INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

ENVIRONMENTAL HEALTH CRITERIA 164

Methylene Chloride

 Second Edition)

 This report contains the collective views of an international group of

 experts and does not necessarily represent the decisions or the stated

 policy of the United Nations Environment Programme, the International

 Labour Organisation, or the World Health Organization.

 Published under the joint sponsorship of

 the United Nations Environment Programme,

 the International Labour Organisation,

 and the World Health Organization

 World Health Organization

 Geneva, 1996

 The International Programme on Chemical Safety (IPCS) is a joint

 venture of the United Nations Environment Programme, the International

 Labour Organisation, and the World Health Organization. The main

 objective of the IPCS is to carry out and disseminate evaluations of

 the effects of chemicals on human health and the quality of the

 environment. Supporting activities include the development of

 epidemiological, experimental laboratory, and risk-assessment methods

 that could produce internationally comparable results, and the

 development of manpower in the field of toxicology. Other activities

 carried out by the IPCS include the development of know-how for coping

 with chemical accidents, coordination of laboratory testing and

 epidemiological studies, and promotion of research on the mechanisms

 of the biological action of chemicals.

 WHO Library Cataloguing in Publication Data

 Methylene chloride.

 (Environmental health criteria; 164)

 1.Methylene chloride - adverse effects 2. Solvents

 I.Series

 ISBN 92 4 157164 0 (NLM Classification: QV 633)

 ISSN 0250-863X

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CONTENTS

 ENVIRONMENTAL HEALTH CRITERIA FOR METHYLENE CHLORIDE

1. SUMMARY

1.1. Identity, physical and chemical properties, and analytical

 methods

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1.2. Sources of human and environmental exposure

1.3. Environmental transport, distribution and transformation

1.4. Environmental levels and human exposure

1.5. Kinetics and metabolism

1.6. Effects on organisms in the environment

1.7. Effects on laboratory mammals and in vitro test systems

1.7.1. Single exposure

1.7.2. Short- and long-term exposure

1.7.3. Skin and eye irritation

1.7.4. Developmental and reproductive toxicity

1.7.5. Mutagenicity and related end-points

1.7.6. Chronic toxicity and carcinogenicity

1.8. Effects on humans

2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL

 METHODS

2.1. Identity

2.2. Physical and chemical properties

2.3. Conversion factors

2.4. Analytical methods

3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

3.1. Natural occurrence

3.2. Anthropogenic sources

3.2.1. Production

3.2.2. Uses

3.2.3. Consumer applications

3.2.4. Sources in the environment

4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION

 Appraisal

4.1. Transport and distribution between media

4.1.1. Water/air

4.1.2. Soil/air

4.1.3. Water/soil

4.1.4. Multicompartment distribution

4.2. Abiotic degradation

4.2.1. Atmosphere

4.2.2. Water

4.2.3. Soil

4.3. Biotransformation

4.3.1. Aerobic

4.3.2. Anaerobic

4.3.3. Bioaccumulation

4.4. Interaction with other physical, chemical or biological

 factors

5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

 Appraisal

5.1. Environmental levels

5.1.1. Atmosphere

 5.1.1.1 Ambient air

 5.1.1.2 Precipitation

5.1.2. Water

5.1.3. Aquatic organisms

5.1.4. Soil and sediment

5.2. Human exposure

5.2.1. General population

 5.2.1.1 Indoor air

 5.2.1.2 Drinking-water

 5.2.1.3 Foodstuffs

 5.2.1.4 Consumer exposure

5.2.2. Occupational exposure

 5.2.2.1 Production

 5.2.2.2 Paint stripping and related activities

 5.2.2.3 Aerosol production and use

 5.2.2.4 Use as a process solvent

 5.2.2.5 Cleaning and degreasing

5.2.3. Occupational exposure limits

5.3. Human monitoring data

5.3.1. Body burden

5.3.2. Occupational exposure studies

5.3.3. Biological exposure indices

6. KINETICS AND METABOLISM

6.1. Absorption

6.1.1. Inhalation exposure

 6.1.1.1 Human studies

 6.1.1.2 Animal studies

6.1.2. Oral exposure

6.1.3. Dermal exposure

6.2. Distribution

6.2.1. Inhalation exposure

 6.2.1.1 Human studies

 6.2.1.2 Animal studies

6.2.2. Oral exposure

6.2.3. Dermal exposure

6.3. Metabolism

6.3.1. In vitro studies

6.3.2. In vivo studies

6.4. Elimination and excretion

6.4.1. Inhalation exposure

 6.4.1.1 Human studies

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 6.4.1.2 Animal studies

6.4.2. Oral exposure

6.4.3. Dermal exposure

7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT

7.1. Microorganisms

7.1.1. Bacteria

 7.1.1.1 Aerobic bacteria

 7.1.1.2 Anaerobic bacteria

7.1.2. Protozoa

7.1.3. Algae

7.2. Aquatic organisms

7.2.1. Plants

7.2.2. Invertebrates

 7.2.2.1 Insects

 7.2.2.2 Crustaceans

 7.2.2.3 Molluscs

7.2.3. Fish

 7.2.3.1 Acute toxicity

 7.2.3.2 Chronic toxicity and reproduction

7.2.4. Amphibians

7.3. Terrestrial organisms

7.4. Population and ecosystem effects

7.4.1. Soil microorganisms

7.4.2. Sediment microorganisms

7.4.3. Microcosms and mesocosms

8. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS

8.1. Single exposure

8.1.1. Acute toxicity data

8.1.2. Oral administration

8.1.3. Inhalation administration

 8.1.3.1 Rat

 8.1.3.2 Mouse

 8.1.3.3 Other animals

8.1.4. Dermal administration

8.1.5. Intraperitoneal administration

8.1.6. Intravenous administration

8.1.7. Subcutaneous administration

8.1.8. Appraisal

8.2. Short-term exposure

8.2.1. Oral administration

8.2.2. Subcutaneous administration

8.2.3. Inhalation administration

 8.2.3.1 Rat

 8.2.3.2 Other animals

8.3. Long-term exposure

8.3.1. Rat

 8.3.1.1 Inhalation exposure

 8.3.1.2 Oral exposure

8.3.2. Mouse

 8.3.2.1 Inhalation exposure

 8.3.2.2 Oral exposure

8.3.3. Other animals

8.3.4. Appraisal

8.4. Skin and eye irritation; skin sensitization

8.4.1. Skin irritation

8.4.2. Eye irritation

8.4.3. Sensitization

8.4.4. Appraisal

8.5. Developmental and reproductive toxicity

8.5.1. Developmental toxicity

8.5.2. Reproductive toxicity

8.5.3. Appraisal

8.6. Mutagenicity and related end-points

8.6.1. In vitro

 8.6.1.1 Bacteria

 8.6.1.2 Fungi and yeasts

 8.6.1.3 Mutation in mammalian cells

 8.6.1.4 Chromosomal effects

 8.6.1.5 DNA damage

 8.6.1.6 DNA binding in vitro

 8.6.1.7 Cell transformation

8.6.2. In vivo

 8.6.2.1 Chromosome damage

 8.6.2.2 Drosophila

 8.6.2.3 DNA damage

 8.6.2.4 DNA binding

 8.6.2.5 Dominant lethal assay

 8.6.2.6 Replicative DNA synthesis

8.6.3. Appraisal

8.7. Chronic toxicity and carcinogenicity

8.7.1. Inhalation exposure

 8.7.1.1 Rat

 8.7.1.2 Mouse

 8.7.1.3 Hamster

8.7.2. Oral administration

 8.7.2.1 Rat

 8.7.2.2 Mouse

8.7.3. Appraisal

8.8. Mechanistic studies

8.8.1. In vitro metabolic studies

8.8.2. In vivo metabolic studies

8.8.3. Pulmonary effects

8.8.4. Studies on oncogene activation

8.8.5. The use of mechanistic studies in extrapolation

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8.8.6. Mammary tumour promotion

8.8.7. Appraisal

8.9. Interspecies and dose extrapolations by kinetic modelling

9. EFFECTS ON HUMANS

9.1. General population exposure

9.1.1. Environmental exposure

9.1.2. Oral exposure

9.2. Occupational exposure

9.2.1. Short-term exposure

 9.2.1.1 Case studies

 9.2.1.2 Skin and eye effects

 9.2.1.3 Laboratory studies

9.2.2. Long-term exposure

 9.2.2.1 Case studies

9.3. Appraisal of human effects

10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT

10.1. Evaluation of human health risks

10.2. Evaluation of effects on the environment

 REFERENCES

 RESUME

 RESUMEN

 NOTE TO READERS OF THE CRITERIA MONOGRAPHS

 Every effort has been made to present information in the criteria

 monographs as accurately as possible without unduly delaying their

 publication. In the interest of all users of the Environmental Health

 Criteria monographs, readers are requested to communicate any errors

 that may have occurred to the Director of the International Programme

 on Chemical Safety, World Health Organization, Geneva, Switzerland, in

 order that they may be included in corrigenda.

 * * *

 A detailed data profile and a legal file can be obtained from the

 International Register of Potentially Toxic Chemicals, Case postale

 356, 1219 Chatelaine, Geneva, Switzerland (Telephone No. 9799111).

 * * *

 This publication was made possible by grant number 5 U01 ES02617-

 15 from the National Institute of Environmental Health Sciences,

 National Institutes of Health, USA, and by financial support from the

 European Commission.

 Environmental Health Criteria

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 The first Environmental Health Criteria (EHC) monograph, on

 mercury, was published in 1976 and since that time an ever-increasing

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 The recommendations of the 1992 UN Conference on Environment and

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 WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR METHYLENE CHLORIDE

 Members

 Dr L.A. Albert, Consultores Ambientales Associados, Xalapa, Veracruz,

 Mexico

 Mr D. Farrar, ICI Chemicals and Polymers, Runcorn, Cheshire, United

 Kingdom (Rapporteur)

 Dr R. Fransson-Steen, Institute of Environmental Medicine, Karolinska

 Institute, Stockholm, Sweden

 Dr S. Henry, US Food and Drug Administration, Washington, DC, USA

 Mr P. Howe, Institute of Terrestrial Ecology, Monks Wood Experimental

 Station, Huntingdon, United Kingdom

 Dr P. Standring, Health and Safety Executive, Bootle, Merseyside,

 United Kingdom

 Dr L. Stayner, Division of Standards Development and Technology

 Transfer, National Institute for Occupational Safety and Health,

 Cincinnati, Ohio, USA

 Dr T. G. Vermeire, Toxicology Advisory Centre, National Institute of

 Public Health and Environmental Hygiene, Bilthoven, The

 Netherlands (Chairman)

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

 Observers

 Dr C. De Rooij, Solvay & Cie S.A., Brussels, Belgium

 Dr T. Green, ICI Chemicals & Polymers Ltd., Runcorn, Cheshire, United

 Kingdom

 Secretariat

 Dr M. Gilbert, International Programme on Chemical Safety, World

 Health Organization, Geneva, Switzerland (Secretary)

 Dr P. Demers, Unit of Analytical Epidemiology, International Agency

 for Research on Cancer, Lyon, France

 ENVIRONMENTAL HEALTH CRITERIA FOR METHYLENE CHLORIDE

 A WHO Task Group on Environmental Health Criteria for Methylene

 Chloride met at the Institute of Terrestrial Ecology, Monks Wood,

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 United Kingdom from 16 to 20 August 1993. Dr S. Dobson welcomed the

 participants on behalf of the host institution, and Dr M. Gilbert

 opened the meeting on behalf of the three cooperating organizations of

 the IPCS (ILO/UNEP/WHO). The Task Group reviewed and revised the draft

 monograph and made an evaluation of the risks for human health and the

 environment from exposure to methylene chloride.

 The first draft of this monograph was prepared by Mr D. Farrar,

 ICI Chemicals and Polymers, Runcorn, United Kingdom.

 Dr M. Gilbert, IPCS, was responsible for the overall scientific

 content of this monograph. After his death in July 1994, this

 responsibility was transferred to Dr P.G. Jenkins, IPCS, who also

 dealt with the technical editing.

 The efforts of all who helped in the preparation and finalization

 of the monograph are gratefully acknowledged.

 ABBREVIATIONS

 ALT alanine aminotransferase

 AST aspartate aminotransferase

 BEI Biological Exposure Index

 CO-Hb carboxyhaemoglobin

 GST glutathione transferase

 LEV local exhaust ventilation

 MATC maximum acceptable toxicant concentration

 NADPH reduced nicotinamide adenine dinucleotide phosphate

 NIOSH National Institute for Occupational Safety and Health (USA)

 SCE sister-chromatid exchange

 SGOT serum glutamic-oxaloacetic transaminase

 SGPT serum glutamic-pyruvic transaminase

 TT toxicity threshold

 TWA time-weighted average

 UDS unscheduled DNA synthesis

1. SUMMARY

1.1 Identity, physical and chemical properties, and analytical methods

 Methylene chloride (dichloromethane) is a clear, highly volatile,

 non-flammable liquid with a penetrating ether-like odour. The pure dry

 compound is very stable. Methylene chloride hydrolyses slowly in the

 presence of moisture, producing small quantities of hydrogen chloride.

 Commercial methylene chloride normally contains small quantities of

 stabilizers to prevent decomposition.

 Analytical methods are available for the determination of

 methylene chloride in biological media and environmental samples. All

 methods involve gas chromatography in combination with a suitable

 detector. In this way, very low detection limits have been reached

 (e.g., in food: 7 ng/sample; water: 0.01 μg/litre; air: 1.76 μg/m3

 (0.5 ppb); blood: 0.022 mg/litre).

1.2 Sources of human and environmental exposure

 World production of methylene chloride is estimated to be

 570 000 tonnes/year. Most applications are based on its solvent

 capacity for grease, plastics and paint binding agents, in combination

 with its volatility and stability. The worldwide usage pattern

 comprises aerosols (20-25%), paint remover (25%), process solvent in

 the chemical and pharmaceutical industry (35-40%), miscellaneous uses

 (e.g., polyurethane foam manufacturing) and metal cleaning (10-15%).

 The usage of methylene chloride is tending to decrease, at least in

 western Europe.

 More than 99% of the atmospheric releases of methylene chloride

 result from its use as an end-product by various industries and the

 use of paint removers and aerosol products at home.

1.3 Environmental transport, distribution and transformation

 Due to its high volatility, most of the methylene chloride

 released to the environment will partition to the atmosphere, where it

 will degrade by reaction with photochemically produced hydroxyl

 radicals with a lifetime of 6 months.

 Abiotic degradation in water is slow compared to evaporation.

 Methylene chloride has been shown to disappear rapidly from soil and

 ground water.

 The aerobic and anaerobic degradation of methylene chloride has

 been established by a variety of different test systems. Complete

 biodegradation, especially by acclimated bacterial cultures under

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

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 acclimated municipal sludge). In bioreactors, up to 10% degradation

 per hour is achievable. There is no evidence of significant

 bioaccumulation or biomagnification.

1.4 Environmental levels and human exposure

 Methylene chloride has been detected in the ambient air of rural

 and remote areas at concentrations of 0.07-0.29 μg/m3. In suburban

 areas, the average concentration is < 2 μg/m3 and in urban areas

 < 15 μg/m3. In the vicinity of hazardous waste sites up to

 43 μg/m3 has been found. Precipitation may also contain methylene

 chloride.

 Methylene chloride enters the aquatic environment through waste

 water discharge from various industries, and methylene chloride has

 been found in surface water, ground water and sediment.

 Exposure of members of the general public to methylene chloride

 will occur from its use in consumer products such as paint removers,

 which can result in relatively high levels being found in indoor air.

 Occupational exposure during production arises primarily during

 filling and packaging (manufacturing is in closed systems). Because of

 its use in paint strippers, occupational exposure to methylene

 chloride occurs during formulation of paint-remover, original

 equipment manufacture, and in commercial furniture refinishing.

 Methylene chloride is widely used as a process solvent in the

 manufacture of a variety of products, in particular in the industries

 mentioned in section 1.2.

 Biological monitoring of methylene chloride exposure can be based

 on measurement of the solvent itself in exhaled air or blood. However,

 as production of carbon monoxide with exposure for more than 3-4 h/day

 appears to be the limiting factor in regard to health risk, biological

 monitoring based upon either analysis of carbon monoxide in exhaled

 air or of carboxyhaemaglobin (CO-Hb) in blood is to be preferred.

 However, this can only be used for non-smoking subjects. Sampling

 should be done at about 0-2 h post-exposure, or after 16 h, i.e. on

 the following morning.

 Post-exposure CO-Hb levels 2 h after exposure ceases are not

 expected to exceed 2-3%, and at 16 h 1%, in the case of an 8-h

 exposure to less than 350 mg methylene chloride/m3 in non-smokers.

1.5 Kinetics and metabolism

 Methylene chloride is rapidly absorbed though the alveoli of the

 lungs into the systemic circulation. It is also absorbed from the

 gastrointestinal tract, and dermal exposure results in absorption but

 at a slower rate than via the other routes of exposure.

 Methylene chloride is quite rapidly excreted, mostly via the lungs

 in the exhaled air. It can cross the blood-brain barrier and be

 transferred across the placenta, and small amounts can be excreted in

 urine or in milk.

 At high concentrations, most of the absorbed methylene chloride is

 exhaled unchanged. The remainder is metabolized to carbon monoxide,

 carbon dioxide and inorganic chloride. Metabolism occurs by either or

 both of two pathways, whose relative contribution to the total

 metabolism is markedly dependent on the dose and on the animal species

 concerned. One pathway involves oxidative metabolism mediated by

 cytochrome P-450 and leads to both carbon monoxide and carbon dioxide.

 This pathway appears to operate similarly in all rodents studied and

 in man. Whilst this is the predominant metabolic route at lower doses,

 saturation occurs at a relatively low dose (around 1800 mg/m3).

 Increasing the dose above the saturation level does not lead to extra

 metabolism by this route.

 The other pathway involves a glutathione transferase (GST), and

 leads via formaldehyde and formate to carbon dioxide. This route seems

 only to become important at doses above the saturation level of the

 "preferred" oxidative pathway. In some species (e.g., the mouse) it

 becomes the major metabolic pathway at sufficiently high doses. In

 contrast, in other species (e.g., hamster, man) it seems to be used

 very little at any dose.

 Species difference in GST metabolism correlates well with the

 observed species difference in carcinogenicity. The extent of

 metabolism by this pathway in relevant species has been used as the

 basis for a kinetic model to describe the metabolic behaviour of

 methylene chloride in various species.

1.6 Effects on organisms in the environment

 Algae and aerobic bacteria show no inhibition of growth below

 500 mg/litre. Bacteria have been identified that are able to grow in

 the presence of methylene chloride at much higher concentrations

 including a saturated solution in water (section 4.2.4.1). Anaerobic

 bacteria are more sensitive; growth inhibition has been observed at

 1 mg/litre in anaerobic biological sludge.

 In soil a concentration of 10 mg/kg strongly decreased the ATP

 content of the biomass including fungi and aerobic bacteria, and

 induced transient inhibition of enzyme activity. The no-observed-

 effect level was 0.1 mg/kg. In earthworms methylene chloride is

 moderately toxic (100-1000 μg/cm2) in the filter-paper contact

 toxicity test. In sediment no toxic effects were observed even at very

 high levels.

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 In higher plants no effects were found after exposure for 14 days

 to 100 mg/m3.

 Adult fish seem to be relatively insensitive to methylene chloride

 even after prolonged exposure (14-day LC50 > 200 mg per litre). The

 effect of methylene chloride on Daphnia is difficult to assess given

 the large variation in the outcome of the studies performed. The

 lowest reported EC50 was 12.5 mg/litre.

 In the aquatic environment, fish and amphibian embryos have been

 shown to be the most sensitive with effects on hatching from

 5.5 mg/litre.

1.7 Effects on laboratory mammals and in vitro test systems

1.7.1 Single exposures

 The acute toxicity of methylene chloride by inhalation and oral

 administration is low. The inhalation 6-h LC50 values for all

 species are between 40 200 and 55 870 mg/m3. Oral LD50 values of

 1410-3000 mg/kg were recorded. Acute effects after methylene chloride

 administration by various routes of exposure are primarily associated

 with the central nervous system (CNS) and the liver, and these

 occurred at high doses. CNS disturbances were found at concentrations

 of 14 100 mg/m3 or more, with slight changes in EEG at 1770 mg/m3.

 Slight histological changes in the liver were found at 17 700 mg/m3

 or more. Occasionally other organs were affected such as the kidney or

 respiratory system. In mice, effects on the lungs were restricted to

 the Clara cells after exposure to 7100 mg/m3. Cardiac sensitization

 to adrenaline-induced arrhythmia has been reported. Cardiovascular

 effects have been seen but the effects were inconsistent.

1.7.2 Short- and long-term exposure

 Prolonged exposure to high concentrations of methylene chloride

 (> 17 700 mg/m3) caused reversible CNS effects, slight eye

 irritation and mortality in several laboratory species. Body weight

 reduction was observed in rats at 3500 mg/m3 and in mice from

 17 700 mg/m3. Slight effects on the liver were noted in dogs

 continuously exposed to 3500 mg/m3 for up to 100 days. After

 intermittent exposure, effects on the liver were observed in rats at

 3500 mg/m3 and in mice at 14 100 mg/m3.

 Other target organs are the lungs and the kidneys.

 No evidence of irreversible neurological damage was seen in rats

 exposed by inhalation to concentrations up to 7100 mg/m3 for 13

 weeks.

 Oral administration of methylene chloride to rats caused effects

 on the liver from about 200 mg/kg per day.

1.7.3 Skin and eye irritation

 Methylene chloride is moderately irritant to the skin and eyes of

 experimental animals.

1.7.4 Developmental and reproductive toxicity

 Methylene chloride is not teratogenic in rats or mice at

 concentrations up to 16 250 mg/m3. No evidence of an effect on the

 incidence of skeletal malformations or other developmental effects

 were observed in three animal studies. Small effects on either fetal

 or maternal body weight were reported at 4400 mg/m3, and on

 postnatal weight gain of male rats at 0.04% in the diet. A two-

 generation reproductive toxicity study in rats exposed to methylene

 chloride by inhalation at concentrations up to 5300 mg/m3, 6 h/day,

 5 days/week for 17 weeks did not show evidence of an adverse effect on

 any reproductive parameter, neonatal survival or neonatal growth in

 either the F0 or F1 generation.

1.7.5 Mutagenicity and related end-points

 Under appropriate exposure conditions, methylene chloride is

 mutagenic in prokaryotic microorganisms with or without metabolic

 activation (Salmonella or Escherichia coil). In eukaryotic systems

 it gives either negative or, in one case, weakly positive results.

 In vitro gene mutation assays and tests for unscheduled DNA

 synthesis (UDS) in mammalian cells were uniformly negative. In vitro

 assays for chromosomal aberrations using different cell types gave

 positive results, whereas negative or equivocal results were obtained

 in tests for sister chromatid exchange (SCE) induction.

 The majority of the in vivo studies reported provided no

 evidence of mutagenicity of methylene chloride (e.g., chromosome

 aberration assay, micronucleus test or UDS assay). Marginal increase

 in frequencies of SCEs and micronuclei in mice has been reported

 following inhalation exposure to high concentrations of methylene

 chloride.

 There was no evidence of binding of methylene chloride to DNA or

 DNA damage in rats or mice given high doses of methylene chloride.

 These studies are potentially the most sensitive in vivo studies,

 the best of which are capable of detecting one alkylation in 106

 nucleotides.

 Within the limitations of the short-term tests currently

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 available, there is no conclusive evidence that methylene chloride in

 genotoxic in vivo.

1.7.6 Chronic toxicity and carcinogenicity

 Methylene chloride is carcinogenic in the mouse, causing both lung

 and liver tumours, following exposure to high concentrations (7100 and

 14 100 mg/m3) of methylene chloride. The incidence of both lung and

 liver tumours was increased in mice exposed to 7100 mg/m3 for 26

 weeks and maintained for a further 78 weeks. There was no substantial

 evidence of associated toxicity or hyperplasia in the target organs.

 Syrian hamsters exposed to methylene chloride by inhalation at

 concentrations up to 12 400 mg/m3 for 2 years showed no evidence of

 a carcinogenic effect related to exposure to methylene chloride.

 Rats exposed to methylene chloride via various routes have shown

 increased incidences of tumours at certain sites. An excess of tumours

 in the region of the salivary gland was reported in female rats

 exposed to either 5300 or 12 400 mg/m3 for 2 years. This excess was

 only evident when the tumours, which were all of mesenchymal origin,

 were grouped together for statistical analysis. As the tumours arose

 from a variety of different cells, the statistical approach adopted

 was inappropriate. Furthermore, it was reported that the rats in the

 study had been infected with a common viral disease (sialoda-

 cryoadenitis) early in the study, an infection that affects primarily

 the salivary gland. It is likely that these tumours were not causally

 related to exposure to methylene chloride but that the exposure had

 exacerbated the response of the infection in the region of the

 salivary gland. The response was not seen in a second study in which

 rats were exposed to either 3500, 7100 or 14 100 mg/m3 for their

 lifetime. A further inhalation study on rats exposed to methylene

 chloride at concentrations up to 1770 mg/m3 for their lifetime

 showed no evidence of carcinogenicity. Rats exposed to methylene

 chloride via their drinking-water or by gavage similarly showed no

 substantive evidence of carcinogenicity.

 An increased incidence of benign mammary tumours in rats exposed

 to methylene chloride has been reported in three studies, two

 following exposure by inhalation and the third by gavage. There are no

 reports of increases in mammary tumour incidence in hamsters or in

 mice receiving methylene chloride at comparable dose levels. The

 dependence of mammary tumours upon pituitary hormones in both male and

 female rats has been established unequivocally. In the rat, prolactin

 acts as both an initiator and promoter of mammary carcinogenesis.

 There is good evidence that increased prolactin levels increase the

 incidence of mammary tumours (e.g., the grafting of multiple pituitary

 glands into Sprague-Dawley rats increases the incidence of mammary

 tumours and there is a positive correlation between elevated blood

 prolactin levels and mammary tumours in aged R-Amsterdam female rats).

 Treatments that induce hyperprolactinaemia in female rats that have

 received carcinogens produce a dramatic increase in tumour incidence.

 These treatments include adrenalectomy, pituitary homografts and high

 dietary fat.

 The mechanism by which methylene chloride induces mammary adenomas

 in the rat is important for human hazard assessment. Female Sprague-

 Dawley rats receiving methylene chloride have a high blood level of

 prolactin. In common with the response to other agents which act via

 hyperprolactinaemia, the methylene chloride-induced response is of

 benign neoplasms only. There is no evidence for the binding of

 methylene chloride to the DNA of other tissues and hence it seems

 unlikely that it will bind to mammary tissue when the primary site of

 metabolism is the liver. It seems most likely, therefore, that the

 increased incidence of mammary adenomas is the result of an indirect

 mechanism operating via hyperprolactinaemia.

 In humans, there is conflicting evidence on whether or not mammary

 tumours are as responsive to prolactin as is the case in the rat. The

 rat has elevated levels of prolactin when fed ad libitum in

 comparison to a restricted dietary regimen and this may explain why

 the mammary tumour incidence is so responsive to a variety of

 environmental and other effects. In the rat, however, prolactin is 

 luteotrophic. An increase in the circulating levels of prolactin will

 lead to an increase in progesterone and exogenous oestrogen levels. It

 is the presence of all three factors that causes tubular-alveolar

 growth of the mammary glands, which ultimately leads to tumour

 development. Prolactin is not luteotrophic in primates. It is

 unlikely, therefore, that this mechanism of tumour development is of

 relevance to man.

 The mechanism of production of mammary tumours in the rat

 involving hyperprolactinaemia will occur only at doses of methylene

 chloride which affect prolactin levels. There is no direct information

 on prolactin levels in rats receiving low doses of methylene chloride,

 but no increase in mammary adenomas has been observed following the

 administration of low doses in either inhalation or drinking-water

 studies (i.e. below 250 mg/kg body weight).

1.8 Effects on humans

 Methylene chloride irritates the skin and eyes especially when

 evaporation is prevented. In these circumstances, prolonged contact

 may cause chemical burns. A case of serious pulmonary oedema has been

 reported after excessive inhalation. Fatalities due to accidental

 inhalation and skin contamination have been reported. The main toxic

 effects of methylene chloride are reversible CNS depression and CO-Hb

 formation. Liver and renal dysfunctions and effects on haematological

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 parameters have also been reported following exposure to methylene

 chloride.

 Neurophysiological and neurobehavioural disturbances have been

 observed in human volunteers exposed to methylene chloride at

 concentrations of 694 mg/m3 for 1.5-3.0 h. No evidence of

 neurological effects was seen in men with exposure for several years

 to methylene chloride at concentrations ranging from 260 to

 347 mg/m3. Similarly, a group of retired airplane strippers with a

 long history of exposure to methylene chloride (22 years) at high but

 unspecified levels performed a battery of neurophysiological and

 psychological tests within the "normal" range, when compared with a

 control group who had a history of either no or only low exposure to

 methylene chloride.

 An increased rate of spontaneous abortion in employees in Finnish

 pharmaceutical industries has been attributed to exposure to methylene

 chloride. A causal relationship was not established because of

 insufficiencies in the design of the study.

 Several mortality studies in relevant cohorts show an inconsistent

 pattern in the causes of death. Excesses in mortality from specific

 diseases (e.g., pancreatic cancer, ischaemic heart disease) were not

 consistently increased, but confined to single studies. These effects

 cannot be attributed to exposure to methylene chloride.

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

2.1 Identity

 Formula: CH2CI2

 CI

 '

 Structure: CI - C - H

 '

 H

 Relative molecular mass: 84.93

 Common name: Methylene chloride

 Synonyms: DCM; dichloromethane; methane

 dichloride; methylene bichloride;

 methylene dichloride; methylenum

 chloratum

 Tradenames: Aerothene MM; Freon 30; Narkotil;

 Solaestin; Solmethine

 CAS name (9 CI): Methane, dichloro-

 CAS registry number: 75-09-2

 EC registry number: 602-004-00-3

 EINECS registry number: 200-838-9

 RTECS registry number: PA 8050000

 Purity of technical 99.9% (analytical grade)

 product:

 Impurities of technical Mostly C1- and C2-chlorinated

 product hydrocarbons (up to 200 mg/kg)

 (ECETOC, 1984)

 Stabilizer: Typically 0.005-0.2% (w/w)

 methanol, ethanol, amylene

 (2-methyl-but-2-ene), cyclohexane

 or tertiary butylamine (ECSA, 1989)

2.2 Physical and chemical properties

 Methylene chloride is a clear, colourless, highly volatile, non-

 flammable liquid with a penetrating ether-like odour. Pure dry

 methylene chloride is a very stable compound and is non-corrosive. In

 the presence of water, it undergoes very slow hydrolysis to produce

 small quantities of hydrogen chloride, which can lead to corrosion,

 e.g., to mild steel. This reaction is accelerated by elevated

 temperatures and the presence of alkalis or metals. In the vapour

 phase under abnormal conditions (elevated temperatures, high UV light

 exposure, flame, sparks, red hot surfaces), methylene chloride may be

 decomposed to give small amounts of hydrogen chloride, carbon monoxide

 and phosgene (ECSA, 1989). Other physical and chemical properties are

 given in Table 1.

 Commercial methylene chloride is normally stabilized (section 2.1)

 to prevent decomposition. Applications in aggressive conditions, such

 as special metal cleaning operations may require more sophisticated

 stabilizer technology. Poorly stabilized methylene chloride can react

 violently with aluminium or other light metals.

2.3 Conversion factors

 Conversion factor for methylene chloride concentrations in air,

 calculated at 20°C and 1.013 hPa are:

 1 mg/m3 = 0.28 ppm

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 1 ppm = 3.53 mg/m3

 and for carbon monoxide:

 1 mg/m3 = 0.86 ppm

 1 ppm = 1.16 mg/m3

2.4 Analytical methods

 Details of sampling and methods of analysis used in biological

 media and environmental samples are given in Tables 2 and 3.

 Table 1. Physical and chemical properties

 

 Parameter, units Value Reference

 

 Boiling temperature (°C at 1.013 hPa) 40 Weast et al. (1988)

 Melting temperature (°C at 1.013 hPa) -95.1 Weast et al. (1988)

 Relative density of liquid D (20) 1.3266 Weast et al. (1988)

 (water at 4°C = 1 kg/m3) 4

 Vapour pressure (hPa at 20°C) 470 ECSA (1989)

 Saturation concentration in air 1.7 Calculated

 (kg/m3 at 20°C)

 Vapour density at 20°C (air = 1) 2.93 IPCS (1984)

 Threshold odour concentration 743 Leonardos et al.

 (mg/m3) (1969)

 (odour: ether-like) 700-1060 DFG (1983)

 880 Amoore & Hautala

 (1983)

 540-2160 Ruth (1986)

 Solubility in water (g/kg at 20°C) 20 Verschueren (1983)

 13.0 Horvath (1982)

 Solubility in alcohol, ether, acetone Weast et al. (1988)

 and benzene

 Partition coefficients, at 20°C 1.25 IPCS (1984)

 log Pow (octanol/water) 1.3 Hansch & Leo

 (1979)

 log Koc 0.89

 calculated from Kow

 (Karickhoff, 1981)

 Henry's Law constant, Pa.m3/mol at 380

 20°C Smith (1989)

 Flash point, closed cup (°C) None ECSA (1989)

 Explosion limits in aira (%) 13-22 ECSA (1989)

 Auto-flammability, ignition temp. (°C) 605 ECSA (1989)

 

a This is with a high energy source; these conditions are unlikely

 to arise in normal operations.

 

 Table 2. Analytical methods for determining methylene chloride in biological monitoring (ATSDR, 1991)

 

 Sample matrix Preparation method Analytical Sample detection Percentage Reference

 methoda limit recovery

 

 Blood Heat sample, collect GC/FID 0.022 mg/litre 49.8±1.33 Di Vincenzo et al.

 headspace vapour (1971)

 Urine Heat sample, collect GC/FID No data 59±2.75 Di Vincenzo et al.

 headspace vapour (1971)

 Breath Heat sample, inject into gas GC/FID 0.706 ± 0.353 No data Di Vincenzo et al.

 sample loop mg/m3 (1971)

 (0.2 ± 0.1 ppm)

 Adipose tissue Hydrolyse with acid, heat GC/FID 1.6 mg/kgb No data Engström & Bjurström

 sample, collect headspace (1977)

 vapour

 Human milk Purge with helium, trap on GC/MS No data No data Pellizzari et al. (1982)

 sorbent trap, desorb thermally

 

a FID = flame ionisation detector; GC = gas chromatography; MS = mass spectrometry

b Lowest reported concentration

 Table 3. Analytical methods for determining methylene chloride in environmental samples (ATSDR, 1991)

 

 Sample Preparation method Analytical Sample detection Percentage Reference

 matrix methoda limit recovery

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 Air Adsorb on charcoal, desorb with GC/FID 88.25μg/m3 90-110c APHA (1977)

 carbon disulfide (25 ppb)b

 Air Adsorb on charcoal, desorb with GC/FID 0.01 mg 95.3 NIOSH (1987)

 carbon disulfide

 Air Adsorb on charcoal, desorb with GC/ECD approx. 1.76 μg/m3 No data Woodrow et al.

 benzyl alcohol (approx. 0.5 ppb) (1988)

 Water Purge with inert gas, trap on sorbent GC/HSD No data 85 US EPA (1989c)

 trap, desorb thermally

 Water Purge with inert gas, trap on sorbent GC/ELCD 0.01 μg/litre 97-100 US EPA (1989)

 trap, desorb thermally

 Water Purge with inert gas, trap on sorbent GC/MS 1.0 μg/litre 99 US EPA (1989b)

 trap, desorb thermally

 Water Purge with inert gas, trap on sorbent HRGC/MS 0.03-0.09 μg/litre 95-97 US EPA (1989a)

 trap, desorb thermally

 Water Purge with inert gas, trap on sorbent HRGC/ELCD 0.01-0.05 μg/litre 97±28 APHA (1989a)

 trap, desorb thermally

 

 Table 3 (Cont'd)

 

 Sample matrix Preparation method Analytical Sample detection Percentage Reference

 methoda limit recovery

 

 Water Purge with inert gas, trap on sorbent HRGC/MS 0.02-0.2 μg/litre 95±5 APHA (1989b)

 trap, desorb thermally

 Water Purge with helium, trap on sorbent GC/MS No data 99-105 Michael et al.

 trap, desorb thermally (1988)

 Waste Purge with inert gas, trap on sorbent GC/HSD 0.25 μg/litre 97.9±2.6 US EPA (1982a)

 water trap, desorb thermally

 Waste Purge with inert gas, trap on sorbent GC/MS 2.8 μg/litre 89±28 US EPA (1982b)

 water trap, desorb thermally

 Soil/solid Purge with inert gas, trap on sorbent GC/MS 5 μg/kg D-221 US EPA (1986a)

 waste trap, desorb thermally

 Soil/solid Purge with inert gas, trap on sorbent GC/HSD No data 25-162 US EPA (1986b)

 waste trap, desorb thermally; or inject

 directly into GC

 Food Equilibrate in heated sodium sulfate GC/ELCD 0.05 ppm No data Page & Charbonneau

 solution, collect headspace vapour (1984)

 Food Isolate solvent by closed system GC/ELCD 7 ng 94 Page & Charbonneau

 vacuum distillation with toluene as (1977)

 carrier solvent

 

 Table 3 (Cont'd)

 

 Sample matrix Preparation method Analytical Sample detection Percentage Reference

 methoda limit recovery

 

 Food Isolate solvent by closed system GC/ECD 7 ng 100 Page & Charbonneau

 vacuum distillation with toluene as (1977)

 carrier solvent

 Food Purge with nitrogen, trap on sorbent GC/ELCD 1.2 mg/kgd 84-96 Heikes (1987)

 trap, elute with hexane

 Food Extract with acetone-water, back GC/ELCD 4 μg/kg 66 Daft (1987)

 extract with iso-octane

 

a ECD = electron capture detector; ELCD = electrolytic conductivity detector; FID = flame ionisation detector; GC = gas chromatography;

 HRGC = high resolution gas chromatography; HSD = halogen-specific detector; MS = mass spectrometry

b Lowest value for various compounds reported during collaborative testing of this method

c Estimated accuracy of the method when the personal sampling pump is calibrated with a charcoal tube in the line

d Lowest reported concentration

 

3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

3.1 Natural occurrence

 Methylene chloride is not known to occur naturally in the

 environment.

3.2 Anthropogenic sources

3.2.1 Production

 Methylene chloride is produced almost exclusively by the Stauffer

 process. Methyl chloride is first produced by the reaction of methanol

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 with hydrogen chloride and is then reacted with chlorine. Chloroform

 and, to a lesser extent, carbon tetrachloride are also produced.

 Historically the direct route to methylene chloride by chlorination of

 methane was also used; this also produced the other three

 chloromethanes in varying proportions depending on the conditions used

 (CEC, 1986; ICI, personal communication to the IPCS).

 World production of methylene chloride in 1980 was estimated to be

 570 000 tonnes (Edwards et al., 1982); a similar figure is considered

 to apply currently (ECSA, 1992). USA production was 229 000 tonnes in

 1988, the demand being 207 000 tonnes. The total amount produced in

 western Europe ranged from 331 500 tonnes in 1986 to 254 200 tonnes in

 1991 (ECSA, 1992).

3.2.2 Uses

 The usage of methylene chloride in Western Europe shows a decrease

 from 200 000 tonnes/year in 1975-1985 (CEFIC, 1986) to

 175 000 tonnes/year in 1989 and to 150 000 tonnes/year in 1992 (CEFIC,

 1993).

 Most of the applications of methylene chloride are based on its

 considerable solvent capacity, especially for grease, plastics and

 various paint-binding agents. Other important properties are its

 volatility and stability; it is also non-flammable. Among its uses are

 (CEFIC, 1983):

 - a component of paint and varnish strippers, and adhesive

 formulations

 - a solvent in aerosol formulations

 - an extractant in food and pharmaceutical industries

 - a process solvent in cellulose ester production and fibre and

 film forming

 - a process solvent in polycarbonate production

 - a blowing agent in flexible polyurethane foams

 - the extraction of fats and paraffins

 - plastics processing, and metal and textile treatment

 - a vapour degreasing solvent in metal-working industries

 An estimated breakdown of usage worldwide before 1985 is given in

 Table 4.

 Table 4. Estimated usage patterns (BUA, 1986)

 

 USA (1985) Western Europe (1984)

 

 Aerosols 25 10

 Paint strippers 23 50

 Degreasing agent 8 13

 Film, electronics industries 7 15

 Blowing agent 5

 Others 35 12

 

 It should be noted that these data apply to the situation

 approximately 10 years ago and may have changed since. Reliable

 reports on present trends are not available.

3.2.3 Consumer applications

 The main use in consumer products is in paint strippers, where

 methylene chloride is the main constituent (70-75%). The second

 important use is in hairspray aerosols, where it acts as a solvent and

 vapour pressure modifier. In the European Community (EC) it may be

 used in such products at concentrations of up to 35% w/w (European

 Council, 1982). The US Food and Drug Administration has banned the use

 of methylene chloride in cosmetic products. It is also used in aerosol

 paints. Other types of methylene chloride-containing products are

 household cleaning products and lubricating, degreasing and automotive

 products, some of which may be in aerosol form. Chemical products

 containing methylene chloride were banned from sale or transfer to

 consumers for their private use in 1993 according to the Swedish Code

 of Statutes. Furthermore, it may not be used for working purposes

 after 1st January 1996 (National Chemical Inspectorate, Sweden,

 personal communication to the IPCS).

3.2.4 Sources in the environment

 Most of the methylene chloride released to the environment results

 from its use as an end-product by various industries, and the use of

 paint removers and aerosol products in the home. Methylene chloride is

 mainly released to the environment in air and, to a lesser extent, in

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 water and soil.

 Methylene chloride is released to the atmosphere during its

 production, storage and transport, but more than 99% of the

 atmospheric releases result from industrial and consumer uses (US EPA,

 1985). It has been estimated that 85% of the total amount of methylene

 chloride produced in the USA is lost to the environment, of which 86%

 is released to the atmosphere (US EPA, 1985). Data reported to the US

 EPA for the 1988 Toxic Chemical Release Inventory indicate that

 approximately 170 000 tonnes of the USA production volume for 1988

 (230 000 tonnes) was lost to the atmosphere; of this, 60 000 tonnes

 resulted from industrial methylene chloride emissions and 110 000

 tonnes from the use of consumer products and from other sources such

 as hazardous waste sites.

