified) being selected to cause maternal toxicity. The dams w ere allowed to deliver and their litters were examined post-natally. A lthough a small change in maternal weight was found, no effects on lit ters were reported (Narotsky et al., 1992).
8.5.2 Reproductive toxicity

When rats received methylene chloride in th e drinking-water at a level of 125 mg/litre during a period of 13 wee ks before mating, no effects were found on the female fertility inde x, litter size, survival of pups at 4 weeks or the number of re sorptions (Bornmann & Loeser, 1967).
A two-generation inhalation study was condu cted to evaluate the effects of inhaled methylene chloride on the re productive capability, neonatal growth and survival of rats (Nitschke et al., 1988b). Groups of 30 male and 30 female 6-week-old F-344 rats (F 0 ) were exposed to 0, 350, 1770 or 5300 mg/m 3 (6 h/day, 5 days/week for 14 weeks).

After this exposure, F 0 animals were allowed to mate using one male and one female of the respective treatment grou ps to produce the F 1 litters. After weaning, 30 males and 30 females (4 weeks old) from each treatment group were randomly selected andassigned to the respective exposure groups. After exposure to t he relevant concentration of methylene chloride (6 h/day, 5 days/week for 17 weeks), the F 1 adults were allowed to mate to produce F 2 litters. Reproductive parameters examined included ferti lity, litter size and neonatal growth and survival. All adults and se lected weanlings were examined for grossly visible lesions. No advers e effects on reproductive parameters, neonatal survival or n eonatal growth were noted in animals exposed to methylene chloride in either the F 0 or F 1 generations. Similarly, there were no treatment-re lated gross pathological changes in F 0 and F 1 adults or F 1 and F 2 weanlings; histopathological examination of tis sues did not reveal any lesions in F 1 and F 2 weanlings attributable to exposure to methylene chloride. Therefore, the results of t his study indicate that exposure to concentrations as high as 5300 mg/m 3 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 m ice at concentrations up to 16 250 mg/m 3. No evidence of an effect on the incidence of skeletal malformations or other devel opmental effects was observed in three animal studies. Small effect s on either fetal or maternal body weights were reported at 4400 mg/ m 3. A two- generation reproductive toxicity study in rats exp osed to methylene chloride by inhalation at concentrations of up to 5300 mg/m 3, 6 h/day, 5 days/week, did not show evidence of an adverse effect on any reproductive parameter, neonatal survival or n eonatal growth in either the F 0or F1 generation.

8.6 Mutagenicity and related end-points
Studies on the mutagenic potential of methy lene chloride have been performed on bacteria, fungi and cultured mamma lian 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 testedusing 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 vapo ur of methylene chloride of undisclosed purity at levels rangin g from 20 100 up to
201 000 mg/m 3 . Metabolic activation by either induced rat liver S9 fraction, cytosol fraction, or microsomal fract ion increased the mutagenicity of methylene chloride (Simmon et a l., 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 w as more active than the microsomal fraction (Green, 1983). A positive r esult was reported in strains TA98 and TA100 with and without 30% ham ster liver S9 (Zeiger et al., 1990) using the vapour phase (desiccato r procedure) protocol.
Negative mutagenicity results were obtainedin studies not using 

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mutagenic activity was found when methylene chl oride 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 (se e section 6.3) indicate that the conjugation of methylene chloride withglutathione (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 wit h methylene chloride only produced a very small increase in mutageni city (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 re sponse, with and without Aroclor-induced rat-liver S9, microsome s or cytosol, was marginally higher at the highest methylene chlo ride 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 exogenou s GSH had only a small effect on the mutagenic response in TA100 or TA 100 gsh in the absence or presence of S9. According to the authors, th ese data suggest that if the interaction between methylene chloride a nd GSH is responsible for the observed mutagenicity, it occurs at ext remely low levels of intracellular GSH and is not significantly affe cted 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 vapo ur 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 th e level of glutathione present in the strain TA100.

Table 18. In vitro mutagenicity assays

Assay Strain/type S9 Dose Result aObservations Refer ence activatio n

Salmonella TA100, ± 10% 100-10 000 -ve Preincubationprotocol 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 protoc ol-dessicator Zeiger et al. typhimurium rat or ml/chamber procedure (1990) hamster liver S9

Salmonella TA100, ±S9 -ve Standard plat e-incorporation protocol Nestmann et al. typhimurium TA1535, (1980) TA1537, TA98, TA1538

Salmonella TA100, +ve 0.5μl in openglass dish within a Nestmann et al. typhimurium TA1535 desiccator; r evertants: 2-fold (1980) increase in T A1535; 6-fold in TA100

Salmonella TA1535, ± S9 up to 750/μl in +ve Active in str ains TA98 and TA100 Gocke et al. typhimurium TA100, a 9 litre (1981) TA1538, desiccator TA98, TA1537

Salmonella TA100 ±S9 0-1.4% +ve Significant i ncreases in mutagenic Jongen et al. typhimurium activity by a ddition of rat liver (1982) cytosol fract ion; marginal increases by addition o f microsomal fraction

Table 18 (Cont'd)

Assay Strain/type S9 Dose Result aObservations Refer ence activatio n

Salmonella TA100, ±S9 0-5% for 2, 4, 6 +ve Vapour phase protocol. Data Dillon et al. typhimurium 100gsh or 48 h suggest inter action between (1992) (glutathione methylene chl oride and GSH deficient) responsible f or 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 t he number of revertants, (1977) desiccator with a mutati on rate over 7-fold (approx 18-318 higher than c ontrols at 320 mg/m 3 ; mg/m 3 ) for 7 h two experiments were con ducted

Salmonella TA98, TA100 ±S9 0, 20, 100 to +ve Vapour phase protocol; S9 prepared Jongen et al. typhimurium 201 000 mg per m 3 from the livers of phenobarbital (1978) (5 concentrations) pretreated r ats; a dose-related 

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Table 18 (Cont'd)

Assay Strain/type S9 Dose Result aObservations Refer ence activatio n

Salmonella TA100 Not stated Vapour phase protocol; time-course Jongen (1984) typhimurium study to eval uate the most appropriate e xposure time for maximum diffe rentiation of the methylene chl oride induced reversion rat es in the presence and absence of a metabolic activation system; the m aximum 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 summari zed in review, original Simmon & typhimurium desiccator data were not available; vapour Kauhanen (1978) (approx 390 mg phase control ; S9) from livers of
per m 3 ) for 6.5 Aroclor-pretreated rats; addition of or 8 h S9 increased the mutation rate 1.5 fold; no furt her 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 pr etreated rats; dose- (1980) 9 litre desiccator related incre ase in the mutation rate, (0-293 mg/m 3 ) with an approximate 10-fold in crease for 8 h for both stra ins at the highest dose used; the pre sence of S9 resulted in a slightly hi gher mutation rate

Table 18 (Cont'd)

Assay Strain/type S9 Dose Result aObservations Refer ence activatio n

Salmonella TA1535, Not stated +ve Same study, b ut no data were given typhimurium TA1537, for the strai ns 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 r evertants 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 rathe r 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 pr etreated rats; a dose- 297 000 mg/m 3 related increase in the mutation rate for 3 days was observed

0 or 98 800 +ve Vapour phase protocol; the presence mg/m 3 for 3 of S9 enhanced the mutatio n rate days 1.19 fold; ondividing the S9 material into microsom al and high-speed supernatant ( cytosolic) fractions, only the high-spee d supernatant enhanced (1.27 fold) t he mutation rate; a small isoto pe effect was observed when 2 H-methylene chloride was substituted f or 1 H-methylene chloride

Table 18 (Cont'd)

Assay Strain/type S9 Dose Result aObservations Refer ence activatio n

Direct examin ation of the metabolism of methylene chloride by the bacterium ind icated that radio- labelled carb on dioxide and trace amounts of ra diolabelled carbon monoxide were formed, together with consider able incorporation of radioactivityinto 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; 

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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 fo rm only; dose-response (1980) (0-38 500 mg relationship (source of S9 not
per m 3 vapour) stated) (negative results were in gas-tight obtained in s tudies not employing chambers gas-tight jar s)

Table 18 (Cont'd)

Assay Strain/type S9 Dose Result aObservations Refer ence activatio n

Salmonella TA1535, +S9 Concentration +ve Vapour phase protocol; study Longstaff et al. typhimurium TA100 range not reported onlybriefly, methylene (1984) stated; exposure chloride bein g used as a positive for 72 h control; S9 p repared from livers of Aroclor pretr eated rats. Mutation rate increased 6-f old for TA1535 (with a 50% methylene chloride-air mixture) and 2.4 fold for TA100 (with a 1% methylene chl oride-air mixture); the increased mut ation rate was reproducible; apparently, one strain at least show ed a positive dose response

Salmonella TA100 0.1-1000μg -ve Plate-incorpo ration 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-incorpo ration assays; S9 from Nestmann et al. typhimurium TA1537, mg/plate, livers of Aro clor-induced rats; tested (1980) TA1538, dissolved in to limit of t oxicity; positive results TA98, TA100 dimethyl-sulfoxide for 3 pro-mut agens were obtained using the same batc h of S9, but not necessarily i n parallel incubations; a second, indep endent assay was conducted ove r a limited concentration range; this w as not a satisfactory demonstrationof a negative response

Table 18 (Cont'd)

Assay Strain/type S9 Dose Result aObservations Refer ence activatio n

Escherichia WP2uvrA, ±S9 0-5% for 2, 4, 6 +ve Vapour phase protocol; data suggest Dillon et al. coli pKM101 or 48 h interaction b etween methylene (1992) chloride and GSH responsible for the mutagenic act ivity

Microscreen E. coli WP2S ±S9 0.78-100 +ve Rossman et al. ( ) μl/well (1991)

Saccharomyces D7 0-209 mM +ve Induced mitio tic gene convertants Callen et al. cerevisiae and recombina nts, 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 Result aObservations Refer ence activatio n 

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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-dependen t 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 mutat ion 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 e xpression time was Jongen et al. cells (V79) 141 000 mg/m 3 reported to have no effect; cell (1981) (4 concentrations survival was reduced by about 20% for 1 h); expression at 141 000 mg /m 3 time of 6 days

Table 18 (Cont'd)

Assay Strain/type S9 Dose Result aObservations Refer ence activatio n

Cell mutation L5178Y Not stated -ve Thilagar et al.

mouse (1984a,b) lymphoma

Cell mutation L5178Y ±S9 0-3000 μl/ml ± Overall quest ionable evaluation of Myhr et al.

mouse activity (1990) lymphoma
Cell Primary 0.5 ml/4.6 litre +ve Enhanced tran sformation 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 Result aObservations Refer ence activatio n

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 glutathion e- S-transferase 5-5 expression in Salmonella typhimurium increases mutation rates caused 

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glutathione- S-transferase 5-5 cDNA, either in the correct or reve rse direction, was transfected into TA1535. The res ulting sense- transformed TA1535 (RSJ 100) expressed the enzy me and enhanced base- pair revertants as compared to the anti-sense s train (TPT 100). Mutagenicity was not seen when GSH, purified gl utathione- S- transferase and a methylene dihalide such as me thylene 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 muta tions in the N-terminal region of the loc I gene of excision repair-pro ficient (Uvr + ) and excision repair-defective (Uvr B
- ) strains of E. coli have been described by Zielenska et al. (1993). A total o f 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 foun d for cultures of Saccharomyces cerevisiae strain D7, but not for strains D4 and D3, exposed to methylene chloride of undisclosed pu rity. However, the mutagenic results in D7 occurred at toxic doses (1270 g/m 3 ) in which survival of the yeast cells was reduced to 42% (Cullen et al., 1980).

When assayed for the induction of mitotic s egregation in Aspergillus nidulans P1, methylene chloride significantly increased the frequency of morphologically abnormal colon ies, which produced euploid whole-chromosome segregants (Crebelli e t al., 1988; Crebelli et al., 1992).

8.6.1.3 Mutation in mammalian cells

Methylene chloride was not mutagenic in sev eral tests in which mammalian somatic or human cells were used (Goc ke et al., 1981; Jongen et al., 1981; Perocco & Prodi, 1981; Andrue & W olff, 1983; Burek et al., 1984).

Both negative (Thilagar et al., 1984a) and questionable results (Myhr et al., 1990) were reported when methylen e chloride was tested for gene mutations in a L5178Y mouse lymphoma a ssay at the thymidine kinuse locus. No increase in micronuclei was fo und when methylene chloride was tested in Chinese hamster V79 cell s (Gu & Wang, 1988).

8.6.1.4 Chromosomal effects

Studies on chromosome morphology in culture d mammalian cells indicate that methylene chloride is clastogenic . Chromosomni alterations (chromatid damage, chromosomni exch anges, but no increase
in sister chromatid exchanges) were observed in CHO cells (Thilagar & Kumaroo, 1983), human lymphocytes and L5178Y ce lls (Thilagar et al., 1984a,b), both with and without metabolic activ ation. A small increase in sister-chromatid exchanges (SCEs), without c lear evidence of a dose-response relationship, was found in V79 ce lls when exposed to gaseous methylene chloride at concentrations up to 5% (Jongen et al., 1981 ). A dose-related increase in SCEs was obs erved in CHO cells after a 24-h exposure to methylene chloride. Th e results were statistically significant only at the highest c oncentration (7%) and exposures of shorter duration (2, 4 or 6 h) wer e without effect (McCaroll et al., 1983). In a more recent study , Anderson et al. (1990) reported no increase in chromosomal aber rations 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 lymphocyt es. Those blood samples possessing metabolic activity (conjugators) wer e inactive in the assay, whereas those samples which were metabol ically 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 chl oride failed to induce unscheduled DNA synthesis (UDS) in cultured rat hepatocyte, although some reduction in replicative DNA synthesis occ urred at the higher doses (Andrae & Wolff, 1983). The absence of ev idence 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 UD S assay, but details were not available. In experiments using no exo genous 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-specifi c but reversible inhibition of replicative DNA synthesis was obs erved in both cell lines, probably due to a metabolic block of syn thesis. 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 o f rat liver S9 (Perocco & Prodi, 1981).