 Estimates of annual global emissions of 500 000 tonnes have been

 reported for methylene chloride (WMO, 1991). The short atmospheric

 lifetime of methylene chloride (see section 4.2.1) implies that

 emissions quantities given on a seasonal as well as on a regional

 basis are more relevant for comparison with atmospheric measurements.

 The total emission into the air in western Europe was estimated to be

 173 000 tonnes for 1989 and 180 000 tonnes in 1991.

4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION

 Appraisal

 Due to its high volatility, most of the methylene chloride

 released to the environment will partition to the atmosphere, where

 it will degrade by reaction with photochemically produced hydroxyl

 radicals with a lifetime of 6 months. Given an intra-hemispheric

 mixing time of approximately 1 month, transport can occur to regions

 far removed from the emission source. The atmospheric lifetime is

 fairly short relative to the inter-hemispheric transport time of 1 to

 1.5 years, resulting in higher concentrations of methylene chloride

 in the northern hemisphere, where most of the emissions occur at

 present.

 Methylene chloride is expected to have no significant impact on

 stratospheric ozone depletion. It will not contribute significantly

 to photochemical smog formation.

 Hydrolysis and photolytically induced degradation in water are

 slow compared to evaporation. Methylene chloride has been shown to

 disappear rapidly from soil and ground water due to bio-

 transformation.

 The aerobic and anaerobic degradation of methylene chloride has

 been proven by a variety of different test systems. Complete

 biodegradation by acclimated bacterial cultures under aerobic

 conditions is rapid. There is no evidence that significant

 bioaccumulation or biomagnification of methylene chloride along the

 food chain will occur.

4.1 Transport and distribution between media

4.1.1 Water/air

 Methylene chloride enters the hydrosphere either directly, via

 aqueous effluents, or indirectly from the atmosphere by dissolution in

 sea water and in rain water. Due to its high volatility (Henry's Law

 constant 380 Pa.m3/mol at 20°C) and low liquid-film transfer

 coefficient (Kp = 0.005 m/h), methylene chloride is rapidly

 transferred from the hydrosphere to the atmosphere.

 Under laboratory conditions, the estimated half-life for

 volatilization of methylene chloride from water at 25°C was 18-25 min

 (when present at 1 mg/litre and stirred at 200 rpm). Removal of 90% of

 the methylene chloride required 60-80 min. When stirring was minimal

 (15 seconds every 5 min), the time required for 50% reduction in the

 concentration was about 90 min. The presence of 3% sodium chloride (as

 in sea water) decreased the evaporation rate by 10% (Dilling et al.,

 1975; Dilling, 1977).

 Various factors have been shown to affect the rate of

 volatilization. For example, the half-life for volatilization of

 methylene chloride from a depth of 1 m has been shown to be 3 h

 (Lyman, 1982). The application of wind across the surface of the water

 caused an increase of 17% in volatilization over a period of 20 min

 compared to the presence of still conditions (Dilling et al., 1975). A

 decrease in the water temperature decreased the rate of

 volatilization. For example, over a period of 30 min, a 28% decrease

 in rate was seen at 1-2°C compared to that at 25°C (Dilling et al.,

 1975).

 When measured under field conditions in experimental ponds, half-

 lives for methylene chloride of 26-28 h have been reported (Merlin et

 al., 1992). Its half-life for evaporation from the river Rhine has

 been estimated to be 33-38 clays (Zoeteman et al., 1980). Further

 estimates of the half-life for its evaporation are between 3 and 48 h

 depending on wind and mixing conditions (Halbartschlager et al.,

 1984). In a further study, methylene chloride was not detected at a

 point 4-8 km from the point of release into an estuarine bay (Helz &

 Hsu, 1978) or at 25 km below its discharge point in a river basin (De

 Walle & Chain, 1978).

 Rain-out is considered to be a limited process for removal of

 methylene chloride from the troposphere. If it is assumed that its

 aqueous-phase concentration is in equilibrium with the background

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 concentration in the northern hemisphere of about 123-134 ng/m3

 (35-38 ppt) (Cox et al., 1976; WMO, 1991), the total amount of

 methylene chloride rained out in the northern hemisphere will be

 700 tonnes/year (assuming a rain fall of 2.5x1014 tonnes/year

 containing 9.9 ng/m3 (2.8 ppt) at 10°C). The same calculation

 performed at 20°C (Henry's constant is 1.57 times higher) would lead

 to a value of 445 tonnes methylene chloride rained out annually in the

 northern hemisphere. For the southern hemisphere, rainout quantities

 of 390 and 248 tonnes methylene chloride can be calculated. The half-

 life for removal by wet deposition is 550 years (Cupitt, 1980).

 In 1978, it was estimated that 2.5% of releases at ground level

 may reach the stratosphere (Derwent & Eggleton, 1978).

4.1.2 Soil/air

 Methylene chloride present in the soil is predicted to evaporate

 from the near-surface layer into the atmosphere because of its high

 vapour pressure (470 hPa at 20°C).

4.1.3 Water/soil

 The adsorption coefficient sediment/water for methylene chloride

 is 8-10 (log Koc = 0.89-1.05). Methylene chloride has a low tendency

 to adsorb to soil (adsorption coefficient 0.25 for a soil containing

 1% organic carbon, Giger et al., 1983). Therefore there is a potential

 for it to leach to ground water.

 The amount of adsorption of methylene chloride to dry granular

 bentonite clay added at a concentration of 375-750 mg/litre was found

 to be 10-22% within 10-30 min. In the presence of 500 mg/litre peat

 moss, about 40% of methylene chloride was absorbed after 10 min. Some

 adsorption by dry-powdered dolomitic limestone was observed, but not

 with silica sand (Dilling et al., 1975).

4.1.4 Multicompartment distribution

 The regional distribution of methylene chloride over water, soil

 and air compartments may be estimated by means of the fugacity model

 developed by MacKay (Slooff & Ros, 1988). Application of this model

 suggests that over 98% of the total emissions of the chemical will be

 found in air, 1 to 2% in water and far less than 1% in soil and ground

 water (BUA, 1986; Slooff & Ros, 1988).

4.2 Abiotic degradation

4.2.1 Atmosphere

 The principal process by which methylene chloride is scavenged

 from the atmosphere is the reaction with hydroxyl rate of methylene

 chloride can be calculated from the rate constant for the initiating

 breakdown reaction with HO. and the varying concentration of these

 radicals in the troposphere. Determination of the rate constant for

 the reaction of methylene chloride with hydroxyl radicals has been the

 subject of various investigations. WMO (1991) recommends the following

 value:

 kOH = 5.8 × 10-12 exp(-1100/T) cm3 molecule-1 s-1

 Other reactive species (e.g., ozone, oxygen atoms, chlorine atoms

 and nitrate radicals) are not thought to contribute significantly to

 the primary attack on methylene chloride (Table 5). As methylene

 chloride does not absorb in the visible or near ultraviolet light

 region (> 290 nm), direct homogeneous gas-phase photolysis in the

 troposphere is of negligible importance.

 Table 5. Primary tropospheric reactions of methylene chloride (other than with .OH)

 

 Reaction k (at 25°C) Global average [X] Lifetime

 with: (cm3 molecule-1 s-1) (molecule cm-3) (years)

 

 .Cl 4.1 × 10-13 103 77

 (IUPAC, 1992) (estimated) (estimated)

 .NO3 <3.2 × 10-17 1.2 × 108 > 8.3

 .O(3p) 6.44 × 10-16 2.5 × 104 approx. 2000

 (Barassin & Cambourieu, 1973)

 .O(1D) < 5 × 10-10 0.5 > 120

 (estimated)

 

 

 Carbon dioxide and hydrogen chloride are the major breakdown

 products and minor quantities of carbon monoxide and phosgene are

 formed (Sanhueza & Heicklen, 1975; Rayez et al., 1987). The breakdown

 reaction can be described as follows:

 CH2Cl2 + HO. --> .CHCl2 + H2O

 .CHCl2 + O2 --> .CHCl2O2

 .CHCl2O2 + NO --> .CHCl2O + NO2

 .CHCl2O --> .Cl + HCOCl or

 .CHCl2O + O2 --> COCl2 + HO2 (minor reaction)

 Formyl chloride may be taken up by cloud droplets, hydrolysed to

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 formic acid and wet deposited as such, or dry deposited to the ocean

 or land surfaces and then hydrolysed. The overall lifetime for wet or

 dry deposition is unlikely to exceed a few months and may be much

 shorter. On the other hand, degradation in the troposphere by

 photolysis or reaction with HO. may possibly be a more rapid process.

 The reaction products would be carbon oxides (CO, CO2) and HCl

 (Libuda et al., 1990).

 Phosgene is known to hydrolyse slowly in the gas phase, but

 rapidly once dissolved in liquid water, to give CO2 and HCl.

 HCl is removed from the troposphere by wet deposition (dissolution

 in atmospheric water droplets and subsequent rain-out) or dry

 deposition (direct uptake by the oceans, land surfaces, vegetation

 etc.) with an average lifetime of about 1 week. The amount of chloride

 deposited in this manner is completely negligible compared to the

 natural atmospheric chloride flux of around 1010 tonnes/year

 primarily from sea-salt aerosols (WMO, 1991).

 In the stratosphere methylene chloride will rapidly degrade by

 photolysis and reaction with chlorine radicals (Derwent et al., 1976).

4.2.2 Water

 Sunlight absorption of water results in the formation of HO. and

 hydrated electrons (eaq). The near surface concentrations of HO.

 and eaq are 4 × 10-16 mol/litre and 5 × 10-17 mol/litre,

 respectively, which corresponds to theoretical half-lives for

 methylene chloride of 400 and 33 days. In water systems these

 reactions are very limited, the reaction with hydroxyl radicals being

 dominant. The total rate constant for the sunlight-induced

 transformation in surface water (with a depth of 2.5 m, a DOC content

 of 4 mg/litre, a chlorophyll a content of 10 μg/litre and a

 suspended matter content of 40 mg/litre) was estimated to be

 2.8 × 10-5 day-1 (half-life 68 years). The HO. causes 90% of this

 transformation (Slooff & Ros, 1988). No direct photolysis of methylene

 chloride was found after visible and UV irridiation for 5 days at 22°C

 (Chodola et al., 1989).

 The half-life of a 1 mg/litre aqueous solution of methylene

 chloride was found to be about 1.5 years when measured in sealed glass

 tubes in the dark at 25°C and pH 7 (Dilling et al., 1975). No

 significant hydrolysis was found at 50°C and pH 4 or 9.2 after 7 days

 in the dark (Chodola et al., 1989). Under acidic and basic conditions

 in the temperature range of 80-150°C, the hydrolysis of methylene

 chloride results in the formation of formaldehyde and HCl (Fells &

 Moelwyn-Hughes, 1958). Extrapolation of these data to 25°C gives a

 long half-life of about 680-704 years (Dilling et al., 1975; Radding

 et al., 1977). As the activation energy for hydrolysis of methylene

 chloride varies with temperature, the extrapolation of rate data from

 80-150°C may not be valid.

 No reductive dehalogenation of methylene chloride in water was

 observed in the presence of sodium sulfide and haematein, a common

 iron porphyrin (Klecka & Gonsior, 1984).

4.2.3 Soil

 As is the case in aqueous systems, hydrolysis is probably not an

 important process in the removal of methylene chloride from soil (see

 section 4.2.2).

 In a lysimeter experiment, a 90% decrease over 2.5 m soil column

 was obtained (Nellor et al., 1985).

 In the report of a spillage, the concentrations of methylene

 chloride were up to 802 mg/m3 and 26 900 mg/m3 near the point of

 leakage. In both cases, methylene chloride could not be detected some

 hundred metres away from the points of contamination even in the

 direction of the groundwater flow (ECSA, 1989). In the neighbourhood

 of polluted areas, an increase of bacterial activity has been found.

 In well-documented cases of accidental spills to soils, methylene

 chloride disappeared rapidly from ground water, probably due to

 (bio)degradation (Baldanf, 1981; Leitfaden für die Beurteilung, 1983).

4.3 Biotransformation

4.3.1 Aerobic

 Negligible oxygen consumption was found in a biochemical oxygen

 demand (BOD) test (Klecka, 1982), and methylene chloride was

 considered to be degradation resistant in a degradation test following

 the Japanese MITI standards (Kawasaki, 1980). However, complete

 degradation occurred during a static-culture flask test (Tabak et al.,

 1981).

 In laboratory studies methylene chloride was almost completely

 transformed within days by bacteria enriched from a primary sewage

 sludge, municipal activated sludge (with or without acclimitization)

 and industrial waste water (Rittmann & McCarty, 1980; Davis et al.,

 1981; Klecka, 1982; Stover & Kincannon, 1983; Halbartschlager et al.,

 1984).

 In field studies it has been shown that methylene chloride is

 efficiently removed from water treatment works (Namkung & Rittmann,

 1987).

 Certain strictly aerobic, facultative methylotrophic bacteria,

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 like Pseudomonas DMI and Hyphomicrobium DM2, both readily isolated

 from contaminated soil and waste-water treatment plants, are capable

 of using methylene chloride as a sole carbon source for growth

 (Brunner et al., 1980; Stucki et al., 1981).

 Secondary substrate utilisation of methylene chloride was

 demonstrated by Pseudomonas sp. strain LP. This strain showed a

 preference towards degrading methylene chloride over acetate, whether

 it was the primary or the secondary substrate (Lapat-Polasko et al.,

 1984).

 In Hyphomicrobium DM2, a glutathione (GSH)-dependent, strongly

 inducible enzyme (a glutathione S-transferase) was found to be

 responsible for the degradation of methylene chloride. It converted

 methylene chloride to formaldehyde via the nucleophilic displacement

 of chloride and the formation of S-chloromethyl glutathione and

 S-hydroxymethyl glutathione. This enzymic dehalogenation in extracts

 of methylene-chloride-grown cells amounted to 1160 mg/g protein per h

 under alkaline (pH 8-9) conditions (Stucki et al., 1981; Leisinger,

 1983).

 Eight other bacteria (mainly Pseudomonads ), capable of growing

 on methylene chloride as their sole carbon source, were isolated from

 enriched cultures. Maximum degradation rates for methylene chloride

 (up to 860 mg/litre per h) were found for an initial saturated

 solution of 14.5 g/litre in a pH-controlled fermenter (flow rate

 10 ml/h). Further increases in degradation rate were limited by the

 high salt concentration resulting from the neutralization of the

 degradation products. In a fluidized bed reactor with bacteria

 immobilized on silica, a degradation rate of methylene chloride of up

 to 1600 mg/litre per h was observed (Gälli and Leisinger, 1985;

 Stucki, 1990).

 Ubiquitous soil- and water-dwelling nitrifying bacteria such as

 Nitrosomonas europaea, which depends for growth on the oxidation of

 ammonia, were able to degrade 1 mg methylene chloride/litre completely

 within 24 h in the presence of ammonia and by 67% in the absence of

 ammonia (Vannelli et al., 1990).

 The removal of methylene chloride from aerobic soil was

 significantly increased following exposure to methane (Henson et al.,

 1988).

 Flathman et al. (1992) described the remediation of ground water

 contaminated with dichloromethane after a leak. Air stripping was used

 initially on water pumped out from the contaminated site, and 97% of

 the contamination was removed in this way. This was followed by the

 first phase of bioremediation, in which contaminated water was

 withdrawn from the site and added to a bioreactor containing bacteria

 acclimated to DCM. The treated water was reinjected on the site

 together with the bacteria. This phase decreased the concentration by

 97% over a period of 40 days. A second phase of bioremediation

 followed some 3 years later, dealing with a subsection of the original

 site. In this case, the indigenous bacteria were used and nutrients

 were added to the site. Concentrations before treatment were up to

 5200 mg/litre; after 10 months these had reduced to < 2 mg/litre. At

 this point active treatment ceased, but the levels of DCM continued to

 decrease, falling below 10 μg/litre at all but one of the sampling

 sites.

 The biodegradation of methylene chloride in contaminated ground

 water can be strongly inhibited in the presence of other contaminants

 such as 1,2 dichloroethane, xylene and ethylbenzene (Scholz-Muramatsu

 et al., 1988).

 Aerobic biodegradation of methylene chloride was observed in a

 variety of surface soils including sand, a sandy loam and a sandy clay

 loam, as well as in subsurface clay soil. Degradation occurred over

 concentrations ranging from approximately 0.1 to 5 mg/litre. The time

 required for 50% disappearance of the parent compound varied between

 1.3 and 191.4 days.

4.3.2 Anaerobic

 Details of studies on the anaerobic biodegradation of methylene

 chloride are given in Table 6.

 Methylene chloride was degraded at a concentration of 200 μg/litre

 in the aqueous phase of natural sediment. Degradation was observed to

 proceed via methyl chloride, although accumulation was not observed

 (Wood et al., 1981). After a varying acclimation period using

 anaerobic digestion in waste water, 86-92% conversion to CO2 will

 occur (Gossett, 1985). The half-life of methylene chloride in an

 anaerobic water/sludge system is 11 days (Bayard et al., 1985).

 Methylene chloride degradation was observed under anaerobic

 conditions in sandy loam soil (Davis & Madsen, 1991).

4.3.3 Bioaccumulation

 The n-octanol/water partition coefficient for methylene chloride

 is 18 (log Pow = 1.25-1.3). As a consequence, its bioaccumulation is

 not expected to be significant. Moreover, its high depuration and

 degradation rate will reduce the probability of bioaccumulation.

 No experimental bioconcentration factor (BCF) for methylene

 chloride is available. Its theoretical BCF ranges between 0.91 and 7.9

 (Veith et al., 1980; Lyman et al., 1982; Veith and Kosian, 1983;

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 Bayard et al., 1985). Further data indicative of bioaccumulation in

 aquatic organisms and human breast milk can be found in sections 5.1.3

 and 5.3.1, respectively.

 There is no evidence of biomagnification.

4.4 Interaction with other physical, chemical or biological factors

 The ozone-depletion potential (ODP) of methylene chloride, as

 compared to the standard ODP of CFC11, can be estimated from the

 numbers of chlorine atoms (2 as compared to 3 for CFC11) and the

 atmospheric lifetime (0.7 years as compared to 60 years). This results

 in an ODP for methylene chloride of 0.4% of that of CFC11.

 Table 6. Aerobic biodegradation of methylene chloride

 

 Test system Condition Duration Degradation Initial concentration Reference

 

Laboratory studies

 Unknown aerobic, BOD 20 days none Klecka (1982)

 Domestic waste aerobic 28 days none Kawasaki (1980)

 water (MITI)

 Domestic waste aerobic, static, 7 days for each 100% 5, 10 mg/litre, loss by Tabak et al. (1981)

 water subcultures taken at days culture transformation volatilization 6.25%

 14 and 21

 Enriched primary aerobic, static, closed 24 h almost 25 mg/litre Rittmann & McCarty

 sewage effluent complete (1980)

 transformation

 Industrial waste aerobic 6 h 92% 50 mg/litre Davis et al. (1981)

 water, municipal transformation,

 activated sludge no metabolites

 Activated sludge aerobic, continuous-flow 2-6 days > 99% 180 mg/litre, loss by Stover & Kincannon

 reactor volatilization 5% (1983)

 Municipal activated aerobic 50 h 49-66% 1, 10, 100 mg/litre Klecka (1982)

 sludge (9-11 days mineralization

 acclimatization)

 Activated sludge aerobic 20-28 mg//litre 264-1300 mg/litre Halbartschlager et al.

 (6 weeks per hour (1984)

 acclimatization) transformation

 

 Table 6 (Cont'd)

 

 Test system Condition Duration Degradation Initial concentration Reference

 

Field studies

 Water treatment aerobic 30-55% removal 50-150 μg/litre Loehr (1987)

 works

 Conventional aerobic 5-6 h 96.0-96.3% Namkung & Rittmann

 activated sludge transformation (1987)

 plant

 

 

 At the current estimated total emission rate of 500 000 tonnes per

 year, the calculated tropospheric chlorine loading due to methylene

 chloride is 35 ppt, i.e. approximately 1% of the total chlorine

 loading of 3600 ppt (WMO, 1991).

 As methylene chloride has a low photochemical ozone creation

 potential in the troposphere (0.9), when compared with chemicals such

 as ethanol (27) or ethylene (100), it will not contribute

 significantly to photochemical smog formation (Derwent & Jenkin,

 1991).

5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

 Appraisal

 As a consequence of release during its production and use,

 methylene chloride is found in biota, water and air. Levels in water

 and air tend to be higher in industrial and urban areas than in rural

 areas. Improved control of emissions has led to lower environmental

 levels of methylene chloride.

 For the general population, air is the major source of exposure

 to methylene chloride. In indoor air, higher levels may result from

 the use of consumer products which contain methylene chloride. High

 levels of methylene chloride may occur for short periods of time when

 paint strippers and aerosols are used.

 Exposure to methylene chloride can occur during its production

 and use as a paint stripper. cleaner, degreaser, process solvent and

 as an aerosol.

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5.1 Environmental levels

 Environmental levels measured before 1980 were summarized in

 EHC 32: Methylene Chloride (IPCS, 1984). This monograph therefore

 focuses on levels measured after 1980.

5.1.1 Atmosphere

 5.1.1.1 Ambient air

 In the ambient air of rural and remote areas, mean background

 levels of methylene chloride are 0.07-0.29 μg/m3 (Table 7). The

 average concentrations in suburban and urban areas, respectively, are

 reported to be < 2 μg/m3 and < 15 μg/m3. In the vicinity of

 hazardous waste sites, up to 43 μg/m3 have been round.

 5.1.1.2 Precipitation

 Rain water sampled in Koblenz (Germany) in 1982-1983 was found to

 contain up to 4 μg methylene chloride/litre (Hellmann, 1984).

5.1.2 Water

 Data on the levels of methylene chloride in water are presented in

 Table 8.

 Table 7. Methylene chloride levels in ambient air

 

 Country/ Location Year of Concentration Reference

 region measurement (μg/m3)

 

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

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

 Netherlands Delft, Vlaardingen (urban 1980-1981 14.1 (max. annual mean) Guicherit & Schulting (1985)

 area)

 Isle of Terschelling (rural, 1980-1981 1.4 (max. annual mean) Guicherit & Schulting (1985)

 suburban area)

 mean concentration in 1980-1981 9 Guicherit & Schulting (1985)

 the country

 USA rural, suburban areas - 0.18-2.1 Shah & Heyerdahl (1988)

 San Francisco Bay area 1984 3.2-9.1 Levaggi et al. (1988)

 urban areas 1980 0.8-6.7 Shah & Heyerdahl (1988)

 Shikiya et al. (1984)

 1980-1981 1.35-6.76 Singh at al. (1982)

 1981 0.8-2.5 Harkov (1984)

 1982 2.4-4.2 Harkov (1984)

 1987 0.95-1.64 Pleil & McClenny (1990)

 1988 0.62-1.80 Pleil & McClenny (1990)

 1989 0.48-1.68 Pleil & McClenny (1990)

 hazardous waste sites 1983-1984 0.3-43 Harkov et al. (1985)

 Arctic Spitzbergen July 1982 0.26±0.04 Hov et al, (1984)

 March 1983 0.29±0.06 Hov et al. (1984)

 Northern eastern Pacific 1981 0.12±0.15 Singh et al. (1983)

 hemisphere

 Southern eastern Pacific 1981 0.07 Singh et al, (1983)

 hemisphere

 

 Table 8. Methylene chloride levels in water

 

 Country Location Year of Concentration Reference

 measurement (μg/litre)

 

Ground water

 Italy Milan 1983 4.5 CEFIC (1986)

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

Surface water

 Germany Mosel 1983 1.5-2.0 Hellmann (1984)

 Neckar 1983 0.6-1.0 Hellmann (1984)

 Elbe 1983 0.7-2.1 Hellmann (1984)

 Elbe 1988 11 (max) LWA (1990)

 Weser 1982-1983 < 0.5 Hellmann (1984)

 Weser 1988 6 (max) LWA (1990)

 Rhine at various sites 1981-1983 < 1 LWA (1981,1982,1983)

 Rhine at Koblenz 1983 5.35-171 (monthly mean) Hellmann (1984)

 Rhine at the Wesel 1983 < 2.0 Hellmann (1984)

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

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 Rhine at various sites 1988 3.3 (max) LWA (1989)

 

 Table 8 (Cont'd)

 

 Country Location Year of Concentration Reference

 measurement (μg/litre)

 

 1989 1.0 (max) LWA (1990)

 1990 1.1-3.9 (90th percentile) LWA (1991)

 1991 < 0.1 (max) LWA (1992)

 1986 0.1 (mean) BUA (1986)

 Main 1985 ± 0.2 Van de Graaff (1986)

 Emscher 1988 8.5 (max) LWA (1989)

 1989 2.5 (max) LWA (1990)

 1990 3.9 (max) LWA (1991)

 1991 < 0.1 (max) LWA (1992)

 Lippe 1988 5.5 (max) LWA (1989)

 1989 < 1 (max) LWA (1990)

 1990 2.4 (90th percentile) LWA (1991)

 1991 < 0.1 (max) LWA (1992)

 Wupper 1988 2.3 (max) LWA (1989)

 1989 13.6 (90th percentile) LWA (1990)

 1990 3.0 (90th percentile) LWA (1991)

 

 Table 8 (Cont'd)

 

 Country Location Year of Concentration Reference

 measurement (μg/litre)

 

 USA Susquehanna river, 1987 10 (mean) Smith (1989)

 Columbia

 Lancaster 1987 4.7 (mean) Smith (1989)

 Ohio river basin (11 1980-1981 > 1 (238 samples) Howard et al. (1990)

 stations, 4972 samples) > 10 (19 samples)

 Sea and estuarine East Pacific Ocean 1981 0.002 (mean) Singh et al. (1983)

 water (30 samples)

 East Sea (German Coast) 1983 1.3-2.6 Hellmann (1984)

 North Sea (German 1983 0.06-0.20 Hellmann (1984)

 Coast)

 

 

 In surface water, levels of methylene chloride have been reported

 to vary from not detectable to 10 μg/litre. According to data recorded

 in the US EPA STORET database, 30% of the samples showed methylene

 chloride levels above the detection limits. A median concentration of

 0.1 μg/litre was estimated (Staples et al., 1985).

 Limited information concerning the contamination of sea water and

 estuaries by methylene chloride is available. It appears that

 methylene chloride can be found at up to 2.6 μg/litre in coastal

 waters of the Baltic Sea. Levels of up to 0.20 μg/litre have been

 found in North Sea coastal waters. Methylene chloride is generally not

 detected in open oceans. A mean concentration of 2.2 ng/litre has been

 reported in the South Pacific Ocean.

 Methylene chloride enters the aquatic environment primarily

 through waste water discharge. An estimated amount of 0.2% of the

 total methylene chloride production is released in waste water

 (Dequinze et al., 1984). The input from air rain-out has been

 estimated for the northern and southern hemisphere (section 4.1.1).

 Waste water from certain industries has been reported to contain

 methylene chloride at average concentrations in excess of

 1000 μg/litre, these being coal mining, aluminium forming,

 photographic equipment and supplies, pharmaceutical manufacture,

 organic chemical/plastics manufacture, paint and ink formulation,

 rubber processing, foundries and laundries. The maximum concentration

 measured was 210 mg/litre in waste water from the paint and ink

 industry and the aluminium-forming industry (US EPA, 1981).

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

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 the samples recorded contained methylene chloride with a median

 concentration of 10 μg/litre (Staples et al., 1985).

 Samples from the outfalls of four municipal treatment plants in

 Southern California, USA, with both primary and secondary treatment,

 contained < 10 to 400 μg methylene chloride/litre (Young et al.,

 1983). In 30 Canadian water-treatment facilities, average

 concentrations of methylene chloride in summer and winter were found

 to be 10 μg/litre and 3 μg/litre, respectively (maximum, 50 μg/litre)

 (Otson et al., 1982).

 In leachate from industrial and municipal landfills, methylene

 chloride concentrations were reported to range from 0.01 to

 184 000 μg/litre (Sabel & Clark, 1984; Brown & Donnelly, 1988;

 Sawhney, 1989).

 Background data on ground water contamination by methylene

 chloride are limited. It is the sixth most frequently detected organic

 contaminant in ground water at hazardous waste disposal sites in the

 CERCLA database (178 sites), the detection frequency being 19% (Plumb,

 1987). In contaminated ground water in Minnesota, USA, up to

 250 μg/litre has been detected (Sabel & Clark, 1984). Levels of up to

 110 μg/litre were found in percolation water from a waste-disposal

 site in Germany. However, methylene chloride was not found

 (< 1 μg/litre) in the ground water below the site (Heil et al.,

 1989).

5.1.3 Aquatic organisms

 Concentrations of methylene chloride in freshwater organisms have

 been reported for oyster and clams from Lake Ponchartrain, Louisiana,

 USA. Levels ranging from 4.5 to 27 μg/kg (wet weight) could be

 detected (Ferrario et al., 1985).

 No methylene chloride was detected in fish taken from the River

 Rhine in 1981 (Binnemann et al., 1983).

 Levels of methylene chloride up to 700 μg/kg wet weight were found

 in marine bottom fish taken from Commencement Bay in the state of

 Washington, USA (Nicola et al., 1987).

 Data on biota collected in the US EPA STORET data base show an

 average level of 660 μg/kg in the 28% of the samples in which

 methylene chloride was detected (Staples et al., 1985).

5.1.4 Soil and sediment

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

 The levels of methylene chloride found in sediment from Lake

 Pontchartrain, Louisiana ranged from not detectable to 3.2 μg/kg wet

 weight (Ferrario et al., 1985).

 Data recorded in the US EPA STORET database revealed a median

 concentration of 13 μg/kg in 20% of 338 sediment sampling data

 (Staples et al., 1985).

 The levels of methylene chloride found in sediments from the river

 Rhine in 1987-1988 varied from non-detectable to 30-40 μg/kg. At one

 site maximum concentrations of 220-2200 μg/kg were measured (BUA,

 1993, personal communication to the IPCS).

5.2 Human exposure

5.2.1 General population

 5.2.1.1 Indoor air

 In buildings where products containing methylene chloride are

 used, air levels of methylene chloride much higher than outdoor levels

 (< 15 μg/m3, see section 5.1.1.1) may be found (Table 7).

 Relatively high levels (mean 670 μg/m3, peak level 5000 μg/m3)

 have been found in the indoor air of residential houses (De Bortoli et

 al., 1986).

 5.2.1.2 Drinking-water

 Methylene chloride has been detected in drinking-water supplies

 (estimations made before 1980) in numerous cities in the USA (Dowty et

 al., 1975; Coleman et al., 1976; Kopfler et al., 1977; Kool et al.,

 1982), the mean concentrations reported being generally less than

 1 μg/litre. An average of 3-10 μg/litre and a maximum of 50 μg/litre

 were observed in a Canadian study of 30 drinkable water treatment

 facilities (Otson et al., 1982).

 Samples from 128 drinking-water wells in the USA showed that 3.1%

 of them had methylene chloride levels of 1-5 μg/litre (Kelley, 1985).

 Rodruigez Rojo et al. (1989) sampled the drinking-water of

 Santiago de Compostela, Spain, in 1987. Methylene chloride was found

 in 98.4% of the samples; the average concentration was 14.1 μg/litre,

 with a range of 1.2-93.2 μg/litre. Other halomethanes were also found

 and measured in the samples at average concentrations ranging from

 9 to 25 μg/litre.

 A wide sampling exercise involving 630 public community water

 supplies (serving 6.9 million people in New Jersey, USA) was carried

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 out in 1984 and 1985 by McGeorge et al. (1987). The percentage of

 positive results for methylene chloride ranged from 2.6 to 7.1%. The

 median concentration ranged from 1.1 to 2.0 μg/litre and the range for

 the whole sampling period was 0.5 to 39.6 μg/litre.

 5.2.1.3 Foodstuffs

 Although methylene chloride is used in food processing (solvent

 extraction of coffee, spices, hops), there is little information on

 its residual levels in food. In the USA, residues of methylene

 chloride were found in decaffeinated coffee beans (0.32 to 0.42 mg/kg)

 whilst a major coffee processor reported levels of 0.01 to 0.1 mg/kg

 (ATSDR, 1992).

 No methylene chloride was detected in ice-cream and yoghurt (BUA,

 1986).

 In seven types of decaffeinated ground coffee the methylene

 chloride content ranged from < 0.05 to 4.04 mg/kg; in eight instant

 coffee samples <0.05 to 0.91 mg/kg was found (Page & Charbonneau,

 1984).

 Heikes & Hopper (1986) analysed samples of grains and intermediate

 grain-based foods for a range of fumigants using a purge-and-trap

 method. Methylene chloride was not found in any of the grain samples,

 nor in uncooked rice or dried lima beans. It was found in some of the

 intermediate foods such as bleached flour (30 μg/kg), yellow corn meal

 (4.7 μg/kg), lasagne noodles (5.4 μg/kg) and yellow cake mix

 (4.6 μg/kg).

 One of the authors (Heikes, 1987) investigated levels of methylene

 chloride in table-ready foods, taken from the US Food and Drug

 Administration's Total Diet Study. Of the 19 foods examined, eight

 contained methylene chloride above the quantification limit (not

 given). Detailed results for six of the foods are given in Table 9.

 Table 9. Dichloromethane content of table ready foods

 (Heikes, 1987)

 

 Food Number of Number Range of

 samples positive concentration

 (μg/kg)

 

 Butter 7 7 1.1-280

 Margarine 7 7 1.2-81

 Ready-to-eat cereal 11 10 1.6-300

 Cheese 8 8 3.9-98

 Peanut butter 7 4 26-49

 Highly processed foodsa 12 10 5-310

 

a e.g., frozen chicken dinner, fish sticks, pot pie

 5.2.1.4 Consumer exposure

 Consumers are exposed to methylene chloride via the use of a

 number of formulated products such as aerosols or paint strippers. A

 USA survey found that 78% of paint removers and 66% of aerosol spray

 paints sold as household products contained methylene chloride (US

 EPA, 1987). Over 100 consumer products in Sweden contain methylene

 chloride (National Chemical Inspectorate, Sweden, personal

 communication to the IPCS). In Norway the number is around 140,

 including 45 paint removers (AKZO, personal communication to the

 IPCS).

 Methylene chloride does not appear to be subject to widespread

 volatile substance abuse. Statistics on deaths resulting from

 substance abuse in the United Kingdom were collected over the period

 1971-1991 and analysed by product type. Of the 1221 deaths recorded,

 five were assigned to the group "paint thinners and paint strippers".

 Methylene chloride is used only in the latter products, the former

 containing solvents such as toluene and xylene which are known to be

 substances of abuse (Flanagan et al., 1990).

 A large do-it-yourself consumer population uses paint strippers

 containing methylene chloride on furniture and woodwork. Formulations

 are available mainly in liquid form, but also, occasionally, as an

 aerosol. Exposures have been estimated on the basis of USA

 investigations of household solvent products. The estimated levels

 ranged from less than 35 mg/m3 to a few short-term exposures of 14

 100 to 21 200 mg/m3. The majority of the concentration estimates

 were below 1770 mg/m3 (US EPA, 1990).

 Methylene chloride exposure was estimated while using a number of

 formulations of paint stripper in a small room. Various ventilation

 conditions were evaluated and a worst possible case was simulated,

 with doors and windows closed. In one test, involving furniture

 stripping in a room with through ventilation, the operator exposure

 was found to be 289 mg/m3 on a 2-h TWA. Peaks of exposure were

 observed during application (460 mg/m3) and during scraping-off

 (710-1410 mg/m3) (ICl, 1988, personal communication to the IPCS).

 A series of paint-stripping exercises were performed in a small

 room. Various ventilation conditions were evaluated while using a

 number of formulations of paint stripper. A worst possible case was

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 simulated with doors and windows closed. Concentrations of methylene

 chloride in the room rose to 14.1-17.6 g/m3 (4000-5000 ppm),

 although it is questionable whether anyone could work in such

 conditions without breathing apparatus. Further exercises with the

 door and windows open (as recommended by suppliers) reduced

 atmospheric concentrations by more than a factor of 10. Exposure to

 methylene choride resulted in an 8-h TWA of 187-226 mg/m3

 (53-64 ppm). The actual stripping operations took between 1 and 1.5 h.

 Maximum exposures occurred during initial application and scraping-

 off. These exposures were of a few minutes duration and concentrations

 never exceeded 3.53 g/m3 (1000 ppm) (ICI, 1990, personal

 communication to the IPCS).

 The effect of variation in the formulation was also investigated.

 During paint stripping, background concentrations in the room varied

 from 710-1410 mg/m3 to less than 350 mg/m3 depending on the

 formulation. However, within 5-6 min of application the level of

 methylene chloride fell to an equilibrium concentration of around

 71 mg/m3, irrespective of the formulation used. The shortest time

 before reaching equilibrium was about 2-2.5 min (ICI, 1990, personal

 communication to the IPCS).

 Studies in the Netherlands have measured peak concentrations in

 salons of 21-106 mg/m3 (6-30 ppm), with an 8-h TWA of

 3.53-17.65 mg/m3 (1-5 ppm) (CEC Scientific committee of

 Cosmetology). The same study measured a peak concentration of

 265 mg/m3 (75 ppm) arising from home use of a hairdressing aerosol

 containing 35% methylene chloride. This equates to a TWA of

 2.65 mg/m3 (0.75 ppm).

 Studies in the United Kingdom simulating consumer exposure during

 salon use yielded values well within the national Maxium Exposure

 Limit (353 mg/m3 or 100 ppm for an 8-h TWA). The hairdresser

 received an exposure of 77.7 mg/m3 (22 ppm, on an 8-h TWA) during

 what is considered to be exceptionally heavy use (i.e. a 10-second

 spray every 15 min for an 8-h period). The customer exposure was found

 to be 106-265 mg/m3 (30-75 ppm) (10-min TWA) (ICI, 1990, personal

 communication to the IPCS).

 The same study simulated home use of personal-care aerosols

 containing methylene chloride. Even adverse conditions (small room, no

 ventilation) resulted in an exposure of 353 mg/m

3 (100 ppm) (10-min

 TWA), equating to 7.06 mg/m3 (2 ppm) on an 8-h TWA (ICI, 1990,

 personal communication to the IPCS). This work was characterized by

 low air changes, virtually no ventilation and a more frequent rate of

 application than that determined by surveys of actual hairdressing

 work in salons.

5.2.2 Occupational exposure

 Exposure to methylene chloride can occur during its production and

 use as a paint stripper, cleaner, degreaser, process solvent and as an

 aerosol. Exposure concentrations that have been reported in various

 industries are presented in Table 10. Below is a brief description of

 exposure conditions in some of the reported industries.

 5.2.2.1 Production

 Production of methylene chloride is normally carried out in a

 closed system, with a relatively small number of people being

 involved. Exposure arises primarily during filling and packing

 operations. Occupational exposures are listed in Table 10. Some

 measured ranges, e.g., 35-81 mg/m3 and 85-244 mg/m3 (HSE, 1992),

 indicate that engineering control techniques can bring 8-h TWA

 exposures below 350 mg/m3.

 5.2.2.2 Paint stripping and related activities

 Workers in the formulation of paint removers are exposed while

 transferring methylene chloride from storage tanks, during mixing

 (blending) operations and while packaging. The extent of exposure will

 depend on the control measures and work practices in force. Exposure

 levels (8-h TWA) range from a low of 0-18 mg/m3 to over 1770 mg/m3

 (US EPA, 1990).

 Table 10. Occupational exposure to methylene chloride

 

 Industry Activity Exposure range Commentsa Reference

 (8-h TWA) (mg/m3)

 

 Production Production actvities 219-374 Maintenance activity with RPE; HSE (1987)

 Results obtained at one plant

 Aircraft Paint stripping 35-81 RPE provided HSE (1992)

 Paint stripping 35-289 Submission to OSHA in 1987. RPE Air Transport Association

 provided (USA)

 Various industries Painting 21-299 See IARC (1986) Chrostek & Levine (1981)

 Paint stripping 18-1765 US EPA (1990)

 Used aerosol < 0-494 Some work areas were congested Fleeger & Lee (1988)

 adhesives

 Pharmaceuticals - 7.1-3749 NCI feasibility study Zahm et al. (1987)

 Production work 0-18 Enclosed process HSE (1992)

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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 Exposure range Commentsa Reference

 (8-h TWA) (mg/m3)

 

 Triacetate Production 180-350 ECSA (1989)

 fibre/film 237-3442 NCI feasibility study Zahm et al. (1987)

 manufacturing 180-2440 Products contain acetone Ott et al. (1983)

 Furniture Paint stripping 25-3810 Many without adequate controls HSE (1992)

 Paint stripping 201-1292 Variable degrees of control McCammon et al. (1991)

 Washing/refinishing 53-780 Variable degrees of control McCammon et al, (1981)

 Spraying adhesive 219-1490 Many without adequate controls HSE (1992)

 General Cleaning, degreasing, 53-141 See IARC (1986) Ruhe et al. (1981)

 manufacturing, etc. < 0-460 See IARC (1986) Ruhe et al. (1982)

 cleaning and

 degreasing

 Foam industry Glue spraying 85-244 LEV HSE (1992)

 Moulding 88-1090 High exposure due to HSE (1992)

 insufficient/inadequate LEV

 < 247 Jernelov & Antonsson

 (1987)

 Unknown 7.1-251 NCI feasibility study Zahm et al. (1987)

 Various jobs 18-580 High exposure experienced by Boeniger (1991)

 sprayers

 Motor vehicle Paint spraying, 7.1-247 LEV and RPE HSE (1992)

 manufacture stripping

 

 Table 10 (Cont'd)

 

 Industry Activity Exposure range Commentsa Reference

 (8-h TWA) (mg/m3)

 

 Quarry Laboratory work, 71-1370 High exposures due to inadequate HSE (1992)

 mineral processing control

 Metal treatment 7.1-790 NCI feasibility study Zahm et al. (1987)

 Nutrition Extraction < 0-106 See IARC (1986) Cohen et al. (1980)

 

a RPE = respiratory protection equipment; LEV = local exhaust ventilation

 

 Paint strippers are widely used in a number of industries:

 automotive, rubber products, furniture and fixtures, plastic, and

 electronic industries. Exposure to methylene chloride takes place

 during application, removal of the substrate soaked in methylene

 chloride, and the disposal of the spent paint remover. Typical

 exposures (8-h TWA) range from 18 mg/m3 to about 1770 mg/m3 (US

 EPA, 1990).

 Exposure of commercial furniture refinishers to methylene chloride

 occurs when stripping involves either the dipping of furniture into a

 tank containing a mixture of solvents including methylene chloride

 (typically 65%) or coating it manually with a brush. Exposure levels

 are highly variable and greatly influenced by the size of the

 organization, engineering controls in place and work practices. Some

 refinishers may operate on a part-time basis and from their homes. In

 some instances where the worker was leaning over the tank or using a

 brush to scrub the surface coating, concentrations of > 7100 mg/m3

 have been recorded (McCammon et al., 1991; HSE, 1992). Better work

 stations and work practices have helped to reduce exposures greatly.

 5.2.2.3 Aerosol production and use

 In the packaging of aerosol cans, exposure arises primarily during

 filling and packing. Levels observed are generally below 180 mg/m3

 (ECSA, 1989). Potential occupational exposure to methylene chloride as

 a result of aerosol products varies according to the use and the work

 undertaken.

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 Consumer exposure due to the use of cosmetic and paint spray

 aerosols is discussed in section 5.2.1.4.

 5.2.2.4 Use as a process solvent

 Methylene chloride is widely used as a process solvent in the

 manufacture of a variety of products. Most of the processes are

 carried out in closed systems, with the exception of triacetate fibre

 and film manufacture. Normally, exposure levels are low, but

 occasionally high exposures (> 350 mg/m3; 10-min TWA) may occur in

 such operations as filter changing, charging and discharging. Some

 industrial processes involve somewhat higher exposure levels; in

 particular, the manufacture of cellulose triacetate fibres and films

 can involve exposure up to 350 mg/m3 (8-h TWA) even when good

 engineering controls are installed (Zahm et al., 1987; ECSA, 1989). As

 part of an epidemiological study of cellulose fibre production

 workers, Ott et al. (1983) reported exposure levels ranging from 177

 to 2436 mg/m3 in the processing area, and exposure ranging from 18

 to 1341 mg/m3 in the preparation area based on sampling performed in

 1978. The range for the entire plant was later reported to be from

 below detectable limits to 6000 mg/m3 (Lanes et al., 1990). As part

 of an epidemiological study of photographic film workers, Friedlander

 et al. (1978) reported exposures ranging from 0 to 1236 mg/m3 based

 on sampling carried out between 1959 and 1975. The highest mean

 exposures at this plant were reported for group leaders (402 mg/m3)

 (Hearne et al., 1987).

 In the pharmaceutical industry, methylene chloride is used as a

 solvent and extraction medium. Sealed processes, high recovery rates

 and careful handling of discharges have helped to keep the exposure

 levels below around 106 mg/m3 (Zahm et al., 1987; HSE, 1992).

 Methylene chloride is also used as an extraction medium in the

 nutrition industry, where the exposure levels are generally low when

 the processes are adequately controlled (Cohen et al., 1980).

 Methylene chloride is used in the foam industry for cleaning

 process equipment, purging spray guns, and as an auxiliary blowing

 agent. It is also used as a releasing agent in the moulding of

 polyurethane products. Exposure levels ranging from a few mg/m3 to

 short-term exposures of over 1770 mg/m3 have been reported (Jernelov

 & Antonsson, 1987; Boeniger, 1991; HSE, 1992).

 The use of methylene chloride as a solvent in adhesives can result

 in occupational exposure, during the application of the adhesive, to

 short-term levels in excess of 350 mg/m3 (Fleeger & Lee, 1988; HSE,

 1992). Processes involving the formulation of adhesives are likely to

 be well controlled.

 Methylene chloride is also used as a solvent in the analysis of

 bitumen samples. This work is normally carried out in small

 laboratories and exposure levels will be high unless adequate control

 measures are used.

 5.2.2.5 Cleaning and degreasing

 In the manufacture of metal products, cleaning (degreasing) is

 required before painting, plating, plastic coating, etc. The degree of

 exposure to methylene chloride will be influenced by many factors,

 including the age of the equipment, type of engineering controls

 available, their maintenance, handling, and drying methods. In

 general, it is possible to reduce exposure levels to below 124 mg/m3

 (Swedish National Board of Occupational Safety & Health, personal

 communication to the IPCS).

5.2.3 Occupational exposure limits

 A listing of some national occupational exposure limits is given

 in Table 11.

 Table 11. Occupational exposure limit valuesa

 

 Country TWA STEL TWA STEL Remarks Reference

 (mg/m3, 20°C)b (ppm) (ppm) (ppm)

 

 Australia 350 - 100 - Suspected carcinogen ILO (1991)

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

 potential

 Belgium 174 - 50 - Suspected human carcinogen ACGIH (1992)

 Czechoslovakia 500 2500 - - ILO (1991)

 Denmark 174 - 50 - Absorption through skin may be ILO (1991)

 significant

 Suspected carcinogen

 Finland 350 870 100 250 ILO (1991)

 France 360 1800 100 500 ILO (1991)

 Germany 360 1800c 100 500 Suspected of carcinogenic DFG (1991)

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 potential

 Italy 174 - 50 - Suspected human carcinogen ACGIH (1992)

 Japan 350 - 100 - ILO (1991)

 Netherlands 350 1750 100 500 Arbeidsinspectie

 (1991)

 Norway 125 - 35 - Carcinogen Arbeidstilsynet

 (1990)

 

 Table 11 (Cont'd)

 

 Country TWA STEL TWA STEL Remarks Reference

 (mg/m3, 20°C)b (ppm) (ppm) (ppm)

 

 Portugal 174 - 50 - ILO (1991)

 Sweden 120 250d 35 70 Classified as a low potent AFS (1990)

 carcinogen

 Switzerland 360 1800 100 500 Classification for teratogenic ILO (1991)

 effects not possible; biological

 monitoring required

 United Kingdom 350 870 100 250 Maximum exposure limit

 USA - ACGIH 174 50 Suspected human carcinogen ACGIH (1992)

 

a TWA = time-weighted average concentration (8-h working period); STEL = short-term exposure limit (15 min, unless specified)

b Official values; some countries use different conversion factors and/or other ambient temperature

c 30 min

d 15 min

 

5.3 Human monitoring data

5.3.1 Body burden

 Methylene chloride was detected in all eight samples of human milk

 from four urban areas (Pellizzari et al., 1982). Hardin & Manson

 (1980) could still find methylene chloride in mother's milk 17 h after

 the end of exposure.

 In 12 male volunteers exposed to 2600 mg/m3, biopsies showed

 adipose tissue levels of 10.2, 8.4 and 1.6 mg/kg at 1, 4, and 22 h,

 respectively, after a 1-h exposure (Engström & Bjurström, 1977). When

 Antoine et al. (1986) analysed whole blood from 250 individuals, the

 mean concentration of methylene chloride was 0.7 μg/litre with a range

 from not detected to 25 μg/litre.

 BUA (1986) monitored saliva and tissues from people living in

 industrialized areas of Beckum, Germany. They reported that no

 methylene chloride was detected.

5.3.2 Occupational exposure studies

 In a cohort of 14 furniture strippers exposed to methylene

 chloride at concentrations of 53 to 1290 mg/m3, post-exposure breath

 concentrations of methylene chloride ranged from 8.1 to 590 mg/m3

 (McCammon et al., 1991).

 Mother's milk in Soviet women manufacturing rubber articles

 contained a mean of 74 μg/kg in 17 out of 28 samples approximately 5 h

 after start of work, the level declining after termination of work

 (Jensen, 1983).

 A group of seven non-smoking workers, who had previously been

 exposed to methylene chloride for several years, were exposed to a

 mean concentration of 635 mg/m3 (in addition, there was exposure to

 154 mg/m3 (mean) chloroform). The pre-exposure average carbon

 monoxide level in alveolar air was 34 mg/m3, increasing to

 58 mg/m3 during exposure; before the next exposure, the carbon

 monoxide level was 27 mg/m3: this corresponded to carboxyhaemoglobin

 (CO-Hb) levels of 4.9%, 8.3% and 3.9%, respectively. The biological

 half-life of CO-Hb was 13 h (Rathey et al., 1974). Although methylene

 chloride does not accumulate following repeated exposure, these data

 clearly indicate that CO-Hb levels will be cumulative if the periods

 between exposure are insufficient to allow the CO-Hb levels to return

 to normal.

 CO-Hb levels in a worker accidentally overcome by methylene

 chloride vapour were found to have increased to 19%. A further worker

 with a history of ischaemic heart disease, who had been exposed

 concurrently with the first patient, had a CO-Hb level of 6% on the

 day following the exposure (Benzon et al., 1978).

 Methylene chloride levels in alveolar air and blood were measured

 in 14 shoe-sole factory workers. The alveolar concentration, mean

 blood levels, and CO-Hb levels, following exposure to 74±28 mg/m3

 methylene chloride, were: 49 mg/m3, 0.41 mg/litre, and 4.0%,

 respectively; upon exposure to 124+42 mg methylene chloride/m3:

 71 mg/m3, 0.99 mg/litre, and 5.2%, respectively; and upon exposure

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 to 339±265 mg methylene chloride/m3: 229 mg/m3, 3.07 mg/litre, and

 6.5%, respectively. In this factory, the methylene chloride exposure

 was highly variable; the data are too limited to allow valid

 extrapolation (Perbellini et al., 1977).

 The relationship between low environmental levels of methylene

 chloride, carbon monoxide in alveolar air and urinary excretion of

 methylene chloride was studied in a group of 20 manufacturing workers

 (12 smokers, 8 non-smokers). A good correlation was observed between

 levels of methylene chloride and urinary excretion of methylene

 chloride. The correlation between alveolar air and levels of methylene

 chloride was poor except when the analysis was restricted to

 nonsmokers (Ghittori et al., 1993)

5.3.3 Biological exposure indices

 Biological Exposure Indices (BEIs) are reference values intended

 for guidelines for the evaluation of potential health hazards in

 industrial hygiene practice. The BEIs for methylene chloride at the

 end of a working shift have been given as: CO-Hb level 5%, blood level

 of methylene chloride 1 mg/litre.

 Biological monitoring of methylene chloride exposure can be based

 on measurement of the solvent itself in exhaled air or blood. However,

 as production of carbon monoxide with exposure for more than 3-4 h/day

 appears to be the limiting factor with respect to health risk,

 biological monitoring based upon either analysis of carbon monoxide in

 exhaled air or of CO-Hb in blood is to be preferred. However, this can

 only be applied in non-smoking subjects. Sampling should be carried

 out either about 0-2 h after exposure or 16 h later, i.e. on the

 following morning.

 In the case of an 8-h exposure to less than 350 mg methylene

 chloride/m3 in non-smokers, CO-Hb levels 2-h after exposure ceases

 are not expected to exceed 2-3%, and after 16 h to exceed 1%.

6. KINETICS AND METABOLISM

 Appraisal

 Methylene chloride is rapidly absorbed though the alveoli of the

 lungs into the systemic circulation. It is also absorbed from the

 gastrointestinal tract and dermal exposure results in absorption but

 at a slower rate than that of the other exposure routes.

 Distribution studies indicate that, via inhalation or dermal

 exposure, methylene chloride distributes to all tissues. It can cross

 the blood-brain barrier and it can be transferred across the

 placenta. Concentrations of methylene chloride rise more slowly in

 adipose tissue and longer exposures are required before these tissue

 levels equal those of the blood. Data indicate that methylene

 chloride and/or its metabolites do not accumulate in tissues.

 Methylene chloride is metabolized to carbon monoxide, carbon

 dioxide and inorganic chloride. Methylene chloride is eliminated from

 the body primarily via the lungs in expired air. Urinary excretion

 plays a minor role in its elimination. As exposure levels increase, a

 large proportion of methylene chloride is exhaled unchanged.

 Metabolism occurs by either or both of two pathways; their relative

 contribution to the total metabolism is markedly dependent on the

 exposure level and on the animal species concerned. One pathway

 involves oxidative metabolism mediated by cytochrome P-450 and leads

 to both carbon monoxide and carbon dioxide. This pathway appears to

 operate similarly in a qualitative and quantitative sense in all

 rodents studied and in humans. This is the predominant metabolic

 route, but saturation occurs at around 1800 mg/m3. Increasing the

 dose above the saturation level does not lead to extra metabolism by

 this route.

 The other pathway involves a glutathione transferase, and leads

 via formaldehyde and formate to carbon dioxide. This route seems only

 to become important at doses above the saturation level of the

 "preferred" oxidative pathway. There are marked species and dose-

 dependent differences in the contribution that this pathway makes to

 the metabolism of methylene chloride.

6.1 Absorption

6.1.1 Inhalation exposure

 6.1.1.1 Human studies

 The principal route of human exposure to methylene chloride is

 inhalation. Evaluation of pulmonary uptake indicated that 70-75% of

 inhaled vapour was absorbed in human subjects exposed to 180, 350, 530

 and 710 mg/m3. Initial absorption was rapid, as indicated by levels

 of methylene chloride in the blood of approximately 0.6 mg/litre in

 the first hour of exposure to levels of 350-710 mg/m3. At a level of

 180 mg/m3, the increase in blood methylene chloride concentration

 was 0.2 mg/litre for the first hour. There was a direct correlation

 between the steady-state blood methylene chloride values and the

 exposure concentration, both during the exposure and for the first 2 h

 after the exposure in all groups. Steady-state blood levels appeared

 to be reached after 4 h and remained constant until the end of

 exposure. Once exposure ceased, methylene chloride was rapidly cleared

 from the blood. Seven hours after exposure, less than 0.1 mg/litre was

 detected following exposure to 180, 350 or 530 mg/m3. Only

 1 mg/litre was detected in the highest group (710 mg/m3) 16 h after

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 exposure. In all other dose groups the blood levels had returned to

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

 In common with other lipophilic organic vapours, methylene

 chloride absorption appears to be influenced by factors other than the

 vapour concentration. Prior to reaching steady state, increased

 physical activity increases the amount of methylene chloride absorbed

 by the body, due to an increase in ventilation rate and cardiac

 output, since these factors increase blood flow through the lungs and

 promote absorption (Di Vincenzo et al., 1972; Astrand et al., 1975).

 Uptake also increases with the body fat percentage, since

 methylene chloride dissolves in fat to a greater extent than it

 dissolves in aqueous media. Therefore, obese subjects will absorb and

 retain more methylene chloride than lean subjects exposed to the same

 vapour concentration. Under controlled conditions, there was a 30%

 greater absorption and retention of methylene chloride by obese

 subjects exposed to 2650 mg/m3 for 1 h as compared to lean subjects

 (Engström & Bjurström, 1977).

 Åstrand et al. (1975) reported that the amount of methylene

 chloride taken up increases with physical workload, whereas the

 retention decreases. With a 50-watt workload, the uptake was twice as

 high, whereas the retention decreased from 55% to 45%. When exposure

 was coupled with workload (physical exercise), the concentration in

 alveolar air was increased during the whole post-exposure period

 compared with exposure under rest conditions.

 6.1.1.2 Animal studies

 Studies of the relationship between inhalation exposures of

 animals and their blood methylene chloride concentrations indicate

 that absorption is proportional to the magnitude and duration of the

 exposure over a methylene chloride concentration range of 350 to

 28 200 mg/m3. This conclusion is based on the monitoring of blood

 methylene chloride concentrations following inhalation exposure in

 dogs and rats (Di Vincenzo et al., 1972; MacEwen et al., 1972; McKenna

 et al., 1982). As is the case with humans, blood methylene chloride

 levels reach a steady-state value which does not increase further as

 the duration of exposure increases (McKenna et al., 1982).

 The data from studies of blood methylene chloride values during a

 6-h exposure of rats to between 180 and 5300 mg/m3 suggest that the

 steady-state blood/air concentration ratio increases as the exposure

 concentration increases. The ratio of the steady-state methylene

 chloride concentrations in plasma to the exposure concentration was

 0.001, 0.005 and 0.006 at levels of 180, 1800 and 5300 mg/m3,

 respectively (McKenna et al., 1982). It is postulated that the

 increased ratio at steady state results from saturation of metabolic

 pathways as exposure increases, rather than from an increased

 absorption coefficient.

 Kim & Carlson (1986)conducted experiments to compare the effects

 of a 12-h exposure schedule to those of an 8-h schedule on the CO-Hb

 formation resulting from methylene chloride inhalation. Rats and mice

 were exposed to 710, 1800 or 3500 mg/m3 8 h/day for 5 days, or

 12 h/day for 4 days. No significant difference in carboxyhaemoglobin

 levels was found. The peak blood methylene chloride level was found to

 be dependent upon the methylene chloride exposure concentration, but

 the half-life was independent of the duration of exposure or the

 concentration of methylene chloride, 

6.1.2 Oral exposure

 No data are available on oral absorption of methylene chloride in

 humans.

 Treatment of mice and rats with methylene chloride in water or in

 corn oil suggests that methylene chloride is easily absorbed from the

 gastrointestinal tract. Methylene chloride levels were measured in gut

 segments up to 40 min after rats were administered single oral doses

 in water. The amounts measured were similar at both dose levels (50 or

 200 mg/kg). Of the administered dose (200 mg/kg), 60% was recovered

 from the upper gastrointestinal tract < 10 min after treatment (20%

 recovery after 40 min). The amount of methylene chloride in the lower

 gastrointestinal tract accounted for < 2% of the administered dose up

 to the 40-min test interval (Angelo et al., 1986b).

 In mice administered oral doses of non-radioactive methylene

 chloride at 10 or 50 mg/kg in water, approximately 25% of the

 administered dose was detected in the upper gastrointestinal tract

 within < 20 min. Similarly, after treatment with methylene chloride

 at 10, 50, or 1000 mg/kg in corn oil, approximately 55% of the

 administered dose was detected in the upper gut segment and remained

 there for 2 h (Angelo et al., 1986a).

6.1.3 Dermal exposure

 Methylene chloride has been shown to be absorbed through human

 skin (Stewart & Dodd, 1964). In this study, a volunteer immersed a

 thumb in methylene chloride for 30 min under conditions which

 precluded the inhalation of vapour. The subsequent alveolar air

 concentration was 3.1 ml/m3; 2 h later it had fallen to

 0.69 ml/m3.

 Various studies on the rate of absorption through animal skin and

 subsequent pharmacokinetics have been reported. Tissue concentrations

 of methylene chloride were measured in various organs (lung, liver,

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 brain, kidney, heart and fat) of 128 white rats, using gas

 chromatography, following immersion of two-thirds of their tails in

 the solvent for 1, 2, 3 or 4 h. Small increases were seen in most

 tissues after 1 or 2 h of exposure, and methylene chloride

 concentrations in fatty tissues increased markedly after 3 h of

 exposure. After 4 h of exposure, methylene chloride concentrations

 remained elevated in fatty tissues and were increased in all other

 tissues studied (Makisimov & Mamleyeva, 1977). The dermal absorption

 rate for methylene chloride through mouse skin in vivo has been

 measured to be 6.58 mg/h per cm2 (Tsuruta, 1975).

 The dermal permeability constant for the absorption of methylene

 chloride vapour through rat skin in vivo has been measured following

 exposure to 106, 212 and 353 g/m3 for 4 h. Blood levels of methylene

 chloride were shown to reach steady state levels after 1 h of exposure

 to the two lower concentrations and after 3 h of exposure to

 353 g/m3. The mean dermal permeability constant was calculated to be

 0.28 mg/h per cm2 (McDougal et al., 1986).

6.2 Distribution

6.2.1 Inhalation exposure

 6.2.1.1 Human studies

 Engström & Bjurström (1977) exposed 12 male subjects (six slim and

 six obese) to 2650 mg methylene chloride/m3 for 1 h. The total

 uptake of methylene chloride in the slim group was 1116 ± 34 mg and in

 the obese group 1446 ± 110 mg/kg. Estimation of methylene chloride in

 needle biopsies showed that the adipose tissues contained

 approximately 8 to 35% of the average total amount absorbed. The

 amount or methylene chloride absorbed was highly correlated with the

 degree of obesity and body weight. In the slim subjects, the

 concentration in the adipose tissue during the 4-h period after

 exposure was approximately twice that in the obese subjects. However,

 despite a lower concentration, the total amount of methylene chloride

 calculated to be in the body fat was greater in obese subjects.

 A survey of the levels of methylene chloride in certain tissues

 from pregnant or nursing women has been reported. The study was

 conducted following observations of disturbances in the pattern of

 pregnancy and lactation in female operatives in an industrial rubber

 article manufacturing facility. The survey was conducted in an

 unspecified number of women who had been exposed to several chemicals

 during their work for at least 3 years. The chemicals included

 gasoline, ethylene dichloride and methylene chloride. An estimate of

 the average workplace concentration of methylene chloride was reported

 to be 85.6 mg/m3. A control group (number unspecified) was

 constituted from women working in the same facility but who had had no

 direct contact with the chemicals. The tissues examined were the

 blood, the fetal membranes and the fetus, all tissue samples being

 obtained at the time of abortion of the fetus. The mean tissue

 concentrations of methylene chloride (54 observations) were reported

 to be 0.66 ± 0.21, 0.34 ± 0.10 and 1.15 ± 0.20 mg/kg for the blood,

 fetal membranes and fetus, respectively, compared to 0.12 ± 0.07,

 0.013 ± 0.01 and 0.016 ± 0.001 mg/kg in the controls. Methylene

 chloride was also detected in 17 out of 28 specimens of breast milk

 taken from exposed nursing women. An average concentration of

 0.074 ± 0.04 μg/litre (n = 40) was found in the breast milk 5-7 h

 after the start of the exposure; an insignificant quantity of

 methylene chloride was reported 17 h after cessation of exposure

 (Vosovaja et al., 1974).

 6.2.1.2 Animal studies

 Distribution studies in rats demonstrate that methylene chloride

 (and/or its metabolites) is present in the liver, kidney, lungs,

 brain, muscle and adipose tissues after inhalation exposures (Carlsson

 & Hultengren, 1975; McKenna et al., 1982). One hour after exposure,

 the highest concentration of radioactive material was found in the

 white adipose tissue, followed by the liver. The concentration in the

 kidney, adrenal and brain were less than half that in the liver.

 Radioactivity in the fat deposits declined rapidly during the first

 2 h after exposure (Carlsson & Hultengren, 1975). Concentrations in

 the other tissues declined more slowly. Whole body autoradiography in

 mice at one hour after inhalation of 10 μl 14C-methylene chloride

 for 10 min showed a rapid and even distribution immediately after

 exposure. A high uptake was noted in brain, body fat, blood, liver and

 kidney. Evaporated sections showed a high retention of non-volatile

 radioactivity, presumably representing metabolites, in the liver,

 bronchi and kidneys. Thirty minutes after inhalation, radioactivity

 started to appear in tissues with a high cell turnover such as bone

 marrow, thymus and gastrointestinal mucosa, and in tissues with a high

 rate of protein synthesis such as the spleen, exocrine pancreas and

 salivary glands (Bergman, 1979).

 On the other hand, after 5 days of exposure to 710 mg/m3 for

 6 h/day, the concentration of methylene chloride in the perirenal fat

 was 6-7 times greater than that in the blood and liver (Savolainen et

 al., 1977). It has been suggested that methylene chloride first

 saturates the blood and extravascular fluid compartment before

 entering the fatty deposits (Di Vincenzo et al., 1972). Thus,

 concentrations of methylene chloride will rise slowly in adipose

 tissues, and longer exposures to methylene chloride will be required

 before adipose tissue levels equal those in the blood. The animal data

 are therefore consistent with the human adipose tissue data discussed

 above.

 Exposure of pregnant rats to methylene chloride leads to exposure

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 of the fetus to both methylene chloride and carbon monoxide (Anders &

 Sunram, 1982).

6.2.2 Oral exposure

 No studies are available regarding distribution of methylene

 chloride in humans following oral exposure.

 In animals, radioactivity from labelled methylene chloride was

 detected in the liver, kidney, lung, brain, epididymal fat, muscle,

 and testes after exposure of rats to a single gavage dose of 1 or

 50 mg/kg. The tissue samples were taken 48 h after dosing. At that

 time, the lowest concentration of radioactivity was found in the fat.

 The highest concentrations were in the liver and kidney. This was true

 for both doses (McKenna & Zempel, 1981).

 Similar results were observed in rats administered methylene

 chloride doses of 50-1000 mg/kg for 14 days. At each dose tested, and

 in each tissue, the label was rapidly cleared during the 240 min

 following each exposure (Angelo et al., 1986b).

6.2.3 Dermal exposure

 No information is available regarding distribution in humans or

 animals following dermal exposure to methylene chloride.

6.3 Metabolism

 Species differences in metabolism and their relevance to

 carcinogenicity are described in section 8.8.2.

6.3.1 In vitro studies

 In vitro experiments using liver fractions, homogenates, slices

 and hepatocytes, mainly from the rat, confirmed the presence of the

 two metabolic pathways. The primary reaction, first described by Kubic

 & Anders (1975), appears to be an oxidative dehalogenation giving

 carbon monoxide and chloride ion. The reaction is catalysed by rat

 liver microsomal fractions and is dependent upon NADPH and molecular

 oxygen. The presence of a binding spectrum and inducers confirmed the

 involvement of the cytochrome P-450 mixed function oxidase system.

 More recent studies have identified the cytochrome P-450 isoenzyme as

 cytochrome P-450 IIEl (Pankow et al., 1991; Guengerich et al., 1992;

 Pankow & Jagielki, 1993). The highest activity was found in liver

 microsomes, which were five times more active than lung microsomes and

 thirty times more active than kidney microsomes. The proposed

 metabolic route involves rearrangement of the primary hydroxylation

 product to formyl chloride followed by decomposition to carbon

 monoxide (Kubic & Anders, 1978). Although the transient intermediates

 have not been isolated or identified, their formation is consistent

 with the enzyme involved and the products formed.

 The effect of pyrazole on methylene chloride metabolism in male

 Wistar rats was investigated by Pankow et al. (1991). Rats received a

 single methylene chloride close of 6.2 mmol/kg (0.4 ml/kg) orally.

 Pyrazole was administered by intraperitoneal injection. The metabolism

 of methylene chloride to carbon monoxide can be stimulated or

 inhibited by pyrazole; the effect depends on the interval between

 pyrazole and methylene chloride administration, and on the dose.

 Stimulation of methylene chloride metabolism to carbon monoxide is due

 to inducers of the isoenzyme cytochrome P-450 (CYP2El) such as

 isoniazid, ethanol and other solvents. The inhibition was observed

 following pre-treatment with high pyrazole doses or following

 simultaneous administration of pyrazole and methylene chloride. The

 inhibition may reflect the competition between pyrazole and methylene

 chloride for oxidation by CYP2El as long as pyrazole is present in the

 blood, or may also reflect the hepatotoxic effect of pyrazole.

 Hepatic cytochrome P-450 levels were not increased in rats exposed

 by inhalation to methylene chloride (5.29-10.59 g/m3 (1500 or

 3000 ppm)) 6 h/day for 3 clays (Toftgard et al., 1982), nor in rats

 exposed to 1.76 or 3.53 g/m3 (500 or 1000 ppm) 6 h/day for 2 weeks

 (Kurppa & Vainio, 1981). Marginal increases were seen in a third study

 (Norpoth et al., 1974) in which rats were exposed to 0.176-17.6 g/m3

 (50-5000 ppm), 5 h/day for 10 days. In the study by Kurppa & Vainio

 (1981) an increase in renal ethoxycoumarin de-ethylase activity was

 reported.

 The second metabolic pathway occurring in rat liver is localized

 in the soluble (cytosolic) fraction (Ahmed & Anders, 1976, 1978). It

 does not require oxygen but is dependent upon glutathione and a

 glutathione- S-transferase enzyme, the products in vitro being

 formaldehyde and chloride ion. The rapid and almost quantitative

 conversion of formaldehyde to formic acid and then carbon dioxide

 known to occur in vivo (Neely, 1964) is consistent with this pathway

 being the source of carbon dioxide exhaled after exposure to methylene

 chloride. The intermediates involved in the metabolism of methylene

 chloride to formaldehyde are unknown, but the nature of the enzyme

 involved and the dependence upon glutathione suggest that

 S-chloromethyl-glutathione is formed and rapidly hydrolysed and

 degraded to glutathione and formaldehyde (Ahmed & Anders, 1978). The

 isoenzyme involved in the metabolism of methylene chloride has been

 identified as a member of glutathione- S-transferase class theta

 (Meyer et al., 1991).

 The chemistry of the S-chloromethyl thioethers (Bohme et al.,

 1949) and the lack of depletion of glutathione during this reaction

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 are consistent with these conjugates being extremely transient.

 Formaldehyde, in addition to its metabolism to carbon dioxide, becomes

 incorporated into the C-1 metabolic pool via formic acid. Therefore,

 exposure to radiolabelled methylene chloride results in the

 incorporation of radioactivity into macromolecules including nucleic

 acids.

 Hallier et al. (1993) described an apparent polymorphism in the

 metabolism of methylene chloride in human blood. The metabolic

 activity was reported to be localized in erythyrocytes (Thier et al.,

 1991) and to be due to the presence of a glutathione- S-transferase

 enzyme (Schroeder et. al., 1992). The work by Schroeder describes the

 detection of enzyme activity in erythrocytes using methyl bromide as a

 substrate, not methylene chloride. Furthermore, in experiments

 investigating the influence of cofactors on enzyme activity,

 glutathione could be replaced by L-cysteine, suggesting that this

 enzyme is not a glutathione- S-transferase. The work by Thier et al.

 (1991) identified metabolic activity in plasma and not, as reported by

 Hallier et al. (1993), in erythrocytes.

6.3.2 In vivo studies

 The metabolism of methylene chloride in various animal species and

 in humans has been studied extensively (e.g., Fodor et al., 1973;

 Kubic et al., 1974; Roth et al., 1975; Lee Rodkey & Collison, 1977;

 Peterson, 1978; McKenna & Zempel, 1981; McKenna et al., 1982; Angelo

 et al., 1986a,b).

 Methylene chloride and the other dihalomethanes are unique in

 being the only class of industrial chemicals known to be metabolized

 to carbon monoxide. This metabolic pathway (dependent on cytochrome

 P-450), first discovered in humans (Stewart et al., 1972), results in

 elevated levels of CO-Hb and in increased levels of carbon monoxide in

 expired air. Subsequent studies in experimental animals and in humans

 established that this pathway is rate-limited by enzyme saturation, so

 that at high doses the levels of CO-Hb become constant and independent

 of dose (Lee Rodkey & Collison, 1977). Later experiments in animals

 using radiolabelled methylene chloride identified carbon dioxide as

 the other major metabolite (Di Vincenzo & Hamilton, 1975). Although

 carbon dioxide is a known metabolite of carbon monoxide, the amount of

 carbon dioxide formed from the monoxide was thought unlikely to

 account for the levels found during exposure to methylene chloride.

 This suggested the presence of a second pathway (dependent on

 glutathione- S-transferase), which was subsequently confirmed in

 experimental animals.

 Two reports have described the effects of pretreatment or co-

 administration of other organic solvents on the metabolism of

 methylene chloride to carbon monoxide. Pankow et al. (1991) described

 increases in CO-Hb levels in rats pretreated with a single gavage dose

 of benzene, toluene or isomers of xylene, up to 32 h prior to a 6-h

 exposure to methylene chloride. CO-Hb levels increased from 9.3%, in

 rats exposed to methylene chloride alone, to 22.7% in rats pretreated

 with m-xylene. Similar increases were seen in rats pretreated with a

 single garage dose of methanol (Pankow & Jagielki, 1993). In both

 studies, the levels of CO-Hb were reduced when the solvents were co-

 administered with methylene chloride. The results of both studies were

 considered to be consistent with the metabolism of methylene chloride

 by cytochrome P-450 IIE1.

 At first sight it might appear that the relative molar amounts of

 carbon monoxide and carbon dioxide exhaled in vivo provide an index

 of the activity of the two metabolic pathways. Studies using metabolic

 inhibitors suggest that significant amounts of carbon dioxide are also

 derived from the oxidative P-450 pathway (Gargas et al., 1986; Reitz

 et al., 1986). Similar studies in mice using metabolic inhibitors have

 confirmed these findings, leading to the conclusion of the authors

 that the cytochrome P-450 pathway is the major route of metabolism of

 methylene chloride within species (Ottenwalder et al., 1989). This

 finding is consistent with either hydrolysis of formyl chloride to

 formic acid or with formyl chloride reacting with glutathione to form

 S-formyl glutathione. The rapid enzymatic and chemical breakdown of

 this conjugate (Uotila & Koivusalo, 1974a,b) would yield formic acid

 and hence carbon dioxide. Thus, a quantitative correlation between the

 amount of carbon monoxide and carbon dioxide exhaled and the activity

 of the two pathways no longer appears to be valid.

 Levels of CO-Hb in the blood, following exposure to methylene

 chloride, are both dose- and time-dependent. Human subjects exposed to

 concentrations of 1770 mg/m3 or less for 1 h have CO-Hb levels of

 1-4%. These levels rose to an average of 10% saturation within 1 h

 after exposure to 3500 mg/m3 for 2 h (Stewart et al., 1972). Hake et

 al. (1975) reported CO-Hb levels in excess of 8% following exposure to

 880 mg/m3 for 7.5 h.

 Human volunteers were exposed to 350 or 1240 mg/m3 for 6 h and

 levels of methylene chloride in blood and exhaled air, CO-Hb and

 exhaled CO were measured. At the end of the 6-h exposure, the CO-Hb

 concentration of the group exposed to 1240 mg/m3 was 1.4 times

 higher than that of the group exposed to the lower dose. Likewise, the

 concentration of exhaled CO in the high-dose group was 2.1 times

 higher than that of the low-dose group. The authors concluded that

 their finding of non-linearity between administered dose and the CO-Hb

 and CO levels is an indication of saturation of the metabolic pathway

 (McKenna et al., 1980).

 Physical exercise performed during exposure to methylene chloride

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 will produce higher blood CO-Hb levels than those found in sedentary

 workers (Åstrand et al., 1975; Di Vincenzo & Kaplan, 1981b). Under a

 moderate workload, an exposure to 350 mg/m3 for 7.5 h may cause a

 CO-Hb saturation of about 5% at the end of the exposure period (Di

 Vincenzo & Kaplan, 1981b). Other factors, including smoking and

 exposure to combustion and automobile exhaust, will increase CO-Hb

 levels.

6.4 Elimination and excretion

6.4.1 Inhalation exposure

 6.4.1.1 Human studies

 Methylene chloride is removed from the body mainly in expired air

 and urine. In four human subjects exposed to methylene chloride

 (350 mg/m3) for 2 h, an average of 22.6 μg methylene chloride was

 excreted in the urine within 24 h after the exposure. In seven

 subjects exposed to 710 mg/m3 for 2 h, the corresponding value was

 81.5 μg (Di Vincenzo et al., 1972). These data show that the amount

 excreted in the urine is insignificant. Methylene chloride excretion

 in expired air was most evident during the first 30 min after

 exposure. Initial post-exposure concentrations of methylene chloride

 in expired breath following 2-and 4-h exposure periods were about

 71 mg/m3 and fell to about 18 mg/m3 at the end of 30 min. Small

 amounts of methylene chloride remained in the expired air at 2.5 h.

 A detailed study of the relationship between the measurements of

 methylene chloride in expired air or blood, carbon monoxide in expired

 air and CO-Hb in blood was undertaken by Di Vincenzo & Kaplan

 (1981a,b). At the end of exposure of non-smoking, sedentary volunteers

 for 7.5 h to methylene chloride vapour concentrations of

 180-710 mg/m3, the mean concentration of the solvent in alveolar air

 and in blood, and the percent CO-Hb saturation were measured, as shown

 in Table 12.

 By 7 h after exposure to any concentration, the expired air

 contained less than 3.5 mg/m3 methylene chloride; at 16 h, only

 negligible levels were detected (Di Vincenzo & Kaplan, 1981a). These

 data suggest that, due to its rapid elimination, measurements of

 methylene chloride in expired air are unsuitable for use as a marker

 of occupational exposure.

 Table 12. Methylene chloride in expired air and blood, and carboxyhaemoglobin

 (CO-Hb) levels of human volunteers following 7.5 h exposure

 (from Di Vincenzo & Kaplan, 1981a)

 

 Methylene Methylene choride Methylene chloride CO-Hb

 chloride exposure in expired air in blood levels

 (mg/m3) (mg/m3) (mg/litre)

 

 180 53 0.3 1.9%

 350 124 0.8 3.4%

 530 194 1.2 5.3%

 710 282 1.8 6.8%

 

 

 Di Vincenzo & Kaplan (1981a) reported that, in a human volunteer

 study, exposure to 180, 350, 530 or 710 mg/m3 for 7.5 h/day (for 5

 days) resulted in peak CO-Hb levels of 1.9, 3.4, 5.3 and 6.8%,

 respectively (Table 12). It was estimated that an 8-h exposure to

 about 530 mg methylene chloride/m3 is equivalent to an 8-h exposure

 to 124 mg carbon monoxide/m3, in as much as either exposure under

 sedentary conditions will increase blood CO-Hb levels to about 5% of

 saturation by the end of the exposure.

 Di Vincenzo & Kaplan (1981b) also investigated the effects of

 exercise and cigarette smoking on the uptake, metabolism and excretion

 of methylene chloride. The effects of smoking and methylene chloride

 exposure on CO-Hb saturation levels were found to be additive.

 Exercise was found to increase the absorption of methylene chloride

 and CO-Hb levels. However, the effects of exercise on CO-Hb were not

 observed to increase with heavy workloads beyond the level achieved

 with moderate work-loads, suggesting a saturation of this effect (see

 also section 5.3.2).

 Engström & Bjurström (1977) found that, during the first 2 h after

 exposure, the concentration in alveolar air tended to be lower and

 declined more rapidly in obese subjects than in slim ones. After this,

 the concentration dropped more slowly in the obese group. During the

 late phase of elimination, the obese subjects tended to have a higher

 concentration in expired air.

 6.4.1.2 Animal studies

 In rats, methylene chloride was excreted in the expired air,

 urine, and faeces following a single 6-h exposure to 180, 1800 or

 53 000 mg methylene chloride/m3 (McKenna et al., 1982). At

 180 mg/m3, only 5% of the exhaled label was found as methylene

 chloride. The remainder was exhaled as CO and CO2. As the exposures

 increased, so did the exhalation of non-metabolized methylene

 chloride. Methylene chloride accounted for 30% of the label from the

 1800 mg/m3 dose and 55% of the label for the 53 000 mg/m3 dose. A

 combination of exhaled methylene chloride, CO2 and CO accounted for

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 58%, 71% and 79% of the inhaled methylene chloride dose for the 180,

 1800 and 53 000 mg/m3 doses, respectively. Urinary excretion

 accounted for 7.2-8.9% of the dose and 1.9-2.3% of the dose was in the

 faeces.

6.4.2 Oral exposure

 Expired air accounted for 78-90% of the excreted dose in rats in

 the 48-h period following a 1 or 50 mg/kg dose of methylene chloride

 in aqueous solution (McKenna & Zempel, 1981). The radiolabel was

 present in the exhaled air as CO and CO2, as well as in expired

 methylene chloride. The amount of methylene chloride in the expired

 air increased from 12% to 72% as the dose was increased from 1 to

 50 mg/kg. Radiolabel in the urine accounted for 2-5% of the dose under

 the above exposure conditions, while 1% or less of the dose was found

 in the faeces. These data indicate that the lungs are the major organ

 of methylene chloride excretion even under oral exposure conditions.

 Mice excreted 40% of the administered dose (100 mg/kg) unchanged in

 expired air within 96 h (Yesair et al., 1977).

6.4.3 Dermal exposure

 No information is available regarding excretion and elimination in

 humans or animals following dermal exposure to methylene chloride.

7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT

 Appraisal

 Algae and aerobic bacteria show no inhibition of growth below

 500 mg/litre. Bacteria which are able to grow in the presence of

 methylene chloride at much higher concentrations (including a

 saturated solution in water) have been identified. Anaerobic bacteria

 are more sensitive; growth inhibition has been observed at 3 mg/litre

 in anaerobic biological sludge.

 In the aquatic environment, fish and amphibian embryos are the

 most sensitive with effects on hatching from 5.5 mg/litre.

 Adult fish are relatively insensitive to methylene chloride. even

 after prolonged exposure (14-day LC50 > 200 mg/litre). The effect

 of methylene chloride on Daphnia is variable; the lowest reported

 EC50 was 135 mg/litre in a closed system.

 In soil, 10 mg/kg strongly decreased the ATP content of the

 biomass, adversely affected the growth of fungi and aerobic bacteria.

 and induced transient inhibition of enzyme activity. The no-observed-

 effect level was 0.1 mg/kg. In earthworms, LC50 values were in the

 range of 300 to more than 1000 μg/cm2 ). In sediment, no toxic

 effects were observed even at very high levels.

 In higher plants, no effects were found after exposure for 14

 days to 100 mg/m3.

7.1 Microorganisms

7.1.1 Bacteria

 7.1.1.1 Aerobic bacteria

 No inhibition of growth was observed at 19.6-19 600 mg/litre

 methylene chloride in Bacillus subtilis, Pseudomonas cepacia and

 Aeromonas hydrophylia (Schubert, 1979). Inhibition of

 bioluminescence of Photobacterium phosphoreum by 50% occurred after

 a 15-min exposure to 2880 mg/litre (Hermens et al., 1985).

 In a standard 16-h growth-inhibition test with Pseudomonas

 putida, a threshold of 500 mg/litre for methylene chloride was

 determined (Bringmann & Kühn, 1977b). The glycolysis of Pseudomonas

 putida was inhibited after a 16-h exposure to 1000 mg/litre

 (Bringmann & Meinck, 1964).

 Nenzda & Seydel (1988) report minimum inhibitory methylene

 chloride concentrations for the bacteria Escherichia coli and

 M. smegmatis of 1049 and 1468 mg/litre, respectively.

 For heterotrophs, 50% inhibition of oxygen consumption occurred at

 320 mg/litre after 24 h (Blum & Speece, 1991).