8.6.1.6 DNA binding in vitro
Several studies have investigated the poten tial of methylene chloride and its metabolites to bind covalently to DNA. Incubation of
14 C-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 14 C- labelled methylene chloride (0.8 μmol/ml), toge ther with hepatic microsomes and a NADPH-generating system, there was no evidence of any 

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an earlier in vitro study which failed to detect any DNA binding of methylene chloride or its metabolites (Anders e t 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. Ne gative 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 f requency of transformation by SA7 virus in a dose-related m anner (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 in crease 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 chl oride (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-Coll ins et al., 1990).
No increase in the frequency of either SCEs or chromosome aberrations was observed in bone marrow cells o f female B6C3F 1 mice after a single subcutaneous injection of methyl ene chloride (2500 or 5000 mg/kg) (Westbrook-Collins et al., 1989; Al len et al., 1990).
Inhalation exposure of female B6C3F 1 mice to 14 000 or 28 000 mg/m 3 for 10 days (6 h/day, 5 days/week) resulted in sli ght increases in the frequency of SCEs in lung cell s and peripheral blood lymphocytes, in chromosome aberrations in lung and bone marrow cells, and in micronuclei in peripheral blood erythroc ytes. The results were statistically significant at 28 200 mg/m 3 for all end-points. At 14 100 mg/m 3 , 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 wa s observed following a 3-month inhalation exposure to 7100 mg/m 3 (Westbrook-Collins et al.,

Table 19. In vivo mutagenicity assays

Assay Strain/type Result aObservations Re ference

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 B6C3F 1 mouse bone -ve s.c., 2500 or 50 00 mg/kg Westbrook-Collins et al. (1989 ); aberration marrow Allen et al. (1990)

Chromosome Female B6C3F 1 mouse bone ± inhalation, 14 100 or 2 8 200 mg/m 3 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/m 3 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 B6C3F 1 mouse bone -ve s.c., 2500 or 50 00 mg/kg Westbrook-Collins et al. (1989 ); exchange marrow Allen et al. (1990)

Sister-chromatid Female B6C3F 1 mouse lung ± inhalation, 14 100 or 2 8 200 mg/m 3 for Westbrook-Collins et al. (1989); exchange cells, peripheral blood 10 days Allen et al, (1990) lymphocytes

Sister-chromatid Female B6C3F 1 mouse lung ± inhalation, 7100 mg/m 3 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 Result aObservations Re ference

Micronucleus Female B6C3F 1 mouse ± inhalation, 14 100 or 2 8 200 mg/m 3 for Westbrook-Collins et al. (1989); peripheral blood 10 days Allen et al. (1990) erythrocytes 

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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 B6C3F 1 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/m 3 for 2 Trueman & Ashby (1987) DNA synthesis male B6C3F 1 mouse or 6 h hepatocytes

Unscheduled Male B6C3F 1 mouse -ve inhalation, 14 100 mg/m 3 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 o il, Raje et al. (1988) 3/week for 4 weeks

inhalation at 350, 530 or 710 mg/m 3 , Basso et al., (1987); 2 h/day, 5 days/week for 6 weeks Raje et al. (1988)
no microscopic lesions of testes or br ain

Table 19 (Cont'd)

Assay Strain/type Result aObservations Re ference

Drosphila Berlin K, Basc -ve 0 to 14 260 mg/m 3 for 6 h, 1 week or 2 Kramers et al. (1991) melanogaster weeks; sex-linked recessive lethal ass ay, and somatic mutation and recombinatio n test
Drosphila Berlin K, Basc +ve 125 or 620 mM; increased frequency o f 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 aberrati on data (excluding gaps) were not reported, and so the increases m ight not be significant. Increases in SCE and micronuclei w ere small (up to 2-fold at 28 000 mg/m 3 ).

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/ m 3 (6 h/day, 5 days/week) for 6 months (Burek et al., 1984).

Inconclusive results were reported when met hylene chloride was tested in C57B1/6 mice for its ability to induc e damage in the synaptosomal complex, an experimental end-point which can reveal induced damage at the meiotic prophase, followi ng intraperitoneal injection of 1500 mg/kg (Westbrook-Collins et a l., 1988).

8.6.2.2 Drosophila

No mutagenicity was detected in the recessi ve lethal test on Drosophila melanogaster fed, or injected with, 1-2% methylene chloride (Abrahamson & Valencia, 1980). A margi nal increase in the number of recessive deaths was found after feed ing 125 or 650 nmol methylene chloride in 2% dimethylsulfoxide to Drosophila melanogaster (Gocke et al., 1981). This study may not be rel iable because control values from different solvent treatments were p ooled, and because the increases seen were significant only when resul ts from the two dose levels were combined.

Methylene chloride was not active in the se x-linked recessive lethal assay or the somatic mutation and recomb ination test carried out with Drosophila melanogaster using inhalation exposure up to 14 260 mg/m 3 (Kramers et al., 1991).

8.6.2.3 DNA damage

Methylene chloride has been evaluated for i ts ability to initiate unscheduled DNA synthesis (UDS) in the livers o f male mice and rats in vivo. Alpk:AP rats were exposed by oral garage to 100, 50 0 or 1000 mg/kg body weight, and hepatocytes were as sessed for UDS via autoradiography 4 and 12 h later (Trueman & Ash by, 1987). In a second study, F-344 rats or B6C3F 1 mice were exposed by inhalation to either 7100 or 14 100 mg/m 3 for 2 or 6 h, and hepatocytes were assessed for UDS immediately after exposure. In both studies, methylene chloride failed to induce UDS. Simila r results were reported by Lefevre & Ashby (1989) following exposure of male B6C3F 1 mice either by oral gavage in corn oil (1000 mg/kg) or by inhalation of an atmosphere containing 14 100 mg/m 3 for 2 h.

Slightly increased DNA damage, measured by alkaline elution of DNA 

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(2.55 g/kg) in corn oil for 24 h, has been repo rted (Kitchin & Brown, 1989).

DNA single strand breaks were found in mous e hepatocytes after in vivo exposure to 14 120 mg/m 3 for 3 or 6 h but not in hepatocytes of similarly exposed rats. It was further obser ved in vitro that the lowest concentration of methylene chloride need ed to induce DNA single strand breaks in mouse hepatocytes was 75 times below that in rat hepatocytes. This DNA damage was not accompanie d 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 poten tial 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/m 3 (Green et al., 1987a,b,c). In both studies the DNA was hydrolysed and analysed by chromatography to di stinguish between alkylation of DNA and incorporation of radioact ivity though the C-1 pool. No evidence of alkylation was found, bothstudies having the power to detect one alkylation per 10 6 nucleotides.

No alkylation of DNA was reported by Ottenw alder & Peter (1989), following a DNA binding assay of methylene chlo ride in rats and mice.

Male mice and hamsters were exposed to 14 1 00 mg/m 3 , 6 h/day for 2 days, followed on the third day by a 6-h expo sure to a decreasing concentration (15 900 to 8800 mg/m 3 ) of 14 C-labelled methylene chloride (Casanova et al., 1992). DNA-protein c ross-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 d id not exclude possible formation in a sub-population of lung cells. Th ese results demonstrate that formaldehyde derived from methylene chlori de can form DNA-protein cross-links in the liver of B6C3F 1 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 i njected subcutaneously 3 times/week for 4 weeks with 5 ml/kg of 5% v/vor 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 7 10 mg/m 3 (2 h/day, 5 days/week for 6 weeks) (Basso et al., 1987; Raj e et al., 1988). Mating was started 1 week later for the injection grou p and 2 days later for the inhalation group, each male mouse being mat ed with a virgin adult female. The mating continued for 2 weeks. The f etuses were examined on

day 17 of gestation. No significant differences in any of the mutagenicity parameters were found between cont rol 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 abil ity of methylene chloride to induce replicative DNA synthesis (S -phase) in the livers and lungs of B6C3F 1 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 80 0 mg/m 3 for 2 h, but not following a single oral dose of 1000 mg/kg (Lefevre & Ashby, 1989). The biological significance of these inc reases is unclear due to similar increases being seen in some control groups.
There were no sustained increases in DNA sy nthesis in the livers of female mice exposed by inhalation to 3.53, 7 .06, 14.12 or 28.24 g/m 3 (1000, 2000, 4000 or 8000 ppm) for up to 4 weeks, nor in female mice exposed to 7.06 g/m 3 for up to 2 years (Foley et al., 1993).

Increases in DNA synthesis were not seen in rats exposed to 1770 mg/m 3 for 6 or 12 months (Nitschke et al., 1988a,b).

Replicative DNA synthesis was measured in t he lungs of female mice exposed to 7.06 or 28.24 g/m 3 for 1, 2, 3 or 4 weeks and in mice exposed to 7.06 g/m 3 for 13 and 26 weeks. Small decreases in the labelling indices were reported at all these po ints (Kanno et al., 1993). In contrast, Foster et al. (1992) report ed 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/m 3 for 13 weeks.

8.6.3 Appraisal

Under appropriate exposure conditions, methylene chloride is mutagenic in prokaryotic microorganisms with or wi thout metabolic activation (S. typhimurium or E. coli). In eukaryotic systems it gives either negative or, in one case, weakly posi tive results. In vitro gene mutation assays and tests for UDS in m ammalian 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 i nduction.

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 t ests using B6C3F 1 

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aberrations and micronuclei in mice have been repo rted following inhalation of high concentrations of methylene chl oride. Increases in hepatic DNA single strand breaks and DNA-protein c rosslinks were seen in mice, but not in rats, exposed to 14 100 mg/m 3

.

Within the limitations of the short-term tests cur rently available, in vivo genetic activity has only been detected in tests using B6C3F 1 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-Daw ley rats (8 weeks old) were exposed by inhalation to 0, 1770, 5300 or 12 400 mg/m 3 methylene chloride (99% pure) 6 h/day, 5 days p er week for 2 years (Table 20). Overall survival in the study, incl uding 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 month s, but not in a dose- related manner. From 12 months, non-neoplastic pathological effects on the liver (increased hepatocellular vacuolizati on consistent with fatty change) were observed in both males and f emales at all exposure levels in a dose-related fashion. In males (530 0 and 12 400 mg/m 3 ) and females (12 400 mg/m 3 ), 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 gla nd tumours in males and females, and tumours in the mid-cervical region close to the salivary gland in males. There was no significant increa se in the proportion of animals with benign or malignant mammary tumour s; however, the total number of benign mammary tumours showed a margi nally 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 obse rved in females (165/96; 218/95; 245/95; 287/97; p < 0.001). Th ere was no indication of an increase in the number or incidence of ma lignant mammary tumours in either males or females. The background hist orical 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 th e inhalation routes

Species Strain/sex Route of Doses Observations Reference administration/pr otocol/ (mg/m 3 ) group size

Rat Sprague-Dawley, Inhalation, 6 h/ day, 5 0, 1770, Increase in various typ es of sarcomas in the Burek et al. male and female days/week for 2 years 5300, mid-cervical area in th e region of the (1984) 95 animals/sex/ group 12 400 salivary gland in mid- and high-dose males (1/92, 0/95, 5/95, 11/9 7). Slight dose- related increase in tot al number of benign mammary tumours in male s (8/95, 6/95, 11/95, 17/97; p =0.046) ; dose-related increase in total numbe r of benign mammary tumours in fema les (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 incidenc e of benign Nitschke et al. male and female days/week for 20 1770 mammary tumours in male s or in females (1988a) months (males) , 24 exposed to 177 or 710 m g/m 3 ; increased months (females ); 90 incidence of benign mammary tumours in males/grou p females at 1770 mg/m 3 ; no increase in the 198 females/g roup number of any malignant tumours; NOAEL 710 mg/m 3

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 anima ls/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/pr otocol/ (mg/m 3 ) 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 fo r 7 benign and/or malignant tumours, in (1988) and after weeks, then 7 h/ day, 5 maternal or offspring a nimals; no pregnancy days per week f or 97 statistically significa nt increase in total weeks; malignant tumours 54 females exp osed 60 female cont rols
Rat Sprague-Dawley, inhalation, prot ocol as 350 As above Maltoni et al. male and female above for 15 an d 104 (1988) offspring from weeks; 60-70 ma les or 12th day in utero females/group, 158 

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controls

Mouse B6C3F 1 inhalation, 6 h/day, 5 7100 Mice exposed for more than one year Kari et al. (1992) days/week for v arious showed an excess of lun g and liver tumours periods up to 104 weeks; killed at end of exposure; 364 f emales exposed, 364 f emale controls

Table 20 (Cont'd)

Species Strain/sex Route of Doses Observations Reference administration/pr otocol/ (mg/m 3 ) group size

Mouse B6C3F 1 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 m ice maintained for 104 weeks; 68 fem ales exposed, 68 fe male controls

Mouse B6C3F 1 , male Inhalation, 6 h/day, 5 0, 710 0, Dose-dependent increases in (1) NTP (1986); and female days/week for 102 14 100 alveolar/bronchiolar ad enomas Mennear et al. weeks; 50 males: 3/50, 19/50, 24/ 50 (1988) animals/sex/g roup females: 2/50, 23/48, 2 8/48 (2) alveolar/bronchiola r carcinomas males: 2/50, 10/50, 28/ 50 females: 1/50, 13/48, 2 9/48 (3) hepatocellular aden oma and carcinoma - combined males: 22/50, 24/49, 33 /49 females: 3/50, 16/48, 4 0/48
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/m a showed an increase in the number of sarcomas in the mid-cervical area in the region of the salivary glands (1 subcutaneous sarcoma in cont rols, n = 92, 2 subcutaneous sarcomas and 3 salivary gland schw annoma at 5300 mg/m 3 , n = 95; and 5 subcutaneous fibrosarcomas and 2 subcutaneous mesofibrosarcomas as well as 2 salivary gland s arcomas at 12 400 mg/m 3 , n = 97). A number of uncertainties affect the toxicological significance of these observation s. As stated by the investigators, all but two of these tumours wer e large and appeared to invade all adjacent tissues in the neck region. Histologically all were sarcomas. Some tumours morphologically res embled the overall type, fibrosarcoma while others resembled anoth er, neurofibrosarcoma, and still others were undifferentiated or pleom orphic. Only two tumours were small enough to be localized in th e 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 fro m mesenchymal tissue they represent a variety of tumour types. There is no explanation for the sex difference observed. Both male and fema le rats in the study had a viral disease (siabclacryoudenitis, whichaffects the salivary glands) during the first 2 months of the study. The infection did not increase mortality and all exposure groups appe ared 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/m 3 revealed salivary gland sarcomas in only two anima ls, a male at 1770 mg/m 3 and a female at 177 mg/m 3 . The historical incidence in this laboratory was reported to ra nge from 0 to 2% in control groups of Sprague-Dawley rats (Nitschke et al., 1988a).