 With other bacteria (Acinetobacter, Alcaligenes, Flavobacterium,

 Pseudomonas cepacia, Aeromonas hydrophila), stimulation of growth

 was observed at 200 mg/litre (Davis et al., 1981).

 The IC50 for inhibition of multiplication of Escherichia coli

 was 37.2 mg/litre (Nendza & Seydel, 1988).

 In the OECD activated sludge respiration-inhibition test (method

 209) using sealed vessels, the EC50 value for methylene chloride was

 more than 1000 mg/litre after 30 min (Volskay & Grady, 1988).

 Concentrations up to 1000 mg/litre had no effect on the oxygen

 consumption or glucose metabolism of activated sludge acclimated to

 methylene chloride for 3 days (Klecka, 1982).

 In methylene chloride-utilizing bacteria, e.g., Hypho-microbium,

 up to 1700 mg/litre did not interfere with growth (Stucki et al.,

 1981).

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 Blum & Speece (1991) found that the IC50 for reduction of

 ammonia was 1.2 mg/litre after 24 h for Nitrosomonas.

 7.1.1.2 Anaerobic bacteria

 Anaerobic bacteria are more sensitive than aerobic bacteria.

 Methanogenesis of mixed rumen microflora was inhibited from

 136 mg/litre (Bauchop, 1967). At 93 mg/litre, the growth of a mixed

 bacterial population from an anaerobic digester was inhibited by 50%

 (Thiel, 1969). Addition of methylene chloride to anaerobic sludge from

 an operating municipal digester showed, after 48 h, a 20% inhibition

 of gas production at 3 mg/litre and a 50% inhibition at 50 mg/litre

 (Hayes & Bailey, 1977). Addition of methylene chloride to the feed of

 a mixed anaerobic culture, developed in the laboratory from seed from

 a sewage treatment plant, decreased the gas production to such an

 extent that at 3.3 mg/litre it had virtually ceased after 5 days,

 compared with 15 days in controls (Vargas & Ahlert, 1987).

 Blum & Speece (1991) determined the toxicity of methylene chloride

 to methanogenic bacteria and found an IC50 for inhibition of gas

 production of 7.2 mg/litre.

 Stuckey et al. (1980) used a batch system in which methylene

 chloride was added in ethanol to sludge from a laboratory digester in

 a Warburg apparatus. Some inhibition was noted at the lowest

 concentration tested (3.16 mg/litre); the concentration for 50%

 inhibition over 60 h was estimated to be 14 mg/litre.

7.1.2. Protozoa

 The bacteriovorous ciliated protozoan Uronema parduczi Chatton-

 Lwoff was not affected after a 20-h exposure to 16 000 mg/litre (EC5

 inhibition cell proliferation) (Bringmann & Kühn, 1980). Also, no

 effects were observed in Microregma heterostoma after a 28-h

 exposure to 1000 mg/litre (Bringmann & Meinck, 1964).

7.1.3 Algae

 In several freshwater green algae (Selenastrum capricornutum,

 Scenedesmus subspicatus. Scenedesmus quadricauda, Chlorella vulgaris,

 Chlamydomonas angulosa), photosynthesis (chlorophyll a content,

 CO2 uptake) and cell number were only affected by methylene chloride

 from 1450 mg/litre (Bringmann & Kühn, 1978; Hutchinson et al., 1978;

 US EPA, 1980). The threshold (7-day EC3) for effects in the

 cyanobacterium (blue-green alga) Microcystis aeruginosa was

 550 mg/litre (Bringmann & Kühn, 1978).

 In the marine diatom Skeletonema costatum methylene chloride

 exposure had no effect on chlorophyll a content or cell number at

 662 mg/litre (US EPA, 1980).

7.2 Aquatic organisms

 The volatility of methylene chloride presents difficulties in

 aquatic toxicity testing. Therefore, care should be taken when

 interpreting results based on nominal concentrations and open static

 systems. Flow-through systems or closed static systems are necessary

 to conduct adequate toxicity studies on volatile substances. However,

 these systems were not always used (Tables 14 and 15).

7.2.1 Plants

 The EC50 for Lemna minor growth was 2000 mg/litre, whilst both

 growth and photosynthesis of the plant Groenlandia densa were

 totally inhibited at this concentration after 7 days (Merlin et al.,

 1992).

7.2.2 Invertebrates

 7.2.2.1 Insects

 The toxicity of methylene chloride for insects was investigated in

 adult Tribolium confusum and grain weevil (Calandra granaria). The

 LC50 for a 5-h exposure in fumigation vessels was 82 and

 380 mg/litre, respectively (Ferguson & Pirie, 1948; Negherbon, 1959).

 Table 13. Acute aquatic toxicity of methylene chloride to algae

 

 Organism Description Method Parametera Concentration Reference

 (mg/litre)

 

 Diatom Skeletonema costatum Chlorophyll a content, 96-h EC50 > 662 US EPA (1980)

 (salt water) cell number

 Green alga Selenastrum Chlorophyll a content, 96-h EC50 > 662 US EPA (1980)

 capricornutum cell number

 Green alga Scenedesmus Cell number 8-day TT 1450 Bringmann & Kühn

 quadricauda (1978)

 Green alga Chlorella vulgaris CO2 uptake 3-h EC50 2292 Hutchinson et al.

 (1978)

 Green alga Chlamydomonas CO2 uptake 3-h EC50 1477 Hutchinson et al.

 angulosa (1978)

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 Cyanobacterium Microcystis aeruginosa Cell number 8-day TT 550 Bringmann & Kühn

 (blue-green alga) (1978)

 

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

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

 

 pH/dissolved Hardness

 Organism Temperature oxygen (mg CaCO3 Flow/ Parameter Concentration References

 (°C) (mg/litre) per litre) Static (mg/litre) and remarks

 

Crustacean

 Water flea 22 7.4-9.4/ 173 Static 48-h LC50 220 Le Blanc (1980)

 (Daphnia magna) 6.5-9.1 48-h NOEC 68 (nominal concentration

 Water flea 20-22 7.6-7.7 Unknown Static 24-h EC50 2100-2270 Bringmann & Kühn

 (Daphnia magna) 24-h NOEC 1550-1707 (1977a, 1982)

 (nominal concentration)

 Water flea Unknown Unknown Unknown 48-h LC50 1250 Bringmann & Meinck

 (Daphnia magna) (1964)

 Water flea Unknown Unknown Unknown Static 24-h EC50 1959 Kühn et al, (1989)

 (Daphnia magna) 48-h EC50 1682 (closed system)

 Water flea Unknown Unknown Unknown Static 48-h EC50 135 Abernethy et al. (1986)

 (Daphnia magna) (closed system)

 Water flea 18-20 8/8.7-8.8 11.7 Unknown 48-h LC50 480 RIVM (1986)

 (Daphnia magna) 48-h NOEC 100 (nominal concentration

 closed system)

Fish

 Goldfish Unknown Unknown Unknown Static 24-h LC50 420 Jenson (1978)

 (Carrassius (nominal concentration)

 auratus)

 

 Table 14 (Cont'd)

 

 pH/dissolved Hardness

 Organism Temperature oxygen (mg CaCO3 Flow/ Parameter Concentration References

 (°C) (mg/litre) per litre) Static (mg/litre) and remarks

 

 Fathead minnow 12 7.8-8.0/ 67 Static 98-h LC50 310 Alexander et al, (1978)

 (Pimephales > 5 Flow 96-h LC50 193 (static test nominal, flow-

 promelas) (adult) 96-h NOEC 66.3 through measured

 concentrations;

 aquarium covered with

 plastic film for the first

 24-h)

 Fathead minnow 25 Unknown 73-82 Flow 96-h LC50 502 Dill et al. (1987)

 (Pimephales (analysed concentration)

 promelas)

 (juvenile)

 Bluegill, 21-23 6.5-7.9/ 32-48 Static 96-h LC50 220 Buccafusco et al. (1981)

 (Lepomis unknown (nominal concentration,

 macrochirus) aquarium not capped)

 

 Table 15. Acute aquatic toxicity of methylene chloride in salt water

 

 Temperature pH/dissolved Hardness Flow/ References

 Organism (°C) oxygen (mg CaCO3 Stat Parameter Concentration and remarks

 (mg/litre) per litre) (mg/litre)

 

Crustacean

 Mysid shrimp Unknown Unknown Unknown Static 96-h LC50 260 US EPA (1980) (nominal

 (Mysidopsis bahia) concentration)

 Grass shrimp 20±2 6.1-8.0 8-12 Static 48-h LC50 108.5 Burton & Fischer (1990)

 (Palaemonetes pugia) > 4

Fish

 Golden orfe Unknown Unknown Unknown Static 48-h LC50 521-528 Juhnke & Lüdemann (1978)

 (Leuciscus idus)

 Killifish (juvenile) 20±2 6.1-8.0/ Unknown Static 48-h LC50 97.0 Burton & Fischer (1990)

 (Fundulus > 4

 heteroclitus)a

 Sheepshead minnow 25-31 Unknown 10-31 Static 98-h LC50 330 Heitmuller et al. (1981)

 (Cyprinodon 98-h NOEC 130 (nominal concentration)

 variegatus)

 

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a fish died within 1 hour; the measured 1-h LC50 was 135 mg/litre. The 48-h value was the average of the initial and final concentrations.

 

 7.2.2.2 Crustaceans

 Data on the toxicity of methylene chloride to crustaceans are

 presented in Table 14. Daniels et al. (1985) and Knie (1988) reported

 a 48-h LC50 of 27 mg/litre and a 24-h LC50 of 12.5 mg/litre; such

 values are lower than those presented in Table 14 by almost one order

 of magnitude. However, no experimental details were given and,

 therefore, the validity of the data cannot be assessed.

 7.2.2.3 Molluscs

 In seawater, metamorphosis was induced in up to 63% of the larvae

 of the nudibranch mollusc (Phestilla sibogae) when exposed to

 8.5-25.5 mg/litre (Pennington & Hadfield, 1989).

7.2.3 Fish

 7.2.3.1 Acute toxicity

 Data on the acute toxicity of methylene chloride to fish are

 presented in Table 14.

 The acute toxicity of methylene chloride to adult fathead minnows

 (Pimephales promelas) has been studied both in a static and a flow-

 though system. The observed effects (loss of equilibrium,

 melanization, narcosis and swollen, haemorrhaging gills) were

 reversible at a sublethal level (Alexander et al., 1978).

 7.2.3.2 Chronic toxicity and reproduction

 Data on the chronic and embryo-larval toxicity of methylene

 chloride to fish are summarized in Table 16.

 In a 32-day embryo-larval test with fathead minnow (Pimephales

 promelas), the larval survival and weight was affected from 209 and

 142 mg/litre, respectively. The maximum acceptable toxicant

 concentration (MATC) based on body weight was calculated to be

 108 mg/litre. The ratio between the acute 8-day LC50 value and the

 32-day embryo-larval MATC is 4.6, indicating a small difference

 between acute and chronic effects of methylene chloride (Dill et al.,

 1987).

7.2.4 Amphibians

 In closed flow-through systems, short-term embryo-larval tests

 were carried out, from 2 to 6 h post-spawning to 4 days post-hatch, on

 amphibian eggs of Rana catesbeiana. R. palustris and Bufo fowleri

 (hatching times ranged from 3 to 4 days). After combining frequencies

 for lethality and teratogenesis, the analytically determined post-

 hatching LC50s were > 32 mg/litre for the pickerel frog

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

 

 Dissolved Hardness Flow/ References and

 Description Temperature pH oxygen (mg CaCO3 Static Parameter Concentration remarks

 (°C) (mg/litre) per litre) (mg/litre)

 

 Chronic toxicity

 Guppy 22±1 ? > 5 25 daily LC50, 14 days 295 Könemann

 (Poecilia reticulata) renewal (1981) (covered

 with glass,

 nominal)

 Fathead minnow 25±1 6.8-8.6 > 9 73-82 flow LC50, 8 days 471 Dill et al.

 (Pimephales promelas) NOEC, (1987)

 (juvenile) 8 days 357 (analytical

 concentration)

 Embryo-larva toxicity

 Fathead minnow 20.4±0.6 7.8 6.5 95 flow LC50

a 34 Black et al.

 (Pimephales promelas) (1982)

 (embryo-larva)

 Fathead minnow 25±1 6.8-8.6 > 9 73-82 flow LOEC, 32 days 209 Dill et al.

 (Pimephales promelas) (survival) (1987)

 (embryo-larva) LOEC, 32 days 142 (analytical

 (weight) concentration)

 Rainbow trout 13.3±0.3 7.8 9.4 106 flow LC50

a,b 13.1 Black et al.

 (Salmo gairdneri) (1982)

 (embryo)

 

 Table 16 (Cont'd)

 

 Dissolved Hardness Flow/ References and

 Description Temperature pH oxygen (mg CaCO3 Static Parameter Concentration remarks

 (°C) (mg/litre) per litre) (mg/litre)

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 Rice fish 25±1 7.6-8.4 4.5-8.8 11.7 renewal LC50, 3 weeks 106 RIVM (1986)

 (Oryzias latipes) 23±2 3 times/ NOEC, 3 weeks 75 (analytical

 (egg-larva) week concentration)

 

a Eggs were exposed from 30 min after fertilization to 4 days post-hatch

b Teratogenic effects were observed at 5.5 mg/litre

 

 (R. palustris) and Fowler's toad (Bufo fowleri) and 17.78 mg/litre

 for the bullfrog (R. catesbeiana). In the latter, anomalous larvae

 and 16% decreased hatching were observed at 6.73 mg/litre. For the

 pickerel frog and Fowler's toad, hatching was decreased by 14 and 20%

 at 10 and 32 mg/litre, respectively. In the hatched populations

 slightly higher incidences of teratogenic effects were observed (Birge

 et al., 1980).

 Black et al. (1982) exposed several amphibian species to methylene

 chloride from 30 min after fertilization to 4 days post-hatch. Post-

 hatching LC50 values ranging from 16.9 to > 48 mg per litre were

 found for the European common frog (Rana temporaria), Northwestern

 salamander (Arabystoma gracile), African clawed frog (Xenopus

 laevis) and the Leopard frog (R. pipiens). The European common

 frog and the Northwestern salamander were the most sensitive to

 methylene chloride.

7.3 Terrestrial organisms

 The toxicity of methylene chloride to higher plants (Phaseolus

 vulgaris, Raphanus sativus radicula, Lepidum sativum, Trifolium

 pratense, Saintpaula ionatha, Petunia hybrida) was evaluated, using

 the LIS (Landesanstalt für Immissionsschutz, Essen) test; no effect

 was observed at 100 mg/m3 exposure over 14 days (Van Haut & Prinz,

 1979).

 In leaves of alfalfa (Medicago sativa), the effect of methylene

 chloride vapour on the photosynthetic fixation of 14CO2 was

 tested; photosynthesis appeared to be reduced from 388 000 mg/m3

 (Lehman & Paech, 1972).

 In a 48-h filter-paper contact toxicity test on the earthworm

 Eisenia fetida, the LC50 was 304 μg/cm2 in one study and

 > 1000 μg/cm2 in another. Therefore, methylene chloride was

 classified as moderately toxic (100-1000 μg/cm2) (Roberts & Dorough,

 1984; Neuhauser et al., 1985).

 In embryos of White leghorn chicken, the LD50 for injection of

 methylene chloride in the yolk sac is 14 mg/egg (Verrett et al.,

 1980).

7.4 Population and ecosystem effects

7.4.1 Soil microorganisms

 When added to brown soil at 10 mg/kg (dry weight), methylene

 chloride decreased the ATP content of the soil biomass by 80-85%,

 compared to controls, and adversely affected the growth of soil fungi

 and aerobic bacteria after 3 days. A slight recovery was observed by

 the end of the 56-day experiment. Anaerobic bacteria were hardly

 influenced and, in the case of the obligate anaerobic Clostridium

 sp., the growth was even increased. The replacement of oxygen

 probably explained the stimulation of growth in the latter case

 (Kanazawa & Filip, 1987).

 Incubation of soil for 2 months with 1-10 mg/kg (dry weight)

 methylene chloride reduced the activity of ß-glucosidase,

 ß-acetylglucosaminidase and proteinase during the first 28 days, with

 recovery after 2 months; no effect was observed at 0.1 mg/kg (Kanazawa

 & Filip, 1986).

7.4.2 Sediment microorganisms

 In sediment from a freshwater stream, methylene chloride did not

 significantly affect the electron transport system (ETS) activity

 during a 1-h enzymatic assay at 66 500 mg/kg. When assayed over an

 11-day period, 1330-66 500 mg/kg caused a fluctuating stimulation of

 ETS activity, which may indicate a marked alteration of the stability

 of the biological activity in the sediment. Microbial respiration,

 measured by CO2 evolution, was inhibited (EC50) after 7 days

 at 15 500 mg/kg. However, when measured by oxygen uptake, it was

 stimulated at up to 26 500 mg/kg (Trevors, 1985).

7.4.3 Microcosms and mesocosms

 Microcosms composed of water plants (Elodea canadensis, Lemna

 minor), algae (Scenedesmus subspicatus) and snails (Physa sp.)

 were exposed to 500 or 1000 mg/litre for 21 days. At 1000 mg/litre a

 decrease in oxygen content of the water was observed, together with

 mortality in snails and algae, as well as necrosis on fronds of Lemna

 minor. The photosynthesis of the plants was inhibited. These effects

 were less at 500 mg/litre, but this concentration was still lethal to

 snails. In outdoor mesocosms, containing a large diversity of species,

 initial concentrations of 137-156 mg/litre did not induce any toxicity

 (Merlin et al., 1992).

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8. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS

8.1 Single exposure

8.1.1 Acute toxicity data

 See Table 17.

8.1.2 Oral administration

 Musculoskeletal disturbances were found in Sprague-Dawley rats at

 doses of 530 mg/kg or more. Hypotension, hypothermia and haematuria

 were also noted (dose threshold not reported). The gastrointestinal

 tract was found to be congested with micro-haemorrhages or partial

 destruction from doses of 530 mg/kg or more. Blood CO-Hb increased in

 a dose-related manner (Laham, 1978).

 Various effects have been reported following the acute

 administration of large oral doses of methylene chloride. The effects

 include a decreased cytochrome P-450 content in liver microsomes of

 Sprague-Dawley rats receiving 1000 mg/kg (Moody et al., 1981), CNS

 effects and evidence of pathological changes in the liver and kidney

 of Wistar rats receiving 2000 mg/kg (Janssen & Pott, 1988a), evidence

 of liver necrosis and increased glucose-6-phosphatase activity in male

 rats (strain unspecified) receiving 2210 mg/kg (Reynolds & Yee, 1967),

 and decreased hepatic secretion of triglycerides followed by an

 increased hepatic triglyceride content in male mice receiving

 2700 mg/kg (Selan & Evans, 1982).

 No liver toxicity was found in male Wistar rats receiving up to

 4400 mg/kg by oral administration (Danni et al., 1981).

 Liver damage was investigated in Sprague-Dawley rats exposed to up

 to 1275 mg/kg given orally. Serum ALT activity was unaffected, as was

 liver cytochrome P-450 and glutathione content. However, increased

 ornithine decarboxylase activity and DNA damage were found in the

 liver (Kitchin & Brown, 1989).

8.1.3 Inhalation administration

 8.1.3.1 Rat

 Behavioural changes and CNS disturbances were found in several

 studies. Decreased running activity was found in rats exposed to

 17 700 mg/m3 for 1.5 h (Heppel & Neal, 1944); hypothermia,

 hypotension and convulsion in Sprague-Dawley rats from 28 200 mg/m3

 (6-h exposure) (Laham, 1978); CNS depression in Alderley-Park rats at

 31 800 mg/m3 (10-min EC50) (Clark & Tinston, 1982) and in rats at

 40 000 mg/m3 (2-h exposure) (Ulanova & Yonovskayo, 1959); and

 dyspnoea and anaesthesia in rats from 53 000 mg/m3 (30-min exposure)

 (Schumacher & Grandjean, 1960; Kashin et al., 1980).

 Table 17. Acute toxicity of methylene chloride

 

 Species Route Vehicle Parameter Concentration Reference

 

 Rat (Wistar, male) oral none LD50 1710-2250 mg/kg Klimmer (1988)

 Rat CDF (F-344) oral none LD50 1530-2524 mg/kg Carreon (1981)

 Rat (Sprague-Dawley) oral none LD50 2120 mg/kg Kimura et al, (1971)

 young male

 Rat (Sprague-Dawley)

 - male oral none LD50 2280 mg/kg Laham (1978)

 - female oral none LD50 1410 mg/kg

 Mouse (CF-1, male) oral unknown LD50 1987 mg/kg Aviado et al. (1977a,b)

 Dog oral mucillage of LD50 3000 mg/kg Barsoum & Saad

 acacia (1934)

 Rat (Alderley Park) inhalation 15-min LC50 197 790 mg/m3 Clark & Tinston (1982)

 Rat (Sprague-Dawley) inhalation 6-h LC50 52 000 mg/m3 Bonnet et al. (1980)

 male

 Rat (Sprague-Dawley) inhalation 6-h LC50 > 28 000 mg/m3 Landry et al, (1981)

 Mouse (CF-1, male) inhalation 20-min LC50 92 680 mg/m3 Aviado et al. (1977a,b)

 Mouse (LF1, female) inhalation 6-h LC50 49 100 mg/m3 Gradiski et al, (1978)

 Mouse (ICR, male) inhalation 6-h LC50 55 870 mg/m3 Scott et al. (1979)

 Guinea-pig inhalation 6-h LC50 40 200 mg/m3 Balmer et al. (1976)

 

 Table 17 (Cont'd)

 

 Species Route Vehicle Parameter Concentration Reference

 

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 Rat (Sprague-Dawley) intratracheal ALDa 350 mg/kg McCarty et al. (1992)

 male

 Mouse (CF-1, male) intraperitoneal unknown LD50 448 mg/kg Aviado et at. (1977a,b)

 Mouse intraperitoneal unknown LD50 500 mg/kg Schumacher &

 Grandjean (1960)

 Mouse (Swiss-Webster, intraperitoneal corn oil LD50 1990 mg/kg Klaassen & Plaa (1966)

 male)

 Dog intraperitoneal corn oil LD50 1260 mg/kg Klaassen & Plaa (1967)

 Mouse subcutaneous olive oil LD50 6500 mg/kg Kutob & Plaa (1962)

 

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

 

 Studies on sleeping patterns in Wistar rats by measuring

 electroencephalographic (EEG) and electromyographic (EMG) activity

 have shown a dose-related increase in total sleeping time and

 intervals between rapid eye movement (REM) sleep when the rats were

 exposed to 1770, 3500 or 10 600 mg/m3 (500, 1000 or 3000 ppm)

 methylene chloride for 3 h. During exposure to 3500 or 10 600 mg/m3,

 the percentage of sleep defined as REM decreased by nearly 20% (Fodor

 & Winneke, 1971; Fodor et al., 1973). Studies using similar techniques

 on the narcotic effects of methylene chloride in rats (strain

 unspecified) have been reported by Berger & Fodor (1968). Rats were

 exposed to a range of concentrations of methylene chloride from 9900

 to 99 000 mg/m3 (2800 to 28000 ppm) for unspecified periods of time.

 There was an initial period of excitation followed by deep narcosis

 with a decrease in muscle tone and EEG activity and subsequent

 breathing difficulties. Cessation of electrical activity was noted

 after a 1.5-h exposure to 88 000 or 99 000 mg/m3 (25 000 or

 28 000 ppm) and after a 6-h exposure to 54 000 or 64 000 mg/m3

 (16000 or 18000 ppm). Following exposure to 17 700 to 31 800 mg/m3

 (5000 to 9000 ppm) methylene chloride, long periods of sleep occurred

 without desynchronization phases. Following exposure to concentrations

 of methylene chloride below 17 700 mg/m3 (5000 ppm), there were no

 measurable effects on either EEG or EMG activity (Berger & Fodor,

 1968).

 Exposure of F-344 rats to 7100 mg/m3 for 2.5 h caused

 statistically significant changes in somatosensory evoked responses

 and EEG. The lack of effect of 157 mg carbon monoxide/m3 (which

 induces a CO-Hb level of 10%, comparable with that produced by

 exposure to 7100 mg methylene chloride/m3) on evoked responses,

 indicated that the effects were probably due to methylene chloride

 itself and not to its principal metabolite carbon monoxide (Mattsson

 et al., 1988). Alterations in somatosensory evoked potentials were

 also observed after a 1-h exposure of F-344 rats to dose levels of

 17 700 mg/m3 or more (Rebert et al., 1989).

 No macroscopic lesions were found in rats at the 6-h LC50 of

 53 000 mg/m3 (Bonnet et al., 1980). Congestion of various organs, as

 well as oedema of the brain, heart, lungs and hip region, was noted

 following exposure to 71 000 mg/m3 (6-h exposure) in Sprague-Dawley

 rats (Laham, 1978).

 Increased CO-Hb levels were found from 1770 mg/m3 in various

 strains of rats (MacEwen et al, 1972; Laham, 1978; Dill et al., 1978;

 Kurppa et al., 1981). Ascorbic acid content of the liver was increased

 in rats exposed to 40 000 mg/m3 for 2 h (Ulanova & Yonovskayo,

 1959), but no effect on cytochrome P-450 or specific liver enzymes was

 noted in Wistar rats after a 3-h exposure to 3500 mg/m3 or in

 Sprague-Dawley rats after a 5-min (repeated 5 times) exposure to

 350 mg/m3, except for increased microsomal and decreased lysosomal

 ß-glucuronidase activity (Kurppa et al., 1981).

 Intratracheal administration of methylene chloride in Sprague-

 Dawley rats showed lethal levels at 350 mg/kg (corresponding to 17.5%

 of the oral LD50), death occurring in a few seconds. This result

 emphasises that aspiration of methylene chloride may present more of a

 hazard than oral ingestion (McCarty et al., 1992).

 8.1.3.2 Mouse

 CNS depression resulting in reversible narcosis was reported in

 mice exposed to between 14 100 and 52 200 mg/m3 methylene chloride

 for 2-6 h (Flury & Zernik, 1931) or to 47 700 mg/m3 for 128 min

 (EC50) (Kashin et al., 1980). This effect was also noted in CF-1

 mice exposed to 35 300 mg/m3 for 20 min (Aviado et al., 1977a,b) or

 in Swiss mice exposed to 45 900 mg/m3 for 7 h (Svirbely et al.,

 1947). Exposure at 35 000 mg/m3 for 2 h was the minimal CNS

 effective concentration found in the mouse (Lazarew, 1929).

 Ability to learn a simple passive avoidance task, 1-4 days after

 exposure, was reduced in Swiss-Webster mice exposed to 168 000 mg/m3

 for 20 seconds (Alexeef & Kiglore, 1983).

 Fatty changes were noted in the liver and, less frequently, in the

 kidney from 56 500 mg/m3 after a 7-h exposure (Svirbely et al.,

 1947). Microscopic lesions in the liver, kidneys, lungs and adrenal

 were found (no dose-response relationship) in mice exposed to lethal

 doses for 6 h (Gradiski et al., 1978).

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 Cardiac sensitization to the effects of adrenaline was reported in

 Swiss mice exposed to 710 000 mg/m3 for 6 min) (Aviado & Belej,

 1974).

 Male NMRI mice were exposed to methylene chloride by inhalation in

 a study of tolerance, i.e. decreased responsiveness to a chemical that

 arises as a result of previous exposure to the same chemical

 (Kjellstrand et al., 1990). Motor activity measured with Doppler radar

 units was used to monitor the behavioural reactions of the animals.

 Motor activity increased on exposure to methylene chloride and

 decreased on constant exposure. Termination of exposure was followed

 by hypoactivity.

 8.1.3.3 Other animals

 After 6 h of exposure to 17 700 mg/m3, the concentration of

 triglycerides was increased in the liver of guinea-pigs, and reduced

 in the serum (Bulmer et al., 1976; Morris et al., 1979).

 Histopathological liver changes, consisting of the appearance of lipid

 droplets, were first seen in guinea-pigs at 17 700 mg/m3 (Morris et

 al., 1979). Slight to moderate vacuolation in the liver of guinea-pigs

 was seen after a 6-h exposure to 38 800 mg/m3. In addition, lungs

 showed congestion and haemorrhage. Behavioural changes were also noted

 (Balmer et al., 1976).

 No effect on blood pressure, heart rate or EEG activity was found

 in rabbits exposed to 90 000 mg/m3 for 2 h (Truhaut et al, 1972).

 Only serum AST was significantly increased.

 In Beagle dogs, ECG changes as well as decreased blood pressure,

 heart and respiratory rate were found at 141 200 mg/m3 (7-h

 exposure). Behavioural effects were already noted after a 1-h exposure

 to 53 000 mg/m3 (reduced reflexes) (Von Oettingen et al., 1950).

 Increased CO-Hb values, but no change in haematocrit, haemoglobin

 concentration or red cell count, were reported in dogs and rhesus

 monkeys exposed for 24 h to 17 700 mg/m3. Slight pathological

 changes in the liver (fatty changes, vacuolization) could be

 attributed to the treatment (MacEwen et al., 1972).

 Cardiac effects such as arrhythmia, tachycardia and hypotension

 were found in monkeys, dogs and rabbits exposed for 1-5 min to levels

 of methylene chloride exceeding 35 300 mg/m3 (Belej et al., 1974;

 Adams & Erickson, 1976; Taylor et al., 1976; Aviado et al., 1977a,b;

 Aviado, 1978).

 One study on rabbits showed allergic reactions after inhalation

 (Shmuter & Kashin, 1978). However, the experimental protocol of this

 study is questionable and the result has not been confirmed.

8.1.4 Dermal administration

 No effect was noted on rats receiving a dermal application of

 methylene chloride of 710 mg/kg for 0.5 to 4 h, except for an increase

 in CO-Hb levels (Makisimov & Mamleyeva, 1977). Only slight behavioural

 effects and macroscopic changes in the liver (swelling) were found in

 Wistar rats receiving 2000 mg/kg under an occlusive dressing for 24 h

 (Janssen & Pot, 1988b). Haemoglobinuria was observed in rats when

 abdominal skin was immersed in methylene chloride for 2-20 min

 (Schutz, 1960).

8.1.5 Intraperitoneal administration

 A single intraperitoneal injection of 510 mg methylene chloride/kg

 in rats slowed down the sciatic motor conduction velocity by 11% and

 gave rise to a CO-Hb level of 6.8% (Pankow et al., 1979).

 Signs of CNS depression were found in Wistar rats and CFI mice

 receiving, respectively, 1060 mg/kg (with phenobarbital pretreatment)

 and 114 mg/kg (Aviado et al., 1977; Masuda et al., 1980).

 Various biological investigations in rats and mice showed a dose-

 related increase in serum AST and/or ALT activity at > 660 mg/kg.

 No effect was observed on glucose-6-phosphatase activity, cytochrome

 P-450 content or BSP retention (Klaassen & Plaa, 1966; Cornish et al.,

 1973; Masuda et al, 1980; Corsi et al., 1983). Increased ALT activity

 was reported in mongrel dogs receiving 800 mg/kg in corn oil (Klaassen

 & Plaa, 1967). No histological changes in the liver could be found in

 rats exposed to < 1300 mg/kg (Cornish et al., 1973; Corsi et al.,

 1983). However, mild hepatic inflammation was noted in Swiss Webster

 mice receiving lethal doses of methylene chloride (2000 mg/kg)

 (Klaassen & Plaa, 1966). Renal tubular changes were noted in F-344

 rats administered intrapertinoneally with 1330 mg/kg in corn oil

 (Kluwe et al., 1982) and in Swiss-Webster mice receiving 2000 mg/kg in

 corn oil (Klaassen & Plaa, 1966). However, no renal histological

 change could be found in Swiss mice receiving 1300 mg/kg in corn oil

 (Plaa & Larson, 1965). Slight histological changes were noted in the

 liver and kidneys of mongrel dogs receiving 1300 mg/kg (Klaassen &

 Plaa, 1967).

8.1.6 Intravenous administration

 The minimum lethal concentration in anaesthetized dogs was found

 to be 200 mg/kg in olive oil after intravenous administration (Barsoum

 & Saad, 1934).

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 rotation in Sprague-Dawley rats during intravenous perfusion of

 5.1 mg/kg per min for 60 min (Tham et al., 1984).

 Following a single intravenous injection of 3.1, 6.2 or

 12.4 mmol/kg, methylene chloride was found to sensitize the myocardium

 of rats to arrhythmia development in response to catecholamines. The

 release by methylene chloride of endogenous catecholamines is possibly

 a cause of these modified cardiovascular actions (Mueller et al.,

 1991).

8.1.7 Subcutaneous administration

 The minimum lethal concentration in the rabbit was found to be

 2700 mg/kg (Barsoum & Saad, 1934). Prolongation of phenobarbital-

 induced sleeping time occurred at 1700 mg/kg in Swiss mice. No effect

 on liver function or histology was observed at up to 5000 mg/kg (Kutob

 & Plaa, 1962).

8.1.8 Appraisal

 The acute toxicity of methylene chloride by inhalation and oral

 administration is low. The inhalation 6-h LC50 values for all

 species lie between 40 200 and 55 870 mg/m3. Oral LD50 values of

 1410-3000 mg/kg have been recorded. Acute effects after methylene

 chloride administration by various routes of exposure are primarily

 associated with the central nervous system (CNS) and the liver. CNS

 disturbances were found at 14 100 mg/m3 or more and slight changes

 in EEG at 1770 mg/m3. Slight histological changes in the liver

 were found at concentrations of 17 700 mg/m3 or more. In the mouse,

 but not in the rat, effects on the lungs restricted to the Clara

 cells were observed after exposure to 7100 mg/m3. Occasionally the

 kidney is affected. Cardiac sensitization to adrenaline-induced

 arrhythmias has been reported, and cardiovascular effects were seen

 at concentrations above 35 000 mg/m3. However, the effects were

 inconsistent.

8.2 Short-term exposure

8.2.1 Oral administration

 CD1 mice given doses of up to 665 mg/kg per day of methylene

 chloride in corn oil by gavage for 14 days did not show any effect on

 liver enzymes. Microscopic examination revealed slight vacuolation in

 the liver from 333 mg/kg. No damage to the kidneys was reported

 (Condie et al., 1983).

8.2.2 Subcutaneous administration

 Reduction of the systolic blood pressure of hypertensive Sprague-

 Dawley rats was reported after subcutaneous exposure to 2000 mg/kg

 twice a week for 17 weeks. No effect was found in normotensive rats.

 Very slight histopathological changes in the liver and lungs of

 hypertensive rats were described (Loyke, 1973).

8.2.3 Inhalation administration

 8.2.3.1 Rat

 Changes in Sprague-Dawley rats were reported after exposure to

 methylene chloride (3500 mg/m3) 2 h/day for 15 days; decreased body

 weight, increased hepatic lipid peroxidation, and high concentrations

 of methylene chloride in the brain, kidney and blood immediately after

 inhalation were observed (Ito et al., 1990). Similar hepatic effects

 (hypertrophic hepatocytes, and increased lipid peroxidation and

 glutathione peroxidase activity) were reported in male Wistar rats

 exposed to 3500 mg/m3 2 h/day for 20 consecutive days (Takashita et

 al., 1991). Biochemical tests were performed on the serum and brain of

 groups of six male Sprague-Dawley rats which were exposed to 250, 1100

 or 3500 mg/m3 6 h/day for 3 days and killed 16-18 h later. A

 selective reduction in dopamine concentration, with changes in

 dopamine turnover in some forebrain dopamine nerve terminal systems,

 was reported. A dose-dependent increase in noradrenaline turnover in

 the anterior periventricular hypothalamic area and dose-dependent

 decreased noradrenaline concentration in the posterior periventricular

 area were observed. No significant changes were reported in the

 secretion of anterior pituitary hormones (Fuxe et al., 1984).

 Groups of five male and five female F-344 rats were exposed to

 methylene chloride at concentrations of 5740, 11 500, 22 900, 45 900

 or 56 500 mg/m3, 6 h/day for 19 days. Intermittent scratching,

 ataxia and hyperactivity were seen in all rats exposed to

 22 900 mg/m3 or more. Dyspnoea and anaesthesia were observed in

 animals exposed to 45 900 mg/m3 or more. Some deaths were also

 observed at these concentrations (NTP, 1986).

 No, or limited, lesions were found in the liver and lungs of F-344

 rats, exposed to 7100 or 14 100 mg/m3, 6 h/day for 10 days, after

 light and electron microscopic examination (Hext et al., 1986). No

 effect on the liver was found in rats (strain unspecified) after

 inhalation of 880 mg/m3, for 5 h/day for 28 days (Norpoth et al.,

 1974)

 Inhalation exposure of Sprague-Dawley rats to methylene chloride

 concentrations of 12 800 mg/m3 (5 h/day, 5 days/week) for 4 weeks

 revealed an inflammatory response and cell damage in the lungs as

 demonstrated by the increase biochemical response (enzymatic and nonMethylene chloride (EHC 164, 1996, 2nd edition) http://www.inchem.org/documents/ehc/ehc/ehc164.htm

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 enzymatic) observed in cell-free lavage effluents from the lungs (Sahn

 & Lowther, 1981).

 8.2.3.2 Other animals

 Increased liver weight and increased mitotic activity in

 hepatocytes were observed in male B6C3F1 mice after 2 weeks of

 repeated exposure to 14 100 mg/m3 6 h/day for up to 3 weeks

 (Eisenbrandt & Reitz, 1986). Necrosis of occasional epithelial cells

 in the bronchi and bronchioles, together with reactive hyperplasia of

 adjacent lymphoid tissue, was observed in a few animals.

 Groups of five male and five female B6C3F1 mice were exposed to

 methylene chloride at concentrations of 5740, 11 500, 22 900, 45 900

 or 56 500 mg/m3, 6 h/day for 19 days. Hyperactivity (dose-related)

 was seen in exposed mice, but no exposure-related pathological

 findings were observed. Some deaths occurred in mice exposed to

 45 900 mg/m3 or more (NTP, 1986).

 CD-1 mice, Golden Syrian hamsters, Sprague-Dawley and CDF (F-344)

 rats were exposed to 0, 8800, 17 700 or 28 200 mg/m3, 6 h/day,

 5 days/week, for a total of 19, 18, 19 or 7 exposures, respectively,

 over a period of 21 to 28 days. Animals exposed to 28 000 mg/m3

 showed anaesthetic effects and a decrease in the body weight of rats.

 At 18 000 mg/m3 there was slight anaesthesia, decreased body weight

 in male rats, increased aspartate aminotransferase (ASAT) in female

 mice and Sprague-Dawley rats, and increased liver weights in female

 mice, hamsters and rats. At 8800 mg/m3, the animals exhibited more

 scratching activity than the controls, but showed no other effects

 attributable to exposure (Nitschke et al., 1981)

 Following exposure to 17 700 mg/m3, a reduction in body weight

 of mice was observed, and relative liver weights were increased up to

 the end of the 7-days of continuous exposure. Fatty infiltration, an

 increase in the triglyceride concentration and hydropic degeneration

 of the endoplasmic reticulum gradually disappeared. Protein synthesis

 was depressed. Necrosis was observed in a few hepatocytes (Weinstein

 et al., 1972).

 Carboxyhaemoglobin levels were raised after continuous exposure of

 monkeys to 88.25 mg/m3 (25 ppm) for 28 days (MacEwen & Vernot, 1972;

 Haun et al., 1972).

 A group of NMRI mice was continuously exposed to 130-1059 mg/m3

 (37-300 ppm) for 30 days, while another was intermittently exposed for

 1-12 h/day to 2118-25 416 mg/m3 (600-7200 ppm) for 30 days,

 corresponding to an average exposure (24-h mean value) of 1059 mg/m3

 (300 ppm). In addition, groups of mice were continuously exposed to

 1059 mg/m3 for 4, 8, 15 and 90 days. The blood level of

 butyrylcholinesterase was significantly increased from 265 mg/m3

 (75 ppm) in continuously exposed male mice and after intermittent

 exposure in male rats. Moreover, liver weight was significantly

 increased in a dose-related manner from 265 mg/m

3 (75 ppm) and

 330 mg/m3 (150 ppm) in male and female mice, respectively. Finally,

 fatty accumulation was found in both sexes at 265 mg/m3 (75 ppm) or

 more. All effects were reversible (Kjellstrand et al., 1986). CO-Hb

 levels were raised after continuous exposure of monkeys to

 88.25 mg/m3 (25 ppm) for 28 days (MacEwen et al., 1972, Haun et al.,

 1972).

8.3 Long-term exposure

8.3.1 Rat

 8.3.1.1 Inhalation exposure

 In a study by Leuschner et al. 1984, 20 male and 20 female

 Sprague-Dawley rats were exposed to 35 g/m3, 6 h/day for 90 days.

 Haematological, clinical chemistry and urinary parameters were

 measured and histological examinations were performed. A slight

 redness of the conjunctiva lasting for 1-10 h was observed after each

 exposure. No other treatment-related signs of toxicity were reported.

 Groups of 10 male and 10 female F-344 rats were exposed to

 methylene chloride at concentrations of 1850, 3700, 7400, 14 800 and

 29 700 mg/m3 for 6 h/day, 5 days/week for 13 weeks. One male and one

 female rat exposed to 29 700 mg/m3 died before the end of the study,

 whereas none of the control rats died. Foreign body pneumonia was

 observed in some rats exposed to > 7410 mg/m3. The mean body

 weight in males and females exposed to 29 700 mg/m3 was lower than

 in controls. Liver lipid to liver weight ratios were statistically

 significantly reduced in both males and females exposed to

 29 700 mg/m3 and in females exposed to 14 800 mg/m3 when compared

 to controls (NTP, 1986).

 Male and female F-344 rats were exposed to 177, 710 or

 7100 mg/m3 6 h/day, 5 days/week for 13 weeks. No treatment-related

 alterations in sensory evoked potentials (flash, auditory brainstem,

 somatosensory or caudal nerve) or neuropathology were observed at any

 of the exposure levels (Mattsson et al., 1990).

 CNS depression was found in rats during each daily session of

 repeated exposure to 35 000 mg/m3 7 h/day, 5 days/week for 6 months

 (Heppel et al., 1944).

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 Rats (sex and strain unspecified) were continuously exposed to

 either 88 or 350 mg/m3 for 100 days. Slight cytoplasmatic

 vacuolization with positive fat stains in the liver and tubular

 degeneration in the kidney were observed (Haun et al., 1972).

 8.3.1.2 Oral exposure

 Rats receiving methylene chloride in the drinking-water at a

 concentration of 125 mg/litre for 13 weeks did not show any effects on

 behaviour, body weight, haematology, urinalysis, blood glucose level,

 plasma free fatty acids, or the oestrous cycle (Bornmann & Loeser,

 1967).

 Groups of 20 male and 20 female Fischer-344 rats were given 0.15,

 0.45, and 1.50% methylene chloride in drinking-water for 3 months,

 equivalent to 166, 420 and 1200 mg/kg per day, respectively, for males

 and 209, 607, and 1469 mg/kg per day for females. Slightly decreased

 body weights were observed in mid-dose males and high-dose females

 throughout the study. There were no differences between treated and

 control animals with regard to mortality, physical observations, food

 consumption or gross necropsy results. There were no exposure-related

 effects observed following the histopathological evaluation of rat

 tissues from the 1 month interim necropsies. However, hepatocellular

 changes were observed following treatment for 3 months; central

 lobular necrosis, granulomatous foci, ceroid or lipofuscin

 accumulation, and cytoplasmic eosinophilic bodies were observed in

 high-dose males and females and in some mid-dose females. A dose-

 dependent increased incidence of hepatocyte vacuolation was also

 observed, many of the vacuoles containing lipid which was generalized

 or concentrated in the central lobular region (Kirschman et al.,

 1986).

8.3.2 Mouse

 8.3.2.1 Inhalation exposure

 Groups of 10 male and 10 female B6C3F, mice were exposed to

 methylene chloride at concentrations of 1850, 3700, 7400, 14 800 or

 29 700 mg/m3 for 6 h/day, 5 days/week for 13 weeks. Exposure-related

 deaths were seen in some mice exposed to 29 700 mg/m3. Hepatic

 centrilobular hydropic degeneration was observed in males and females

 exposed to 29 700 mg/m3 and in females exposed to 14 800 mg/m3.

 Both mean body weights and liver lipid to liver weight ratios were

 reduced in males and females exposed to 29 700 mg/m3 when compared

 to controls (NTP, 1986).

 Mice (strain and sex unspecified) were continuously exposed to

 either 88 or 350 mg/m3 for 100 days. Slight cytoplasmatic

 vacuolization was found at both dose levels, and a decrease in the

 microsomal cytochrome P-450 content was found in the liver of mice

 exposed to methylene chloride at 350 mg/m3 (Haun et al., 1972).

 Female ICR mice were continuously exposed to 350 mg/m3 for 10

 weeks. Fatty infiltration, vacuolization and enlarged hepatocyte

 nuclei persisted up to the end of the exposure period. A reversible

 increase in plasma triglycerides was also observed (Weinstein &

 Diamond, 1972).

 8.3.2.2 Oral exposure

 Groups of 20 male and 20 female B6C3F1 mice were given 0.15,

 0.45 and 1.50% methylene chloride in drinking-water for 3 months,

 equivalent to 226, 587 and 1911 mg/kg per day, respectively, for males

 and 231, 586 and 2030 mg/kg per day for females. Slightly lower body

 weights were observed in mid-dose and high-dose males and in females

 from week 6 to the end of the study. Treated and control animals did

 not differ with respect to physical and ophthalmological observations

 or food consumption. There were no exposure-related effects observed

 following the histopathological evaluation of mice following a 1-month

 exposure. However, after 3 months of exposure, subtle centrilobular

 fatty changes in the liver were observed, these being most prominent

 in mice receiving either 587 or 1911 mg/kg per day. No other exposure-

 related changes were reported (Kirschmann et al., 1986).

8.3.3 Other animals

 Heppel et al. (1944) did not find organ lesions related to

 exposure at 17 700 mg/m3 (7 h/day, 5 days/week for 6 months) in

 studies on dogs, monkeys, rats, rabbits and guinea-pigs, with the

 exception of moderate centrilobular fatty degeneration of the liver

 and pneumonia in 3 out of 14 guinea-pigs. CNS depression was found in

 all species following exposure to 35 000 mg/m3; all animals became

 inactive, sometimes after initial excitement. At 35 000 mg/m3, dogs

 also showed fatty degeneration of the liver.

 Hepatic changes (slight cytoplasmatic vacuolation) and vacuolar

 changes in the renal tubules were found in dogs exposed continuously

 to 3500 mg/m3 for up to 100 days. Abnormal haematology and increased

 activity of serum enzymes were reported after 4 weeks. Oedema of the

 brain was observed at a concentration of 17 350 mg/m3 (Haun et al.,

 1972).

 Three male and three female beagle dogs were exposed to

 17 700 mg/m3, 6 h/day for 90 days. Haematology, clinical chemistry

 and urinary parameters were measured and ECG and circulatory functions

 were examined. At the end of the study, histological examinations were

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 performed. Slight sedation was induced throughout the exposure period

 and all dogs had slight erythema, lasting up to 10 h after exposure.

 No deaths and no other signs of toxicity were observed (Leuschner et

 al., 1984).

 Decreased levels of neurotransmitter amino acids were observed in

 gerbil brains after continuous inhalation exposure to methylene

 chloride (340 mg/m3) for 3 months (Briving et al., 1986). In gerbils

 exposed by inhalation to 1240 mg/m3 for 3 months, followed by a 4-

 month solvent-free period, increased brain concentrations of two

 astroglial proteins and decreased levels of DNA in the hippocampus and

 cerebellum were observed (Rosengren et al., 1986). Decreased

 hippocampal DNA levels were also observed in gerbils exposed to

 740 mg/m3 (Rosengren et al., 1986; Karlsson et al., 1987). It was

 suggested by the authors that this effect may have been the result of

 the loss of nerve cells.

8.3.4 Appraisal

 Prolonged exposure to high concentrations of methylene chloride

 (> 17 700 mg/m3 ) caused reversible CNS effects, slight eye

 irritation and mortality in several laboratory species. Body weight

 reduction was observed in rats at 3500 mg/m3 and in mice at

> 17 700 mg/m3 . After intermittent exposure, effects on the liver

 were observed in rats at 3500 mg/m3 and in mice at 14 100 mg/nz3 .

 After continuous exposure, slight cytoplasmatic vacuolization in the

 liver of both rats and mice were found at 88 and 350 mg/m3.

 No evidence of irreversible neurological damage was seen in rats

 exposed by inhalation to concentrations of < 7100 mg/m3 for 13

 weeks.

 Oral administration of methylene chloride to rats caused effects

 on the liver with a no-observed-effect level of 125 mg/m3.

8.4 Skin and eye irritation; sensitization

8.4.1 Skin irritation

 Application of 0.5 ml methylene chloride to rabbits for 24 h,

 under a semi-occlusive patch on abraded or intact skin, caused severe

 erythema and oedema with necrosis and acanthosis (Duprat et al.,

 1976). Rabbits exposed to 0.5 ml methylene chloride for 4 h under

 occlusive patch test condition, either with or without simultaneous

 exposure to other chlorinated solvents, showed moderate skin

 irritation but no corrosive effect (Van Beek, 1990).

8.4.2 Eye irritation

 Duprat et al. (1976) and Ballantyne et al. (1976) exposed rabbits

 once to 0.1 ml methylene chloride by ocular instillation. Moderate to

 severe changes were seen in the conjunctiva, together with increased

 corneal thickness and intra-ocular tension. All effects were

 reversible. Vapour exposure of the eyes to 17 700 mg/m3 caused

 slight increases in corneal thickness and intra-ocular tension.

8.4.3 Sensitization

 No data are available.

8.4.4 Appraisal

 Liquid methylene chloride is moderately irritant to the skin and

 eyes in experimental animals.

8.5 Developmental and reproductive toxicity

8.5.1 Developmental toxicity

 When groups of Sprague-Dawley rats and Swiss-Webster mice were

 exposed to methylene chloride at a concentration of 4400 mg/m3 on

 days 6-15 of pregnancy for 7 h/day, maternal body weight in the mice

 was increased and the dams of both rats and mice had CO-Hb levels as

 high as 12.5% during exposure. In both species, an increased incidence

 of minor skeletal anomalies was observed, i.e. dilated renal pelvis in

 rats and extra sternebrae in mice (Schwetz et al., 1975). No

 significant teratogenic or fetotoxic effects were observed in either

 species.

 Groups of 18 rats were exposed before and/or during 17 days of

 pregnancy to a methylene chloride concentration of 16 250 mg/m3 for

 6 h/day. The exposed dams exhibited increased blood CO-Hb levels,

 ranging from 7.1 to 10.1%, and increased relative and absolute liver

 weights. Fetal body weight was decreased, but no increase in the

 incidences of dead fetuses and/or resorptions nor any skeletal and/or

 visceral malformations were observed (Hardin & Manson, 1980). After

 exposure to methylene chloride using the same experimental conditions,

 litters from four groups of 10 rats were used for behavioural testing.

 Body weight gain, food and water consumption, wheel running activity

 and avoidance learning were all unaffected by the exposure. However,

 changes in the general activity of pups were found in both sexes

 starting at the age of 10 days, and were still present in male

 offspring at the age of 150 days. The effects cannot be definitely and

 directly attributed to methylene chloride, however, since elevated

 maternal CO-Hb- or methylene chloride-induced changes in maternal-

 litter interactions could have been contributing factors (Bornschein

 et al., 1980).

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 Groups of seven female Wistar rats were fed methylene chloride at

 levels of 0.04, 0.4 and 4.0% in their diet from days 0-20 of

 pregnancy. Fetuses were examined on day 20 and neonatal growth was

 measured for 8 weeks after birth. Maternal body weight was

 significantly reduced in the 4.0% group. Although a reduction in the

 fetal weight of the females in the 0.4% group was observed, there were

 no differences in any group in the number of implantations and

 resorptions. No external malformations were observed by fetal,

 skeletal and visceral examination, and no differences were observed in

 any group in the frequency of delayed ossifications or in the dilation

 of the renal pelvis. A decrease in postnatal weight gain and in

 absolute liver weight was found in the 0.04% group males at the 8th

 week after birth (Nishio et al., 1984).

 Groups of F-344 rats (number not specified) received methylene

 chloride by gavage (dose level not specified) in corn oil on gestation

 days 6- 15. The compound was tested with at least two dose levels plus

 a concurrent control group, the high dose (not specified) being

 selected to cause maternal toxicity. The dams were allowed to deliver

 and their litters were examined post-natally. Although a small change

 in maternal weight was found, no effects on litters were reported

 (Narotsky et al., 1992).

8.5.2 Reproductive toxicity

 When rats received methylene chloride in the drinking-water at a

 level of 125 mg/litre during a period of 13 weeks before mating, no

 effects were found on the female fertility index, litter size,

 survival of pups at 4 weeks or the number of resorptions (Bornmann &

 Loeser, 1967).

 A two-generation inhalation study was conducted to evaluate the

 effects of inhaled methylene chloride on the reproductive capability,

 neonatal growth and survival of rats (Nitschke et al., 1988b). Groups

 of 30 male and 30 female 6-week-old F-344 rats (F0) were exposed to

 0, 350, 1770 or 5300 mg/m3 (6 h/day, 5 days/week for 14 weeks).

 After this exposure, F0 animals were allowed to mate using one male

 and one female of the respective treatment groups to produce the F1

 litters. After weaning, 30 males and 30 females (4 weeks old) from

 each treatment group were randomly selected and assigned to the

 respective exposure groups. After exposure to the relevant

 concentration of methylene chloride (6 h/day, 5 days/week for 17

 weeks), the F1 adults were allowed to mate to produce F2 litters.

 Reproductive parameters examined included fertility, litter size and

 neonatal growth and survival. All adults and selected weanlings were

 examined for grossly visible lesions. No adverse effects on

 reproductive parameters, neonatal survival or neonatal growth were

 noted in animals exposed to methylene chloride in either the F0 or

 F1 generations. Similarly, there were no treatment-related gross

 pathological changes in F0 and F1 adults or F1 and F2

 weanlings; histopathological examination of tissues did not reveal any

 lesions in F1 and F2 weanlings attributable to exposure to

 methylene chloride. Therefore, the results of this study indicate that

 exposure to concentrations as high as 5300 mg/m3 does not affect the

 normal reproductive function of rats (Nitschke et al., 1988b).

8.5.3 Appraisal

 Methylene chloride is not teratogenic in rats or mice at

 concentrations up to 16 250 mg/m3 . No evidence of an effect on the

 incidence of skeletal malformations or other developmental effects

 was observed in three animal studies. Small effects on either fetal

 or maternal body weights were reported at 4400 mg/m

3 . A two-

 generation reproductive toxicity study in rats exposed to methylene

 chloride by inhalation at concentrations of up to 5300 mg/m3 ,

 6 h/day, 5 days/week, did not show evidence of an adverse effect on

 any reproductive parameter, neonatal survival or neonatal growth in

 either the F0 or F1 generation.

8.6 Mutagenicity and related end-points

 Studies on the mutagenic potential of methylene chloride have been

 performed on bacteria, fungi and cultured mammalian cells. Results

 from in vivo studies on mice and rats have also been reported.

8.6.1 In vitro

 8.6.1.1 Bacteria

 Methylene chloride is mutagenic when tested using the Ames

 protocol in Salmonella typhimurium TA98, TA100 and TA 1535

 (Table 18). The number of revertants increased 3- to 7-fold in a dose-

 related manner when plates were exposed to vapour of methylene

 chloride of undisclosed purity at levels ranging from 20 100 up to

 201 000 mg/m3. Metabolic activation by either induced rat liver S9

 fraction, cytosol fraction, or microsomal fraction increased the

 mutagenicity of methylene chloride (Simmon et al., 1977; Jongen et

 al., 1978, 1982; McGregor, 1979; Kirwin et al., 1980; Barber et al.,

 1980; Nestmann et al., 1980, 1981; Gocke et al., 1981; Dillon et al.,

 1990). In this respect the cytosolic fraction was more active than the

 microsomal fraction (Green, 1983). A positive result was reported in

 strains TA98 and TA100 with and without 30% hamster liver S9 (Zeiger

 et al., 1990) using the vapour phase (desiccator procedure) protocol.

 Negative mutagenicity results were obtained in studies not using

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 vapour phase exposure (Rapson et al., 1980; Nestmann et al., 1980). No

 mutagenic activity was found when methylene chloride was tested in

 Salmonella typhimurium strains TA100, TA1535, TA1537, TA97 and TA98

 with or without the addition of 10% or 30% rat/hamster liver S9, using

 the preincubation protocol (Zeiger et al., 1990).

 Metabolic studies of methylene chloride (see section 6.3) indicate

 that the conjugation of methylene chloride with glutathione (GSH),

 catalysed by cytosolic glutathione- S-transferase, may play a role in

 the observed mutagenicity of methylene chloride in Salmonella.

 However, the direct reaction of glutathione with methylene chloride

 only produced a very small increase in mutagenicity (Jongen et al.,

 1982). In another study, Salmonella typhimurium TA100 and the GSH-

 deficient strain TA100 gsh were exposed to 0-5% methylene chloride

 using a vapour phase protocol. The mutagenic response, with and

 without Aroclor-induced rat-liver S9, microsomes or cytosol, was

 marginally higher at the highest methylene chloride concentrations.

 Salmonella typhimurium TA100 gsh was slightly less responsive than

 TA100 at high doses in the absence of S9. This difference was not seen

 in the presence of S9. The addition of exogenous GSH had only a small

 effect on the mutagenic response in TA100 or TA100 gsh in the absence

 or presence of S9. According to the authors, these data suggest that

 if the interaction between methylene chloride and GSH is responsible

 for the observed mutagenicity, it occurs at extremely low levels of

 intracellular GSH and is not significantly affected by exogenous GSH

 (Dillon et al., 1992).

 The mutagenicity of methylene chloride has also been studied in a

 variety of other microbial systems using a vapour phase protocol.

 Mutagenic effects were observed in E. coli WP2uvrA and pKM101 which

 were exposed to 0-5% methylene chloride using a vapour phase protocol

 (Dillon et al., 1992). These strains of E. coli are deficient in

 glutathione, containing approximately 25% of the level of glutathione

 present in the strain TA100.

 Table 18. In vitro mutagenicity assays

 

 Assay Strain/type S9 Dose Resulta Observations Reference

 activation

 

 Salmonella TA100, ± 10% 100-10 000 -ve Preincubation protocol Zeiger et al.

 typhimurium TA1535, or 30% μg//plate (1990)

 TA1537, rat or

 TA97, TA98 hamster

 liver S9

 Salmonella TA100, TA98 ± 30% 0.1-1.0 +ve Vapour protocol-dessicator Zeiger et al.

 typhimurium rat or ml/chamber procedure (1990)

 hamster

 liver S9

 Salmonella TA100, ±S9 -ve Standard plate-incorporation protocol Nestmann et al.

 typhimurium TA1535, (1980)

 TA1537,

 TA98, TA1538

 Salmonella TA100, +ve 0.5μl in open glass dish within a Nestmann et al.

 typhimurium TA1535 desiccator; revertants: 2-fold (1980)

 increase in TA1535; 6-fold in TA100

 Salmonella TA1535, ± S9 up to 750/μl in +ve Active in strains TA98 and TA100 Gocke et al.

 typhimurium TA100, a 9 litre (1981)

 TA1538, desiccator

 TA98, TA1537

 Salmonella TA100 ±S9 0-1.4% +ve Significant increases in mutagenic Jongen et al.

 typhimurium activity by addition of rat liver (1982)

 cytosol fraction; marginal increases

 by addition of microsomal fraction

 

 Table 18 (Cont'd)

 

 Assay Strain/type S9 Dose Resulta Observations Reference

 activation

 

 Salmonella TA100, ±S9 0-5% for 2, 4, 6 +ve Vapour phase protocol. Data Dillon et al.

 typhimurium 100gsh or 48 h suggest interaction between (1992)

 (glutathione methylene chloride and GSH

 deficient) responsible for the mutagenic

 activity

 Salmonella TA100 S9 2.8 v/v +ve Green (1983)

 typhimurium

 Salmonella TA100 -S9 0, 50-800/μl +ve Vapour phase protocol; dose-related Simmon et al.

 typhimurium per 9 litre increase in the number of revertants, (1977)

 desiccator with a mutation rate over 7-fold

 (approx 18-318 higher than controls at 320 mg/m3;

 mg/m3) for 7 h two experiments were conducted

 Salmonella TA98, TA100 ±S9 0, 20, 100 to +ve Vapour phase protocol; S9 prepared Jongen et al.

 typhimurium 201 000 mg per m3 from the livers of phenobarbital (1978)

 (5 concentrations) pretreated rats; a dose-related

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 for 48 h increase of up to 5-8 fold (mean

 value for 3 experiments) was seen,

 slightly higher in the presence of

 S9; toxicity was noted at the

 highest dose level

 

 Table 18 (Cont'd)

 

 Assay Strain/type S9 Dose Resulta Observations Reference

 activation

 

 Salmonella TA100 Not stated Vapour phase protocol; time-course Jongen (1984)

 typhimurium study to evaluate the most

 appropriate exposure time for

 maximum differentiation of the

 methylene chloride induced

 reversion rates in the presence and

 absence of a metabolic activation

 system; the maximum differentation

 was obtained following 4-6 h

 exposure, and, consequently, an

 exposure time of 6 h was used by

 these workers in the study of Jongen

 et al. (1982)

 Salmonella TA100 ±S9 0 or 1 ml/9 litre +ve Study summarized in review, original Simmon &

 typhimurium desiccator data were not available; vapour Kauhanen (1978)

 (approx 390 mg phase control; S9) from livers of

 per m3) for 6.5 Aroclor-pretreated rats; addition of

 or 8 h S9 increased the mutation rate 1.5

 fold; no further details are available

 Salmonella TA98, TA100 ±S9 0,125, 250, 500 +ve Vapour phase control; S9 from livers Rapson et al.

 typhimurium or 750/μl per of Aroclor pretreated rats; dose- (1980)

 9 litre desiccator related increase in the mutation rate,

 (0-293 mg/m3) with an approximate 10-fold increase

 for 8 h for both strains at the highest dose

 used; the presence of S9 resulted in

 a slightly higher mutation rate

 

 Table 18 (Cont'd)

 

 Assay Strain/type S9 Dose Resulta Observations Reference

 activation

 

 Salmonella TA1535, Not stated +ve Same study, but no data were given

 typhimurium TA1537, for the strains studied; there is no

 TA1538 evidence for an independent

 confirmatory experiment

 Salmonella TA1535, 0.5 ml/desiccator -ve Vapour phase protocol; result Nestmann et al.

 typhimurium TA1537, (volume unknown) doubling of revertants for strain (1980)

 TA1538, TA1535 and a 6-fold increase for

 TA98, TA100 strain TA100 when 0.5 ml methylene

 chloride was added directly to the

 culture rather than a seperate dish

 Salmonella TA100 ±S9 0, 98 800, +ve Vapour phase control; S9 from livers Green (1983)

 typhimurium 177 000, or of Aroclor pretreated rats; a dose-

 297 000 mg/m3 related increase in the mutation rate

 for 3 days was observed

 0 or 98 800 +ve Vapour phase protocol; the presence

 mg/m3 for 3 of S9 enhanced the mutation rate

 days 1.19 fold; on dividing the S9 material

 into microsomal and high-speed

 supernatant (cytosolic) fractions, only

 the high-speed supernatant enhanced

 (1.27 fold) the mutation rate;

 a small isotope effect was observed

 when 2H-methylene chloride was

 substituted for 1H-methylene chloride

 

 Table 18 (Cont'd)

 

 Assay Strain/type S9 Dose Resulta Observations Reference

 activation

 

 Direct examination of the metabolism

 of methylene chloride by the

 bacterium indicated that radio-

 labelled carbon dioxide and trace

 amounts of radiolabelled carbon

 monoxide were formed, together

 with considerable incorporation of

 radioactivity into endogenous

 materials

 Salmonella TA 1535 0, 24 700, +ve Vapour phase protocol; data McGregor (1979)

 typhimurium 42 400, 81 200, available in summary form only;

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 162 400 or dose-response relationship; TA100

 331 800 mg per m3; was reported to give a more marked

 condensation response

 of methylene

 chloride onto

 agar plates

 occurred

 Salmonella TA1535, ±S9 0, 38, 76, 96 or +ve Vapour phase protocol data available Barber et al.

 typhimurium TA98, TA100 115 μmol/plate in summary form only; dose-response (1980)

 (0-38 500 mg relationship (source of S9 not

 per m3 vapour) stated) (negative results were

 in gas-tight obtained in studies not employing

 chambers gas-tight jars)

 

 Table 18 (Cont'd)

 

 Assay Strain/type S9 Dose Resulta Observations Reference

 activation

 

 Salmonella TA1535, +S9 Concentration +ve Vapour phase protocol; study Longstaff et al.

 typhimurium TA100 range not reported only briefly, methylene (1984)

 stated; exposure chloride being used as a positive

 for 72 h control; S9 prepared from livers of

 Aroclor pretreated rats. Mutation rate

 increased 6-fold for TA1535 (with a

 50% methylene chloride-air mixture)

 and 2.4 fold for TA100 (with a 1%

 methylene chloride-air mixture); the

 increased mutation rate was

 reproducible; apparently, one strain

 at least showed a positive dose

 response

 Salmonella TA100 0.1-1000μg -ve Plate-incorporation assays; Rapson et al.

 typhimurium per plate (5 insufficient information was given to (1980)

 concentrations), evaluate the result

 one plate per

 concentration

 Salmonella TA1535, ± S9 Up to 26 -ve Plate-incorporation assays; S9 from Nestmann et al.

 typhimurium TA1537, mg/plate, livers of Aroclor-induced rats; tested (1980)

 TA1538, dissolved in to limit of toxicity; positive results

 TA98, TA100 dimethyl-sulfoxide for 3 pro-mutagens were obtained using

 the same batch of S9, but not

 necessarily in parallel incubations; a

 second, independent assay was

 conducted over a limited concentration

 range; this was not a satisfactory

 demonstration of a negative

 response

 

 Table 18 (Cont'd)

 

 Assay Strain/type S9 Dose Resulta Observations Reference

 activation

 

 Escherichia WP2uvrA, ±S9 0-5% for 2, 4, 6 +ve Vapour phase protocol; data suggest Dillon et al.

 coli pKM101 or 48 h interaction between methylene (1992)

 chloride and GSH responsible for the

 mutagenic activity

 Microscreen E. coli WP2S ±S9 0.78-100 +ve Rossman et al.

 ( ) μl/well (1991)

 Saccharomyces D7 0-209 mM +ve Induced mitiotic gene convertants Callen et al.

 cerevisiae and recombinants, and, to a lesser (1980)

 extent, gene revertants

 Aspergillus Diploid P1 0-0.8 v/v +ve Crebelli et al.

 nidulans (1988, 1992)

 Sister Human +ve Thilagar et al.

 chromatid peripheral (1984a,b)

 exchange lymphocytes,

 CHO cells,

 mouse

 lymphoma

 L5178Y cells

 Sister Chinese ±S9 0-5000 μg/ml -ve Standard (25-29 h after treatment) Anderson et al.

 chromatid hamster harvest time (1990)

 exchange ovary (CHO)

 cells

 

 Table 18 (Cont'd)

 

 Assay Strain/type S9 Dose Resulta Observations Reference

 activation

 

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 Sister CHO cells ±S9 0-15 μl/ml -ve Thilagar &

 chromatid Kumaroo (1983)

 exchange

 Sister Chinese 0-4% ± Marginal (< 2-fold), reproducible Jongen et al.

 chromatid hamster V79 increases in frequency; not dose- (1981)

 exchange cells related

 Chromosome Human Not stated +ve Thilagar et al.

 aberration peripheral (1984a,b)

 lymphocytes,

 CHO; mouse

 lymphoma

 L5178Y cells

 Chromosome CHO cells ±S9 0-15 μl/ml +ve Dose-dependent increase Thilagar &

 aberration Kumaroo (1983)

 Chromosome CHO cells ±S9 0-5000 μg/ml -ve Standard (10-14 h after treatment) Anderson et al.

 aberration harvest time (1990)

 Cells in vitro Chinese 0.5-5% v/v -ve Forward mutation to 6-thioguanine Jongen et al.

 hamster cells resistance; mutation rate was (1981)

 corrected for survival

 Cells in vitro Epithelial -S9 0, 35 (300- -ve Varying the expression time was Jongen et al.

 cells (V79) 141 000 mg/m3 reported to have no effect; cell (1981)

 (4 concentrations survival was reduced by about 20%

 for 1 h); expression at 141 000 mg/m3

 time of 6 days

 

 Table 18 (Cont'd)

 

 Assay Strain/type S9 Dose Resulta Observations Reference

 activation

 

 Cell mutation L5178Y Not stated -ve Thilagar et al.

 mouse (1984a,b)

 lymphoma

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

 mouse activity (1990)

 lymphoma

 Cell Primary 0.5 ml/4.6 litre +ve Enhanced transformation by SA7 Hatch et al.

 transformation Syrian chamber adenovirus (1983)

 hamster

 embyro cells

 HGPRT- Chinese 0-4% -ve Jongen et al.

 deficient hamster V79 (1981)

 cells

 Micronucleus Chinese Not stated -ve Gu & Wang

 hamster V79 (1988)

 cells

 Unscheduled Primary rat Not stated -ve Trueman et al.

 DNA synthesis hepatocytes (1987)

 Unscheduled Primary rat Not stated ± Thilagar et al.

 DNA synthesis hepatocytes (1984a)

 Cell BALB/C-3T3 0.01% -ve Price et al.

 transformation mouse (1978)

 

 Table 18 (Cont'd)

 

 Assay Strain/type S9 Dose Resulta Observations Reference

 activation

 

 Cell C3H-10T1/2 Not stated -ve Thilagar et al.

 transformation CL8 mouse (1984a)

 Unscheduled Primary rat ± A "marginal" positive result reported Thilagar et al.

 DNA synthesis hepatocytes (1984a)

 Unscheduled Human ±S9 2.5-10.0 μl/ml -ve Perocco & Prodi

 DNA synthesis lymphocytes (1981)

 Unscheduled Chinese 0-5% -ve Jongen et al.

 DNA synthesis hamster V79 (1981)

 cells

 DNA repair Primary rat 0.7-16.0 mM -ve Andrae & Wolff

 synthesis hepatocytes (1983)

 

a +ve = positive; -ve = negative; ± = equivocal or inconclusive

 

 A recent study demonstrated that glutathione- S-transferase 5-5

 expression in Salmonella typhimurium increases mutation rates caused

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 by methylene chloride. The plasmid pKK233-2 containing rat

 glutathione- S-transferase 5-5 cDNA, either in the correct or reverse

 direction, was transfected into TA1535. The resulting sense-

 transformed TA1535 (RSJ 100) expressed the enzyme and enhanced base-

 pair revertants as compared to the anti-sense strain (TPT 100).

 Mutagenicity was not seen when GSH, purified glutathione- S-

 transferase and a methylene dihalide such as methylene bromide were

 added to the pre-incubation mixture with TA1535. Formaldehyde did not

 produce mutations in any of the three strains (Thier et al., 1993).

 The nature and distribution of forward mutations in the N-terminal

 region of the loc I gene of excision repair-proficient (Uvr+) and

 excision repair-defective (Uvr B-) strains of E. coli have been

 described by Zielenska et al. (1993). A total of 116 locI-d

 mutations were characterized.

 8.6.1.2 Fungi and yeasts

 A dose-related increase in the frequency of gene conversions,

 mitotic recombinations, and reversions was found for cultures of

 Saccharomyces cerevisiae strain D7, but not for strains D4 and D3,

 exposed to methylene chloride of undisclosed purity. However, the

 mutagenic results in D7 occurred at toxic doses (1270 g/m3) in which

 survival of the yeast cells was reduced to 42% (Cullen et al., 1980).

 When assayed for the induction of mitotic segregation in

 Aspergillus nidulans P1, methylene chloride significantly increased

 the frequency of morphologically abnormal colonies, which produced

 euploid whole-chromosome segregants (Crebelli et al., 1988; Crebelli

 et al., 1992).

 8.6.1.3 Mutation in mammalian cells

 Methylene chloride was not mutagenic in several tests in which

 mammalian somatic or human cells were used (Gocke et al., 1981; Jongen

 et al., 1981; Perocco & Prodi, 1981; Andrue & Wolff, 1983; Burek et

 al., 1984).

 Both negative (Thilagar et al., 1984a) and questionable results

 (Myhr et al., 1990) were reported when methylene chloride was tested

 for gene mutations in a L5178Y mouse lymphoma assay at the thymidine

 kinuse locus. No increase in micronuclei was found when methylene

 chloride was tested in Chinese hamster V79 cells (Gu & Wang, 1988).

 8.6.1.4 Chromosomal effects

 Studies on chromosome morphology in cultured mammalian cells

 indicate that methylene chloride is clastogenic. Chromosomni

 alterations (chromatid damage, chromosomni exchanges, but no increase

 in sister chromatid exchanges) were observed in CHO cells (Thilagar &

 Kumaroo, 1983), human lymphocytes and L5178Y cells (Thilagar et al.,

 1984a,b), both with and without metabolic activation. A small increase

 in sister-chromatid exchanges (SCEs), without clear evidence of a

 dose-response relationship, was found in V79 cells when exposed to

 gaseous methylene chloride at concentrations up to 5% (Jongen et al.,

 1981 ). A dose-related increase in SCEs was observed in CHO cells

 after a 24-h exposure to methylene chloride. The results were

 statistically significant only at the highest concentration (7%) and

 exposures of shorter duration (2, 4 or 6 h) were without effect

 (McCaroll et al., 1983). In a more recent study, Anderson et al.

 (1990) reported no increase in chromosomal aberrations or SCEs in CHO

 cells exposed to up to 5 mg/ml.

 Hallier et al. (1993) described an apparent polymorphism in human

 blood samples used to measure SCEs in lymphocytes. Those blood samples

 possessing metabolic activity (conjugators) were inactive in the

 assay, whereas those samples which were metabolically inactive (non-

 conjugators) produced significant increases in SCEs (see also section

 6.3.1).

 8.6.1.5 DNA damage

 Concentrations of up to 16 mM methylene chloride failed to induce

 unscheduled DNA synthesis (UDS) in cultured rat hepatocyte, although

 some reduction in replicative DNA synthesis occurred at the higher

 doses (Andrae & Wolff, 1983). The absence of evidence of methylene

 chloride-induced UDS in primary rat hepatocytes was also reported by

 Trueman et al. (1987). Thilagar et al. (1984a) reported a "marginal"

 positive result in a primary rat hepatocytes UDS assay, but details

 were not available. In experiments using no exogenous activation

 systems, exposure of hamster V79 cells or human fibroblasts (AH cells)

 to methylene chloride concentrations of between 0.5 and 5% did not

 induce UDS (Jongen et al., 1981). A non-specific but reversible

 inhibition of replicative DNA synthesis was observed in both cell

 lines, probably due to a metabolic block of synthesis. Methylene

 chloride in doses of 2.5, 5 or 10 μ/ml did not induce UDS in human

 lymphocytes in either the presence or absence of rat liver S9 (Perocco

 & Prodi, 1981).

 8.6.1.6 DNA binding in vitro

 Several studies have investigated the potential of methylene

 chloride and its metabolites to bind covalently to DNA. Incubation of

14C-labelled methylene chloride failed to detect any DNA binding,

 but binding to proteins and lipids was observed (Cunningham et al.,

 1981). After incubation of calf thymus DNA in vitro with 14C-

 labelled methylene chloride (0.8 μmol/ml), together with hepatic

 microsomes and a NADPH-generating system, there was no evidence of any

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 DNA alkylation (Di Renzo et al., 1982). These studies confirm those of

 an earlier in vitro study which failed to detect any DNA binding of

 methylene chloride or its metabolites (Anders et al., 1977).

 8.6.1.7 Cell transformation

 Methylene chloride has been tested for its ability to induce

 transformation in a variety of cell systems. Negative results were

 obtained in C3H-10T1/2 CL8 mouse cells at 10 μl/ml (Thilagar et al.,

 1984a) and in Balb/C-3T3 mouse cells at 0.01% (Price et al., 1978).

 Methylene chloride significantly enhanced the frequency of

 transformation by SA7 virus in a dose-related manner (Hatch et al.,

 1983).

8.6.2 In vivo

 8.6.2.1 Chromosome damage

 Large doses of methylene chloride (425, 850 and 1700 mg/kg) given

 twice intraperitoneally to NMRI mice did not increase micronuclei in

 the bone marrow micronucleus test (Gocke et al., 1981). Doses of up to

 4 g/kg body weight (the maximum tolerated dose) administered by gavage

 to C57BL/6J/Alpk mice also failed to induce any increase in bone

 marrow micronuclei (Sheldon et al., 1987). The results of in vivo

 mutagenicity assays are presented in Table 19.

 Intraperitoneal injections of methylene chloride (100, 1000, 1500

 or 2000 mg/kg) did not increase the frequencies of either SCEs or

 chromosome aberrations in bone marrow cells of male C57BL/6J mice

 (Wesforook-Collins et al., 1988; Wesforook-Collins et al., 1990).

 No increase in the frequency of either SCEs or chromosome

 aberrations was observed in bone marrow cells of female B6C3F1 mice

 after a single subcutaneous injection of methylene chloride (2500 or

 5000 mg/kg) (Westbrook-Collins et al., 1989; Allen et al., 1990).

 Inhalation exposure of female B6C3F1 mice to 14 000 or

 28 000 mg/m3 for 10 days (6 h/day, 5 days/week) resulted in slight

 increases in the frequency of SCEs in lung cells and peripheral blood

 lymphocytes, in chromosome aberrations in lung and bone marrow cells,

 and in micronuclei in peripheral blood erythrocytes. The results were

 statistically significant at 28 200 mg/m3 for all end-points. At

 14 100 mg/m3, statistical significance was reached only for the SCE

 frequency in lung cells. A marginal increase in lung cell SCEs and

 micronuclei in peripheral blood erythrocytes was observed following a

 3-month inhalation exposure to 7100 mg/m3 (Westbrook-Collins et al.,

 Table 19. In vivo mutagenicity assays

 

 Assay Strain/type Resulta Observations Reference

 

 Chromosome Male C57BL/6 mouse bone -ve i.p., 0-2000 mg/kg Westbrook-Collins et al. (1988);

 aberration marrow Westbrook-Collins et al. (1990)

 Chromosome Female B6C3F1 mouse bone -ve s.c., 2500 or 5000 mg/kg Westbrook-Collins et al. (1989);

 aberration marrow Allen et al. (1990)

 Chromosome Female B6C3F1 mouse bone ± inhalation, 14 100 or 28 200 mg/m3 for Westbrook-Collins et al. (1989);

 aberration marrow 10 days Allen et al. (1990)

 Chromosome Rat bone marrow -ve inhalation, 1770, 3500 or 12 400 mg/m3 Burek et al. (1984)

 aberration 6 h/day, 5 days/week for 6 months

 Sister-chromatid Male C57BL/6 mouse bone -ve i.p. 0-2000 mg/kg Westbrook-Collins et al. (1988);

 exchange marrow Westbrook-Collins et al. (1990)

 Sister-chromatid Female B6C3F1 mouse bone -ve s.c., 2500 or 5000 mg/kg Westbrook-Collins et al. (1989);

 exchange marrow Allen et al. (1990)

 Sister-chromatid Female B6C3F1 mouse lung ± inhalation, 14 100 or 28 200 mg/m3 for Westbrook-Collins et al. (1989);

 exchange cells, peripheral blood 10 days Allen et al, (1990)

 lymphocytes

 Sister-chromatid Female B6C3F1 mouse lung ± inhalation, 7100 mg/m3 for 3 months; Westbrook-Collins et al. (1989);

 exchange cells small increase Allen et al. (1990)

 Synaptonemal Male C57BL/6 mouse ± i.p., 0-1500 mg/kg Westbrook-Collins et al. (1988)

 complex

 (damage at

 meiotic

 prophase)

 

 Table 19 (Cont'd)

 

 Assay Strain/type Resulta Observations Reference

 

 Micronucleus Female B6C3F1 mouse ± inhalation, 14 100 or 28 200 mg/m3 for Westbrook-Collins et al. (1989);

 peripheral blood 10 days Allen et al. (1990)

 erythrocytes

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 Micronucleus Female B6C3F1 mouse ± inhalation, 7100 mg/m3 for 3 months Westbrook-Collins et al. (1988);

 peripheral blood Allen et al. (1990)

 erythrocytes

 Micronucleus C57BL/6 mouse bone -ve oral in corn oil, up to 4 g/kg Sheldon et al. (1987)

 marrow

 Unscheduled Male Alpk: AP rat -ve oral gavage, 100, 500 or 1000 mg/kg; Trueman et al. (1987)

 DNA synthesis hepatocytes autoradiography 4 and 12 h after

 treatment

 Unscheduled Male B6C3F1 mouse -ve oral gavage, 100 mg/kg in corn oil Lefevre & Ashby (1989)

 DNA synthesis hepatocytes

 Unscheduled Male Fischer-344 rat, and -ve inhalation, 7100 or 14 100 mg/m3 for 2 Trueman & Ashby (1987)

 DNA synthesis male B6C3F1 mouse or 6 h

 hepatocytes

 Unscheduled Male B6C3F1 mouse -ve inhalation, 14 100 mg/m3 for 2 h Lefevre & Ashby (1989)

 DNA synthesis hepatocytes

 Dominant lethal Male Swiss-Webster mouse -ve s.c., 5 ml/kg, 5% or 10% v/v in corn oil, Raje et al. (1988)

 3/week for 4 weeks

 inhalation at 350, 530 or 710 mg/m3, Basso et al., (1987);

 2 h/day, 5 days/week for 6 weeks Raje et al. (1988)

 no microscopic lesions of testes or brain

 

 Table 19 (Cont'd)

 

 Assay Strain/type Resulta Observations Reference

 

 Drosphila Berlin K, Basc -ve 0 to 14 260 mg/m3 for 6 h, 1 week or 2 Kramers et al. (1991)

 melanogaster weeks; sex-linked recessive lethal assay,

 and somatic mutation and recombination

 test

 Drosphila Berlin K, Basc +ve 125 or 620 mM; increased frequency of Gocke et al. (1981)

 melanogaster recessive lethals in Basc test

 

a +ve = positive; -ve = negative; ± = equivocal or inconclusive

 

 1989; Allen et al., 1990). Background data and positive control

 results were not available. Chromosome aberration data (excluding

 gaps) were not reported, and so the increases might not be

 significant. Increases in SCE and micronuclei were small (up to 2-fold

 at 28 000 mg/m3).

 No increase in chromosomal aberrations was observed in bone marrow

 cells of Sprague-Dawley rats (5 of each sex per group) following

 inhalation exposure to 1770, 3500 or 12 400 mg/m

3 (6 h/day,

 5 days/week) for 6 months (Burek et al., 1984).

 Inconclusive results were reported when methylene chloride was

 tested in C57B1/6 mice for its ability to induce damage in the

 synaptosomal complex, an experimental end-point which can reveal

 induced damage at the meiotic prophase, following intraperitoneal

 injection of 1500 mg/kg (Westbrook-Collins et al., 1988).

 8.6.2.2 Drosophila

 No mutagenicity was detected in the recessive lethal test on

 Drosophila melanogaster fed, or injected with, 1-2% methylene

 chloride (Abrahamson & Valencia, 1980). A marginal increase in the

 number of recessive deaths was found after feeding 125 or 650 nmol

 methylene chloride in 2% dimethylsulfoxide to Drosophila melanogaster

 (Gocke et al., 1981). This study may not be reliable because control

 values from different solvent treatments were pooled, and because the

 increases seen were significant only when results from the two dose

 levels were combined.

 Methylene chloride was not active in the sex-linked recessive

 lethal assay or the somatic mutation and recombination test carried

 out with Drosophila melanogaster using inhalation exposure up to

 14 260 mg/m3 (Kramers et al., 1991).