Groups of 90 male and 108 female Sprague-Da wley rats (6-8 weeks old) were exposed to 0, 177, 710 or 1770 mg/m 3 6 h/day, 5 days/week for 20 and 24 months, respectively. Exposure-re lated increases in CO-Hb levels were found. Exposure-related histo -pathological changes were limited to the liver of male and female ra ts and mammary glands of female rats exposed to 1770 mg/m 3 . An increased incidence of hepatocellular vacuolization was found in males and females exposed to 1770 mg/m 3 . Females exposed to this dose level also had an in creased incidence of multinucleated hepatocytes. The in cidence of benign mammary tumours in female rats exposed to 177, 710 and 1770 mg/m 3 was comparable to historical control values (79 -82%), but the number (2.2) of benign mammary tumours per tumour-bear ing female rat exposed to 1770 mg/m 3 was greater than that of controls (1.8) (p < 0.05)

. There were no increases in the incidence of mal ignant 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-Dawl ey rats was judged to be 710 mg/m 3 (Nitschke et al., 1988a).

A group of 54 pregnant Sprague-Dawley rats was exposed to 350 mg/m 3 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 

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were exposed to 212 mg/m 3 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 bearin g benign and/or malignant tumours. Although there was a slight increase in total malignant rumours in rats exposed to 350 mg/m 3 for 104 weeks, this was not deemed to be statistically significant (Maltoni et al., 1988). The Task Group noted the absence of relevant da ta on the design of the experiment such as the statistical methods, hou sing 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/m 3 methylene chloride (99% pure) 6 h/day, 5 days p er week, for 102 weeks (NTP, 1986), body weight gains for both exposedmales and females were comparable to those of the control group. The s urvival of exposed male rats was also comparable to that of the control s, although the survival of all groups of males at the end of t he study was low. Similarly, the survival of female rats was comp arable with that of the controls, with the exception of the high-dose g roup. Non-neoplastic treatment-related increases in the incidence of renal tubular cell degeneration, splenic fibrosis and (females onl y) nasal cavity squamous metaplasia were observed.

Increased incidences of benign tumours of t he mammary gland (all fibroadenomas, except for one adenoma in the hi gh-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 h istorical control incidence of mammary gland fibroadenomas in thi s laboratory for the male rat was 0/50 to 6/49 and for the female ra t 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 B6C3F 1 mice, 8-9 weeks old, were exposed to 0, 7100 or 14 100 mg/m 3 (> 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 male s 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 lun g neoplasia in both

sexes, significant dose-related increases in lu ng and liver tumours having been found. The incidences of alveolar/b ronchiolar 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). In cidence 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 i ncreases were observed in the incidences of testicular atrophy in male mice and uterine and ovarian atrophy in female mice; these effects w ere considered to be secondary responses to neoplasia (NTP, 1986; Me nnear et al., 1988).

Studies have been conducted on the B6C3F 1 mouse to study the time-dependency of exposure to methylene chlori de leading to tumour formation. Groups of 364 female B6C3F 1 mice were exposed to either air or 7100 mg/m 3 methylene chloride for 6 h/day, 5 days/week for 104 weeks. Following exposure for either 462, 4 94, 515, 529, 571, 597, 618, 639 or 660 days, 10 exposed and 5 control mice were randomly selected and sacrificed for the purposes of fol lowing the progression of neoplasia in the lung and the liver. Increas ed incidences of alveolar/bronchiolar adenomas and carcinomas an d 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 mi nimal indications of proliferative lesions in the liver. Mice expose d for longer than one year showed a progressive increase in the incid ence of both alveolar/bronchiolar adenomas and carcinomas. T he incidence of liver tumours in mice exposed for longer than one yea r was relatively constant (40-60%), although a time-dependant in crease in the total liver tumour burden per animal was reported (Ka ri et al., 1993).

Groups of 68 female B6C3F 1 mice were exposed to 100 mg/m 3 6 h/day, 5 days/week for either 26, 52 or 78 we eks. All groups of mice were maintained for 104 weeks, at which time th ey were sacrificed. The exposures were split into two phases, i.e. expo sure to either air or to methylene chloride for the specified period at the beginning or the end of the total exposure period. Further group s of 68 female mice were exposed either to air or to 7100 mg/m 3 methylene chloride for the full 104 weeks. The percentage incidences o f lung adenomas or adenocarcinomas or liver adenomas or adenocarci nomas are given in Table 21. The percentage incidence of both lungand liver tumours in mice exposed to methylene chloride during the f irst 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 o bserved following 78 weeks of exposure to methylene chloride. The st udy also showed that lung rumours appeared earlier in methylene chlo ride-exposed animals than did liver tumours. Comparison of the resul ts of this study with

those presented in the previous paragraph sugge sted that the percentage incidence of lung tumours but not li ver tumours increased 

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when the mutagenicity of tumours was taken into account (Kari et al., 1993).

Table 21. The percentage incidence of lungand liver tumours in female mice exposed to methylene chloride for different tim e periods

Exposure a% incidence o f tumours (adenomas or carcinomas)

Phase I Phase II Lung Liver

air 104 weeks 7.5 27

methylene chloride 26 weeks air 78 weeks 31 b40

methylene chloride 52 weeks air 52 weeks 63 b44 b

methylene chloride 78 weeks air 26 weeks 56 b62 b

methylene chloride 104 weeks 63 b69 b

air 26 weeks methylene ch loride 78 weeks 19 b48 b

air 52 weeks methylene ch loride 52 weeks 15 31

air 78 weeks methylene ch loride 26 weeks 4 34

a exposure to methylene chloride was 7100 mg/m 3 , 6 h/day, 5 days/week
bstatistically 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/m 3 6 h/day, 5 days/week for 2 years, no exposure-rela ted changes were observed in mean body weight and m ean 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 p er 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 gr oup received the treatment for 104 weeks. Additional groups of 5 0 male and 50 female F-344 rats received methylene chloride at a con centration 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 live r of rats receiving 250 mg/kg per day. There were no increases in t he incidence of any tumour in treated rats when compared to control s (Serota et al., 1986a).

When groups of 50 male and 50 female Spragu e-Dawley rats were given 0, 100 or 500 mg/kg methylene chloride (p urity 99.97%) by gavage in olive oil, 4-5 days/week for 64 weeks, exces s mortality was observed in rats of each sex receiving 500 mg/k g. A slightly higher incidence of adenocarcinomas of the mammary gla nd 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 wa s no effect on the total tumour incidence in the exposed groups (M altoni et al., 1988). The Task Group noted the short length of the ex posure period as well as the absence of relevant data on the design o f the experiment, such as the statistical methods applied, housing con ditions, histopathological techniques and pathology sche dule.

Table 22. Carcinogenicity studies by oral route

Species Strain/type Route of Doses Observations Reference administration /protocol/ group/s ize

Rat Fischer-344, Drinking-water adlibitum for 0, 5, 50, 125 No increase in incidence of neoplasms; Serota et al. 

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per dose; schedul ed kills: 5 at per day by methylene chlor ide; significant 26 weeks, 10 at 5 2 weeks, 20 decreases in bo dy weight gain at 125 at 78 weeks; also additional and 250 mg/kg p er day and evidence of groups of control s and liver damage at doses above 50 mg/kg 250 mg/kg (50/sex ) which per day received methylen e chloride for 78 weeks only

Rat Sprague-Dawley, Gavage in olive o il for 64 0, 100 or 500 No effect on to tal tumour incidence in Maltoni et male and female weeks; 50 animals /sex/dose; mg/kg per exposed rats. H igher incidence, not al. (1988) additional contro l group (not day statistically s ignificant, of malignant dosed) of 20 male s and 26 mammary tumours in high-dose females females; surviv al decreased in high dose males and femal es

Mouse B6C3F 1 , male Drinking water ad libitum for 0, 60, 125, 185 No increase in incidence of neoplas ms; Serota et al. and female 104 weeks; group size; or 250 mg/kg evidence of sli ght liver damage at 250 (1986b) 125 m, 100 f (con trols) 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/s ize

Mouse Swiss, male and Gavage in olive o il for 64 0, 100 or Decrease in bod y weight in exposed Maltoni et female weeks 500 mg/kg males and femal es after 36-40 weeks; al, (1988) 50 animals/sex/do se per day dose-related in crease in pulmonary 60 animals/sex/co ntrols tumours in male s - not significant without conside ring mortality rate; significant (p< 0.05) taking into account the mortality r ate; no treatment-related increase in the percentage of animals bearing benign and malignant tumours, or of animals b earing malignant tumours, or of the number of total malignant tumou rs per 100 animals

8.7.2.2 Mouse

Groups of male and female B6C3F 1 mice (7 weeks old) received methylene chloride in the drinking-water for 10 4 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. Hist opathological changes were observed in the liver of mice receiving 25 0 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 mal e mice dying between 52 and 78 weeks, an increase in the incidence of p ulmonary adenomas was observed (0/27 in controls, 4/41 low dose level , 7/33 high dose level) although the effect was not statistically signi ficant in exposed male mice when mortality was not taken into account. There was no increase in the total tumour burden in exposed mice. A d ecrease in body weight was observed in exposed males and females, comp ared 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, c ausing both lung and liver tumours, following exposure to highconcentrations (7100 and 14 100 mg/m 3). The incidence of both lung and liver tumours was increased in mice exposed to 7100 mg/m 3for 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 inhalati on at concentrations up to 12 400 mg/m 3for 2 years showed no evidence of a carcinogenic effect related to exposure to methy lene chloride.

Rats exposed to methylene chloride via various rou tes 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/m 3for 2 years. This excess was only evident when the tumours, which were all of m esenchymal origin, were grouped together for statistical analysis. Asthe tumours arose from a variety of different tissues, the statistic al 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/m 3throughout their lifetime. A further inhalation st udy in rats exposed to methylene chloride at concentra tions up to 1770 mg/m 3throughout their lifetime showed no evidence of

carcinogenicity. These studies, taken together wit h the absence of 

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doubts regarding their biological and toxicologica l significance. Rats exposed to methylene chloride via the drinkin g-water or by gavage similarly showed no substantive evidence of carcinogenicity.

An increase in either the incidence or the multipl icity of benign mammary tumours (fibroadenomas) in rats exposed tomethylene 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 h ighest dose (1770 mg/m 3) group. No effects were observed in males or the other dose groups of females. A dose-related increased i ncidence in benign mammary tumours was observed in female F-344/N rat s, although the incidences were in the range of historical inciden ce (10 to 25%). An increased incidence in high dose (14 100 mg/m 3) males was also within the historical incidence range of 0 to 11%.

In one study, a slight increase in the incidence o f 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 t he mouse or hamster by inhalation or oral administration.

8.8 Mechanistic studies

8.8.1 In vitro metabolic studies

There are three transient reactive intermed iates in the metabolism of methylene chloride. Two of them, formyl chlo ride and S- chloromethyl-glutathione, are assumed to be present on the basis of knowledge of the metabolic pathways; the thi rd, 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 alky lating 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). Formy l chloride is highly unstable, existing chemically only at low tempe ratures (-80°C) in inert solvents (Staab & Datta, 1964). Formaldeh yde 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 add itional formaldehyde derived from methylene would be metabolized by the same efficient pathways.
Comparisons of the rates of metabolism of m ethylene 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 meta bolism in these pathways in vitro had similar differences to those seen in vivo in rats and mice, and enabled a comparison to be m ade with those species (hamster and man) where in vivo data was not available.

Rates of metabolism in human liver have bee n measured for both glutathione- S-transferase (Green et al., 1987a,b,c; Reitz et al., 1989; Bogaards et al., 1993) and cytochrome P-4 50 (Green et al., 1987a,b,c; Reitz et al., 1989) pathways of meth ylene chloride metabolism. A total of 33 human liver samples h ave 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, and8 samples had activity in the range 0.82-1.23 (1.03 ± 0.14) nmol/min p er 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 leas t 1.4 lower than that in rat liver cytosol. Two human lung samples we re 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 activi ty 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 correlati on was obtained in DNA damage tests (section 8.6.2.3.) No such correla tion exists for the cytochrome P-450 pathway where, for example, th e metabolic rate in the hamster is very similar to that in the mouse. C ytochrome P-450- catalysed metabolism of methylene chloride coul d be detected in lung tissue from all three animal species, the relat ive 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 ch loride by the glutathione- S-transferase pathway in human liver samples has been attributed to a deficiency in the transferase i soenzyme 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). 

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A comparative kinetic profile of methylene chloride and its metabolites was determined in B6C3F 1 mice and F-344 rats both during and after a 6-h exposure to atmospheres contain ing various concentrations from 350 to 14 100 mg/m 3 (Green et al., 1986b, 1987a,b,c, 1988). Blood levels of methylene chl oride and CO-Hb and the rates of elimination of methylene chloride, car bon monoxide and carbon dioxide in exhaled air were measured. Stable is otopes were used to quantify the amount of carbon dioxide from eachpathway at dose levels of 350, 1770 and 14 100 mg/m 3 , but only in the mouse. The steady- state blood levels of methylene chloride duringexposure were up to 5 times higher in rats than in mice at the high er dose levels. A comparison of the CO-Hb levels in blood and car bon monoxide levels in expired air showed that rate of metabolism by t he 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/m 3 , resulting in maximal CO-Hb levels of 16% (Green et al., 1987 a,b,c).
Saturation of the cytochrome P-450 pathway in mice was also clearly shown by a 5-10 fold increase in the bl ood levels of methylene chloride when the inhaled concentration was dou bled from 1770 to 3500 mg/m 3 (Green et al., 1987a,b,c).

The stable isotope studies demonstrated tha t the cytochrome P-450 pathway was the major source of carbon dioxide at low exposure levels (350 mg/m 3 ) whereas at high levels ( 14 100 mg/m 3 ) the glutathione- S-transferase pathway was the principal source of car bon dioxide. A comparison of the rate of eliminatio n of the carbon dioxide by rats and mice at the top dose level showed t he glutathione- S- transferase pathway to be 10-12 times more acti ve in mice than rats. The higher rate of metabolic conversion of meth ylene 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 a t 1770 mg/m 3 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/m 3 dose level being an order of magnitude greater than in rats.

3. In all, 33 human liver samples have been as sayed for glutathione-

S-transferase activity. In all cases the levels of ac tivity were lower than those measured in rat liver.
4. Methylene chloride metabolism is dose-depen dent. The utilization of the two pathways is significantly differ ent at the dose levels used in the carcinogenicity studies than at low dose levels.
5. These studies provided the metabolic rate c onstants used in the physiologically based pharmacokinetic model s described in section 8.9.

8.8.3 Pulmonary effects

A number of studies have examined the effec ts 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 concentra tions of 7100 mg/m 3 or more, a specific lesion characterized by mar ked 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 abilit y to metabolize methylene chloride by the two pathways. Cytochr ome 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 significa nt increase in the number of cells in S-phase was observed in bothbronchiolar 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 observedin male B6C3F 1 mice 1 day after a single 6-h exposure to 14 100 mg/ m 3 (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 B6C3F 1 mouse into the role of oncogene activation as a potential mech anism of action of the carcinogenic effect of methylene chloride. A gr oup of 145 female B6C3F 1 mice was exposed to 7100 mg/m 3 for 6 h/day, 5 days/week for up to 27 months. Another group of 235 females a cted as controls. These mice were used for the purpose of providing spo ntaneous and methylene- chloride-induced tumour tissue from both the li ver and the lung for the analysis of proto-oncogene activation and t umour suppressor gene inactivation. The DNA recovered from 54 methyle ne-chloride-induced B6C3F 1 lung tumours and from 7 spontaneous B6C3F 1 lung tumours was 

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amplified DNA fragments of the K-ras gene for f irst 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 l iver tumours and screened by the oligonucleotide hybridization o f PCR (polymerase chain reaction) amplified H-ras gene fragments for co don 61 mutations. The mutation profile of the H-ras gene was similar in the tumour tissue derived from both control and treated mice (Dev ereux et al., 1993).

Mutations of the p53 rumour suppresser gene were examined in lung tumours from female mice exposed to 7100 mg/m 3 (2000 ppm) methylene chloride for 2 years (Hegi et al., 1993). The l imited number of p53 mutations identified in this study and the smal l number of spontaneous tumours precluded any conclusions concerning th e mutagenic spectrum or possible genotoxicity of methylene chloride.

8.8.5 The use of mechanistic studies in extrapolat ion
Studies using liver fractions from rats, mi ce, hamsters and humans have confirmed the existence of two pathways fo r the metabolism of methylene chloride (the cytochrome P-450 pathwa y and the glutathione- S-transferase pathway) and have established substanti al differences between species in the utilization of these pat hways. The rates of metabolism of methylene chloride by the two enz ymes in the liver fractions from a few species have been establis hed. The activities of the cytochrome P-450 pathway in the mouse and h amster 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 bei ng a 10-fold difference in activity between the mouse and the rat. Rate s of metabolism by this pathway in hamsters and humans were even lower than in rats.

The results of a full pharmacokinetic analy sis 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 m ice at dose levels of less than 1770 mg/m 3 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 betwee n mice and rats that had been observed in vitro. The higher metabolic rates in mice accounted for the lower blood levels seen in th is species compared to the rat.

Exposure of mice to atmospheric concentrati ons of 7100 mg/m 3 or more led to recurrent cytotoxicity, increases i n DNA synthesis ( S-phase) and changes in the metabolic complement of m ouse lung Clara cells. All the effects were specific to t he mouse and to the Clara cells. Several of the changes (cytotoxici ty and S-phase) were frequently associated with the development of t umours. However the
significance of these findings with respect to the development of lung tumours in mice exposed to methylene chloride r emains to be established. Effects on chromosomes have also b een reported in the mouse lung.
Studies on the potential role of activation of Ras oncogenes in the development of methylene-chloride-induced l ung and liver tumours have been unable to distinguish between the tum ours seen in methylene- chloride-treated animals and those occurring sp ontaneously in control animals.

Studies in which mice were exposed for diff erent intervals of a 2-year carcinogenicity study established that l ung tumours developed quicker and after shorter exposures than liver tumours. Whether this indicates a different mechanism in the lung fro m 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 g lutathione-deficient strains and strains in which mammalian transfer ase enzymes have been expressed have established the role of this pat hway 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/m 3 . 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 lun g.
There is a consistency between the bacteria l mutagenicity assays, the pharmacokinetic data and the studies of DNAsingle 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 m ouse 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 a nd pharmaco-kinetics of methylene chloride in the rat, mouse and hamste r also provided a plausible explanation for the species differenc es in the carcino- genicity of this chemical. The differing metabo lic 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 pat hway are not. These 

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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 we re observed. At the lower dose levels used by Serota et al. (1986b) and Maltoni et al.
(1986), methylene chloride would have been meta bolized mainly by cytochrome P-450, and glutathione- S-transferase metabolism would have been minimal. Consequently there were no s ignificant increases in either lung or liver tumours in these studies.

8.8.6 Mammary tumour promotion

The dependence of mammary tumours upon pitu itary hormones in both male and female rats has been established unequ ivocally (Welsch & Nagasawa, 1977; Welsch 1985). In the rat, prola ctin acts as a promoter of mammary carcinogenesis. There is good eviden ce that increased prolactin levels increase the incidence of mamm ary 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 i nduces mammary adenomas in the rat is important for human hazard assess ment. Female Sprague- Dawley rats receiving methylene chloride have a high blood level of prolactin (Breslin & Landry, 1986). When male a nd female Sprague- Dawley rats were exposed to 10 600 mg/m 3 (3000 ppm) methylene chloride for 15 to 19 consecutive days, a signi ficant increase (2.3 x) in basal serum prolactin levels was observed in female rats. No significant effect was observed in male rats (B reslin & Landry, 1986).

In humans, there is conflicting evidence on whether or not mammary rumours are as responsive to prolactin as in th e case of rats (Sinha, 1981). The rat has elevated levels of prolactin when fed ad libitum in comparison to a restricted dietary regimen a nd this may explain why the mammary tumour incidence is so easily respo nsive to a variety of environmental and other effects. In the rat, ho wever, prolactin is luteotrophic. An increase in the circulating le vels of prolactin will lead to an increase in progesterone and exogeno us oestrogen levels. The presence of all three factors that causes t ubular-alveolar growth of the mammary glands may ultimately lead to tu mour development. Prolactin is not luteotrophic in primates (Neum ann, 1991).

The mechanism of production of mammary tumo urs in the rat involving hyperprolactinaemia will probably occ ur only at doses of methylene chloride which affect prolactin level s. There is no direct information on prolactin levels in rats receivi ng low doses of methylene chloride, but no increase in mammary adenomas has been observed following the administration of low do ses in either inhalation or drinking-water studies (i.e., bel ow 250 mg/kg body weight or 1770 mg/m 3 ).
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 ch loride to the lung and liver. This explanation is based on the existe nce of an isoenzyme of glutathione- S- transferase which specifically metabolizes methylene chloride to the reactive intermediates r esponsible for tumour induction in the mouse. Markedly lower leve ls 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 bact eria and in the B6C3F 1 mouse, which shows a very high level of activity of the isoenzyme, reveal positive effects, whereas mutage nicity has not been demonstrated in standard in vivo mutagenicity assays using other systems. These observations are consistent with th e 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, a nd the correlation between the activity of this pathway and the speci es differences in carcinogenic response in lung and liver, has led t o its use as the dose surrogate in physiologically based pharmacoki netic models used for human health risk assessment.

The pharmacokinetics of methylene chloride and theresponse seen in B6C3F1 mice suggest that this species is a poor model onwhich to base human hazard assessment to methylene chlor ide.

The mechanism of mammary tumour formation in the r at is probably related to the effect of methylene chloride on pro lactin levels in this species.

8.9 Interspecies and dose extrapolations by kineti c modelling
Two physiologically based pharmacokinetic ( PB-PK) models (Andersen et al., 1987; ECETOC, 1988) have been developedand provide quantitative estimates of the levels of methyle ne chloride metabolites in four mammalian species (mice, rats, hamsters and humans) following inhalation exposure. The models use information on various physiological parameters and metabolic constant s for the cytochrome P-450 and glutathione- S- transferase pathways in the lung and liver, 

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mouse, with values for rats, hamsters and human s scaled from the mouse data. The metabolic constants for these models were obtained from the data described in section 8.8 and from gas upta ke studies described by

Andersen et al. (1987). The models were validat ed against other human experimental data such as the elimination of ca rbon monoxide and blood levels of methylene chloride. Time-course conce ntration data from the model were compared to experimental results in F-344 rats, Syrian Golden hamsters, B6C3F 1 mice and human volunteers. The predicted values for each of the four species were in agr eement with the experimental data. The models were also shown t o predict the appearance and elimination of methylene chlorid e metabolites. A similar model was used by Andersen et al. (1991 ) to predict the time course of the disappearance of CO-Hb after expo sure 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 assumpti ons, 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 potentia l risk, it should be recognized that they are based on a set of assu mptions with varying degrees of certainty. Portier & Kaplan (1989) i nvestigated the impact of varying the intra-population values of the b iological parameters used in the model developed by Andersen et al. (1987) using Monte Carlo and resampling statistical methods. The r esults from this analysis indicated that the estimates of "effec tive" doses in humans may vary widely if variability of the parameter s is taken into account in the PBPK model.

Dankovic & Baiter (1993) investigated the i mpact of exercise and human inter-subject variability on the estimate s 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 fo r 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 refle ct light work conditions increased the glutathion- S-transferase pathway metabolic contribution by a factor of 2.9 for the liver a nd 2.4 for the lung. When the model was also modified to reflect met abolic inter-individual (n = 4) variability in humans, the glutathione- S-transferase pathway contribution estimates were increased by as muc h as 5.4-fold for the liver and 3.6-fold for the lung. More recent da ta have become available on human metabolic parameters, and ar e summarized in section 8.8.1. Based on 33 individual liver samples, th ey suggest that the Portier & Kaplan estimates which assume 200% va riation for metabolic variability may be exaggerated. Since Dankovik & Bailer's calculations were based on the four actual values, their est imates would not change; however, three of their values represen t 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 exa mine the relationship between birth weight in Monroe County and expos ure to emissions of methylene chloride from manufacturing processes of the Eastman Kodak Company in Rochester, New York, USA. County cen sus tracts were categorized as high, moderate, low or no methyl ene chloride exposure, based on the Kodak Air Monitoring Program. Birt h weight and information on variables known to influence bir th weight were obtained from 91 302 birth certificates of white, single births to Monroe County residents from 1976 to 1987. At the leve l of methylene chloride exposure (highest predicted average concentrati on, 50 μg/m 3 ), no significant adverse effect of exposure on birthweight was found, although several problems in the method of esti mation of exposure were identified.

9.1.2 Oral exposure

A 56-year-old woman was found deeply uncons cious and cyanosed after ingesting approximately 300 ml of a paint remover containing mainly methylene chloride and methanol. Approxi mately 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 day s and dropped below 1% thereafter. The woman regained consciousness af ter 14 h, but over the following 3 weeks her condition was complicatedby progressive renal failure, pneumonia, pancreatitis, on-going gast rointestinal 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 

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A number of case-reports have been publishe d regarding short-term exposure to methylene chloride in the occupatio nal environment.

Hall & Rumack (1990) described four cases o f serious methylene chloride poisoning, including two fatalities, i n 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 seco nd-degree burns were reported in areas having direct contact with th e methylene chloride- based paint-stripping compound, and measured CO -Hb levels did not exceed 8.6%. In each case, no respiratory prote ction was worn and ventilation was inadequate, but exposure levels were not known. The authors concluded that the toxic effects were d ue to the anaesthetic properties of methylene chloride.

A 67-year-old male who had been using a pai nt stripper in a poorly ventilated location was brought to a hospital e mergency 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 re call of either recent or past events, and was disorientated to time ( ATSDR, 1993).
These and other case studies of methylene c hloride poisoning during paint-stripping operations have demonstr ated that inhalation can be fatal to humans (Hall & Rumack, 1990; No vak & Hain, 1990; Leikin et al., 1990; Manno et al., 1992). In th e majority of cases reported, quantitative estimates of exposure le vels have not been reported, although methylene chloride was detec ted in various tissues. In one case (Manno et al., 1989), air samples c ollected a few hours later from a well in which two men were found d ead, were analysed by gas chromatography/mass spectrometry and were f ound to contain up to 583 mg methylene chloride/litre and much lower or trace amounts of other solvents; blood levels collected at necro psy 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 c larified. However, in the report of five victims, including two death s, described by Leikin et al. (1990), the authors concluded that the c ause of death was due to solvent-induced narcosis and not carbon mono xide poisoning.

Signs of CNS depression, narcosis, irritati on of the eyes and respiratory tract, lung oedema and sometimes de ath were found after accidental exposures to methylene chloride or p aint remover containing this compound (Moskowitz & Shapiro, 1952; Hughe s, 1954; Bonventre et al., 1977; Fagin et al., 1980). Three myocardia l infarctions in one subject were reported to have followed three ex posures to a paint remover containing methylene chloride over a pe riod of approximately 8 months. The subject was exposed in a poorly ven tilated room, and concentrations were up to 4511 mg/m 3 in the breathing zones (Stewart et al., 1976). Electrocardiographic changes res embling those after carbon monoxide poisoning were found in an expo sed man with a history

suggesting ischaemic heart disease (Benzon et a l., 1978). Three probable cases of phosgene poisoning occurred a fter the use of methylene-chloride-based paint remover near a s ource of heat (Gerritsen & Buschmann, 1960; English, 1964).