 8.6.2.3 DNA damage

 Methylene chloride has been evaluated for its ability to initiate

 unscheduled DNA synthesis (UDS) in the livers of male mice and rats

 in vivo. Alpk:AP rats were exposed by oral garage to 100, 500 or

 1000 mg/kg body weight, and hepatocytes were assessed for UDS via

 autoradiography 4 and 12 h later (Trueman & Ashby, 1987). In a second

 study, F-344 rats or B6C3F1 mice were exposed by inhalation to

 either 7100 or 14 100 mg/m3 for 2 or 6 h, and hepatocytes were

 assessed for UDS immediately after exposure. In both studies,

 methylene chloride failed to induce UDS. Similar results were reported

 by Lefevre & Ashby (1989) following exposure of male B6C3F1 mice

 either by oral gavage in corn oil (1000 mg/kg) or by inhalation of an

 atmosphere containing 14 100 mg/m3 for 2 h.

 Slightly increased DNA damage, measured by alkaline elution of DNA

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 from hepatocytes of rats orally exposed to methylene chloride

 (2.55 g/kg) in corn oil for 24 h, has been reported (Kitchin & Brown,

 1989).

 DNA single strand breaks were found in mouse hepatocytes after in

 vivo exposure to 14 120 mg/m3 for 3 or 6 h but not in hepatocytes

 of similarly exposed rats. It was further observed in vitro that the

 lowest concentration of methylene chloride needed to induce DNA single

 strand breaks in mouse hepatocytes was 75 times below that in rat

 hepatocytes. This DNA damage was not accompanied by cytotoxicity. The

 relation of these findings to the mechanism for carcinogenic effects

 is discussed in section 8.8.1.

 8.6.2.4 DNA binding

 Several studies have investigated the potential of methylene

 chloride to bind covalently to DNA after in vivo exposure. DNA has

 been isolated from the livers and lungs of mice and rats exposed to

 14 100 mg/m3 (Green et al., 1987a,b,c). In both studies the DNA was

 hydrolysed and analysed by chromatography to distinguish between

 alkylation of DNA and incorporation of radioactivity though the C-1

 pool. No evidence of alkylation was found, both studies having the

 power to detect one alkylation per 106 nucleotides.

 No alkylation of DNA was reported by Ottenwalder & Peter (1989),

 following a DNA binding assay of methylene chloride in rats and mice.

 Male mice and hamsters were exposed to 14 100 mg/m3, 6 h/day for

 2 days, followed on the third day by a 6-h exposure to a decreasing

 concentration (15 900 to 8800 mg/m3) of 14C-labelled methylene

 chloride (Casanova et al., 1992). DNA-protein cross-links (DPX) were

 detected in mouse liver, but not in mouse lung or hamster liver or

 lung. The failure to detect DPX in mouse lung did not exclude possible

 formation in a sub-population of lung cells. These results demonstrate

 that formaldehyde derived from methylene chloride can form DNA-protein

 cross-links in the liver of B6C3F1 mice, the formation of DPX being

 dependent on the activity of the GST pathway.

 8.6.2.5 Dominant lethal assay

 Groups of 20 Swiss-Webster male mice were injected subcutaneously

 3 times/week for 4 weeks with 5 ml/kg of 5% v/v or 10% v/v methylene

 chloride in corn oil (Raje et al., 1988). Other groups of Swiss-

 Webster male mice were exposed to 350, 530 or 710 mg/m3 (2 h/day, 5

 days/week for 6 weeks) (Basso et al., 1987; Raje et al., 1988). Mating

 was started 1 week later for the injection group and 2 days later for

 the inhalation group, each male mouse being mated with a virgin adult

 female. The mating continued for 2 weeks. The fetuses were examined on

 day 17 of gestation. No significant differences in any of the

 mutagenicity parameters were found between control and treated groups.

 No microscopic lesions were found in the testes (Basso et al., 1987;

 Raje et al., 1988) or brain (Basso et al., 1987) of the treated males.

 8.6.2.6 Replicative DNA synthesis

 A number of studies have evaluated the ability of methylene

 chloride to induce replicative DNA synthesis (S-phase) in the livers

 and lungs of B6C3F1 mice and in the livers of Sprague-Dawley rats. A

 small, but statistically significant, increase in DNA synthesis was

 observed in the livers of mice exposed to 13 800 mg/m3 for 2 h, but

 not following a single oral dose of 1000 mg/kg (Lefevre & Ashby,

 1989). The biological significance of these increases is unclear due

 to similar increases being seen in some control groups.

 There were no sustained increases in DNA synthesis in the livers

 of female mice exposed by inhalation to 3.53, 7.06, 14.12 or

 28.24 g/m3 (1000, 2000, 4000 or 8000 ppm) for up to 4 weeks, nor in

 female mice exposed to 7.06 g/m3 for up to 2 years (Foley et al.,

 1993).

 Increases in DNA synthesis were not seen in rats exposed to

 1770 mg/m3 for 6 or 12 months (Nitschke et al., 1988a,b).

 Replicative DNA synthesis was measured in the lungs of female mice

 exposed to 7.06 or 28.24 g/m3 for 1, 2, 3 or 4 weeks and in mice

 exposed to 7.06 g/m3 for 13 and 26 weeks. Small decreases in the

 labelling indices were reported at all these points (Kanno et al.,

 1993). In contrast, Foster et al. (1992) reported significant

 increases in the number of cells in S-phase in both the bronchiolar

 and alveolar epithelium of male mice exposed to 14.12 g/m3 for

 13 weeks.

8.6.3 Appraisal

 Under appropriate exposure conditions, methylene chloride is

 mutagenic in prokaryotic microorganisms with or without metabolic

 activation (S. typhimurium or E. coli). In eukaryotic systems it

 gives either negative or, in one case, weakly positive results. In

 vitro gene mutation assays and tests for UDS in mammalian cells were

 uniformly negative. In vitro assays for chromosomal aberrations

 using different cell types gave positive results, whereas negative or

 equivocal results were obtained in tests for SCE induction.

 The majority of the in vivo studies reported have provided no

 evidence of mutagenicity of methylene chloride (e.g., chromosome

 aberration assay,micronucleus test or UDS assay). Where positive

 responses have been seen, they are restricted to tests using B6C3F1

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 mice. Marginal increases in the frequencies of SCEs, chromosomal

 aberrations and micronuclei in mice have been reported following

 inhalation of high concentrations of methylene chloride. Increases in

 hepatic DNA single strand breaks and DNA-protein crosslinks were seen

 in mice, but not in rats, exposed to 14 100 mg/m3.

 Within the limitations of the short-term tests currently

 available, in vivo genetic activity has only been detected in tests

 using B6C3F1 mice.

8.7 Chronic toxicity and carcinogenicity

8.7.1 Inhalation exposure

 8.7.1.1 Rat

 Groups of 95 male and 95 female Sprague-Dawley rats (8 weeks old)

 were exposed by inhalation to 0, 1770, 5300 or 12 400 mg/m3

 methylene chloride (99% pure) 6 h/day, 5 days per week for 2 years

 (Table 20). Overall survival in the study, including that of controls,

 was poor. Mortality among high-dose females was statistically

 increased from the 18th month when compared to controls. Increases in

 CO-Hb were found in treated groups from 6 months, but not in a dose-

 related manner. From 12 months, non-neoplastic pathological effects on

 the liver (increased hepatocellular vacuolization consistent with

 fatty change) were observed in both males and females at all exposure

 levels in a dose-related fashion. In males (5300 and 12 400 mg/m3)

 and females (12 400 mg/m3), a decrease in the incidence (females)

 and severity (males) of age-associated chronic progressive

 glomerulonephrotoxicity was observed. The only reported increases in

 tumour incidence occurred in benign mammary gland tumours in males and

 females, and tumours in the mid-cervical region close to the salivary

 gland in males. There was no significant increase in the proportion of

 animals with benign or malignant mammary tumours; however, the total

 number of benign mammary tumours showed a marginally significant dose-

 related increase in males (controls, 8/95; low-dose, 6/95; mid-dose

 11/95; high-dose, 17/97; p = 0.046) and a dose-related increase in the

 total number of benign mammary tumours was observed in females

 (165/96; 218/95; 245/95; 287/97; p < 0.001). There was no indication

 of an increase in the number or incidence of malignant mammary tumours

 in either males or females. The background historical incidence for

 Sprague-Dawley rats in the laboratory normally exceeds 80% in females

 and is about 10% in males (Burek et al., 1984).

 Table 20. Carcinogenicity studies using the inhalation routes

 

 Species Strain/sex Route of Doses Observations Reference

 administration/protocol/ (mg/m3)

 group size

 

 Rat Sprague-Dawley, Inhalation, 6 h/day, 5 0, 1770, Increase in various types of sarcomas in the Burek et al.

 male and female days/week for 2 years 5300, mid-cervical area in the region of the (1984)

 95 animals/sex/group 12 400 salivary gland in mid- and high-dose males

 (1/92, 0/95, 5/95, 11/97). Slight dose-

 related increase in total number of benign

 mammary tumours in males (8/95, 6/95,

 11/95, 17/97; p =0.046); dose-related

 increase in total number of benign

 mammary tumours in females (165/96,

 218/95, 245/95, 287/97; p<O.001)

 Rat Sprague-Dawley, Inhalation, 6 h/day, 5 O, 177, 710, No increase in incidence of benign Nitschke et al.

 male and female days/week for 20 1770 mammary tumours in males or in females (1988a)

 months (males), 24 exposed to 177 or 710 mg/m3; increased

 months (females); 90 incidence of benign mammary tumours in

 males/group females at 1770 mg/m3; no increase in the

 198 females/group number of any malignant tumours; NOAEL

 710 mg/m3

 Rat Fischer-344/N, Inhalation, 6 h/day, 5 O, 3500, Dose-dependent increase in benign NTP (1986)

 male and female days/week for 102 7100, mammary tumours; male: 0/50, 0/50, 2/50, Mennear et al.

 weeks; 50 animals/sex 14 100 5/50; female: 5/50, 11/50, 13/50, 23/50 (1988)

 group

 

 Table 20 (Cont'd)

 

 Species Strain/sex Route of Doses Observations Reference

 administration/protocol/ (mg/m3)

 group size

 

 Rat Sprague-Dawley, Inhalation, 4 h/day, 5 350 No effect on percentage of animals bearing Maltoni et al.

 females, during days/week for 7 benign and/or malignant tumours, in (1988)

 and after weeks, then 7 h/day, 5 maternal or offspring animals; no

 pregnancy days per week for 97 statistically significant increase in total

 weeks; malignant tumours

 54 females exposed

 60 female controls

 Rat Sprague-Dawley, inhalation, protocol as 350 As above Maltoni et al.

 male and female above for 15 and 104 (1988)

 offspring from weeks; 60-70 males or

 12th day in utero females/group, 158

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 male and 149 female

 controls

 Mouse B6C3F1 inhalation, 6 h/day, 5 7100 Mice exposed for more than one year Kari et al. (1992)

 days/week for various showed an excess of lung and liver tumours

 periods up to 104

 weeks; killed at end of

 exposure; 364 females

 exposed, 364 female

 controls

 

 Table 20 (Cont'd)

 

 Species Strain/sex Route of Doses Observations Reference

 administration/protocol/ (mg/m3)

 group size

 

 Mouse B6C3F1 Inhalation to 6 h/day, 7100 All exposed groups showed excesses of Kari et al. (1992)

 5 days/week for either lung and liver tumours

 26, 52, 28 or 104

 weeks; all mice

 maintained for 104

 weeks; 68 females

 exposed, 68 female

 controls

 Mouse B6C3F1, male Inhalation, 6 h/day, 5 0, 7100, Dose-dependent increases in (1) NTP (1986);

 and female days/week for 102 14 100 alveolar/bronchiolar adenomas Mennear et al.

 weeks; 50 males: 3/50, 19/50, 24/50 (1988)

 animals/sex/group females: 2/50, 23/48, 28/48

 (2) alveolar/bronchiolar carcinomas

 males: 2/50, 10/50, 28/50

 females: 1/50, 13/48, 29/48

 (3) hepatocellular adenoma and carcinoma -

 combined

 males: 22/50, 24/49, 33/49

 females: 3/50, 16/48, 40/48

 Hamster Syrian Golden, Inhalation, 6 h/day, 5 0, 1770, No significant increase in incidence of Burek et al. 

 male and female days/week for 2 years; 5300, benign tumours (1984)

 95 animals/sex/group 12 400

 

 

 Males exposed to 5300 or 12 400 mg/ma showed an increase in the

 number of sarcomas in the mid-cervical area in the region of the

 salivary glands (1 subcutaneous sarcoma in controls, n = 92, 2

 subcutaneous sarcomas and 3 salivary gland schwannoma at 5300 mg/m3,

 n = 95; and 5 subcutaneous fibrosarcomas and 2 subcutaneous

 mesofibrosarcomas as well as 2 salivary gland sarcomas at

 12 400 mg/m3, n = 97). A number of uncertainties affect the

 toxicological significance of these observations. As stated by the

 investigators, all but two of these tumours were large and appeared to

 invade all adjacent tissues in the neck region. Histologically all

 were sarcomas. Some tumours morphologically resembled the overall

 type, fibrosarcoma while others resembled another, neurofibrosarcoma,

 and still others were undifferentiated or pleomorphic. Only two

 tumours were small enough to be localized in the salivary gland,

 appearing to arise in interstitial and capsular tissue. Based on these

 findings the author suggested that all probably arose from the

 salivary gland, although appearing to arise from mesenchymal tissue

 they represent a variety of tumour types. There is no explanation for

 the sex difference observed. Both male and female rats in the study

 had a viral disease (siabclacryoudenitis, which affects the salivary

 glands) during the first 2 months of the study. The infection did not

 increase mortality and all exposure groups appeared to be affected to

 the same degree (Burek et al., 1984).

 A later study in the same laboratory where the highest dose level

 was 1770 mg/m3 revealed salivary gland sarcomas in only two animals,

 a male at 1770 mg/m3 and a female at 177 mg/m3. The historical

 incidence in this laboratory was reported to range from 0 to 2% in

 control groups of Sprague-Dawley rats (Nitschke et al., 1988a).

 Groups of 90 male and 108 female Sprague-Dawley rats (6-8 weeks

 old) were exposed to 0, 177, 710 or 1770 mg/m3 6 h/day, 5 days/week

 for 20 and 24 months, respectively. Exposure-related increases in

 CO-Hb levels were found. Exposure-related histo-pathological changes

 were limited to the liver of male and female rats and mammary glands

 of female rats exposed to 1770 mg/m3. An increased incidence of

 hepatocellular vacuolization was found in males and females exposed to

 1770 mg/m3. Females exposed to this dose level also had an increased

 incidence of multinucleated hepatocytes. The incidence of benign

 mammary tumours in female rats exposed to 177, 710 and 1770 mg/m3

 was comparable to historical control values (79-82%), but the number

 (2.2) of benign mammary tumours per tumour-bearing female rat exposed

 to 1770 mg/m3 was greater than that of controls (1.8) (p < 0.05).

 There were no increases in the incidence of malignant tumours at any

 site in female rats, nor of benign or malignant tumours in male rats,

 exposed to methylene chloride. The no-observed-adverse-effect level

 for chronic inhalation exposure of Sprague-Dawley rats was judged to

 be 710 mg/m3 (Nitschke et al., 1988a).

 A group of 54 pregnant Sprague-Dawley rats was exposed to

 350 mg/m3 4 h/day, 5 days/week for 7 weeks and subsequently for

 7 h/day, 5 days/week for a further 97 weeks. A further group of 60

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 rats served as controls. In addition, groups of 60-70 males or females

 were exposed to 212 mg/m3 from the 12th day in utero for a total

 of either 15 or 104 weeks; there were 158 male and 149 female

 controls. In exposed maternal or offspring rats, methylene chloride

 did not affect the percentage of animals bearing benign and/or

 malignant tumours. Although there was a slight increase in total

 malignant rumours in rats exposed to 350 mg/m3 for 104 weeks, this

 was not deemed to be statistically significant (Maltoni et al., 1988).

 The Task Group noted the absence of relevant data on the design of the

 experiment such as the statistical methods, housing conditions and

 histopathological techniques.

 When groups of 50 male and 50 female F-344/N rats, 7-8 weeks old,

 were exposed by inhalation to 0, 3500, 7100 or 14 100 mg/m3

 methylene chloride (99% pure) 6 h/day, 5 days per week, for 102 weeks

 (NTP, 1986), body weight gains for both exposed males and females were

 comparable to those of the control group. The survival of exposed male

 rats was also comparable to that of the controls, although the

 survival of all groups of males at the end of the study was low.

 Similarly, the survival of female rats was comparable with that of the

 controls, with the exception of the high-dose group. Non-neoplastic

 treatment-related increases in the incidence of renal tubular cell

 degeneration, splenic fibrosis and (females only) nasal cavity

 squamous metaplasia were observed.

 Increased incidences of benign tumours of the mammary gland (all

 fibroadenomas, except for one adenoma in the high-dose group) were

 observed in treated females (control, 5/50; low-dose, 11/50; mid-dose,

 13/50; high-dose, 23/50; p < 0.001). There was a positive trend in

 the incidence of benign tumours of the mammary gland in males (0/50,

 0/50, 2/50, 5/50; p < 0.01). The range of the historical control

 incidence of mammary gland fibroadenomas in this laboratory for the

 male rat was 0/50 to 6/49 and for the female rat was 5/50 to 24/49.

 There were no other exposure-related increases in tumour incidence

 (NTP, 1986; Mennear et al., 1988).

 8.7.1.2 Mouse

 Groups of 50 male and 50 female B6C3F1 mice, 8-9 weeks old, were

 exposed to 0, 7100 or 14 100 mg/m3 (> 99% pure) 6 h/day,

 5 days/week for 102 weeks and killed after 104 weeks of study.

 Survival to the end of the study period in males was: control, 39/50;

 low-dose, 24/50; and high-dose, 11/50; that in females was: 25/50,

 25/49 and 8/49. This reduced survival may have been due to the

 chemically induced development of liver and lung neoplasia in both

 sexes, significant dose-related increases in lung and liver tumours

 having been found. The incidences of alveolar/bronchiolar adenomas

 were: males - 3/50, 19/50 and 24/50 (p < 0.001); and females - 2/50,

 23/48 and 28/48 (p < 0.001). Those for alveolar/bronchiolar

 carcinomas were: males - 2/50, 10/50 and 28/50 (p < 0.001); and

 females - 1/50, 13/48 and 29/48 (p < 0.001). Incidence of

 hepatocellular adenomas or carcinomas (combined) were increased in

 high-dose males and dosed females (males: 22/50, 24/49 and 33/49;

 females: 3/50, 16/48 and 40/48). Dose-related increases were observed

 in the incidences of testicular atrophy in male mice and uterine and

 ovarian atrophy in female mice; these effects were considered to be

 secondary responses to neoplasia (NTP, 1986; Mennear et al., 1988).

 Studies have been conducted on the B6C3F1 mouse to study the

 time-dependency of exposure to methylene chloride leading to tumour

 formation. Groups of 364 female B6C3F1 mice were exposed to either

 air or 7100 mg/m3 methylene chloride for 6 h/day, 5 days/week for

 104 weeks. Following exposure for either 462, 494, 515, 529, 571, 597,

 618, 639 or 660 days, 10 exposed and 5 control mice were randomly

 selected and sacrificed for the purposes of following the progression

 of neoplasia in the lung and the liver. Increased incidences of

 alveolar/bronchiolar adenomas and carcinomas and of hepatocellular

 adenomas and carcinomas in mice exposed for 104 weeks were confirmed.

 In mice sacrificed following less than one year's exposure (368 days),

 there were no lung lesions observed and only minimal indications of

 proliferative lesions in the liver. Mice exposed for longer than one

 year showed a progressive increase in the incidence of both

 alveolar/bronchiolar adenomas and carcinomas. The incidence of liver

 tumours in mice exposed for longer than one year was relatively

 constant (40-60%), although a time-dependant increase in the total

 liver tumour burden per animal was reported (Kari et al., 1993).

 Groups of 68 female B6C3F1 mice were exposed to 100 mg/m3

 6 h/day, 5 days/week for either 26, 52 or 78 weeks. All groups of mice

 were maintained for 104 weeks, at which time they were sacrificed. The

 exposures were split into two phases, i.e. exposure to either air or

 to methylene chloride for the specified period at the beginning or the

 end of the total exposure period. Further groups of 68 female mice

 were exposed either to air or to 7100 mg/m3 methylene chloride for

 the full 104 weeks. The percentage incidences of lung adenomas or

 adenocarcinomas or liver adenomas or adenocarcinomas are given in

 Table 21. The percentage incidence of both lung and liver tumours in

 mice exposed to methylene chloride during the first phase increased

 with the duration of exposure. In mice exposed to methylene chloride

 during the second phase, significant increases in the percentage

 incidence of lung and liver rumours were only observed following 78

 weeks of exposure to methylene chloride. The study also showed that

 lung rumours appeared earlier in methylene chloride-exposed animals

 than did liver tumours. Comparison of the results of this study with

 those presented in the previous paragraph suggested that the

 percentage incidence of lung tumours but not liver tumours increased

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 following withdrawal from exposure to methylene chloride, particularly

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

 1993).

 Table 21. The percentage incidence of lung and liver tumours in female mice exposed to

 methylene chloride for different time periods

 

 Exposurea % incidence of tumours

 (adenomas or carcinomas)

 Phase I Phase II Lung Liver

 

 air 104 weeks 7.5 27

 methylene chloride 26 weeks air 78 weeks 31b 40

 methylene chloride 52 weeks air 52 weeks 63b 44b

 methylene chloride 78 weeks air 26 weeks 56b 62b

 methylene chloride 104 weeks 63b 69b

 air 26 weeks methylene chloride 78 weeks 19b 48b

 air 52 weeks methylene chloride 52 weeks 15 31

 air 78 weeks methylene chloride 26 weeks 4 34

 

a exposure to methylene chloride was 7100 mg/m3, 6 h/day, 5 days/week

b statistically significant p < 0.05

 

 8.7.1.3 Hamster

 When groups of 95 male and 95 female Syrian golden hamsters

 (8 weeks old) were exposed by inhalation to 0, 1770, 5300 or

 12 400 mg/m3 6 h/day, 5 days/week for 2 years, no exposure-related

 changes were observed in mean body weight and mean organ weights.

 CO-Hb levels were increased but this effect was not dose-related. The

 numbers of animals surviving to the end of the study were 16, 20, 11

 and 14 for males and 0, 4, 10 and 9 for females, respectively. The

 incidence of lymphosarcomas was slightly higher in treated females

 than in controls (control, 1/91; low-dose, 6/92; mid-dose, 3/91; high-

 dose, 7/91; p = 0.032). The increased incidence of benign tumours was

 considered to be related to the higher survival of the exposed

 hamsters and not a direct result of exposure to methylene chloride

 (Burek et al., 1984).

8.7.2 Oral administration

 8.7.2.1 Rat

 Groups of 85 male and 85 female F-344 rats (7 weeks old) were

 administered methylene chloride in the drinking-water at

 concentrations of 0, 5, 50, 125 and 250 mg/kg per day for 104 weeks

 (Table 22). Interim sacrifices were carried out at 26, 52 and 78

 weeks, such that 50 males and 50 females per group received the

 treatment for 104 weeks. Additional groups of 50 male and 50 female

 F-344 rats received methylene chloride at a concentration of 250 mg/kg

 per day for 78 weeks, with further groups of 50 male or female rats

 serving as controls. Small changes in mean body weight and food/water

 consumption were seen in rats receiving either 125 or 250 mg/kg per

 day. Dose-related increases were noted in mean haematocrit,

 haemoglobin levels and red blood cell counts at the three highest

 doses. Decreases in serum alkaline phosphatase activity and in

 creatinine, blood urea nitrogen, serum protein and cholesterol levels

 in both sexes were found at 250 mg/kg per day. Treatment-related

 histopathological changes were seen in the liver of rats receiving

 250 mg/kg per day. There were no increases in the incidence of any

 tumour in treated rats when compared to controls (Serota et al.,

 1986a).

 When groups of 50 male and 50 female Sprague-Dawley rats were

 given 0, 100 or 500 mg/kg methylene chloride (purity 99.97%) by gavage

 in olive oil, 4-5 days/week for 64 weeks, excess mortality was

 observed in rats of each sex receiving 500 mg/kg. A slightly higher

 incidence of adenocarcinomas of the mammary gland was observed in

 females receiving 500 mg/kg (4/50, 3/50, and 9/50 in the controls,

 low-dose and high-dose, respectively). There was no effect on the

 total tumour incidence in the exposed groups (Maltoni et al., 1988).

 The Task Group noted the short length of the exposure period as well

 as the absence of relevant data on the design of the experiment, such

 as the statistical methods applied, housing conditions,

 histopathological techniques and pathology schedule.

 Table 22. Carcinogenicity studies by oral route

 

 Species Strain/type Route of Doses Observations Reference

 administration/protocol/

 group/size

 

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

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 mate and female 104 weeks; 85 animals/sex or 250 mg/kg survival and other findings not affected (1986a)

 per dose; scheduled kills: 5 at per day by methylene chloride; significant

 26 weeks, 10 at 52 weeks, 20 decreases in body weight gain at 125

 at 78 weeks; also additional and 250 mg/kg per day and evidence of

 groups of controls and liver damage at doses above 50 mg/kg

 250 mg/kg (50/sex) which per day

 received methylene chloride

 for 78 weeks only

 Rat Sprague-Dawley, Gavage in olive oil for 64 0, 100 or 500 No effect on total tumour incidence in Maltoni et

 male and female weeks; 50 animals/sex/dose; mg/kg per exposed rats. Higher incidence, not al. (1988)

 additional control group (not day statistically significant, of malignant

 dosed) of 20 males and 26 mammary tumours in high-dose

 females females; survival decreased in high dose

 males and females

 Mouse B6C3F1, male Drinking water ad libitum for 0, 60, 125, 185 No increase in incidence of neoplasms; Serota et al.

 and female 104 weeks; group size; or 250 mg/kg evidence of slight liver damage at 250 (1986b)

 125 m, 100 f (controls) per day mg/kg per day

 200 m, 100 f (60 mg/kg)

 100 m, 50 f (125 mg/kg)

 100 m, 50 f (185 mg/kg)

 125 m, 50 f (250 mg/kg)

 

 Table 22 (Cont'd)

 

 Species Strain/type Route of Doses Observations Reference

 administration/protocol/

 group/size

 

 Mouse Swiss, male and Gavage in olive oil for 64 0, 100 or Decrease in body weight in exposed Maltoni et

 female weeks 500 mg/kg males and females after 36-40 weeks; al, (1988)

 50 animals/sex/dose per day dose-related increase in pulmonary

 60 animals/sex/controls tumours in males - not significant

 without considering mortality rate;

 significant (p<0.05) taking into account

 the mortality rate; no treatment-related

 increase in the percentage of animals

 bearing benign and malignant tumours,

 or of animals bearing malignant

 tumours, or of the number of total

 malignant tumours per 100 animals

 

 

 8.7.2.2 Mouse

 Groups of male and female B6C3F1 mice (7 weeks old) received

 methylene chloride in the drinking-water for 104 weeks at levels of 0

 (control groups 60/65 males, 50/50 females), 60 (200 males, 100

 females), 125 (100 males, 50 females), 185 (100 males, 50 females) or

 250 (125 males, 50 females) mg/kg per day. Histopathological changes

 were observed in the liver of mice receiving 250 mg/kg per day. There

 was no increase in the incidence of tumours in any of the exposed

 groups, when compared to controls (Serota et al., 1986b).

 Groups of 50 male and 50 female Swiss mice received either 100 or

 500 mg/kg methylene chloride (purity 99.97%) in olive oil by gavage on

 4-5 days/week for 64 weeks. Groups of 60 males and 60 females served

 as controls and received only olive oil. In male mice dying between 52

 and 78 weeks, an increase in the incidence of pulmonary adenomas was

 observed (0/27 in controls, 4/41 low dose level, 7/33 high dose level)

 although the effect was not statistically significant in exposed male

 mice when mortality was not taken into account. There was no increase

 in the total tumour burden in exposed mice. A decrease in body weight

 was observed in exposed males and females, compared to controls after

 weeks 36-40. No other exposure-related findings were reported (Maltoni

 et al., 1988)

8.7.3 Appraisal

 Methylene chloride is carcinogenic in the mouse, causing both

 lung and liver tumours, following exposure to high concentrations

 (7100 and 14 100 mg/m3 ). The incidence of both lung and liver

 tumours was increased in mice exposed to 7100 mg/m3 for 26 weeks

 and maintained for a further 78 weeks. Associated toxicity or

 hyperplasia in the target organs was not observed.

 Hamsters exposed to methylene chloride by inhalation at

 concentrations up to 12 400 mg/m3 for 2 years showed no evidence of

 a carcinogenic effect related to exposure to methylene chloride.

 Rats exposed to methylene chloride via various routes have shown

 increased incidences of tumours at certain sites. An excess of

 tumours in the region of the salivary gland was reported in male rats

 exposed to either 5300 or 12 400 mg/m3 for 2 years. This excess was

 only evident when the tumours, which were all of mesenchymal origin,

 were grouped together for statistical analysis. As the tumours arose

 from a variety of different tissues, the statistical approach of

 combining tumours was inappropriate. The response was not seen in a

 second study in which rats were exposed to either 3500, 7100 or

 14 100 mg/m3 throughout their lifetime. A further inhalation study

 in rats exposed to methylene chloride at concentrations up to

 1770 mg/m3 throughout their lifetime showed no evidence of

 carcinogenicity. These studies, taken together with the absence of

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 effect on the salivary gland in all other inhalation studies, raise

 doubts regarding their biological and toxicological significance.

 Rats exposed to methylene chloride via the drinking-water or by

 gavage similarly showed no substantive evidence of carcinogenicity.

 An increase in either the incidence or the multiplicity of benign

 mammary tumours (fibroadenomas) in rats exposed to methylene chloride

 via inhalation has been reported in three studies. Increases in

 multiplicity were dose-related in male and female Sprague-Dawley rats

 (historical incidence 10% males and 80% females). The increase in

 multiplicity was observed only in females of the highest dose

 (1770 mg/m3 ) group. No effects were observed in males or the other

 dose groups of females. A dose-related increased incidence in benign

 mammary tumours was observed in female F-344/N rats, although the

 incidences were in the range of historical incidence (10 to 25%). An

 increased incidence in high dose (14 100 mg/m3 ) males was also

 within the historical incidence range of 0 to 11%.

 In one study, a slight increase in the incidence of

 adenocarcinomas in the mammary gland was observed in female Sprague-

 Dawley rats receiving 500 mg/kg by the oral route. A study in

 Fischer-344 rats with dose levels up to 250 mg/kg by the oral route

 (drinking-water) showed no carcinogenic effects.

 No increases in mammary tumours were observed in the mouse or

 hamster by inhalation or oral administration.

8.8 Mechanistic studies

8.8.1 In vitro metabolic studies

 There are three transient reactive intermediates in the metabolism

 of methylene chloride. Two of them, formyl chloride and

 S-chloromethyl-glutathione, are assumed to be present on the basis

 of knowledge of the metabolic pathways; the third, formaldehyde, has

 been identified in vitro. All three have the reactivity necessary to

 bind covalently to macromolecules. Of these S-chloromethyl-

 glutathione is potentially the most potent alkylating agent, a

 conclusion based on the known reactivity of the halothioethers (Bohme

 et al., 1949), structural similarities to the mutagenic glutathione

 conjugates of the 1,2-dihaloethanes, and on the outcome of several

 studies using different liver fractions in the Salmonella mutation

 assay (Jongen et al., 1982; Green, 1983). Formyl chloride is highly

 unstable, existing chemically only at low temperatures (-80°C) in

 inert solvents (Staab & Datta, 1964). Formaldehyde is a common

 metabolic product in vivo which is efficiently metabolized in the

 liver to formic acid. The endogenous formation and metabolism of

 formaldehyde occurs at a high rate, and the additional formaldehyde

 derived from methylene would be metabolized by the same efficient

 pathways.

 Comparisons of the rates of metabolism of methylene chloride by

 each pathway in liver fractions from rats, mice, hamsters and man have

 been carried out (Green et al., 1986b,c; Reitz et al., 1989). These

 experiments demonstrated that the rates of metabolism in these

 pathways in vitro had similar differences to those seen in vivo in

 rats and mice, and enabled a comparison to be made with those species

 (hamster and man) where in vivo data was not available.

 Rates of metabolism in human liver have been measured for both

 glutathione- S-transferase (Green et al., 1987a,b,c; Reitz et al.,

 1989; Bogaards et al., 1993) and cytochrome P-450 (Green et al.,

 1987a,b,c; Reitz et al., 1989) pathways of methylene chloride

 metabolism. A total of 33 human liver samples have been assayed for

 glutathione- S-transferase activity and a range of activities

 reported. In the work by Bogaards et al. (1993), 3 samples had no

 activity, a further group of 11 had activity in the range of 0.20-0.41

 (mean 0.31 ± 0.08) nmol/min per mg protein, and 8 samples had activity

 in the range 0.82-1.23 (1.03 ± 0.14) nmol/min per mg protein. The

 rates measured by Green et al. (1987a,b,c) were within the range found

 by Bogaards et al., (1993) (0.05-0.93 nmol/min per mg protein; mean

 0.42 ± 0.32; n = 7) and those found by Reitz et al. (1989) were

 slightly higher (range 0-3.03 nmol/min per mg; mean 2.09 ± 1.40). The

 activity in all the samples assayed was at least 1.4 lower than that

 in rat liver cytosol. Two human lung samples were assayed and found to

 be lacking in glutathione- S-transferase activity (Reitz et al.,

 1989). However, enzyme activities of this type are significantly lower

 in the lung than the liver, and any such activity may not be detected

 by the currently available assays.

 The 10-fold difference in glutathione- S-transferase activity

 measured in vivo in mice and rats was also found in vitro. There

 is an excellent correlation between glutathione- S-transferase

 metabolism and the outcome of the 2-year cancer studies in the three

 animal species. More support for this correlation was obtained in DNA

 damage tests (section 8.6.2.3.) No such correlation exists for the

 cytochrome P-450 pathway where, for example, the metabolic rate in the

 hamster is very similar to that in the mouse. Cytochrome P-450-

 catalysed metabolism of methylene chloride could be detected in lung

 tissue from all three animal species, the relative activities being

 similar to those in the livers. Glutathione- S-transferase activity

 was detectable only in mouse lung fractions.

 The low rates of metabolism of methylene chloride by the

 glutathione- S-transferase pathway in human liver samples has been

 attributed to a deficiency in the transferase isoenzyme responsible.

 The same liver samples had similar activity to rat liver when assayed

 with an alternative substrate for these enzymes (Green et al.,

 1986b,c).

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8.8.2 In vivo metabolic studies

 A comparative kinetic profile of methylene chloride and its

 metabolites was determined in B6C3F1 mice and F-344 rats both during

 and after a 6-h exposure to atmospheres containing various

 concentrations from 350 to 14 100 mg/m3 (Green et al., 1986b,

 1987a,b,c, 1988). Blood levels of methylene chloride and CO-Hb and the

 rates of elimination of methylene chloride, carbon monoxide and carbon

 dioxide in exhaled air were measured. Stable isotopes were used to

 quantify the amount of carbon dioxide from each pathway at dose levels

 of 350, 1770 and 14 100 mg/m3, but only in the mouse. The steady-

 state blood levels of methylene chloride during exposure were up to

 5 times higher in rats than in mice at the higher dose levels. A

 comparison of the CO-Hb levels in blood and carbon monoxide levels in

 expired air showed that rate of metabolism by the cytochrome P-450

 pathway was similar in both rats and mice. The pathway was saturated

 in both species at exposures of less that 1770 mg/m3, resulting in

 maximal CO-Hb levels of 16% (Green et al., 1987a,b,c).

 Saturation of the cytochrome P-450 pathway in mice was also

 clearly shown by a 5-10 fold increase in the blood levels of methylene

 chloride when the inhaled concentration was doubled from 1770 to

 3500 mg/m3 (Green et al., 1987a,b,c).

 The stable isotope studies demonstrated that the cytochrome P-450

 pathway was the major source of carbon dioxide at low exposure levels

 (350 mg/m3) whereas at high levels ( 14 100 mg/m3) the

 glutathione- S-transferase pathway was the principal source of carbon

 dioxide. A comparison of the rate of elimination of the carbon dioxide

 by rats and mice at the top dose level showed the glutathione- S-

 transferase pathway to be 10-12 times more active in mice than rats.

 The higher rate of metabolic conversion of methylene chloride by mice

 when compared to rats largely accounts for the low blood levels of the

 parent chemical in this species.

 In summary, the in vitro and in vivo studies have provided

 evidence for the following:

 1. The cytochrome P-450 pathway is saturated at 1770 mg/m3 and is

 quantitatively similar in rats and mice in vivo and in rat,

 mouse, hamster and human livers in vitro.

 2. The glutathione- S-transferase pathway is a major pathway only in

 mice, its activity at the 14 100 mg/m3 dose level being an order

 of magnitude greater than in rats.

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

 S-transferase activity. In all cases the levels of activity were

 lower than those measured in rat liver.

 4. Methylene chloride metabolism is dose-dependent. The utilization

 of the two pathways is significantly different at the dose levels

 used in the carcinogenicity studies than at low dose levels.

 5. These studies provided the metabolic rate constants used in the

 physiologically based pharmacokinetic models described in section

 8.9.

8.8.3 Pulmonary effects

 A number of studies have examined the effects of methylene

 chloride on the mouse and rat lung (Eisenbrandt & Reitz, 1986; Hext et

 al., 1986; Green et al., 1987a, Foster et al., 1992; Kanno et al.,

 1993). Following a single exposure at concentrations of 7100 mg/m3

 or more, a specific lesion characterized by marked vacuolization of

 Clara cells were seen in the mouse, but not the rat. No other cell

 types were affected in the mouse (Green et al., 1987a; Foster et al.,

 1992). The morphological damage in Clara cells recovered after 5 days

 of repeated 6-h exposures (Foster et al., 1992). The damage to Clara

 cells was accompanied by a change in the ability to metabolize

 methylene chloride by the two pathways. Cytochrome P-450 metabolism

 was suppressed while glutathione- S-transferase remained unchanged.

 In a thirteen week study (Foster et al., 1992), damage to the Clara

 cells was seen following the first exposure of each week of the study.

 Between days 2 and 9 of this study, a significant increase in the

 number of cells in S-phase was observed in both bronchiolar and

 alveolar epithelium. A similar study (Kanno et al., 1993) failed to

 detect an increase in the number of cells in S-phase.

 Significant pulmonary lesions were observed in male B6C3F1 mice

 1 day after a single 6-h exposure to 14 100 mg/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 B6C3F1 mouse into the

 role of oncogene activation as a potential mechanism of action of the

 carcinogenic effect of methylene chloride. A group of 145 female

 B6C3F1 mice was exposed to 7100 mg/m3 for 6 h/day, 5 days/week for

 up to 27 months. Another group of 235 females acted as controls. These

 mice were used for the purpose of providing spontaneous and methylene-

 chloride-induced tumour tissue from both the liver and the lung for

 the analysis of proto-oncogene activation and tumour suppressor gene

 inactivation. The DNA recovered from 54 methylene-chloride-induced

 B6C3F1 lung tumours and from 7 spontaneous B6C3F1 lung tumours was

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 analysed by the direct sequencing of PCR (polymerase chain reaction)

 amplified DNA fragments of the K-ras gene for first and second exon

 mutations. Twelve mutations were identified in the tumours from

 exposed mice, 5 in exon one and 7 in exon two. There was no difference

 in the frequency of K-ras activation in tumour tissue derived from

 both exposed mice when compared to controls. DNA was isolated from 49

 spontaneous and 50 methylene-chloride-induced liver tumours and

 screened by the oligonucleotide hybridization of PCR (polymerase chain

 reaction) amplified H-ras gene fragments for codon 61 mutations. The

 mutation profile of the H-ras gene was similar in the tumour tissue

 derived from both control and treated mice (Devereux et al., 1993).

 Mutations of the p53 rumour suppresser gene were examined in lung

 tumours from female mice exposed to 7100 mg/m3 (2000 ppm) methylene

 chloride for 2 years (Hegi et al., 1993). The limited number of p53

 mutations identified in this study and the small number of spontaneous

 tumours precluded any conclusions concerning the mutagenic spectrum or

 possible genotoxicity of methylene chloride.

8.8.5 The use of mechanistic studies in extrapolation

 Studies using liver fractions from rats, mice, hamsters and humans

 have confirmed the existence of two pathways for the metabolism of

 methylene chloride (the cytochrome P-450 pathway and the glutathione-

 S-transferase pathway) and have established substantial differences

 between species in the utilization of these pathways. The rates of

 metabolism of methylene chloride by the two enzymes in the liver

 fractions from a few species have been established. The activities of

 the cytochrome P-450 pathway in the mouse and hamster were similar,

 whereas those in the rat and human were lower. In marked contrast, the

 activity of the glutathione- S-transferase in the mouse was very high

 when compared with the other species; there being a 10-fold difference

 in activity between the mouse and the rat. Rates of metabolism by this

 pathway in hamsters and humans were even lower than in rats.

 The results of a full pharmacokinetic analysis of the behaviour of

 methylene chloride and its metabolites in vivo were consistent with

 the species differences observed in vitro. Saturation of the

 cytochrome P-450 pathway occurred in rats and mice at dose levels of

 less than 1770 mg/m3 and resulted in maximal CO-Hb levels of 16% in

 both species. Comparisons of the glutathion- S-transferase pathway

 based on expired carbon dioxide levels at high exposure concentrations

 found the same 10- to 12-fold difference between mice and rats that

 had been observed in vitro. The higher metabolic rates in mice

 accounted for the lower blood levels seen in this species compared to

 the rat.

 Exposure of mice to atmospheric concentrations of 7100 mg/m3 or

 more led to recurrent cytotoxicity, increases in DNA synthesis

 ( S-phase) and changes in the metabolic complement of mouse lung

 Clara cells. All the effects were specific to the mouse and to the

 Clara cells. Several of the changes (cytotoxicity and S-phase) were

 frequently associated with the development of tumours. However the

 significance of these findings with respect to the development of lung

 tumours in mice exposed to methylene chloride remains to be

 established. Effects on chromosomes have also been reported in the

 mouse lung.

 Studies on the potential role of activation of Ras oncogenes in

 the development of methylene-chloride-induced lung and liver tumours

 have been unable to distinguish between the tumours seen in methylene-

 chloride-treated animals and those occurring spontaneously in control

 animals.