A serious case of pulmonary oedema with bil ateral exudative pleural effusions was reported in a 34-year-oldman who presented with respiratory distress. Buie et al. (1986) specul ated that hydrochloric acid, a product of dichloromethane under warm, moist conditions, may have played a role in this patient's parenchyma l abnormalities. Recovery was complete with the exception of neu ropsychiatric 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 ro om. This patient presented with an array of signs and symptoms r anging from liver enzyme elevations to poorly localized abdominal pain. Renal studies and biopsy confirmed the diagnosis of acute tub ular necrosis. Histological studies demonstrated plasma membra ne changes in addition to mitochondrial effects suggestive of anoxic d amage. Serum enzyme changes noted during the patient's stay in hosp ital suggested that hepatocellular injury accompanied the nephrotox ic sequelae.
Another case of chemically induced hepatiti s resulting from accidental exposure to methylene chloride alone has been described by Cordes et al. (1988). The liver was palpable bu t not enlarged or tender. The results of initial tests were norma l except for a leukocyte count of 4900 μl with a left shift, a nd elevated serum enzyme levels of alkaline phosphatase 142, lact ic dehydrogenase and serum aspartate aminotransferase (ALAT). Five d ays after admission, the patient was discharged from hospital. Labor atory tests for hepatitis A and B antibodies were negative.

Further evidence for hepatic effects of met hylene chloride were reported by Puurunen & Sotaniemi (1985). One we ek after a brief but extensive body exposure to methylene chloride, serum ALAT was elevated three-fold in a 24-year-old male chemical facto ry 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). 

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aerosol-spray deodorants were used twice a day for 12 weeks by 75 men and women (Meltzer et al., 1977). On direct con tact, methylene chloride caused a burning sensation and pain (S tewart & Dodd, 1964).

Weber et al. (1990) reported a case of an i ndividual who fell into a vat containing methylene chloride and methano l. After being immersed for about 15 min, the subject suffered extensiv e lesions, including skin burns of superficial and deep severe epide rmal 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 bui lt up and he became unconscious, overturning the bucket as he slump ed into the bottom of the vessel. After about 30 min, the man was res cued. During the time that he was in the vessel, the man sustained se cond and third degree burns to both legs, the areas affected being th ose 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 l ow exposure level after volunteers were exposed to 694 mg/m 3 for 1.5-3 h. Vigilance disturbance and impaired combined tracking moni toring 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/m 3 (Fodor & Winneke, 1971). Visually evoked responses (one of the surrogate methods of measuring visual fu nctions) were altered after 1 h of exposure to 2400 mg/m 3 , while exposed subjects experienced lightheadedness. Blood and urine va riables, except CO-Hb levels, were normal in this study after 1-2 h o f exposure to levels of methylene chloride between 739 and 3420 mg/m 3 . No eye, nose, or throat irritation was observed (Stewart et al., 1972). Most neurobehavioural effects observed were less pro nounced or absent, with carbon monoxide exposures resulting in comparab le CO-Hb levels (Putz et al., 1976).

In a double-blind laboratory experiment, a short inhalation exposure to 2.5 mg methylene chloride/litre didnot impair vigilance performance in human volunteers (time of exposu re and number of subjects not stated) (Kozena et al., 1990).

A clinical laboratory evaluation of 266 exp osed volunteer workers and 251 reference volunteer workers from two ce llulose 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, haemoglo bin levels and haematocrit among white women exposed to a meth ylene chloride level of approximately 1650 mg/m 3 . 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 sub jects of both sexes. A group of 24 exposed male volunteers and 26 refe rence male volunteers from the above two industries was also selectedfor 24-h electro- cardiographic monitoring. Three exposed and 8 r eference workers had reported a history of heart disease. Neither in creased ventricular or supraventricular ectopic activity nor increasedepisodic ST-segment depression was found to be associated with meth ylene 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 di agnosed in one man who had been exposed for 5 years to methylene chlor ide at levels ranging from 2290 to 12 500 mg/m 3 (Weiss, 1967). Another man, exposed for 3 years to levels of methylene chloride rangingfrom 1735 to 3470 mg/m 3 showed a bilateral temporal lobe degeneration (Barrowcliff & Knell, 1979). A case of deliriumand seizures was reported in a man who was exposed to methylene chloride for 4 years. The man reported a 12-month history of intermit tent headache, nausea, blurred vision, shortness of breath, and transi ent memory disturbances. Neuropsychological and EEG examin ations revealed a dysfunction of the right hemisphere. All sympto ms 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 a t the Greater Cincinnati Occupational Health Centre. The mean value of the exposure was reported to be 240 mg/m 3 , ranging from 11 to 544 mg/m 3

. Although the primary complaint of these employe es 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 co unts were reported in workers who used methylene chloride in bonding operations which also resulted in possible skin exposure. It is uncer tain 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 g roups of occupationally exposed subjects. 

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114 mg/m 3 had CO-Hb levels of between 0.8 and 2.5%. No effec ts were found on clinical chemistry, haematology or ele ctrocardio-grams (Di Vincenzo & Kaplan, 1981a). Cherry et al. (1981) did not find any
exposure-related, long-term damage in 29 subjec ts as shown by subjective symptoms, neurobehavioural tests, mo tor nerve conduction velocity, electrocardiograms and clinical exami nations. The men had been exposed for several years to levels of met hylene chloride ranging from 260 to 347 mg/m 3 . 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 av erage of 2 years. Digestive disorders were reported by one-third of these workers. Formic acid was found in the urine. No other de viations were found during the internal, nervous system, eye and la boratory examinations. The methylene chloride concentrations measured varied between 100 and 17 000 mg/m 3 (Kuzelova & Vlasak, 1966).
A group of 1758 retired airline maintenance workers was surveyed by mail and telephone to identify a cohort of w orkers with more than 22 years of methylene chloride exposure followi ng the stripping of paint from airplanes. A cohort of 25 exposed an d 21 non-exposed retirees met the criteria and were tested exten sively (Becker & Lash, 1990; Lash et al., 1991). Following a specially prepared battery of neuropsychological and neurophysio-logical test s performed by professionals without prior knowledge of exposu re status of the employees, exposed and control outcome measures were all within the "normal" range. No statistically significant di fference was found between exposed and control groups, although su btle differences in attention and memory were detected.

In 46 subjects exposed to methylene chlorid e concentrations of 6-34 mg/m 3 for several years, an excess (not significant) of digestive disorders and hypotonia was found ove r controls, while symptoms of gall bladder pathology and swollen liver were frequent. No details were given concerning drinking or smoki ng 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 ou tcome was collected from hospital data, and data on exposures from health personnel at the factories. The odds ratio for methylene chlorid e 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 r atios were also increased for exposures to many other solvents. For those exposed to methylene chloride less than once a week the od ds ratio was 2.0 (95% CI = 0.6-6.6); whereas for those exposed more t han 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 hadworked for at least 10 years in an area where average exposures were 1 677 mg/m 3 were compared to an unexposed group of workers (n = 260) with regards to symptoms and blood chemistry. The methylene chl oride workers were also exposed to acetone and methanol (900 ppm and 10 0 ppm 8-h TWAs, respectively). Health history and blood samples had been collected as part of a company-sponsored health monitoring p rogramme in which both exposed and unexposed workers were participants . No remarkable or statistically significant differences were obse rved in the selected symptoms (including irregular heartbeat, dizzin ess or loss of memory) or in SGPT, bilirubin or haematocrit. The only noticeable difference was in SGOT, where the non-exposed group had hi gher 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 act ive 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 (S MR) study, and a proportionate mortality ratio (PMR) study of me n exposed to methylene chloride at a Kodak photographic film productio n 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 retrosp ective 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 subsequent ly expanded the study population to include all workers (n = 1013) wh o were exposed for at least one year between 1964 and 1970 (Hearne et al., 1987). In a more recent publication Hearne et al. (1990) extende d follow-up of this expanded cohort to 1988.
The Friedlander et al. (1978) study was ini tially conducted to test the hypothesis that exposure to methylene chloride increases the risk of ischaemic heart disease. This hypothesi s was based on the fact that methylene chloride is metabolized to carbo n 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 b een shown to induce electrocardiographic changes in exercising pati ents with pre-existing coronary artery disease. An excess of ischaemic heart disease mortality has also been reported in a cohort of tunnel workers exposed 

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also considered a priori hypotheses in the subsequent articles by Hearne et al., based on the results from the an imal bioassay data described in chapter 8.

Comparisons in both studies (Friedlander et al., 1978 and Hearne et al., 1990) were made with mortality rates (o r proportions) from New York State and from an internal unexposed cohor t from the Kodak facility. Extensive industrial hygiene measurem ents were available for this cohort from after 1980, which indicated th at 8-h TWA exposure concentrations for different occupational class ifications ranged from approximately 35.3-402 mg/m 3 (10-114 ppm) and the mean exposure was 91.8 mg/m 3 (26 ppm) (Hearne et al., 1987). An exposure-respon se 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 obs erved, 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 (laten cy) 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 case s were identified with additional follow-up, and with the additional d ata 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. (19 78) and the subsequent studies by Hearne et al. (1987,1990) failed to detect a significantly increased risk of ischaemic heart disease, lungcancer, liver cancer or other cancers among methylene-chloride-expos ed workers. It also important to recognize that workers at the Koda k facility (T. Hearne, personal communication to the IPCS) were not pe rmitted 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 co hort and thus adjustments for this potential bias could not b e made.

Ott et al. (1983) evaluated a cohort of wor kers exposed to methylene chloride in the production of triacet ate fibre at a manufacturing plant in Rock Hill, South Carolin a, USA. This cohort included 1271 males and female workers who were employed for at least 3 months, sometime between 1954 and 1977, Worke rs from another textile facility that were not exposed to methylene chl oride, 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 J une 1977. Eight-hour TWA methylene chloride exposures in this cohort were estimated to range from 494 to 1677 mg/m 3 (140 to 475 ppm) from a survey conducted in 1977 and 1978. Workers in this stu dy were also reported to have been exposed to methanol and acetone. T he mortality experience of the exposed cohort was compared with the mor tality of the USA population using a modified life-table approach(SMRs). Direct comparisons were also made between the mortalit y of the exposed and unexposed cohorts. Mortality from cardiovascula r disease or any other cause was not found to be significantly increas ed 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 no ted that 8 of the 14 ischaemic heart disease deaths among exposed wh ite men occurred among workers who were actively employed. Although Ot t et al. (1983) did not report any increase in cancer mortality, this s tudy was not designed to evaluate cancer and only included seven mali gnant neoplasms.

The follow-up of the exposed cohort (but no t the unexposed one) studied by Ott et al. (1983) was subsequently e xtended 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 e xcess of cardiovascular or ischaemic heart disease. A significant exces s of cancers of the biliary passages and liver (SMR = 5.75, 95% CI = 1.82-13.78) was observed. Three of the cancers were cholangioca rcinomas of various biliary sites while the fourth was a liver aden ocarcinoma. The SMR for biliary cancer was estimated using mortality ra tes 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 observ ed 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 wi th a potential occupational etiology. One of the cases had onl y 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 wer e 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 stud y of workers from a Hoechst-Celanese cellulose acetate fibre plant in Cumberland, Maryland, USA was reported by Gibbs (1992). Thi s study included 3211 cellulose fibre workers employed in or after 19 70 and followed until 1989. The cohort was divided into three groups: high (> 1235 mg/m 3 , > 350 ppm) methylene chloride exposure, low exp osure (176-350 mg/m 3 , 50-100 ppm), and no exposure. Comparisons were made with USA, Maryland and county mortality rates. Estimates of exposu re levels for this population were based on industrial hygiene mea surements from the plant studied by Ott et al. (1983), which used simila r production methods. 

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particularly among those with long latency and with high levels of methylene chloride exposure. A significant exce ss of cervical cancer was observed among women in the low exposure gr oup, based on Maryland rates (SMR = 4.75 based upon 5 observed), but t here was no evidence of a dose-response relationship. Two cases (exp = 1.40) of biliary cancer were observed among the combined high and low e xposure groups. An
excess of ischaemic heart disease mortality was observed among workers in all three groups when comparisons were basedupon Maryland rates, but not when local county rates were used. Mort ality from lung, pancreatic, liver/biliary and other cancers was not observed to be significantly elevated in this study. As with t he Kodak study, workers at the Hoechst-Celanese facilities were not per mitted to smoke at their workstations. Again this fact may have in duced a negative bias in these studies, particularly with respect to lung cancer or cardiovascular disease.

A cohort study of chemical workers includeda sub-cohort of 226 men employed for at least one year in chlorinat ed methanes production (Ott et al., 1983). Methylene chloride is princ ipally produced by a method involving the hydrochlorination of metha nol which also results in the production of chloroform and carbon tetr achloride (IARC, 1986). The men had been employed between 1940 and 1969 and were followed for mortality until 1979. In all, 42 deaths were ob served and no excesses of respiratory cancer (SMR = 0.70, based on 3 o bserved) 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 betwe en 1942 and 1946; two had been employed for less than 5 years, the th ird for 6 years. No further information on exposure for the individ uals or the sub-cohort was given and the mixed exposure to methylene c hloride, chloroform, and carbon tetrachloride limits the interpretat ion of the results with respect to methylene chloride.

Finally, Heineman et al. (1994) reported th e results of a case- control study of astrocytic brain cancer and oc cupational exposure to chlorinated aliphatic hydrocarbons. The study i ncluded 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 10 8 controls were classified as being potentially exposed to meth ylene chloride in this study. The risk was reported to increase with t he probability of exposure (Odds Ratio (OR) - 2.4 for high probab ility, 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 expo sed to methylene chloride after adjustment for other solvent exp osures. The exposure information used in this study is weaker than t hat generally used in the retrospective cohort mortality studies desc ribed above and the results should therefore be viewed more cautiou sly.
9.3 Appraisal of human effects

The main toxic effects of methylene chloride are r eversible CNS depression and CO-Hb formation. Liver and renal dy sfunctions and effects on haematological parameters have also bee n reported following exposure to methylene chloride. Methylen e chloride will irritate the skin and eyes especially when evapora tion is prevented. Prolonged contact may cause chemical burns.

Neurophysiological and neurobehavioural disturbanc es have been observed in human volunteers exposed to methylene chloride at concentrations of 694 mg/m 3for 1.5-3.0 h. No evidence of neurological effects was seen in men exposed to me thylene chloride for several years at concentrations ranging from 2 60 to 347 mg/m 3

. Similarly, the performance of a group of retired a irplane strippers, with a long history of exposure to methylene chlor ide (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 oe dema has been reported after excessive inhalation.

An increased rate of spontaneous abortion in employees in Finnish pharmaceutical industries has been attributed to e xposure to methylene chloride. This isolated finding from a l imited 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 a nd cardiovascular disease. None of the studies demonstrated a relati onship between exposure to methylene chloride and lung or liver c ancer mortality. With regard to the lung cancers, the lack of smoki ng histories hampers the interpretability of the results. An ex cess 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 st udies no new pancreatic cancer cases were identified with addit ional follow-up; with the additional data the excess was not statis tically significant. It should be noted that the size of t hese studies resulted in very low statistical power to detect a n excess particularly for rare cancers such as liver and bi liary tract tumours. 

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70 of 102 3/23/2016 4:29 PMAssociations between exposure to methylene chlorid e and prostate

and cervical cancers have been reported in studies , each of which had its limitations. An association between the potent ial for exposure to methylene chloride and other organic solvents and brain cancer was

found in a case-control study which classified exp osure to methylene chloride using a job exposure matrix. This finding should be viewed with caution.

The results from these studies have been contradic tory with respect to mortality from ischaemic heart disease. A role for methylene chloride in the induction of ischaemic h eart disease is plausible based on the fact that methylene chlorid e is metabolized to carbon monoxide and induces the formation of carbo xyhaemoglobin in humans. An excess of cardiovascular disease was reported in one of the mortality studies. The fact that further studi es did not provide any compelling evidence of an increased risk of ca rdiovascular 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 s tudies may bias the results towards the null (i.e. no effect) due to t he "healthy worker effect" which is particularly strong for cardiovas cular diseases. This bias may have been further exacerbated by thefact that workers were not permitted to smoke at their workstations.

The currently available epidemiological studies ar e inadequate for drawing any firm conclusions with regard to ei ther cancer or cardiovascular disease risk.

10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS O N THE ENVIRONMENT

10.1 Evaluation of human health risks

Human exposure to methylene chloride is mai nly by inhalation of the vapour. Exposure of the general population to methylene chloride depends strongly on the indoor air concentratio n. Owing to the use of products containing methylene chloride, peak co ncentrations of up to 4000 μg/m 3 have been reported. However, 24-h average exposure s are in general below 50 μg/m 3

. Methylene chloride is rapidly absorbed thro ugh the lung and also from the gastrointestinal tract. It is absorbedvia the skin, but at a much slower rate than by the other routes. Once absorbed, methylene chloride is distributed throughout the body andwill cross both the placenta and the blood-brain barrier. It is rap idly 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, on e involving cytochrome P-450 and the second involving glutathione- S-transferase. Clear species differences exist in the relative contr ibutions 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, th us enhancing the value of results from animal studies in human health risk assessment. This approach has been used in assessing the human c ancer 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 effect s are reversible. Other target organs can be the liver and, occasionall y, the kidney. The odour threshold concentration of methylene chlo ride is reported to be 540 mg/m 3 or more. Mild CNS effects have been reported follo wing exposure to concentrations as low as 694 mg/m 3 for 1.5-3 h (behavioural disturbances). More significant ef fects occur at concentrations in excess of 2000 mg/m 3 . Narcosis has been reported to occur following a 0.5-h exposure to 69 000 m g/m 3 . Metabolism to carbon monoxide leads to increases in blood CO- Hb levels following acute exposure to the vapour, a process which b ecomes saturable following exposures to high levels of methylene chloride. Exposure to either 100 or 530 mg/m 3 for 7.5 h leads to CO-Hb levels of 3.4% and 5.3%, respectively, in human volunteers. This e ffect forms the basis of most, if not all, published occupational exp osure 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/m 3 by inhalation. No evidence of irreversible neurological damage was seen in ra ts exposed to methylene chloride by inhalation at concentrations up to 7100 mg/m 3 for 13 weeks. Additional target organs reported in various species chronically exposed to methylene chloride inclu de the liver and, occasionally, the kidney. The no-observed-adver se-effect level (NOAEL) for chronic intermittent inhalation exposure wa s judged to be 710 mg/m 3 in rats. After continuous exposure, slight cytopla smic vacuolization in the liver of both mice and rat s was observed at 88-350 mg/m 3

. A single study has reported the presence of methylene chloride in the placenta, fetus and breast milk of women fo llowing occupational exposure. The teratogenic potential of methylen e chloride has been assessed in three animal studies. Small effects on either fetal or maternal body weights were reported, but no evi dence of an effect on 

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was seen. A well-conducted two-generation repro ductive toxicity study in rats exposed to methylene chloride by inhala tion at concentrations up to 5300 mg/m 3 , 6 h/day, for 5 days/week showed no evidence of an adverse effect on any reproductive parameter, n eonatal survival or neonatal growth in either the F 0 or F 1 generations.

Under appropriate exposure conditions, meth ylene chloride is mutagenic in prokaryotic microorganisms (Salmonella or E. coli) with or without metabolic activation. In eukary otic systems it gave either negative or, in one case, weakly positiv e 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 ty pes 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 mutagenici ty 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 concentra tions 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 te sts currently available, there is no conclusive evidence that methylene chloride is genotoxic in vivo.

Methylene chloride is carcinogenic in the m ouse, causing both lung and liver tumours, following lifetime exposure to high concentrations (7100 and 14 100 mg/m 3 ). These tumours were not seen in the rat or the hamster.
Increased incidence of benign mammary tumou rs in female rats was observed in one study, and increased incidence and multiplicity were observed in two other rat studies. The increase d incidence of these tumours was within the historical control range ; nevertheless there was a dose-response relationship within one stu dy. It is considered that an increase in a tumour type, which occurs with high and variable incidences in control animals, which does not p rogress to malignancy, and which may be related to changes in prolacti n levels, is of little importance in human hazard assessment.

In vitro and in vivo metabolism and biochemical studies, and mutagenicity assays in bacteria and B6C3F 1 mice have provided a plausible explanation for the mechanism of acti on and the species differences in the carcinogenicity of methylene chloride to the lung and liver. This explanation is based on the exi stence of an isoenzyme of glutathione- S-transferase which specifically metabolizes methylene chloride to the reactive intermediate s responsible for tumour induction in the mouse. Markedly lower l evels 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 v ariability of the current estimates of this enzyme activity in hu man 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 expect ed to be lower than in human liver. The carcinogenic potency of methyl ene chloride in man is expected to be low.
Mutagenicity studies on methylene chloride in bacteria and in the B6C3F 1 mouse, which shows a very high level of activity o f the isoenzyme, reveal positive effects, whereas mut agenicity has not been demonstrated in standard in vivo mutagenicity assays using other systems. These observations are consistent withthe above hypothesis and provide a mechanistic basis for the inducti on 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 pathwa y and the species differences in carcinogenic response, has led t o its use as the dose surrogate in physiologically based pharmacokine tic models used for human health risk assessment.

Overall, animal inhalation studies have sho wn effects on the liver from 710 mg/m 3 and on other organs from 1700 mg/m 3 . However, these effects have not been observed in epidemiologic al 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 exposu re limits of the order of 177 mg/m 3

.
10.2 Evaluation of effects on the environment
Due to its high volatility methylene chlori de 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 hy droxyl radicals giving carbon dioxide and hydrogen chloride as major b reakdown products. Based on a lifetime in the troposphere of about 6 months it may be assumed that only a few percent, if any, of met hylene chloride will reach the stratosphere. No significant impact o n 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 0.07-0.2 9 μg/m 3 have been 

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72 of 102 3/23/2016 4:29 PMmeasured. In suburban and urban areas levels up to 2 and 15 μg/m 3

have been found.
Concentrations of methylene chloride in the surface water of rivers in industrialized areas stay generally b elow 10 μg/litre. In industrial effluents, outfalls of municipal wat er treatment plants and leachates of landfills, concentrations of methy lene chloride of up to 200 mg/litre have been measured.

In the aquatic environment, fish and amphib ian embryos have been shown to be the most sensitive to methylene chl oride, with effects on hatching from 5.5 mg/litre; adult aquatic organ isms are relatively insensitive even after prolonged exposure. Ther e is no evidence to suggest that methylene chloride and/or its meta bolites bioaccumulate in the environment. Given the concentrations ob served in surface water (< 10 μg/litre) and those in contaminated efflu ents (< 200 mg/litre), no significant impact on the aquatic environment is expected.

Localized contamination of soils will not s ignificantly disperse despite the mobility of methylene chloride; in groundwaters and soils, biological degradation processes have been iden tified capable of mineralizing methylene chloride in a few days. From the limited information on soil organisms, it may be assume d that contamination of soil has only a local and transient effect.

Apart from accidental spills, it is conclud ed that the present use of methylene chloride has no significant impact on the environment.

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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 possèd e une puissante odeur éthérée. Lorsqu'il est pur et anhydre, ce compo sé est très stable. Le chlorure de méthylène s'hydrolyse lentement en présence d'humidité, pour donner une petite quantité de chlorure d'h ydrogène. Le chlorure de méthylène du commerce est généralement addit ionné de petites quantités de stabilisants afin d'en éviter la d écomposition.
Il existe des méthodes d'analyse pour le do sage du chlorure de méthylène dans les milieux biologiques et les é chantillons prélevés dans l'environnement. Dans tous les cas, on fai t appel à la chromatographie en phase gazeuse avec un détect eur convenable. On obtient ainsi des limites de détection très bas ses (par exemple dans les denrées alimentaires 7 ng/échantillon; dans l'eau 0,01/μg/litre; dans l'air 1,76/μg/m 3 (0,5 ppb); dans le sang 0,022 mg/litre).

2. Sources d'exposition humaine et environnemental e
On estime à 570 000 tonnes la production an nuelle mondiale de chlorure de méthylène. On l'utilise la plupart du temps comme solvant des graisses, des matières plastiques et des li ants pour peinture, en particulier à cause de sa volatilité et de sa s tabilité. Dans l'ensemble du monde, il est utilisé à 20-25% da ns des aérosols, à 25% comme décapant des peintures, à 35-40% comme so lvant au cours des différentes opérations de l'industrie chimique et pharmaceutique, et enfin à 10-15% dans diverses applications allan t 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 surface s peintes et comme constituant des bombes aérosols à usage domesti que.
3. Transport, distribution et transformation dans l'environnement
En raison de sa forte volatilité, la majeur e partie du chlorure de méthylène libéré dans l'environnement se répart it dans l'atmosphère où il est décomposé en l'espace de six mois par ré action avec des radicaux hydroxyles d'origine photochimique.
Dans l'eau, il est décomposé par voie abiot ique beaucoup plus lentement qu'il ne s'évapore. On a montré que l e chlorure de méthylène disparaissait rapidement du sol et des eaux sou terraines.
Grâce à divers systèmes d'épreuve on a pu é tablir les modalités de la décomposition aérobic et anaérobie du chloru re 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 boue s d'effluents municipaux acclimatées). Dans les réacteurs bio logiques, la décomposition peut atteindre 10% à l'heure. Rie n n'indique qu'il y ait une bioaccumulation ou une bioamplification imp ortante.
4. Concentrations dans l'environnement et expositi on humaine
On a décelé la présence de chlorure de méth ylène dans l'air ambiant de régions rurales et de zones écartées , à la concentration de 0,07-0,29/μg/m 3 . Dans les zones de banlieue, la concentration moyenne est inférieure à 2 μ/m 3 et dans les zones urbaines, elle est inférieure à 15 μg/m 3 . A proximité de décharges jugées dangereuses, on a trouvé des concentrations allant jusqu'à 4 3 μg/m 3 . Les précipitations peuvent également contenir du ch lorure de méthylène.
Le chlorure de méthylène pénètre dans l'env ironnement aquatique par suite de la décharge d'eaux résiduaires pro venant des diverses industries et on en a retrouvé dans les eaux su perficielles et souterraines ainsi que dans les sédiments.
S'il y a exposition au chlorure de méthylèn e de personnes appartenant à la population générale, c'est par suite de son utilisation dans certains produits de consommat ion tels que les décapants pour peinture, dont l'emploi peut ent raîner la présence de teneurs relativement importantes dans l'air int érieur. En ce qui concerne l'exposition professionnelle au cours de la production de chlorure de méthylène, elle se produit essentie llement au cours du remplissage et du conditionnement (la fabricati on s'effectue en circuit fermé). Du fait de l'utilisation de ce composé comme décapant pour peinture, il peut également y avoir exposi tion professionnelle lors de la préparation de ces décapants, lors d e la fabrication de certains équipements et également lorsqu'on pro cède au décapage du mobilier. Le chlorure de méthylène est largemen t 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 solvan t lui-même dans l'air expiré ou dans le sang. Toutefois, étant donné que la production de monoxyde de carbone constitue le facteur limita nt du risque en cas d'exposition supérieure à 3 ou 4 heures par jou r, il vaut mieux que la surveillance biologique s'effectue soit par dos age du monoxyde de 

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(CO-Hb) dans le sang. Toutefois cette méthode n e 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 heu res, 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 m 3 chez un non fumeur.
5. Cinétique et métabolisme chez les animaux de la boratoire et l'homme
Le chlorure de méthylène est rapidement abs orbé 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 d e toutes.
Le chlorure de méthylène est très rapidemen t excrété, en majeure partie dans l'air expiré. Il peut traverser la barrière hémato- encéphalique ainsi que le placenta et on peut l e 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 res te est métabolisé en monoxyde, dioxyde de carbone et chlorures minér aux. La métabolisation s'effectue selon l'une ou l'autre de ces deux v oies 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 ce s voies comporte un processus oxydatif, par l'intermédiaire du cyto chrome P-450, et alle conduit à la production de monoxyde et de dioxy de 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/m 3 ). Même si la dose dépasse la valeur de saturation, il n'y pas accroissement de la métabolisation par cett e voie.
Dans l'autre voie métabolique intervient un e 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 "préf érentielle". Chez certaines espèces (par exemple la souris), elle devient la voie principale lorsque la dose est suffisamment éle vée. En revanche, chez d'autres espèces (par exemple le hamster et l'h omme), elle semble n'être que peu utilisée, qu'elle que soit la do se.
Les différentes interspécifiques touchant l e métabolisme par la voie de la GST sont en bonne corrélation avec l es différences observées selon les espèces, notamment en ce qu i concerne la cancérogénicité du chlorure de méthylène. Le ta ux de métabolisation selon cette voie chez les espèces concernées es t utilisé comme modèle cinétique pour la description du comportement m étabolique du chlorure de méthylène chez diverses espèces.