 Studies in which mice were exposed for different intervals of a

 2-year carcinogenicity study established that lung tumours developed

 quicker and after shorter exposures than liver tumours. Whether this

 indicates a different mechanism in the lung from the liver or reflects

 the cytotoxicity and cell division seen in the lungs of mice is

 unknown at the present time.

 The more recent studies in bacteria using glutathione-deficient

 strains and strains in which mammalian transferase enzymes have been

 expressed have established the role of this pathway in mutagenesis.

 Consistent with this are findings of DNA single strand breaks and DNA-

 protein cross-links in the livers of mice, but not rats or hamsters,

 exposed in vivo to 14 000 mg/m3. Both the single strand beaks and

 cross-links have been shown to be derived from metabolites of the

 glutathione- S-transferase pathway. At the present time, these

 effects have not been demonstrated in mouse lung.

 There is a consistency between the bacterial mutagenicity assays,

 the pharmacokinetic data and the studies of DNA single strand breaks

 and DNA protein cross-links which leads to the conclusion that the

 liver tumours seen in mice are derived from an interaction between

 metabolites of the glutathione- S-transferase pathway and DNA. The

 same level of detail is not available for the mouse lung. However the

 responses seen in the Clara cells and in mouse lung chromosomes are

 not inconsistent with this mechanism.

 The studies of the comparative metabolism and pharmaco-kinetics of

 methylene chloride in the rat, mouse and hamster also provided a

 plausible explanation for the species differences in the carcino-

 genicity of this chemical. The differing metabolic rates by the

 glutathione- S-transferase pathway are consistent with the outcome of

 the cancer studies whereas the blood levels of the parent chemical and

 the metabolic rates by the cytochrome P-450 pathway are not. These

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 results are also consistent with the different responses seen in the

 three mouse cancer bioassays (NTP, 1986; Serota et al. 1986b; Maltoni

 et al., 1986). At the high dose levels used in the NTP study, the

 glutathione- S-transferase pathway would have been the major

 metabolic pathway and high tumour incidences were observed. At the

 lower dose levels used by Serota et al. (1986b) and Maltoni et al.

 (1986), methylene chloride would have been metabolized mainly by

 cytochrome P-450, and glutathione- S-transferase metabolism would

 have been minimal. Consequently there were no significant increases in

 either lung or liver tumours in these studies.

8.8.6 Mammary tumour promotion

 The dependence of mammary tumours upon pituitary hormones in both

 male and female rats has been established unequivocally (Welsch &

 Nagasawa, 1977; Welsch 1985). In the rat, prolactin acts as a promoter

 of mammary carcinogenesis. There is good evidence that increased

 prolactin levels increase the incidence of mammary tumours (Welsch et

 al., 1970), and there is a positive correlation between elevated blood

 prolactin levels and mammary rumours in aged R-Amsterdam female rats

 (Kwa et al., 1974).

 The mechanism by which methylene chloride induces mammary adenomas

 in the rat is important for human hazard assessment. Female Sprague-

 Dawley rats receiving methylene chloride have a high blood level of

 prolactin (Breslin & Landry, 1986). When male and female Sprague-

 Dawley rats were exposed to 10 600 mg/m3 (3000 ppm) methylene

 chloride for 15 to 19 consecutive days, a significant increase (2.3 x)

 in basal serum prolactin levels was observed in female rats. No

 significant effect was observed in male rats (Breslin & Landry, 1986).

 In humans, there is conflicting evidence on whether or not mammary

 rumours are as responsive to prolactin as in the case of rats (Sinha,

 1981). The rat has elevated levels of prolactin when fed ad libitum

 in comparison to a restricted dietary regimen and this may explain why

 the mammary tumour incidence is so easily responsive to a variety of

 environmental and other effects. In the rat, however, prolactin is

 luteotrophic. An increase in the circulating levels of prolactin will

 lead to an increase in progesterone and exogenous oestrogen levels.

 The presence of all three factors that causes tubular-alveolar growth

 of the mammary glands may ultimately lead to tumour development.

 Prolactin is not luteotrophic in primates (Neumann, 1991).

 The mechanism of production of mammary tumours in the rat

 involving hyperprolactinaemia will probably occur only at doses of

 methylene chloride which affect prolactin levels. There is no direct

 information on prolactin levels in rats receiving low doses of

 methylene chloride, but no increase in mammary adenomas has been

 observed following the administration of low doses in either

 inhalation or drinking-water studies (i.e., below 250 mg/kg body

 weight or 1770 mg/m3).

8.8.7 Appraisal

 In vitro and in vivo metabolism and biochemical studies and

 mutagenicity assays in bacteria and B6C3F1 mice have provided a

 plausible explanation for the mechanism of action and the species

 differences in the carcinogenicity of methylene chloride to the lung

 and liver. This explanation is based on the existence of an isoenzyme

 of glutathione-S- transferase which specifically metabolizes

 methylene chloride to the reactive intermediates responsible for

 tumour induction in the mouse. Markedly lower levels of this enzyme

 in rats and hamsters are consistent with the fact that these tumours

 do not appear in these species. The levels of the enzyme are lower in

 human liver than those of the rat or hamster.

 Mutagenicity studies on methylene chloride in bacteria and in the

 B6C3F1 mouse, which shows a very high level of activity of the

 isoenzyme, reveal positive effects, whereas mutagenicity has not been

 demonstrated in standard in vivo mutagenicity assays using other

 systems. These observations are consistent with the above hypothesis

 and provide a mechanistic basis for the induction of tumours in the

 mouse.

 The role of the glutathione-S- transferase isoenzyme in the

 mediation of the demonstrated mutagenic effects, and the correlation

 between the activity of this pathway and the species differences in

 carcinogenic response in lung and liver, has led to its use as the

 dose surrogate in physiologically based pharmacokinetic models used

 for human health risk assessment.

 The pharmacokinetics of methylene chloride and the response seen

 in B6C3F1 mice suggest that this species is a poor model on which

 to base human hazard assessment to methylene chloride.

 The mechanism of mammary tumour formation in the rat is probably

 related to the effect of methylene chloride on prolactin levels in

 this species.

8.9 Interspecies and dose extrapolations by kinetic modelling

 Two physiologically based pharmacokinetic (PB-PK) models (Andersen

 et al., 1987; ECETOC, 1988) have been developed and provide

 quantitative estimates of the levels of methylene chloride metabolites

 in four mammalian species (mice, rats, hamsters and humans) following

 inhalation exposure. The models use information on various

 physiological parameters and metabolic constants for the cytochrome

 P-450 and glutathione- S-transferase pathways in the lung and liver,

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 measured in vitro for four species and measured in vivo for the

 mouse, with values for rats, hamsters and humans scaled from the mouse

 data. The metabolic constants for these models were obtained from the

 data described in section 8.8 and from gas uptake studies described by

 Andersen et al. (1987). The models were validated against other human

 experimental data such as the elimination of carbon monoxide and blood

 levels of methylene chloride. Time-course concentration data from the

 model were compared to experimental results in F-344 rats, Syrian

 Golden hamsters, B6C3F1 mice and human volunteers. The predicted

 values for each of the four species were in agreement with the

 experimental data. The models were also shown to predict the

 appearance and elimination of methylene chloride metabolites. A

 similar model was used by Andersen et al. (1991) to predict the time

 course of the disappearance of CO-Hb after exposure to methylene

 chloride. This model was also shown to predict CO-Hb levels in rats

 and humans exposed to methylene chloride.

 Several authors have discussed the assumptions, range and

 variability of the data used to construct these models. While these

 kinetic models have been extremely useful in improving the

 characterization of human exposure and potential risk, it should be

 recognized that they are based on a set of assumptions with varying

 degrees of certainty. Portier & Kaplan (1989) investigated the impact

 of varying the intra-population values of the biological parameters

 used in the model developed by Andersen et al. (1987) using Monte

 Carlo and resampling statistical methods. The results from this

 analysis indicated that the estimates of "effective" doses in humans

 may vary widely if variability of the parameters is taken into account

 in the PBPK model.

 Dankovic & Baiter (1993) investigated the impact of exercise and

 human inter-subject variability on the estimates of dose derived from

 the PBPK model. The model developed by Andersen et al. (1987) and

 Reitz et al. (1989) assumed resting values for the parameters

 governing cardiac output, alveolar ventilation and blood flow to the

 tissues. The authors modified these parameters to reflect light

 working conditions. The metabolic parameters for humans used in the

 Andersen et al. (1987) model were based on the average of four

 individual liver samples. The authors examined the impact of using the

 individual values rather than the average value in the PBPK model.

 Modifying the physiological parameters to reflect light work

 conditions increased the glutathion- S-transferase pathway metabolic

 contribution by a factor of 2.9 for the liver and 2.4 for the lung.

 When the model was also modified to reflect metabolic inter-individual

 (n = 4) variability in humans, the glutathione- S-transferase pathway

 contribution estimates were increased by as much as 5.4-fold for the

 liver and 3.6-fold for the lung. More recent data have become

 available on human metabolic parameters, and are summarized in section

 8.8.1. Based on 33 individual liver samples, they suggest that the

 Portier & Kaplan estimates which assume 200% variation for metabolic

 variability may be exaggerated. Since Dankovik & Bailer's calculations

 were based on the four actual values, their estimates would not

 change; however, three of their values represent the three highest

 values observed for human glutathione- S-transferase activity.

9. EFFECTS ON HUMANS

9.1 General population exposure

9.1.1 Environmental exposure

 Bell et al. (1991) conducted a study to examine the relationship

 between birth weight in Monroe County and exposure to emissions of

 methylene chloride from manufacturing processes of the Eastman Kodak

 Company in Rochester, New York, USA. County census tracts were

 categorized as high, moderate, low or no methylene chloride exposure,

 based on the Kodak Air Monitoring Program. Birth weight and

 information on variables known to influence birth weight were obtained

 from 91 302 birth certificates of white, single births to Monroe

 County residents from 1976 to 1987. At the level of methylene chloride

 exposure (highest predicted average concentration, 50 μg/m3), no

 significant adverse effect of exposure on birth weight was found,

 although several problems in the method of estimation of exposure were

 identified.

9.1.2 Oral exposure

 A 56-year-old woman was found deeply unconscious and cyanosed

 after ingesting approximately 300 ml of a paint remover containing

 mainly methylene chloride and methanol. Approximately one hour after

 ingestion the CO-Hb level measured was found to be 9%. This level

 varied between 2.5-12% over the following 2 days and dropped below 1%

 thereafter. The woman regained consciousness after 14 h, but over the

 following 3 weeks her condition was complicated by progressive renal

 failure, pneumonia, pancreatitis, on-going gastrointestinal

 haemorrhage and sepsis, which eventually led to death some 25 days

 following ingestion. It was considered that the corrosive properties

 of the formulation rather than the formation of CO-Hb were responsible

 for the lethal outcome (Hughes & Tracey, 1993).

 In an earlier poisoning case with the same paint remover

 formulation, there was recovery after ingestion of 0.5-1 litre

 (Roberts & Marshall, 1976).

9.2 Occupational exposure

9.2.1 Short-term exposure

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 9.2.1.1 Case studies

 A number of case-reports have been published regarding short-term

 exposure to methylene chloride in the occupational environment.

 Hall & Rumack (1990) described four cases of serious methylene

 chloride poisoning, including two fatalities, in small-scale

 furniture-stripping shops in Denver, Colorado, USA. In the three

 patients discovered while still alive, cardiac irregularities were

 recorded. Corneal burns with first- and/or second-degree burns were

 reported in areas having direct contact with the methylene chloride-

 based paint-stripping compound, and measured CO-Hb levels did not

 exceed 8.6%. In each case, no respiratory protection was worn and

 ventilation was inadequate, but exposure levels were not known. The

 authors concluded that the toxic effects were due to the anaesthetic

 properties of methylene chloride.

 A 67-year-old male who had been using a paint stripper in a poorly

 ventilated location was brought to a hospital emergency room

 complaining of headache and chest pain; he was also confused,

 disorientated, had a progressive loss of mental alertness, increased

 fatigue and lethargy, slurred speech, little recall of either recent

 or past events, and was disorientated to time (ATSDR, 1993).

 These and other case studies of methylene chloride poisoning

 during paint-stripping operations have demonstrated that inhalation

 can be fatal to humans (Hall & Rumack, 1990; Novak & Hain, 1990;

 Leikin et al., 1990; Manno et al., 1992). In the majority of cases

 reported, quantitative estimates of exposure levels have not been

 reported, although methylene chloride was detected in various tissues.

 In one case (Manno et al., 1989), air samples collected a few hours

 later from a well in which two men were found dead, were analysed by

 gas chromatography/mass spectrometry and were found to contain up to

 583 mg methylene chloride/litre and much lower or trace amounts of

 other solvents; blood levels collected at necropsy contained 571 and

 601 mg methylene chloride/litre and only trace to a few mg/litre of

 other solvents. The CO-Hb levels were about 30% in blood taken 24 h

 after death.

 In most cases, the cause of death was not clarified. However, in

 the report of five victims, including two deaths, described by Leikin

 et al. (1990), the authors concluded that the cause of death was due

 to solvent-induced narcosis and not carbon monoxide poisoning.

 Signs of CNS depression, narcosis, irritation of the eyes and

 respiratory tract, lung oedema and sometimes death were found after

 accidental exposures to methylene chloride or paint remover containing

 this compound (Moskowitz & Shapiro, 1952; Hughes, 1954; Bonventre et

 al., 1977; Fagin et al., 1980). Three myocardial infarctions in one

 subject were reported to have followed three exposures to a paint

 remover containing methylene chloride over a period of approximately 8

 months. The subject was exposed in a poorly ventilated room, and

 concentrations were up to 4511 mg/m3 in the breathing zones (Stewart

 et al., 1976). Electrocardiographic changes resembling those after

 carbon monoxide poisoning were found in an exposed man with a history

 suggesting ischaemic heart disease (Benzon et al., 1978). Three

 probable cases of phosgene poisoning occurred after the use of

 methylene-chloride-based paint remover near a source of heat

 (Gerritsen & Buschmann, 1960; English, 1964).

 A serious case of pulmonary oedema with bilateral exudative

 pleural effusions was reported in a 34-year-old man who presented with

 respiratory distress. Buie et al. (1986) speculated that hydrochloric

 acid, a product of dichloromethane under warm, moist conditions, may

 have played a role in this patient's parenchymal abnormalities.

 Recovery was complete with the exception of neuropsychiatric

 abnormalities thought to be related to exposure to methylene chloride.

 Miller et al. (1985) reported the case of a 19-year-old man using

 a tile remover, again in a poorly ventilated room. This patient

 presented with an array of signs and symptoms ranging from liver

 enzyme elevations to poorly localized abdominal pain. Renal studies

 and biopsy confirmed the diagnosis of acute tubular necrosis.

 Histological studies demonstrated plasma membrane changes in addition

 to mitochondrial effects suggestive of anoxic damage. Serum enzyme

 changes noted during the patient's stay in hospital suggested that

 hepatocellular injury accompanied the nephrotoxic sequelae.

 Another case of chemically induced hepatitis resulting from

 accidental exposure to methylene chloride alone has been described by

 Cordes et al. (1988). The liver was palpable but not enlarged or

 tender. The results of initial tests were normal except for a

 leukocyte count of 4900 μl with a left shift, and elevated serum

 enzyme levels of alkaline phosphatase 142, lactic dehydrogenase and

 serum aspartate aminotransferase (ALAT). Five days after admission,

 the patient was discharged from hospital. Laboratory tests for

 hepatitis A and B antibodies were negative.

 Further evidence for hepatic effects of methylene chloride were

 reported by Puurunen & Sotaniemi (1985). One week after a brief but

 extensive body exposure to methylene chloride, serum ALAT was elevated

 three-fold in a 24-year-old male chemical factory worker. The serum

 ALAT returned to normal within 2 weeks.

 9.2.1.2 Skin and eye effects

 The irritating action of methylene chloride on the eyes and skin

 has been shown in several cases (see section 9.2.1.1).

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 Slight erythema was found when methylene-chloride-containing

 aerosol-spray deodorants were used twice a day for 12 weeks by 75 men

 and women (Meltzer et al., 1977). On direct contact, methylene

 chloride caused a burning sensation and pain (Stewart & Dodd, 1964).

 Weber et al. (1990) reported a case of an individual who fell into

 a vat containing methylene chloride and methanol. After being immersed

 for about 15 min, the subject suffered extensive lesions, including

 skin burns of superficial and deep severe epidermal damage and a

 severe kerato-conjunctivitis.

 Wells & Waldron (1984) briefly reported on a young employee who

 climbed into a small open vessel with a bucket of about two litres of

 methylene chloride in order to clean the walls. The concentration of

 methylene chloride vapour within the vessel built up and he became

 unconscious, overturning the bucket as he slumped into the bottom of

 the vessel. After about 30 min, the man was rescued. During the time

 that he was in the vessel, the man sustained second and third degree

 burns to both legs, the areas affected being those which were bearing

 the weight of his body while he was unconscious. On his discharge from

 hospital, these areas were dry and required no skin grafting (Wells &

 Waldron, 1984).

 9.2.1.3 Laboratory studies

 Neurobehavioural changes were observed at low exposure level after

 volunteers were exposed to 694 mg/m3 for 1.5-3 h. Vigilance

 disturbance and impaired combined tracking monitoring performance were

 found (Putz et al., 1976). The critical flicker frequency, one of the

 measures for visual function, was reduced after 95 min of exposure to

 1040 mg/m3 (Fodor & Winneke, 1971). Visually evoked responses (one

 of the surrogate methods of measuring visual functions) were altered

 after 1 h of exposure to 2400 mg/m3, while exposed subjects

 experienced lightheadedness. Blood and urine variables, except CO-Hb

 levels, were normal in this study after 1-2 h of exposure to levels of

 methylene chloride between 739 and 3420 mg/m3. No eye, nose, or

 throat irritation was observed (Stewart et al., 1972). Most

 neurobehavioural effects observed were less pronounced or absent, with

 carbon monoxide exposures resulting in comparable CO-Hb levels (Putz

 et al., 1976).

 In a double-blind laboratory experiment, a short inhalation

 exposure to 2.5 mg methylene chloride/litre did not impair vigilance

 performance in human volunteers (time of exposure and number of

 subjects not stated) (Kozena et al., 1990).

 A clinical laboratory evaluation of 266 exposed volunteer workers

 and 251 reference volunteer workers from two cellulose di- and

 tri-acetate plants in the USA, which took into account smoking habits,

 race, sex, age, intensity of exposure, and time of venepuncture,

 revealed increases in red cell counts, haemoglobin levels and

 haematocrit among white women exposed to a methylene chloride level of

 approximately 1650 mg/m3. CO-Hb levels were elevated in all exposed

 groups at all exposure levels (section 5.3). A dose-related increase

 was observed in serum bilirubin for exposed subjects of both sexes. A

 group of 24 exposed male volunteers and 26 reference male volunteers

 from the above two industries was also selected for 24-h electro-

 cardiographic monitoring. Three exposed and 8 reference workers had

 reported a history of heart disease. Neither increased ventricular or

 supraventricular ectopic activity nor increased episodic ST-segment

 depression was found to be associated with methylene chloride exposure

 (Ott et al., 1983).

9.2.2 Long-term exposure

 9.2.2.1 Case studies

 Irreversible damage to the central nervous system with acoustic

 and optical illusions and hallucinations was diagnosed in one man who

 had been exposed for 5 years to methylene chloride at levels ranging

 from 2290 to 12 500 mg/m3 (Weiss, 1967). Another man, exposed for

 3 years to levels of methylene chloride ranging from 1735 to

 3470 mg/m3 showed a bilateral temporal lobe degeneration

 (Barrowcliff & Knell, 1979). A case of delirium and seizures was

 reported in a man who was exposed to methylene chloride for 4 years.

 The man reported a 12-month history of intermittent headache, nausea,

 blurred vision, shortness of breath, and transient memory

 disturbances. Neuropsychological and EEG examinations revealed a

 dysfunction of the right hemisphere. All symptoms and signs cleared

 with removal from the workplace (Tariot, 1983).

 Between December 1984 and June 1986, 34 men with occupational

 exposure to methylene chloride were evaluated at the Greater

 Cincinnati Occupational Health Centre. The mean value of the exposure

 was reported to be 240 mg/m3, ranging from 11 to 544 mg/m3.

 Although the primary complaint of these employees involved problems

 associated with central nervous dysfunction, 8 of the 34 complained of

 testicular, epididymal or lower abdominal pain, and had clinical

 histories relating to infertility. Low sperm counts were reported in

 workers who used methylene chloride in bonding operations which also

 resulted in possible skin exposure. It is uncertain whether the effect

 was due to methylene chloride since the workers were also exposed to

 other chemicals (Kelly, 1988).

 a) Morbidity studies

 The few reports available deal with small groups of occupationally

 exposed subjects.

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 Workers exposed occupationally to a time-weighted average of

 114 mg/m3 had CO-Hb levels of between 0.8 and 2.5%. No effects were

 found on clinical chemistry, haematology or electrocardio-grams (Di

 Vincenzo & Kaplan, 1981a). Cherry et al. (1981) did not find any

 exposure-related, long-term damage in 29 subjects as shown by

 subjective symptoms, neurobehavioural tests, motor nerve conduction

 velocity, electrocardiograms and clinical examinations. The men had

 been exposed for several years to levels of methylene chloride ranging

 from 260 to 347 mg/m3. Age-matched controls were used. In a study

 without a control group, neurasthenic disorders and irritation of the

 eyes and respiratory passages were experienced by half of the 33

 workers exposed to methylene chloride for an average of 2 years.

 Digestive disorders were reported by one-third of these workers.

 Formic acid was found in the urine. No other deviations were found

 during the internal, nervous system, eye and laboratory examinations.

 The methylene chloride concentrations measured varied between 100 and

 17 000 mg/m3 (Kuzelova & Vlasak, 1966).

 A group of 1758 retired airline maintenance workers was surveyed

 by mail and telephone to identify a cohort of workers with more than

 22 years of methylene chloride exposure following the stripping of

 paint from airplanes. A cohort of 25 exposed and 21 non-exposed

 retirees met the criteria and were tested extensively (Becker & Lash,

 1990; Lash et al., 1991). Following a specially prepared battery of

 neuropsychological and neurophysio-logical tests performed by

 professionals without prior knowledge of exposure status of the

 employees, exposed and control outcome measures were all within the

 "normal" range. No statistically significant difference was found

 between exposed and control groups, although subtle differences in

 attention and memory were detected.

 In 46 subjects exposed to methylene chloride concentrations of

 6-34 mg/m3 for several years, an excess (not significant) of

 digestive disorders and hypotonia was found over controls, while

 symptoms of gall bladder pathology and swollen liver were frequent. No

 details were given concerning drinking or smoking habits (Kashin et

 al., 1980).

 A case-control study on 44 women who had a spontaneous abortion

 was performed within a cohort of female workers employed in Finnish

 pharmaceutical factories during 1973 or 1975 to 1980. Three controls

 matched for age at conception within 2.5 years were chosen for every

 case except two. Information about pregnancy outcome was collected

 from hospital data, and data on exposures from health personnel at the

 factories. The odds ratio for methylene chloride exposure, based on 11

 exposed cases, was of borderline significance (2.3 with a 95%

 confidence interval, 1.0-5.7; p = 0.06). Odds ratios were also

 increased for exposures to many other solvents. For those exposed to

 methylene chloride less than once a week the odds ratio was 2.0 (95%

 CI = 0.6-6.6); whereas for those exposed more than once a week the

 odds ratio was 2.8 (95% CI = 0.8-9.5) (Taskinen et al., 1986).

 A group of active workers (n = 150) who had worked for at least 10

 years in an area where average exposures were 1677 mg/m3 were

 compared to an unexposed group of workers (n = 260) with regards to

 symptoms and blood chemistry. The methylene chloride workers were also

 exposed to acetone and methanol (900 ppm and 100 ppm 8-h TWAs,

 respectively). Health history and blood samples had been collected as

 part of a company-sponsored health monitoring programme in which both

 exposed and unexposed workers were participants. No remarkable or

 statistically significant differences were observed in the selected

 symptoms (including irregular heartbeat, dizziness or loss of memory)

 or in SGPT, bilirubin or haematocrit. The only noticeable difference

 was in SGOT, where the non-exposed group had higher levels than the

 exposed group (means of 28.2 versus 25.1, p = 0.06). A limitation to

 this study is that both groups consisted of active healthy workers.

 The age and sex distribution of the two groups was reported to be

 similar, but was not given Soden, 1993).

 b) Mortality studies

 Several studies have evaluated the effects of long-term exposure

 to methylene chloride on mortality of workers. The first study was by

 Friedlander et al. (1978), who performed both a retrospective cohort

 mortality, i.e. standardized mortality ratio (SMR) study, and a

 proportionate mortality ratio (PMR) study of men exposed to methylene

 chloride at a Kodak photographic film production facility in

 Rochester, New York. The PMR study included 334 deaths that occurred

 between 1956 and 1976 among former workers who were exposed to

 methylene chloride at the facility. The retrospective cohort mortality

 study included 751 workers employed during 1964 and involved follow-up

 of this cohort up to 1976. Hearne & Friedlander (1981) extended the

 follow-up of this cohort to 1980 and subsequently expanded the study

 population to include all workers (n = 1013) who were exposed for at

 least one year between 1964 and 1970 (Hearne et al., 1987). In a more

 recent publication Hearne et al. (1990) extended follow-up of this

 expanded cohort to 1988.

 The Friedlander et al. (1978) study was initially conducted to

 test the hypothesis that exposure to methylene chloride increases the

 risk of ischaemic heart disease. This hypothesis was based on the fact

 that methylene chloride is metabolized to carbon monoxide and induces

 the formation of CO-Hb in humans (Stewart et al., 1972). Increases in

 CO-Hb as low as 2% (Allred et al., 1989) have been shown to induce

 electrocardiographic changes in exercising patients with pre-existing

 coronary artery disease. An excess of ischaemic heart disease

 mortality has also been reported in a cohort of tunnel workers exposed

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 to carbon monoxide (Stern et al., 1988). Liver and lung cancer were

 also considered a priori hypotheses in the subsequent articles by

 Hearne et al., based on the results from the animal bioassay data

 described in chapter 8.

 Comparisons in both studies (Friedlander et al., 1978 and Hearne

 et al., 1990) were made with mortality rates (or proportions) from New

 York State and from an internal unexposed cohort from the Kodak

 facility. Extensive industrial hygiene measurements were available for

 this cohort from after 1980, which indicated that 8-h TWA exposure

 concentrations for different occupational classifications ranged from

 approximately 35.3-402 mg/m3 (10-114 ppm) and the mean exposure was

 91.8 mg/m3 (26 ppm) (Hearne et al., 1987). An exposure-response

 analysis, which was presented by Hearne et al. (1987), failed to

 demonstrate an increasing risk for these causes of death with

 increasing methylene chloride exposure. Hearne et al. (1987) observed

 an excess of pancreatic cancer mortality (8 observed, SMR = 2.58, 95%

 confidence interval (CI) = 1.11-5.08). Mirer et al. (1988) published a

 letter suggesting that the excess of pancreatic cancer mortality

 increased with time since first exposure (latency) and was greatest

 among workers in the highest exposure (750 ppm-years) and latency

 (> 30 years) categories (4 observed, SMR = 4.49, 95%

 CI = 1.22-11.49). No new pancreatic cancer cases were identified with

 additional follow-up, and with the additional data the excess was not

 statistically significant (SMR = 1.90, 95% CI = 0.82-3.75) (Hearne et

 al., 1990). The study by Friedlander et al. (1978) and the subsequent

 studies by Hearne et al. (1987,1990) failed to detect a significantly

 increased risk of ischaemic heart disease, lung cancer, liver cancer

 or other cancers among methylene-chloride-exposed workers. It also

 important to recognize that workers at the Kodak facility (T. Hearne,

 personal communication to the IPCS) were not permitted to smoke at

 their workstations and that this fact may have induced a negative bias

 in these studies, particularly with respect to lung cancer or

 cardiovascular disease. Unfortunately detailed information on

 cigarette smoking was not available for this cohort and thus

 adjustments for this potential bias could not be made.

 Ott et al. (1983) evaluated a cohort of workers exposed to

 methylene chloride in the production of triacetate fibre at a

 manufacturing plant in Rock Hill, South Carolina, USA. This cohort

 included 1271 males and female workers who were employed for at least

 3 months, sometime between 1954 and 1977, Workers from another textile

 facility that were not exposed to methylene chloride, but met the same

 inclusion criteria as the exposed cohort, were also included for

 comparison purposes. All workers (both exposed and unexposed) were

 followed for vital status ascertainment up to June 1977. Eight-hour

 TWA methylene chloride exposures in this cohort were estimated to

 range from 494 to 1677 mg/m3 (140 to 475 ppm) from a survey

 conducted in 1977 and 1978. Workers in this study were also reported

 to have been exposed to methanol and acetone. The mortality experience

 of the exposed cohort was compared with the mortality of the USA

 population using a modified life-table approach (SMRs). Direct

 comparisons were also made between the mortality of the exposed and

 unexposed cohorts. Mortality from cardiovascular disease or any other

 cause was not found to be significantly increased relative to the USA

 population. However, the authors did observe a significant increase in

 the risk of ischaemic heart disease (RR = 3.1, p < 0.05) among white

 men in the analysis when the mortality rates of the exposed and

 unexposed cohorts were compared. It was also noted that 8 of the 14

 ischaemic heart disease deaths among exposed white men occurred among

 workers who were actively employed. Although Ott et al. (1983) did not

 report any increase in cancer mortality, this study was not designed

 to evaluate cancer and only included seven malignant neoplasms.

 The follow-up of the exposed cohort (but not the unexposed one)

 studied by Ott et al. (1983) was subsequently extended to 1986 by

 Lanes et al. (1990). The analyses presented in this paper were solely

 based upon comparisons with the USA population and did not include any

 direct comparisons with the unexposed cohort as did the study by Ott

 et al. (1983). This study failed to detect an excess of cardiovascular

 or ischaemic heart disease. A significant excess of cancers of the

 biliary passages and liver (SMR = 5.75, 95% CI = 1.82-13.78) was

 observed. Three of the cancers were cholangiocarcinomas of various

 biliary sites while the fourth was a liver adenocarcinoma. The SMR for

 biliary cancer was estimated using mortality rates from 1973 to 1977.

 The SMR for biliary cancer alone was 20 (95% CI = 5.2-56). Three of

 the four liver and biliary cancer deaths observed in this study were

 thought to have occurred among workers with 10 or more years of

 employment and at least 20 years since first employment (0.35

 expected, SMR = 11.43), a pattern consistent with a potential

 occupational etiology. One of the cases had only been exposed to

 methylene chloride for one year. Lanes et al. (1993) recently extended

 the follow-up of the cohort for an additional 4 years. Although no

 additional cases of liver or biliary cancer were observed, an excess

 from the previous study persisted (SMR = 2.98, 95% CI = 0.81-7.63].

 This latest report did not include an analysis for biliary cancer

 alone.

 Another retrospective cohort mortality study of workers from a

 Hoechst-Celanese cellulose acetate fibre plant in Cumberland,

 Maryland, USA was reported by Gibbs (1992). This study included 3211

 cellulose fibre workers employed in or after 1970 and followed until

 1989. The cohort was divided into three groups: high (> 1235 mg/m3,

 > 350 ppm) methylene chloride exposure, low exposure (176-350 mg/m3,

 50-100 ppm), and no exposure. Comparisons were made with USA, Maryland

 and county mortality rates. Estimates of exposure levels for this

 population were based on industrial hygiene measurements from the plant

 studied by Ott et al. (1983), which used similar production methods.

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 Cancer of the prostate was significantly elevated among men, and

 particularly among those with long latency and with high levels of

 methylene chloride exposure. A significant excess of cervical cancer

 was observed among women in the low exposure group, based on Maryland

 rates (SMR = 4.75 based upon 5 observed), but there was no evidence of

 a dose-response relationship. Two cases (exp = 1.40) of biliary cancer

 were observed among the combined high and low exposure groups. An

 excess of ischaemic heart disease mortality was observed among workers

 in all three groups when comparisons were based upon Maryland rates,

 but not when local county rates were used. Mortality from lung,

 pancreatic, liver/biliary and other cancers was not observed to be

 significantly elevated in this study. As with the Kodak study, workers

 at the Hoechst-Celanese facilities were not permitted to smoke at

 their workstations. Again this fact may have induced a negative bias

 in these studies, particularly with respect to lung cancer or

 cardiovascular disease.

 A cohort study of chemical workers included a sub-cohort of 226

 men employed for at least one year in chlorinated methanes production

 (Ott et al., 1983). Methylene chloride is principally produced by a

 method involving the hydrochlorination of methanol which also results

 in the production of chloroform and carbon tetrachloride (IARC, 1986).

 The men had been employed between 1940 and 1969 and were followed for

 mortality until 1979. In all, 42 deaths were observed and no excesses

 of respiratory cancer (SMR = 0.70, based on 3 observed) or circulatory

 disease (SMR = 0.68, based on 18 observed) were seen. The results for

 liver and biliary cancer were not reported, but three cases of

 pancreatic cancer were observed (0.9 expected). All three persons had

 worked in chlorinated methanes production between 1942 and 1946; two

 had been employed for less than 5 years, the third for 6 years. No

 further information on exposure for the individuals or the sub-cohort

 was given and the mixed exposure to methylene chloride, chloroform,

 and carbon tetrachloride limits the interpretation of the results with

 respect to methylene chloride.

 Finally, Heineman et al. (1994) reported the results of a case-

 control study of astrocytic brain cancer and occupational exposure to

 chlorinated aliphatic hydrocarbons. The study included 300 cases with

 a hospital diagnosis of astrocytic brain cancer and 320 controls

 matched on age, year of death and geographical area. A job-exposure

 matrix was used to classify cases and controls in terms of potential

 exposure to chlorinated aliphatic hydrocarbons including methylene

 chloride (Gomez et al., 1994); 119 cases and 108 controls were

 classified as being potentially exposed to methylene chloride in this

 study. The risk was reported to increase with the probability of

 exposure (Odds Ratio (OR) - 2.4 for high probability, 95%

 CI = 0.9-6.4) and duration of employment (OR = 1.9 for > 20 years,

 95% CI = 0.7-5.2) in jobs considered to be exposed to methylene

 chloride after adjustment for other solvent exposures. The exposure

 information used in this study is weaker than that generally used in

 the retrospective cohort mortality studies described above and the

 results should therefore be viewed more cautiously.

9.3 Appraisal of human effects

 The main toxic effects of methylene chloride are reversible CNS

 depression and CO-Hb formation. Liver and renal dysfunctions and

 effects on haematological parameters have also been reported

 following exposure to methylene chloride. Methylene chloride will

 irritate the skin and eyes especially when evaporation is prevented.

 Prolonged contact may cause chemical burns.

 Neurophysiological and neurobehavioural disturbances have been

 observed in human volunteers exposed to methylene chloride at

 concentrations of 694 mg/m3 for 1.5-3.0 h. No evidence of

 neurological effects was seen in men exposed to methylene chloride

 for several years at concentrations ranging from 260 to 347 mg/m3.

 Similarly, the performance of a group of retired airplane strippers,

 with a long history of exposure to methylene chloride (22 years) at

 high but unspecified levels, in a battery of neurophysiological and

 psychological tests was within the "normal" range when compared with

 a control group who had a history of either no or only low exposure

 to methylene chloride.

 Fatalities due to excessive oral exposure to methylene chloride

 have been reported. A case of serious pulmonary oedema has been

 reported after excessive inhalation.

 An increased rate of spontaneous abortion in employees in Finnish

 pharmaceutical industries has been attributed to exposure to

 methylene chloride. This isolated finding from a limited study makes

 it difficult to interpret the significance of the observations.

 Five mortality studies on methylene chloride have been conducted

 and evaluated specifically with regard to cancer and cardiovascular

 disease. None of the studies demonstrated a relationship between

 exposure to methylene chloride and lung or liver cancer mortality.

 With regard to the lung cancers, the lack of smoking histories

 hampers the interpretability of the results. An excess of mortality

 from biliary cancer was reported in one study, but this was not

 corroborated by other studies. Two studies showed an excess in

 mortality from pancreatic cancer. In one of the studies no new

 pancreatic cancer cases were identified with additional follow-up;

 with the additional data the excess was not statistically

 significant. It should be noted that the size of these studies

 resulted in very low statistical power to detect an excess

 particularly for rare cancers such as liver and biliary tract

 tumours.

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 Associations between exposure to methylene chloride and prostate

 and cervical cancers have been reported in studies, each of which had

 its limitations. An association between the potential for exposure to

 methylene chloride and other organic solvents and brain cancer was

 found in a case-control study which classified exposure to methylene

 chloride using a job exposure matrix. This finding should be viewed

 with caution.

 The results from these studies have been contradictory with

 respect to mortality from ischaemic heart disease. A role for

 methylene chloride in the induction of ischaemic heart disease is

 plausible based on the fact that methylene chloride is metabolized to

 carbon monoxide and induces the formation of carboxyhaemoglobin in

 humans. An excess of cardiovascular disease was reported in one of

 the mortality studies. The fact that further studies did not provide

 any compelling evidence of an increased risk of cardiovascular

 disease might be attributable to their reliance on comparisons with

 the general population as the referent group. The use of general

 mortality rates in occupational cohort mortality studies may bias the

 results towards the null (i.e. no effect) due to the "healthy worker

 effect" which is particularly strong for cardiovascular diseases.

 This bias may have been further exacerbated by the fact that workers

 were not permitted to smoke at their workstations.

 The currently available epidemiological studies are inadequate

 for drawing any firm conclusions with regard to either cancer or

 cardiovascular disease risk.

10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT

10.1 Evaluation of human health risks

 Human exposure to methylene chloride is mainly by inhalation of

 the vapour. Exposure of the general population to methylene chloride

 depends strongly on the indoor air concentration. Owing to the use of

 products containing methylene chloride, peak concentrations of up to

 4000 μg/m3 have been reported. However, 24-h average exposures are

 in general below 50 μg/m3.

 Methylene chloride is rapidly absorbed through the lung and also

 from the gastrointestinal tract. It is absorbed via the skin, but at a

 much slower rate than by the other routes. Once absorbed, methylene

 chloride is distributed throughout the body and will cross both the

 placenta and the blood-brain barrier. It is rapidly excreted, the

 majority being exhaled unchanged via the lungs. The remainder is

 metabolized to carbon monoxide, carbon dioxide and inorganic chloride.

 Two metabolic pathways have been identified, one involving cytochrome

 P-450 and the second involving glutathione- S-transferase. Clear

 species differences exist in the relative contributions of these two

 pathways. These differences have been used as a basis for a

 physiologically based pharmaco-kinetic (PB-PK) model for methylene

 chloride, which allows interspecies comparison of the concentrations

 of active metabolites at the target tissues, thus enhancing the value

 of results from animal studies in human health risk assessment. This

 approach has been used in assessing the human cancer risk associated

 with exposure to methylene chloride.

 The acute toxicity of methylene chloride is low. The predominant

 effects in human beings are CNS depression and elevated blood

 carboxyhaemoglobin (CO-Hb) levels. These effects are reversible. Other

 target organs can be the liver and, occasionally, the kidney. The

 odour threshold concentration of methylene chloride is reported to be

 540 mg/m3 or more. Mild CNS effects have been reported following

 exposure to concentrations as low as 694 mg/m3 for 1.5-3 h

 (behavioural disturbances). More significant effects occur at

 concentrations in excess of 2000 mg/m3. Narcosis has been reported

 to occur following a 0.5-h exposure to 69 000 mg/m3. Metabolism to

 carbon monoxide leads to increases in blood CO-Hb levels following

 acute exposure to the vapour, a process which becomes saturable

 following exposures to high levels of methylene chloride. Exposure to

 either 100 or 530 mg/m3 for 7.5 h leads to CO-Hb levels of 3.4% and

 5.3%, respectively, in human volunteers. This effect forms the basis

 of most, if not all, published occupational exposure limits, where a

 level of 5.0% is judged to be acceptable.

 The predominant effects following repeated or long-term exposure

 to methylene chloride are the same as for acute exposure. Reversible

 symptoms of CNS depression are seen in several species, including

 humans. The lowest-observed-effect level (LOEL) for this effect in all

 animal species is 7100 mg/m3 by inhalation. No evidence of

 irreversible neurological damage was seen in rats exposed to methylene

 chloride by inhalation at concentrations up to 7100 mg/m3 for

 13 weeks. Additional target organs reported in various species

 chronically exposed to methylene chloride include the liver and,

 occasionally, the kidney. The no-observed-adverse-effect level (NOAEL)

 for chronic intermittent inhalation exposure was judged to be

 710 mg/m3 in rats. After continuous exposure, slight cytoplasmic

 vacuolization in the liver of both mice and rats was observed at

 88-350 mg/m3.

 A single study has reported the presence of methylene chloride in

 the placenta, fetus and breast milk of women following occupational

 exposure. The teratogenic potential of methylene chloride has been

 assessed in three animal studies. Small effects on either fetal or

 maternal body weights were reported, but no evidence of an effect on

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 the incidence of skeletal malformations or other developmental effects

 was seen. A well-conducted two-generation reproductive toxicity study

 in rats exposed to methylene chloride by inhalation at concentrations

 up to 5300 mg/m3, 6 h/day, for 5 days/week showed no evidence of an

 adverse effect on any reproductive parameter, neonatal survival or

 neonatal growth in either the F0 or F1 generations.

 Under appropriate exposure conditions, methylene chloride is

 mutagenic in prokaryotic microorganisms (Salmonella or E. coli)

 with or without metabolic activation. In eukaryotic systems it gave

 either negative or, in one case, weakly positive results. In vitro

 gene mutation assays and tests for unscheduled DNA synthesis (UDS) in

 mammalian cells were uniformly negative. In vitro assays for

 chromosomal aberrations using different cell types gave positive

 results, whereas negative or equivocal results were obtained in tests

 for sister chromatid exchange (SCE) induction. The majority of the in

 vivo studies reported provided no evidence of mutagenicity of

 methylene chloride (e.g., chromosome aberration assay, micronucleus

 test or UDS assay). A very marginal increase in frequencies of SCEs,

 chromosomal aberrations and micronuclei in mice has been reported

 following inhalation exposure to high concentrations of methylene

 chloride. The significance of these results is questionable due to

 methodological deficiencies in the statistical analysis. There was no

 evidence of binding of methylene chloride to DNA or DNA damage in rats

 or mice given high doses.