6. Effets sur les êtres vivants dans leur milieu n aturel
Aux concentrations inférieures à 500 mg/lit re, il n'y a aucune inhibition de la croissance des algues et des b actéries aérobies. Il existe des bactéries qui sont capables de croît re en présence de chlorure de méthylène à des concentrations beau coup 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 i nhibition transitoire de l'activité enzymatique. La dose sans effets observables était dans ce cas de 0,1 mg/kg. Le chlorure de méthylène e st modérément toxique pour les lombrics (100 à 1000 μg/cm 2 ) 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 élevée s.
Chez les plantes supérieures, on n'a pas co nstaté d'effets après une exposition de 14 jours à la dose de 100 mg/ m 3

. Les poissons adultes semblent être relative ment insensibles au chlorure de méthylène, même après une expositio n prolongée (la CL 50 à 14 jours est supérieure à 200 mg/litre). Il e st difficile d'apprécier l'effet du chlorure de méthylène su r les daphnies en raison de la très grande dispersion des résulta ts fournis par les différentes études. La CE 50 la plus basse qui ait été rapportée était égale à 12,5 mg/litre.

En milieu aquatique, c'est chez les embryon s 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/li tre.

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 toxic ité aiguë. Ainsi, les valeurs de la CL 50 à 6 heures pour l'ensemble des espèces étudiées, se situent entre 40 200 et 55 870 mg/m 3 . En ce qui concerne la 

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1410-3000 mg/kg. Les effets aigus observés aprè s administration de chlorure de méthylène par diverses voies d'expo sition affectent essentiellement le système nerveux central (SNC ) et le foie, et tous les effets observés l'ont été à des doses élevé es. Des troubles neurologiques ont été observés à des concentrat ions supérieures ou

égaies à 14 100 mg/m 3 , avec de légères altérations du tracé électro- encéphalographique à la dose de 1770 mg/m 3 . De légères modifications histologiques ont été également constatées dans le foie aux concentrations supérieures ou égaies à 17 700 m g/m 3 . Il est également arrivé que d'autres organes soient to uchés, comme les reins ou le système respiratoire. Chez la souris, les effets au niveau pulmonaire étaient limités, après exposition à 7100 mg/m 3 , aux cellules de Clara. On a également fait état d'u ne 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 concen trations de chlorure de méthylène ( > 17 700 mg/m 3 ) a causé des effets réversibles sur le SNC, une légère irritation oculaire et une cert aine mortalité chez diverses espèces d'animaux de laboratoire. Chez le rat on a observé à 3500 mg/m a une réduction du poids corporel, cet effet étant également observé chez la souris à partir de 17 700 mg/m 3 . De légers effets hépatiques ont été également observés ch ez des chiens exposés de manière continue à des doses de 3500 mg/m 3 pendant des périodes allant jusqu'à 100 jours. Après exposition inte rmittente, des effets ont également été observés au niveau du foie ch ez des rats à la dose de 3500 mg/m 3 et chez des souris à 14 100 mg/m 3

. Les autres organes cibles sont les poumons et les reins.
Chez des rats exposés à du chlorure de méth ylène pendant 13 semaines par voie respiratoire à des concentrat ions allant jusqu'à 7100 mg/m 3 , on a observé aucun signe de lésion neurologique irréversible.
L'administration de chlorure de méthylène p ar 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 modéréme nt irritant pour la peau et les yeux chez les animaux de laboratoir e.
7.4 Effets toxiques sur le développement et la rep roduction
Le chlorure de méthylène n'est pas tératogè ne chez le rat ou la souris à des concentrations allant jusqu'à 16 2 50 mg/m 3 . Trois études effectuées sur ces animaux n'ont pas per mis de relever d'effets se traduisant par l'apparition de malformations squelettiques ou autres anomalies du développement. Un léger eff et, se manifestant par une modification du poids foetal ou maternel, a été relevé à la dose de 4400 mg/m 3 , et l'on a constaté qu'après la naissance, le gainde
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/m 3 , et cela pendant 17 semaines, 6 heures par jour et 5 jours par semaine. Aucun effet nocif, de q uelque nature ce que soit, n'a été relevé sur la reproduction, ni su r la survie ou la croissance des ratons nouveaux-nés appartenant à la génération F 0 ou F 1
7.5 Mutagénicité et points d'aboutissement corresp ondants
Dans certaines conditions d'exposition, le chlorure de méthylène se révèle mutagène pour les microorganismes pro caryotes, avec ou sans activation métabolique (Salmonella ou Escherichia coli) Dans les systèmes eucaryotes, il est sans effet mais, da ns un cas, il a donné des résultats légèrement positifs. On a égaleme nt obtenu des résultats uniformément négatifs lors des tests de mutatio n génique in vitro et des tests de recherche d'une synthèse non progr ammée de l'ADN sur cellules mammaliennes. La recherche d'aberratio ns chromosomiques in vitro sur différents types de cellules a donné des résult ats positif s; en revanche, des tests visant à mettre en év idence l'induction d'échanges entre chromatides soeurs ont été soi t négatif s, soit ambigus.
Dans leur majorité, les études in vivo publiées n'ont pas fourni d'éléments en faveur d'une mutagénicité du chlo rure 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 d e 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 so uris de fortes doses de chlorure de méthylène, on n'a pas constaté de l iaison de ce composé à FADN ni de lésions de l'ADN. Ces méthodes pourr aient être les plus sensibles in vivo et les meilleures d'entre elles sont capables de déceler un site d'alkylation sur 10 6 nucléotides. 

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permet de conclure que le chlorure de méthylène soit génotoxique in vivo.
7.6 Toxicité chronique et cancérogénicité
Le chlorure de méthylène est cancérogène po ur la souris et il provoque l'apparition de rumeurs du poumon et d u 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/m 3 , on a constaté que l'incidence des tumeurs pulmonaire s et hépatiques

augmentait lorsqu'on poursuivait l'exposition p endant 78 semaines supplémentaires. Rien n'indique la présence d'e ffets 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/m 3 , 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 4 00 mg/m 3 , on a constaté un excès de la fréquence tumorale au n iveau des glandes salivaires. Cet excès n'apparaissait que lorsqu e les rumeurs, toutes d'origine mésenchymateuse, étaient regroupées e n vue de l'analyse statistique. Etant donné que ces rumeurs trouve nt leur origine dans des cellules de types différents, l'analyse sta tistique utilisée s'est révélée inappropriée. En outre, il a été précis é que les rats utilisés pour cette étude avaient contracté une virose c ommune (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'exp osition 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 deuxiè me 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/m 3 . Une autre étude au cours de laquelle des rats ont é té également exposés à du chlorure de méthylène par la voie respiratoi re et durant toute leur vie, à des concentrations allant jusqu'à 1770 m g/m 3 , n'a pas révélé de signe de cancérogénicité. Aucun signe notabl e 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 pa r mélange à leur eau de boisson.
Trois études font état d'une augmentation d e l'incidence des tumeurs mammaires bénignes chez des rats exposé s 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 faisa nt état d'un accroissement des tumeurs mammaires chez des ha msters ou des souris qui avaient reçu du chlorure de méthylène à des doses comparables. On a établi sans aucun doute possible que les tume urs mammaires étaient liées aux hormones hypophysaires tant chez les rats mâles que chez les femelles. Chez le rat, la prolactine se comport e à la fois comme un initiateur et comme un promoteur des cancers ma mmaires. On a de bonnes raisons de penser qu'un accroissement du taux d e 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'exi stence d'une corrélation positive entre un taux sanguin élev é de prolactine et la présence de tumeurs mammaires chez des rattes â gées de souche R-Amsterdam). Après administration de composés cancérogènes à des rattes, une hyperprolactinémie provoquée chez c es animaux entraîne un accroissement spectaculaire de l'incidence tumo rale. On peut notamment provoquer l'hyperprolactinémie par surrénalecto mie, homogreffe d'hypophyse et régime alimentaire hyperlipidiqu e.
Il est important pour l'évaluation du risqu e chez l'homme, de connaître le mécanisme par lequel le chlorure d e méthylène provoque l'apparition d'adénomes mammaires chez le rat. Les rattes Sprague- Dawley à qui l'on a administré du chlorure de m éthylène présentent un taux sanguin élevé de prolactine. De même qu'av ec les autres composés qui agissent par l'intermédiaire d'une hyperpro lactinémie, 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 mamm aires alors qu'il est principalement métabolisé dans le foie. Il para ît 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 quest ion de savoir si les tumeurs mammaires sont sous la dépendance de la prolactine comme chez le rat, apparaissent contradictoires. Le rat pr ésente un taux élevé de prolactine lorsqu'on le laisse s'alimenter ad libitum plutôt que de le soumettre à un régime strict et cela pourrai t expliquer pourquoi l'incidence des tumeurs mammaires est si clépen dante des divers effets environnementaux ou autres. Chez le rat toutefo is, la prolactine a un caractère lutéotrope. L'augmentation du taux de prolactine dans le sang circulant conduit à une augmentation du ta ux de progestérone et d'oestrogène exogènes. C'est la présence de l'e nsemble de ces trois facteurs qui provoque la croissance tubulo-alvé olaire des glandes mammaires et qui finit par déboucher sur la for mation de tumeurs. Ce 

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pris en considération chez l'homme.

Le mécanisme de formation de tumeurs mammai res chez le rat par l'intermédiaire d'une hyperprolactinémie n'inte rvient qu'à des doses où le chlorure de méthylène agit sur les taux d e prolactine. On ne dispose pas de données de première main sur les taux de prolactine chez des rats soumis à de faibles doses de chlo rure de méthylène, mais l'administration de faibles doses de ce composé , soit par inhalation, soit par mélange à l'eau de boisson (doses infé rieures à 250 mg/kg de poids corporel) ne conduit pas, selon les étude s 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. D ans ces conditions, un contact prolongé peut entraîner des brûlures ch imiques. On a signalé un cas grave d'oedème pulmonaire consécutif à u ne inhalation excessive de chlorure de méthylène. On a également signal é 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 dépre ssion réversible du système nerveux central et dans la formation de carboxyhémoglobine. On a également rapporté des cas d'insuffisance hép atique et rénale avec anomalies hématologiques, à la suite d'une expo sition à du chlorure de méthylène.
Chez des volontaires humains exposés pendan t une heure et demie à trois heures à du chlorure de méthylène à la co ncentration de 694 mg/m 3 , on a observé des troubles neurophysiologiques et neuro- comportementaux. En revanche, aucun signe d'eff et neurologique n'a été observé chez des hommes exposés plusieurs année s à du chlorure de méthylène à des concentrations allant de 260 à 347 mg/m 3 . De même, on a soumis à une batterie de tests neurophysio logiques et psychologiques un groupe d'anciens décapeurs d' aéronefs qui avaient été longtemps exposés à du chlorure de méthylèn e (22 ans), à des doses élevées mais non précisées; comparés à ceux d'u n groupe témoin qui n'avait jamais été exposé à du chlorure de méth ylène ou du moins uniquement à de faibles doses, les résultats ob tenus par les ouvriers se sont révélés "normaux".
On a attribué à une exposition au chlorure de méthylène une augmentation du taux d'avortements spontanés co nstatée chez des employées des industries pharmaceutiques finlan daises. L'étude en question présentait cependant des défauts de co nception qui n'ont pas permis d'établir une relation causale.

Plusieurs éludes de mortalité sur des cohor tes exposées au chlorure de méthylène font ressortir une absenc e d'uniformité dans les causes de décès. La surmortalité due à certaine s maladies (par exemple cancer du pancréas, cardiopathies ischémiques) ne se manifeste pas de façon uniforme, mais seulement dans certaines é ludes. Ces effets ne peuvent être attribués à l'exposition au chloru re de méthylène.

RESUMEN
1. Identidad, propiedades físicas y químicas, y mé todos analíticos

El cloruro de metileno (diclorometano) es u n liquido claro, altamente volátil y no inflamable, con un olor penetrante parecido al del éter. El compuesto puro en polvo es muy est able. El cloruro de metileno se hidroliza lentamente en presencia d e 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 μ g/m 3 (0,5 ppb); sangre: 0,022 mg/litro).

2. Fuentes de exposición humana y ambiental
Se estima que la producción mundial de clor uro de metileno asciende a 570 000 toneladas/año. La mayoría su s aplicaciones se basan en su capacidad para disolver grasas, plásticos y agentes aglutinantes de pintura, así como en su volatilidad y estabi lidad. Su uso a nivel mundial se reparte del siguiente modo: aerosole s (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%). E l uso de cloruro de metileno tiende a disminuir, al menos en Europa occidental.

Más del 99% del cloruro de metileno liberad o a la atmósfera procede de diversas industrias que lo emiten co mo 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 part e 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 ag ua es lenta en comparación con la evaporación. Se ha comprobad o que el cloruro de 

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Se han utilizado diversos sistemas de ensay o 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., min eralización del 49%-66%

en 50 horas en fangos urbanos tratados). En los biorreactores se puede alcanzar una degradación de hasta un 10% por ho ra. No hay indicios de una bioacumulación o biomagnificación important es.
4. Niveles medioambientales y exposición humana

Se ha detectado cloruro de metileno en el a ire ambiente de zonas rurales y remotas a concentraciones comprendida s entre 0,07 y 0,29 μg/m 3 . En zonas suburbanas la concentración promedio es <2 μg/m 3 , y en zonas urbanas, <15 μg/m 3 . En las proximidades de vertederos de desechos peligrosos se han hallad o hasta 43 μg/m 3 . Las precipitaciones también contienen a veces cloru ro de metileno.
El cloruro de metileno penetra en el medio acuático a través de las descargas de aguas residuales de diversas i ndustrias, habiéndose detectado su presencia en aguas superficiales, aguas subterráneas y sedimentos.
La población general se expone al cloruro d e metileno cuando utiliza productos de consumo tales como los qui tapinturas, cuyo empleo puede acompañarse de la presencia de niveles re lativamente altos en el aire del interior de los domicilios. La exposic ión ocupacional durante la producción tiene lugar sobre todo durante el llenado y envasado (la fabricación se lleva a cabo en sistemas cerrado s). Tratándose de un compuesto usado en los quitapinturas, la exposi ción laboral al cloruro de metileno se produce durante la elaboración d e quitapinturas, la fabricación de material para ordenadores y el a cabado comercial de muebles. El cloruro de metileno es ampliamente empleado como disolvente industrial en la elaboración de dive rsos productos, sobre todo en las industrias que se mencionan en la s ección 1.2.
La vigilancia biológica de la exposición al cloruro de metileno puede basarse en la medición del propio disolve nte en el aire espirado o en la sangre. No obstante, dado que la produc ción 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 riesg os para la salud, es preferible basar la vigilancia biológica en el análisis bien del monóxido de carbono presente en el aire espirad o, o bien de la carboxihemoglobina (CO-Hb) en sangre. Así y tod o, esto sólo se puede aplicar a las personas no fumadoras. Deben toma rse muestras antes de transcurridas aproximadamente dos horas tras la exposición, o bien al cabo de 16 horas, esto es, a la mañana siguient e.
Los niveles postexposición de CO-Hb a las d os 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/m 3 de cloruro de metileno.
5. Cinética y metabolismo
El cloruro de metileno es absorbido rápidam ente por los alvéolos pulmonares, a través de los cuales llega a la c irculación sistémica. Es absorbido también por el tracto gastrointest inal, así como por vía cutánea, si bien en este último caso la velocid ad de absorción es menor que por otras vías de exposición.

El cloruro de metileno se excreta con consi derable 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 o rina y la leche.
A altas concentraciones la mayoría del clor uro de metileno absorbido se espira inalterado. El resto es met abolizado en monóxido de carbono, dióxido de carbono y cloruro inorgá nico. Hay dos vías posibles de metabolización, cuya contribución r elativa al metabolismo total depende en gran medida de la dosis y de l a especie animal considerada. Una vía consiste en un proceso de metabolismo oxidativo mediado por el citocromo P-450, que conduce a l a producción tanto de monóxido de carbono como de dióxido de carbono. Esta vía funciona de manera parecida en todos los roedores estudiado s 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/m 3 ). Aumentar la dosis por encima de ese nivel de sa turación 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 est a vía sólo adquiere importancia a dosis superiores al nivel de satu ración 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 se a la dosis.
Las diferencias interespecies del metabolis mo mediado por la GTF guardan una clara relación con las diferencias interespecies observadas en lo que respecta a la carcinogenic idad. Analizando la intensidad del metabolismo mediado por esta vía en determinadas especies, se ha elaborado un modelo cinético de l metabolismo del cloruro de metileno en diversas especies.
6. Efectos en organismos presentes en el medio amb iente 

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crecimiento de algas y de bacterias aerobias. S e han descubierto bacterias capaces de crecer en presencia de clo ruro de metileno a concentraciones mucho mayores, incluida una sol ución saturada en agua

(sección 4.2.4.1 ). Las bacterias anaerobias so n más sensibles; se ha observado inhibición del crecimiento a una conc entración de 1 mg/litro en fangos biológicos anaerobios.
En el suelo, se observó que una concentraci ón de 10 mg/kg reducía considerablemente el contenido de ATP de la bio masa, incluidos hongos y bacterias aerobias, e inducía una inhibición transitoria de la actividad enzimática. El nivel sin efectos obse rvados fue de 0,1 mg/kg. En las lombrices de tierra el clorur o de metileno tiene un efecto moderadamente tóxico (100-1000 μg/cm 2 ), como demuestra la prueba de toxicidad de contacto con papel de fi ltro. En sedimentos no se observaron efectos tóxicos ni siquiera a con centraciones muy altas.

En plantas superiores no se observaron efec tos al cabo de 14 días de exposición a 100 mg/m 3

. Los peces adultos parecen relativamente ins ensibles al cloruro de metileno, incluso después de una exposición pro longada (14 días, CL 50 > 200 mg/litro). El efecto del cloruro de metileno en Daphnia resulta difícil de evaluar porque hay grandes d iferencias entre los resultados de los estudios realizados. La CE 50 más baja notificada es de 12,5 mg/litro.

En cuanto al entorno acuático, se ha demost rado que los embriones de peces y anfibios son los más sensibles, obse rvándose efectos sobre la incubación a partir de 5,5 mg/litro.

7. Efectos en mamíferos de laboratorio y en sistem as 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 CL 50 -6h por inhalación está comprendida en todas las especies entre 40 200 y 55 870 mg/m 3 . Se han registrado DL 50 orales de 1410-3000 mg/kg. Los efectos agudos de l a administración de cloruro de metileno por diver sas vías de exposición se manifiestan fundamentalmente en el sistema n ervioso central (SNC) y en el hígado, y se producen a dosis altas. Se h an observado trastornos del SNC a concentraciones de 14 100 mg/m 3 o más, con ligeras variaciones del EEG a 1770 mg/m 3 . A concentraciones de 17 700 mg/m 3 o más se observaron leves cambios histológicos en el hígado. Ocasionalmente se vieron afectados otro s órganos, tales como el riñón o el sistema respiratorio. En el ratón , los efectos sobre los pulmones se limitaron a las células de Clara de spués de una exposición a 7100 mg/m 3 . Se ha notificado la aparición de sensibilización cardiaca a la arritmia inducida por adrenalina. Se han observado efectos cardiovasculares, si bien de manera irr egular.
7.2 Exposición a corto y a largo plazo
La exposición prolongada a concentraciones altas de cloruro de metileno ( >17 700 mg/m 3 ) 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/m 3 , y en ratones a partir de 17 700 mg/m 3 . El hígado de perros expuestos continuamente a 3500 mg/m 3 por espacio de hasta 100 días se vio ligeramente afectado. Se observaron asimismo efectos en el hígado tras la exposición intermitente a 3500 m g/m 3 en la rata, y a 14 100 mg/m 3 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 h asta 7100 mg/m 3 durante 13 semanas.

La administración oral de cloruro de metile no 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 irr itante 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/m 3 . En tres estudios realizados con animales no se observaron indicios de varia ción de la incidencia de malformaciones esqueléticas ni otros efectos sobre el desarrollo. Se notificaron efectos leves sobre el peso corp oral fetal o materno a una concentración de 4400 mg/m 3 , así como sobre el aumento de peso postnatal de ratas macho a una concentración de l 0,04% en la dieta. Un estudio de toxicidad reproductiva llevado a cab o en dos generaciones de ratas expuestas a cloruro de metileno por in halación a concentraciones de hasta 5300 mg/m 3 , 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 generaci ones, F 0 o F 1 . 

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En condiciones de exposición adecuadas el c loruro de metileno tiene efectos mutágenos en microorganismos proc ariotas, con o sin activación metabólica (Salmonella o Escherichia coli). En los sistemas eucariotas los resultados son negativo s, 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 cromosómi cas en diferentes tipos de células dieron resultados positivos, mientra s 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 clo ruro 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 d e 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 administrar on dosis altas de cloruro de metileno no se observaron indicios d e unión del cloruro de metileno al ADN ni de lesiones de éste. Son ést os los estudios in vivo potencialmente más sensibles, el mejor de los cuale s permite detectar una alquilación por cada 10 6 nucleótidos.

Dentro de las limitaciones de las pruebas a corto plazo actualmente disponibles, no hay pruebas concluy entes 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 1 00 mg/m 3 ) es causa de tumores tanto pulmonares como hepáticos. La inc idencia de esos dos tipos de tumores aumentó en ratones expuestos a 7100 mg/m 3 durante 26 semanas y estudiados durante 78 semanas más. No se observaron signos claros de toxicidad o hiperplasia asocia das en los órganos diana.
La exposición de hámsters sirios a cloruro de metileno por inhalación a concentraciones de hasta 12 400 mg /m 3 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 tu mores 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/m 3 durante dos años. Ese exceso sólo se hizo paten te cuando se procedió a agrupar los tumores, todos ellos de origen mese nquimatoso, con fines estadísticos. El método estadístico utilizado e ra inapropiado dado que los tumores procedían de células de diverso tip o. Además, se señaló que las ratas utilizadas se habían visto infect adas al principio del estudio por un virus causante de una enfermedadcomún, la sialodacrioadenitis, que afecta sobre todo a la glándula salival. Probablemente los tumores no estaban relacionad os causalmente con la exposición al cloruro de metileno, y la exposic ión se limitó a
exacerbar la respuesta a la infección en la reg ión de la glándula salival. El efecto no se reprodujo en un segund o estudio realizado con ratas expuestas a 3500, 7100 ó 14 100 mg/m 3 durante su ciclo de vida. Un estudio ulterior realizado con ratas e xpuestas por inhalación a concentraciones de hasta 1770 mg/m 3 de cloruro de metileno durante todo su ciclo de vida no reveló indicios de car cinogenicidad. En ratas expuestas al cloruro de metileno a través del a gua que consumían o de alimentos administrados con sonda tampoco se ob servaron indicios significativos de carcinogenicidad.
Tres estudios han puesto de manifiesto un a umento de la incidencia de tumores mamarios benignos en ratas expuestas a cloruro de metileno, en dos de los casos por inhalación y en el terc ero por administración forzada. No se ha notificado ningún aumento de la incidencia de tumores mamarios en hámsters o en ratones somet idos a dosis comparables de cloruro de metileno. La dependen cia de los tumores mamarios de las hormonas hipofisarias en la rat a, tanto macho como hembra, es un dato incontrovertible. En la rata , la prolactina actúa como iniciador y como promotor de la carcinogén esis mamaria. Hay datos convincentes de que el aumento de los niveles d e prolactina incrementa la incidencia de tumores mamarios (p. ej., el i njerto de varias hipófisis en ratas Sprague-Dawley aumenta la in cidencia de tumores mamarios, y se ha observado además una correlac ión positiva entre la existencia de niveles elevados de prolactina en sangre y la incidencia de tumores mamarios en ratas hembra R-Amsterdamviejas). En las ratas hembra que han recibido carcinógenos, los trata mientos inductores de hiperprolactinemia dan lugar a un aumento espec tacular 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 inducc ión de adenomas mamarios por el cloruro de metileno en la rata es importante para poder evaluar los riesgos para el hombre. Las r atas Sprague-Dawley hembras sometidas a cloruro de metileno present an 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 s e limita a la aparición 

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cloruro de metileno al ADN de otros tejidos, po r lo que parece improbable que pueda unirse al tejido mamario, tanto más cuanto que su metabolismo se produce fundamentalmente en el h ígado. Es más probable, por tanto, que el aumento de la incidencia de a denomas mamarios se deba a un mecanismo indirecto en el que interve nga la hiperprolac- tinemia.
En cuanto al hombre, hay datos contradictor ios respecto a si los tumores mamarios son tan sensibles a la prolact ina como en la rata. Este animal presenta niveles elevados de prolac tina cuando es alimentado ad libitum en lugar de sometido a una dieta restringida,
lo cual explica quizá la gran sensibilidad de l a incidencia de tumores mamarios a diversos efectos ambientales y de ot ro tipo. En la rata, no obstante, la prolactina es luteotrófica. Un aum ento de la prolactina circulante da lugar a un aumento de los niveles de progesterona y de estrógenos exógenos. Es la coincidencia de esto s tres factores lo que causa el crecimiento túbulo-alveolar de las glá ndulas mamarias y, finalmente, el desarrollo del tumor. La prolact ina no es luteotrófica en los primates; es improbable, por tanto, que este mecanismo de desarrollo tumoral pueda tener importancia en e l hombre.

En la rata, el mecanismo de desarrollo de t umores mamarios mediado por la hiperprolactinemia sólo entra en juego a las dosis de cloruro de metileno que alteran los niveles de prolacti na. No se dispone de información directa sobre los niveles de prolac tina en ratas sometidas a dosis bajas de cloruro de metileno, pero no s e ha observado ningún aumento de la incidencia de adenomas mamarios t ras la administración de dosis bajas por inhalación o a través del ag ua 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 quí micas. Se ha notificado un caso de edema pulmonar grave por inhalación excesiva. Se han producido también defunciones en casos de inhal ación o contaminación cutánea accidentales. Los principales efectos t óxicos del cloruro de metileno son la depresión reversible del SNC y la formación de CO-Hb. Se ha señalado también la aparición de disfunci ones hepáticas y renales y de trastornos hematológicos tras la e xposición al producto.
Se han observado problemas neurofisiológico s y neurocomporta- mentales en voluntarios humanos expuestos a con centraciones de cloruro de metileno de 694 mg/m 3 durante 1,5-3,0 horas. No se han observado efectos neurológicos en hombres expuestos duran te varios años a concentraciones del producto comprendidas entre 260 y 347 mg/m 3 . De forma parecida, un grupo de raspadores de pintu ra de aviones ya jubilados con antecedentes de una larga (22 año s) exposición a concentraciones altas, si bien no especificadas , de cloruro de metileno obtuvieron resultados "normales" en un a 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 espontáneo s entre empleadas de la industria farmacéutica finlandesa se ha atribui do a la exposición a cloruro de metileno. Sin embargo, el diseño inc orrecto del estudio ha impedido establecer una relación causal.

Varios estudios de mortalidad realizados en cohortes pertinentes muestran resultados dispares en cuanto a las ca usas de defunción. Se ha observado un aumento de la mortalidad por en fermedades especificas (como por ejemplo el cáncer pancreático o la ca rdiopatía isquémica), pero de forma irregular y sólo en determinados estudios. Estos efectos no se pueden atribuir a la exposición al clorur o de metileno.

See Also:

Toxicological Abbreviations Methylene chloride (EHC 32, 1984, 1st edition) Methylene chloride (HSG 6, 1987) Methylene chloride (PIM 343) 

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