 Within the limitations of the short-term tests currently

 available, there is no conclusive evidence that methylene chloride is

 genotoxic in vivo.

 Methylene chloride is carcinogenic in the mouse, causing both lung

 and liver tumours, following lifetime exposure to high concentrations

 (7100 and 14 100 mg/m3). These tumours were not seen in the rat or

 the hamster.

 Increased incidence of benign mammary tumours in female rats was

 observed in one study, and increased incidence and multiplicity were

 observed in two other rat studies. The increased incidence of these

 tumours was within the historical control range; nevertheless there

 was a dose-response relationship within one study. It is considered

 that an increase in a tumour type, which occurs with high and variable

 incidences in control animals, which does not progress to malignancy,

 and which may be related to changes in prolactin levels, is of little

 importance in human hazard assessment.

 In vitro and in vivo metabolism and biochemical studies, and

 mutagenicity assays in bacteria and B6C3F1 mice have provided a

 plausible explanation for the mechanism of action and the species

 differences in the carcinogenicity of methylene chloride to the lung

 and liver. This explanation is based on the existence of an isoenzyme

 of glutathione- S-transferase which specifically metabolizes

 methylene chloride to the reactive intermediates responsible for

 tumour induction in the mouse. Markedly lower levels of this enzyme in

 rats and hamsters are consistent with the fact that these tumours do

 not appear in these species. The levels of the enzyme in the liver are

 lower in humans than in rats or hamsters. The variability of the

 current estimates of this enzyme activity in human liver is low, but

 the possibility of wider variation existing in subpopulations cannot

 be discounted. Although the currently available information on enzyme

 activity in human lung is limited, it is expected to be lower than in

 human liver. The carcinogenic potency of methylene chloride in man is

 expected to be low.

 Mutagenicity studies on methylene chloride in bacteria and in the

 B6C3F1 mouse, which shows a very high level of activity of the

 isoenzyme, reveal positive effects, whereas mutagenicity has not been

 demonstrated in standard in vivo mutagenicity assays using other

 systems. These observations are consistent with the above hypothesis

 and provide a mechanistic basis for the induction of tumours in the

 mouse.

 The role of the glutathione- S-transferase isoenzyme in the

 mediation of the demonstrated mutagenic effects, together with the

 correlation between the activity of this pathway and the species

 differences in carcinogenic response, has led to its use as the dose

 surrogate in physiologically based pharmacokinetic models used for

 human health risk assessment.

 Overall, animal inhalation studies have shown effects on the liver

 from 710 mg/m3 and on other organs from 1700 mg/m3. However, these

 effects have not been observed in epidemiological studies. Effects on

 the CNS have been observed in both animals and humans and a threshold

 in humans has been defined, based on the level of the metabolite

 carbon monoxide in the blood, leading to exposure limits of the order

 of 177 mg/m3.

10.2 Evaluation of effects on the environment

 Due to its high volatility methylene chloride released to the

 environment will end up in the atmosphere where it can be transported

 to regions far removed from the emission source. Methylene chloride is

 degraded in the troposphere by reaction with hydroxyl radicals giving

 carbon dioxide and hydrogen chloride as major breakdown products.

 Based on a lifetime in the troposphere of about 6 months it may be

 assumed that only a few percent, if any, of methylene chloride will

 reach the stratosphere. No significant impact on stratospheric ozone

 depletion is expected. Methylene chloride will also not contribute

 significantly to photochemical smog formation. In ambient air in rural

 and remote areas, background levels of 0.07-0.29 μg/m3 have been

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 measured. In suburban and urban areas levels up to 2 and 15 μg/m3

 have been found.

 Concentrations of methylene chloride in the surface water of

 rivers in industrialized areas stay generally below 10 μg/litre. In

 industrial effluents, outfalls of municipal water treatment plants and

 leachates of landfills, concentrations of methylene chloride of up to

 200 mg/litre have been measured.

 In the aquatic environment, fish and amphibian embryos have been

 shown to be the most sensitive to methylene chloride, with effects on

 hatching from 5.5 mg/litre; adult aquatic organisms are relatively

 insensitive even after prolonged exposure. There is no evidence to

 suggest that methylene chloride and/or its metabolites bioaccumulate

 in the environment. Given the concentrations observed in surface water

 (< 10 μg/litre) and those in contaminated effluents

 (< 200 mg/litre), no significant impact on the aquatic environment is

 expected.

 Localized contamination of soils will not significantly disperse

 despite the mobility of methylene chloride; in groundwaters and soils,

 biological degradation processes have been identified capable of

 mineralizing methylene chloride in a few days. From the limited

 information on soil organisms, it may be assumed that contamination of

 soil has only a local and transient effect.

 Apart from accidental spills, it is concluded that the present use

 of methylene chloride has no significant impact on the environment.

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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 possède une puissante odeur

 éthérée. Lorsqu'il est pur et anhydre, ce composé est très stable. Le

 chlorure de méthylène s'hydrolyse lentement en présence d'humidité,

 pour donner une petite quantité de chlorure d'hydrogène. Le chlorure

 de méthylène du commerce est généralement additionné de petites

 quantités de stabilisants afin d'en éviter la décomposition.

 Il existe des méthodes d'analyse pour le dosage du chlorure de

 méthylène dans les milieux biologiques et les échantillons prélevés

 dans l'environnement. Dans tous les cas, on fait appel à la

 chromatographie en phase gazeuse avec un détecteur convenable. On

 obtient ainsi des limites de détection très basses (par exemple dans

 les denrées alimentaires 7 ng/échantillon; dans l'eau 0,01/μg/litre;

 dans l'air 1,76/μg/m3 (0,5 ppb); dans le sang 0,022 mg/litre).

2. Sources d'exposition humaine et environnementale

 On estime à 570 000 tonnes la production annuelle mondiale de

 chlorure de méthylène. On l'utilise la plupart du temps comme solvant

 des graisses, des matières plastiques et des liants pour peinture, en

 particulier à cause de sa volatilité et de sa stabilité. Dans

 l'ensemble du monde, il est utilisé à 20-25% dans des aérosols, à 25%

 comme décapant des peintures, à 35-40% comme solvant au cours des

 différentes opérations de l'industrie chimique et pharmaceutique, et

 enfin à 10-15% dans diverses applications allant de la fabrication de

 mousse de polyuréthane au décapage des métaux. Son utilisation tend à

 augmenter, tout au moins en Europe de l'Ouest.

 Les émissions atmosphériques de chlorure de méthylène proviennent

 à plus de 99% de son utilisation comme produit final par diverses

 industries ou encore comme décapant des surfaces peintes et comme

 constituant des bombes aérosols à usage domestique.

3. Transport, distribution et transformation dans l'environnement

 En raison de sa forte volatilité, la majeure partie du chlorure de

 méthylène libéré dans l'environnement se répartit dans l'atmosphère où

 il est décomposé en l'espace de six mois par réaction avec des

 radicaux hydroxyles d'origine photochimique.

 Dans l'eau, il est décomposé par voie abiotique beaucoup plus

 lentement qu'il ne s'évapore. On a montré que le chlorure de méthylène

 disparaissait rapidement du sol et des eaux souterraines.

 Grâce à divers systèmes d'épreuve on a pu établir les modalités de

 la décomposition aérobic et anaérobie du chlorure de méthylène. Sa

 biodécomposition complète est rapide, notamment en aérobiose, sous

 l'action de cultures bactériennes acclimatées (par exemple 49 à 66% de

 minéralisation en 50 heures en présence de boues d'effluents

 municipaux acclimatées). Dans les réacteurs biologiques, la

 décomposition peut atteindre 10% à l'heure. Rien n'indique qu'il y ait

 une bioaccumulation ou une bioamplification importante.

4. Concentrations dans l'environnement et exposition humaine

 On a décelé la présence de chlorure de méthylène dans l'air

 ambiant de régions rurales et de zones écartées, à la concentration de

 0,07-0,29/μg/m3. Dans les zones de banlieue, la concentration

 moyenne est inférieure à 2 μ/m3 et dans les zones urbaines, elle est

 inférieure à 15 μg/m3. A proximité de décharges jugées dangereuses,

 on a trouvé des concentrations allant jusqu'à 43 μg/m3. Les

 précipitations peuvent également contenir du chlorure de méthylène.

 Le chlorure de méthylène pénètre dans l'environnement aquatique

 par suite de la décharge d'eaux résiduaires provenant des diverses

 industries et on en a retrouvé dans les eaux superficielles et

 souterraines ainsi que dans les sédiments.

 S'il y a exposition au chlorure de méthylène de personnes

 appartenant à la population générale, c'est par suite de son

 utilisation dans certains produits de consommation tels que les

 décapants pour peinture, dont l'emploi peut entraîner la présence de

 teneurs relativement importantes dans l'air intérieur. En ce qui

 concerne l'exposition professionnelle au cours de la production de

 chlorure de méthylène, elle se produit essentiellement au cours du

 remplissage et du conditionnement (la fabrication s'effectue en

 circuit fermé). Du fait de l'utilisation de ce composé comme décapant

 pour peinture, il peut également y avoir exposition professionnelle

 lors de la préparation de ces décapants, lors de la fabrication de

 certains équipements et également lorsqu'on procède au décapage du

 mobilier. Le chlorure de méthylène est largement utilisé comme solvant

 lors de la préparation de différents produits, en particulier dans les

 industries citées à la section 1.2.

 La surveillance biologique de l'exposition au chlorure de

 méthylène peut s'effectuer par dosage du solvant lui-même dans l'air

 expiré ou dans le sang. Toutefois, étant donné que la production de

 monoxyde de carbone constitue le facteur limitant du risque en cas

 d'exposition supérieure à 3 ou 4 heures par jour, il vaut mieux que la

 surveillance biologique s'effectue soit par dosage du monoxyde de

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 carbone dans l'air expiré, soit par dosage de la carboxyhémoglobine

 (CO-Hb) dans le sang. Toutefois cette méthode ne vaut que pour les

 sujets non fumeurs. Les prélèvements doivent s'effectuer environ 0 à 2

 heures après l'exposition, ou au bout de 16 heures, c'est-à-dire le

 matin suivant.

 Les taux de CO-Hb, 2 heures après cessation de l'exposition, ne

 devraient pas dépasser 2 à 3%, et au bout de 16 heures 1%, dans le cas

 d'une exposition de 8 heures à moins de 350 mg de chlorure de

 méthylène par m3 chez un non fumeur.

5. Cinétique et métabolisme chez les animaux de laboratoire et l'homme

 Le chlorure de méthylène est rapidement absorbé au niveau des

 alvéoles pulmonaires et pénètre dans le courant sanguin. Il est

 également absorbé dans les voies digestives et aussi par voie

 percutanée, mais cette voie est la plus lente de toutes.

 Le chlorure de méthylène est très rapidement excrété, en majeure

 partie dans l'air expiré. Il peut traverser la barrière hémato-

 encéphalique ainsi que le placenta et on peut le retrouver en petites

 quantités dans les urines ou le lait.

 A fortes concentrations, la majeure partie du chlorure de

 méthylène absorbée est expirée tel quel. Le reste est métabolisé en

 monoxyde, dioxyde de carbone et chlorures minéraux. La métabolisation

 s'effectue selon l'une ou l'autre de ces deux voies ou les deux à la

 fois, et la prédominance de l'une ou de l'autre dépend largement de la

 dose et de l'espèce animale en cause. Une de ces voies comporte un

 processus oxydatif, par l'intermédiaire du cytochrome P-450, et alle

 conduit à la production de monoxyde et de dioxyde de carbone. Il

 semble que cette voie soit identique chez tous les rongeurs étudiés et

 chez l'homme. Il s'agit de la voie prédominante aux faibles doses,

 mais il y a saturation à des doses relativement modérées (autour de

 1800 mg/m3). Même si la dose dépasse la valeur de saturation, il n'y

 pas accroissement de la métabolisation par cette voie.

 Dans l'autre voie métabolique intervient une glutathion-

 transférase (GST) qui conduit à la formation de dioxyde de carbone par

 l'intermédiaire du formaldéhyde et du formiate. Il semble que cette

 voie ne prenne de l'importance que lorsque les doses dépassent la

 valeur de saturation de la voie oxydative "préférentielle". Chez

 certaines espèces (par exemple la souris), elle devient la voie

 principale lorsque la dose est suffisamment élevée. En revanche, chez

 d'autres espèces (par exemple le hamster et l'homme), elle semble

 n'être que peu utilisée, qu'elle que soit la dose.

 Les différentes interspécifiques touchant le métabolisme par la

 voie de la GST sont en bonne corrélation avec les différences

 observées selon les espèces, notamment en ce qui concerne la

 cancérogénicité du chlorure de méthylène. Le taux de métabolisation

 selon cette voie chez les espèces concernées est utilisé comme modèle

 cinétique pour la description du comportement métabolique du chlorure

 de méthylène chez diverses espèces.

6. Effets sur les êtres vivants dans leur milieu naturel

 Aux concentrations inférieures à 500 mg/litre, il n'y a aucune

 inhibition de la croissance des algues et des bactéries aérobies. Il

 existe des bactéries qui sont capables de croître en présence de

 chlorure de méthylène à des concentrations beaucoup plus élevées et

 notamment en solution aqueuse (section 4.2.4.1 ). Les bactéries

 anaérobies sont beaucoup plus sensibles; ainsi on a observé un blocage

 de la croissance à la dose de 1 mg/litre, dans des boues biologiques

 anaérobies.

 A la concentration de 10 mg/kg de terre, on a constaté une forte

 diminution de la teneur en ATP de la biomasse, notamment des

 champignons et des bactéries aérobies, et une inhibition transitoire

 de l'activité enzymatique. La dose sans effets observables était dans

 ce cas de 0,1 mg/kg. Le chlorure de méthylène est modérément toxique

 pour les lombrics (100 à 1000 μg/cm2) soumis au test de toxicité par

 contact sur papier filtre. Dans les sédiments, on n'a pas observé

 d'effets toxiques, même à des doses très élevées.

 Chez les plantes supérieures, on n'a pas constaté d'effets après

 une exposition de 14 jours à la dose de 100 mg/m

3.

 Les poissons adultes semblent être relativement insensibles au

 chlorure de méthylène, même après une exposition prolongée (la CL50

 à 14 jours est supérieure à 200 mg/litre). Il est difficile

 d'apprécier l'effet du chlorure de méthylène sur les daphnies en

 raison de la très grande dispersion des résultats fournis par les

 différentes études. La CE50 la plus basse qui ait été rapportée

 était égale à 12,5 mg/litre.

 En milieu aquatique, c'est chez les embryons de poissons et

 d'amphibiens que l'on a constaté la sensibilité la plus élevée avec

 des effets sur l'éclosion â partir de 5,5 mg/litre.

7. Effets sur les mammifères de laboratoire et les systèmes d'épreuve

 in vitro

7.1 Exposition unique

 Lorsqu'il est absorbé par inhalation ou par voie orale, le

 chlorure de méthylène présente une faible toxicité aiguë. Ainsi, les

 valeurs de la CL50 à 6 heures pour l'ensemble des espèces étudiées,

 se situent entre 40 200 et 55 870 mg/m3. En ce qui concerne la

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 DL50 par voie orale, on a obtenu des valeurs allant de

 1410-3000 mg/kg. Les effets aigus observés après administration de

 chlorure de méthylène par diverses voies d'exposition affectent

 essentiellement le système nerveux central (SNC) et le foie, et tous

 les effets observés l'ont été à des doses élevées. Des troubles

 neurologiques ont été observés à des concentrations supérieures ou

 égaies à 14 100 mg/m3, avec de légères altérations du tracé électro-

 encéphalographique à la dose de 1770 mg/m3. De légères modifications

 histologiques ont été également constatées dans le foie aux

 concentrations supérieures ou égaies à 17 700 mg/m3. Il est

 également arrivé que d'autres organes soient touchés, comme les reins

 ou le système respiratoire. Chez la souris, les effets au niveau

 pulmonaire étaient limités, après exposition à 7100 mg/m3, aux

 cellules de Clara. On a également fait état d'une sensibilisation

 cardiaque à l'arythmie induite par l'adrénaline. D'autres effets

 cardio-vasculaires ont également été observés, mais pas de façon

 systématique.

7.2 Exposition à court et à long terme

 Une exposition prolongée à de fortes concentrations de chlorure de

 méthylène (> 17 700 mg/m3) a causé des effets réversibles sur le

 SNC, une légère irritation oculaire et une certaine mortalité chez

 diverses espèces d'animaux de laboratoire. Chez le rat on a observé à

 3500 mg/ma une réduction du poids corporel, cet effet étant

 également observé chez la souris à partir de 17 700 mg/m3. De légers

 effets hépatiques ont été également observés chez des chiens exposés

 de manière continue à des doses de 3500 mg/m3 pendant des périodes

 allant jusqu'à 100 jours. Après exposition intermittente, des effets

 ont également été observés au niveau du foie chez des rats à la dose

 de 3500 mg/m3 et chez des souris à 14 100 mg/m3.

 Les autres organes cibles sont les poumons et les reins.

 Chez des rats exposés à du chlorure de méthylène pendant 13

 semaines par voie respiratoire à des concentrations allant jusqu'à

 7100 mg/m3, on a observé aucun signe de lésion neurologique

 irréversible.

 L'administration de chlorure de méthylène par voie orale à des

 rats a entraîné des effets hépatiques à partir d'une dose journalière

 d'environ 200 mg/kg.

7.3 Irritation cutanée et oculaire

 Le chlorure de méthylène se révèle modérément irritant pour la

 peau et les yeux chez les animaux de laboratoire.

7.4 Effets toxiques sur le développement et la reproduction

 Le chlorure de méthylène n'est pas tératogène chez le rat ou la

 souris à des concentrations allant jusqu'à 16 250 mg/m3. Trois

 études effectuées sur ces animaux n'ont pas permis de relever d'effets

 se traduisant par l'apparition de malformations squelettiques ou

 autres anomalies du développement. Un léger effet, se manifestant par

 une modification du poids foetal ou maternel, a été relevé à la dose

 de 4400 mg/m3, et l'on a constaté qu'après la naissance, le gain de

 poids des rats mâles était également affecté à la dose de 0,04% dans

 la nourriture. Une étude toxicologique portant sur deux générations a

 été effectuée sur des rats qui ont été exposés par la voie

 respiratoire à du chlorure de méthylène, à des concentrations allant

 jusqu'à 5300 mg/m3, et cela pendant 17 semaines, 6 heures par jour

 et 5 jours par semaine. Aucun effet nocif, de quelque nature ce que

 soit, n'a été relevé sur la reproduction, ni sur la survie ou la

 croissance des ratons nouveaux-nés appartenant à la génération F0 ou

 F1

7.5 Mutagénicité et points d'aboutissement correspondants

 Dans certaines conditions d'exposition, le chlorure de méthylène

 se révèle mutagène pour les microorganismes procaryotes, avec ou sans

 activation métabolique (Salmonella ou Escherichia coli) Dans les

 systèmes eucaryotes, il est sans effet mais, dans un cas, il a donné

 des résultats légèrement positifs. On a également obtenu des résultats

 uniformément négatifs lors des tests de mutation génique in vitro et

 des tests de recherche d'une synthèse non programmée de l'ADN sur

 cellules mammaliennes. La recherche d'aberrations chromosomiques in

 vitro sur différents types de cellules a donné des résultats positif

 s; en revanche, des tests visant à mettre en évidence l'induction

 d'échanges entre chromatides soeurs ont été soit négatif s, soit

 ambigus.

 Dans leur majorité, les études in vivo publiées n'ont pas fourni

 d'éléments en faveur d'une mutagénicité du chlorure de méthylène (par

 exemple recherche d'aberrations chromosomiques, recherche de

 micronoyaux ou synthèse non programmée de l'ADN). Après avoir fait

 inhaler à des souris de fortes concentrations de chlorure de

 méthylène, on a observé un accroissement minime de la fréquence des

 échanges entre chromatides soeurs et du nombre de micronoyaux.

 Après administration à des rats et à des souris de fortes doses de

 chlorure de méthylène, on n'a pas constaté de liaison de ce composé à

 FADN ni de lésions de l'ADN. Ces méthodes pourraient être les plus

 sensibles in vivo et les meilleures d'entre elles sont capables de

 déceler un site d'alkylation sur 106 nucléotides.

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 Dans les limites des épreuves à court terme actuelles, rien ne

 permet de conclure que le chlorure de méthylène soit génotoxique

 in vivo.

7.6 Toxicité chronique et cancérogénicité

 Le chlorure de méthylène est cancérogène pour la souris et il

 provoque l'apparition de rumeurs du poumon et du foie après exposition

 à des concentrations élevées (7100 et 14 100 mg/m3). Chez les souris

 qui avaient été exposées 26 semaines à la dose de 7100 mg/m3, on a

 constaté que l'incidence des tumeurs pulmonaires et hépatiques

 augmentait lorsqu'on poursuivait l'exposition pendant 78 semaines

 supplémentaires. Rien n'indique la présence d'effets toxiques

 concomitants ou d'une hyperplasie au niveau des organes cibles.

 Des hamsters dorés exposés pendant deux ans à du chlorure de

 méthylène, à des concentrations allant jusqu'à 12 400 mg/m3, n'ont

 présenté aucun signe d'effets cancérogènes qui soient attribuables à

 ce composé.

 Chez des rats exposés par différentes voies à du chlorure de

 méthylène, on a constaté un accroissement de l'incidence tumorale à

 certaines localisations. Ainsi, chez des rattes exposées pendant deux

 ans à des doses égaies soit à 5300, soit à 12 400 mg/m3, on a

 constaté un excès de la fréquence tumorale au niveau des glandes

 salivaires. Cet excès n'apparaissait que lorsque les rumeurs, toutes

 d'origine mésenchymateuse, étaient regroupées en vue de l'analyse

 statistique. Etant donné que ces rumeurs trouvent leur origine dans

 des cellules de types différents, l'analyse statistique utilisée s'est

 révélée inappropriée. En outre, il a été précisé que les rats utilisés

 pour cette étude avaient contracté une virose commune

 (sialodacryoadénite) au début de l'expérience, affection qui concerne

 essentiellement les glandes salivaires. Il est donc probable qu'il n'y

 a pas de lien causal entre ces tumeurs et l'exposition au chlorure de

 méthylène, mais que l'exposition à ce composé a exacerbé la réaction à

 l'infection au niveau de la glande salivaire. D'ailleurs cette

 réaction n'a pas été observée lors d'une deuxième étude au cours de

 laquelle des rats ont été exposés pendant toute la durée de leur vie à

 des doses respectivement égaies à 3500, 7100 et 14 100 mg/m3. Une

 autre étude au cours de laquelle des rats ont été également exposés à

 du chlorure de méthylène par la voie respiratoire et durant toute leur

 vie, à des concentrations allant jusqu'à 1770 mg/m3, n'a pas révélé

 de signe de cancérogénicité. Aucun signe notable de cancérogénicité

 n'a été non plus observé chez des rats à qui l'on avait administré du

 chlorure de méthylène, soit par gavage, soit par mélange à leur eau de

 boisson.

 Trois études font état d'une augmentation de l'incidence des

 tumeurs mammaires bénignes chez des rats exposés à du chlorure de

 méthylène; dans une des études le composé a été administré par garage

 tandis que dans les deux autres l'exposition a eu lieu par la voie

 respiratoire. Il n'y a aucune publication faisant état d'un

 accroissement des tumeurs mammaires chez des hamsters ou des souris

 qui avaient reçu du chlorure de méthylène à des doses comparables. On

 a établi sans aucun doute possible que les tumeurs mammaires étaient

 liées aux hormones hypophysaires tant chez les rats mâles que chez les

 femelles. Chez le rat, la prolactine se comporte à la fois comme un

 initiateur et comme un promoteur des cancers mammaires. On a de bonnes

 raisons de penser qu'un accroissement du taux de prolactine augmente

 l'incidence des tumeurs mamamaires (par exemple la greffe de plusieurs

 hypophyses à des rats Sprague-Dawley augmente l'incidence des tumeurs

 mammaires chez ces animaux et on a relevé l'existence d'une

 corrélation positive entre un taux sanguin élevé de prolactine et la

 présence de tumeurs mammaires chez des rattes âgées de souche

 R-Amsterdam). Après administration de composés cancérogènes à des

 rattes, une hyperprolactinémie provoquée chez ces animaux entraîne un

 accroissement spectaculaire de l'incidence tumorale. On peut notamment

 provoquer l'hyperprolactinémie par surrénalectomie, homogreffe

 d'hypophyse et régime alimentaire hyperlipidique.

 Il est important pour l'évaluation du risque chez l'homme, de

 connaître le mécanisme par lequel le chlorure de méthylène provoque

 l'apparition d'adénomes mammaires chez le rat. Les rattes Sprague-

 Dawley à qui l'on a administré du chlorure de méthylène présentent un

 taux sanguin élevé de prolactine. De même qu'avec les autres composés

 qui agissent par l'intermédiaire d'une hyperprolactinémie, la réaction

 au chlorure de méthylène se traduit uniquement par l'apparition de

 néoformations à caractère bénin. Rien n'indique que le chlorure de

 méthylène se lie à l'ADN d'autres tissus et par conséquent il paraît

 improbable qu'il se lie à l'ADN des tissus mammaires alors qu'il est

 principalement métabolisé dans le foie. Il paraît donc probable que

 l'accroissement de l'incidence des adénomes mammaires résulte d'un

 mécanisme indirect agissant par l'intermédiaire d'une

 hyperprolactinémie.

 Chez l'homme, les faits relatifs à la question de savoir si les

 tumeurs mammaires sont sous la dépendance de la prolactine comme chez

 le rat, apparaissent contradictoires. Le rat présente un taux élevé de

 prolactine lorsqu'on le laisse s'alimenter ad libitum plutôt que de

 le soumettre à un régime strict et cela pourrait expliquer pourquoi

 l'incidence des tumeurs mammaires est si clépendante des divers effets

 environnementaux ou autres. Chez le rat toutefois, la prolactine a un

 caractère lutéotrope. L'augmentation du taux de prolactine dans le

 sang circulant conduit à une augmentation du taux de progestérone et

 d'oestrogène exogènes. C'est la présence de l'ensemble de ces trois

 facteurs qui provoque la croissance tubulo-alvéolaire des glandes

 mammaires et qui finit par déboucher sur la formation de tumeurs. Ce

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 mécanisme de formation tumorale n'a donc vraisemblablement pas à être

 pris en considération chez l'homme.

 Le mécanisme de formation de tumeurs mammaires chez le rat par

 l'intermédiaire d'une hyperprolactinémie n'intervient qu'à des doses

 où le chlorure de méthylène agit sur les taux de prolactine. On ne

 dispose pas de données de première main sur les taux de prolactine

 chez des rats soumis à de faibles doses de chlorure de méthylène, mais

 l'administration de faibles doses de ce composé, soit par inhalation,

 soit par mélange à l'eau de boisson (doses inférieures à 250 mg/kg de

 poids corporel) ne conduit pas, selon les études effectuées, à un

 accroissement des adénomes mammaires.

8. Effets sur l'homme

 Le chlorure de méthylène irrite la peau et les yeux, en

 particulier lorsqu'il ne peut pas s'évaporer. Dans ces conditions, un

 contact prolongé peut entraîner des brûlures chimiques. On a signalé

 un cas grave d'oedème pulmonaire consécutif à une inhalation excessive

 de chlorure de méthylène. On a également signalé des cas de décès par

 suite de l'inhalation accidentelle de chlorure de méthylène ou d'un

 contact cutané avec ce composé. Les principaux effets toxiques du

 chlorure de méthylène consistent dans une dépression réversible du

 système nerveux central et dans la formation de carboxyhémoglobine. On

 a également rapporté des cas d'insuffisance hépatique et rénale avec

 anomalies hématologiques, à la suite d'une exposition à du chlorure de

 méthylène.

 Chez des volontaires humains exposés pendant une heure et demie à

 trois heures à du chlorure de méthylène à la concentration de

 694 mg/m3, on a observé des troubles neurophysiologiques et neuro-

 comportementaux. En revanche, aucun signe d'effet neurologique n'a été

 observé chez des hommes exposés plusieurs années à du chlorure de

 méthylène à des concentrations allant de 260 à 347 mg/m3. De même,

 on a soumis à une batterie de tests neurophysiologiques et

 psychologiques un groupe d'anciens décapeurs d'aéronefs qui avaient

 été longtemps exposés à du chlorure de méthylène (22 ans), à des doses

 élevées mais non précisées; comparés à ceux d'un groupe témoin qui

 n'avait jamais été exposé à du chlorure de méthylène ou du moins

 uniquement à de faibles doses, les résultats obtenus par les ouvriers

 se sont révélés "normaux".

 On a attribué à une exposition au chlorure de méthylène une

 augmentation du taux d'avortements spontanés constatée chez des

 employées des industries pharmaceutiques finlandaises. L'étude en

 question présentait cependant des défauts de conception qui n'ont pas

 permis d'établir une relation causale.

 Plusieurs éludes de mortalité sur des cohortes exposées au

 chlorure de méthylène font ressortir une absence d'uniformité dans les

 causes de décès. La surmortalité due à certaines maladies (par exemple

 cancer du pancréas, cardiopathies ischémiques) ne se manifeste pas de

 façon uniforme, mais seulement dans certaines éludes. Ces effets ne

 peuvent être attribués à l'exposition au chlorure de méthylène.

RESUMEN

1. Identidad, propiedades físicas y químicas, y métodos analíticos

 El cloruro de metileno (diclorometano) es un liquido claro,

 altamente volátil y no inflamable, con un olor penetrante parecido al

 del éter. El compuesto puro en polvo es muy estable. El cloruro de

 metileno se hidroliza lentamente en presencia de humedad, dando lugar

 a pequeñas cantidades de ácido clorhídrico. Al compuesto comercial se

 agregan pequeñas cantidades de estabilizadores para prevenir su

 descomposición.

 Existen métodos analíticos para determinar el cloruro de metileno

 en medios biológicos y muestras ambientales; en todos ellos se utiliza

 cromatografía de gases y un detector apropiado. De esta manera se han

 alcanzado limites de detección muy bajos (p. ej., alimentos:

 7 ng/muestra; agua: 0,01 μg/litro; aire: 1,76 μg/m3 (0,5 ppb);

 sangre: 0,022 mg/litro).

2. Fuentes de exposición humana y ambiental

 Se estima que la producción mundial de cloruro de metileno

 asciende a 570 000 toneladas/año. La mayoría sus aplicaciones se basan

 en su capacidad para disolver grasas, plásticos y agentes aglutinantes

 de pintura, así como en su volatilidad y estabilidad. Su uso a nivel

 mundial se reparte del siguiente modo: aerosoles (20%-25%),

 quitapinturas (25%), disolvente en la industria química y farmacéutica

 (35%-40%), usos varios (p. ej., la fabricación de espuma de

 poliuretano) y limpieza de metales (10%-15%). El uso de cloruro de

 metileno tiende a disminuir, al menos en Europa occidental.

 Más del 99% del cloruro de metileno liberado a la atmósfera

 procede de diversas industrias que lo emiten como producto fina], o es

 el resultado del uso doméstico de quitapinturas y aerosoles.

3. Transporte, distribución y transformación en el medio ambiente

 Debido a su alta volatilidad, la mayor parte del cloruro de

 metileno liberado al medio pasa a la atmósfera, donde se degrada

 reaccionando con radicales hidroxilo de origen fotoquímico; su tiempo

 de permanencia es de seis meses.

 La degradación abiótica del compuesto en agua es lenta en

 comparación con la evaporación. Se ha comprobado que el cloruro de

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 metileno desaparece rápidamente del suelo y de las aguas subterráneas.

 Se han utilizado diversos sistemas de ensayo para determinar la

 degradación aerobia y anaerobia del cloruro de metileno. La

 biodegradación completa, sobre todo en cultivos bacterianos tratados y

 en condiciones aerobias, es rápida (p. ej., mineralización del 49%-66%

 en 50 horas en fangos urbanos tratados). En los biorreactores se puede

 alcanzar una degradación de hasta un 10% por hora. No hay indicios de

 una bioacumulación o biomagnificación importantes.

4. Niveles medioambientales y exposición humana

 Se ha detectado cloruro de metileno en el aire ambiente de zonas

 rurales y remotas a concentraciones comprendidas entre 0,07 y

 0,29 μg/m3. En zonas suburbanas la concentración promedio es

 <2 μg/m3, y en zonas urbanas, <15 μg/m3. En las proximidades de

 vertederos de desechos peligrosos se han hallado hasta 43 μg/m3. Las

 precipitaciones también contienen a veces cloruro de metileno.

 El cloruro de metileno penetra en el medio acuático a través de

 las descargas de aguas residuales de diversas industrias, habiéndose

 detectado su presencia en aguas superficiales, aguas subterráneas y

 sedimentos.

 La población general se expone al cloruro de metileno cuando

 utiliza productos de consumo tales como los quitapinturas, cuyo empleo

 puede acompañarse de la presencia de niveles relativamente altos en el

 aire del interior de los domicilios. La exposición ocupacional durante

 la producción tiene lugar sobre todo durante el llenado y envasado (la

 fabricación se lleva a cabo en sistemas cerrados). Tratándose de un

 compuesto usado en los quitapinturas, la exposición laboral al cloruro

 de metileno se produce durante la elaboración de quitapinturas, la

 fabricación de material para ordenadores y el acabado comercial de

 muebles. El cloruro de metileno es ampliamente empleado como

 disolvente industrial en la elaboración de diversos productos, sobre

 todo en las industrias que se mencionan en la sección 1.2.

 La vigilancia biológica de la exposición al cloruro de metileno

 puede basarse en la medición del propio disolvente en el aire espirado

 o en la sangre. No obstante, dado que la producción de monóxido de

 carbono con una exposición de más de 3-4 horas/día parece ser el

 factor limitante en lo que respecta a los riesgos para la salud, es

 preferible basar la vigilancia biológica en el análisis bien del

 monóxido de carbono presente en el aire espirado, o bien de la

 carboxihemoglobina (CO-Hb) en sangre. Así y todo, esto sólo se puede

 aplicar a las personas no fumadoras. Deben tomarse muestras antes de

 transcurridas aproximadamente dos horas tras la exposición, o bien al

 cabo de 16 horas, esto es, a la mañana siguiente.

 Los niveles postexposición de CO-Hb a las dos horas de interrumpir

 la exposición no deben sobrepasar el 2%-3%, y a las 16 horas el 1%, en

 los no fumadores expuestos durante ocho horas a menos de 350 mg/m3

 de cloruro de metileno.

5. Cinética y metabolismo

 El cloruro de metileno es absorbido rápidamente por los alvéolos

 pulmonares, a través de los cuales llega a la circulación sistémica.

 Es absorbido también por el tracto gastrointestinal, así como por vía

 cutánea, si bien en este último caso la velocidad de absorción es

 menor que por otras vías de exposición.

 El cloruro de metileno se excreta con considerable rapidez,

 fundamentalmente a través del aire espirado por los pulmones. Puede

 atravesar la barrera hematoencefálica, así como la placenta, y se

 excreta también en pequeñas cantidades por la orina y la leche.

 A altas concentraciones la mayoría del cloruro de metileno

 absorbido se espira inalterado. El resto es metabolizado en monóxido

 de carbono, dióxido de carbono y cloruro inorgánico. Hay dos vías

 posibles de metabolización, cuya contribución relativa al metabolismo

 total depende en gran medida de la dosis y de la especie animal

 considerada. Una vía consiste en un proceso de metabolismo oxidativo

 mediado por el citocromo P-450, que conduce a la producción tanto de

 monóxido de carbono como de dióxido de carbono. Esta vía funciona de

 manera parecida en todos los roedores estudiados y en el hombre. Si

 bien es la vía metabólica predominante a dosis bajas, se satura

 también a dosis relativamente bajas (en torno a 1800 mg/m3).

 Aumentar la dosis por encima de ese nivel de saturación no conlleva un

 mayor metabolismo a través de esa vía.

 La otra vía está mediada por una glutatión-transferasa (GTF) y

 conduce, previa producción de formaldehído y de formato, a la

 formación de dióxido de carbono. Al parecer esta vía sólo adquiere

 importancia a dosis superiores al nivel de saturación de la vía

 oxidativa «preferente». En algunas especies (p. ej., el ratón)

 constituye la principal vía metabólica a dosis suficientemente altas.

 Por el contrario, en otras especies (p. ej., el hámster o el hombre)

 esta vía apenas es utilizada, cualquiera que sea la dosis.

 Las diferencias interespecies del metabolismo mediado por la GTF

 guardan una clara relación con las diferencias interespecies

 observadas en lo que respecta a la carcinogenicidad. Analizando la

 intensidad del metabolismo mediado por esta vía en determinadas

 especies, se ha elaborado un modelo cinético del metabolismo del

 cloruro de metileno en diversas especies.

6. Efectos en organismos presentes en el medio ambiente

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 Por debajo de 500 mg/litro no se observa inhibición del

 crecimiento de algas y de bacterias aerobias. Se han descubierto

 bacterias capaces de crecer en presencia de cloruro de metileno a

 concentraciones mucho mayores, incluida una solución saturada en agua

 (sección 4.2.4.1 ). Las bacterias anaerobias son más sensibles; se ha

 observado inhibición del crecimiento a una concentración de 1 mg/litro

 en fangos biológicos anaerobios.

 En el suelo, se observó que una concentración de 10 mg/kg reducía

 considerablemente el contenido de ATP de la biomasa, incluidos hongos

 y bacterias aerobias, e inducía una inhibición transitoria de la

 actividad enzimática. El nivel sin efectos observados fue de

 0,1 mg/kg. En las lombrices de tierra el cloruro de metileno tiene un

 efecto moderadamente tóxico (100-1000 μg/cm2), como demuestra la

 prueba de toxicidad de contacto con papel de filtro. En sedimentos no

 se observaron efectos tóxicos ni siquiera a concentraciones muy altas.

 En plantas superiores no se observaron efectos al cabo de 14 días

 de exposición a 100 mg/m3.

 Los peces adultos parecen relativamente insensibles al cloruro de

 metileno, incluso después de una exposición prolongada (14 días,

 CL50 > 200 mg/litro). El efecto del cloruro de metileno en Daphnia

 resulta difícil de evaluar porque hay grandes diferencias entre los

 resultados de los estudios realizados. La CE50 más baja notificada

 es de 12,5 mg/litro.

 En cuanto al entorno acuático, se ha demostrado que los embriones

 de peces y anfibios son los más sensibles, observándose efectos sobre

 la incubación a partir de 5,5 mg/litro.

7. Efectos en mamíferos de laboratorio y en sistemas de prueba

 in vitro

7.1 Exposiciones aisladas

 La toxicidad aguda del cloruro de metileno por vía respiratoria y

 por vía oral es baja. La CL50-6h por inhalación está comprendida en

 todas las especies entre 40 200 y 55 870 mg/m3. Se han registrado

 DL50 orales de 1410-3000 mg/kg. Los efectos agudos de la

 administración de cloruro de metileno por diversas vías de exposición

 se manifiestan fundamentalmente en el sistema nervioso central (SNC) y

 en el hígado, y se producen a dosis altas. Se han observado trastornos

 del SNC a concentraciones de 14 100 mg/m3 o más, con ligeras

 variaciones del EEG a 1770 mg/m3. A concentraciones de

 17 700 mg/m3 o más se observaron leves cambios histológicos en el

 hígado. Ocasionalmente se vieron afectados otros órganos, tales como

 el riñón o el sistema respiratorio. En el ratón, los efectos sobre los

 pulmones se limitaron a las células de Clara después de una exposición

 a 7100 mg/m3. Se ha notificado la aparición de sensibilización

 cardiaca a la arritmia inducida por adrenalina. Se han observado

 efectos cardiovasculares, si bien de manera irregular.

7.2 Exposición a corto y a largo plazo

 La exposición prolongada a concentraciones altas de cloruro de

 metileno (>17 700 mg/m3) causó efectos reversibles sobre el SNC,

 ligera irritación ocular y mortalidad en varias especies de

 laboratorio. Se observó una reducción del peso corporal en ratas a

 3500 mg/m3, y en ratones a partir de 17 700 mg/m3. El hígado de

 perros expuestos continuamente a 3500 mg/m3 por espacio de hasta 100

 días se vio ligeramente afectado. Se observaron asimismo efectos en el

 hígado tras la exposición intermitente a 3500 mg/m3 en la rata, y a

 14 100 mg/m3 en el ratón.

 Otros órganos diana son los pulmones y los riñones.

 No se hallaron indicios de daño neurológico irreversible en ratas

 expuestas por inhalación a concentraciones de hasta 7100 mg/m3

 durante 13 semanas.

 La administración oral de cloruro de metileno a ratas causó

 efectos hepáticos a partir de 200 mg/kg al día.

7.3 Irritación cutánea y ocular

 El cloruro de metileno es moderadamente irritante para la piel y

 los ojos de animales experimentales.

7.4 Toxicidad para el desarrollo y la reproducción

 El cloruro de metileno no es teratógeno en la rata o el ratón a

 concentraciones de hasta 16 250 mg/m3. En tres estudios realizados

 con animales no se observaron indicios de variación de la incidencia

 de malformaciones esqueléticas ni otros efectos sobre el desarrollo.

 Se notificaron efectos leves sobre el peso corporal fetal o materno a

 una concentración de 4400 mg/m3, así como sobre el aumento de peso

 postnatal de ratas macho a una concentración del 0,04% en la dieta. Un

 estudio de toxicidad reproductiva llevado a cabo en dos generaciones

 de ratas expuestas a cloruro de metileno por inhalación a

 concentraciones de hasta 5300 mg/m3, 6 h/día, 5 días/semana durante

 17 semanas no puso de manifiesto ningún efecto adverso en lo tocante a

 los parámetros reproductivos, la supervivencia neonatal o el

 crecimiento neonatal en ninguna de las generaciones, F0 o F1.

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7.5 Mutagenicidad y criterios de evaluación relacionados

 En condiciones de exposición adecuadas el cloruro de metileno

 tiene efectos mutágenos en microorganismos procariotas, con o sin

 activación metabólica (Salmonella o Escherichia coli). En los

 sistemas eucariotas los resultados son negativos, salvo en un caso en

 que fueron débilmente positivos. Los ensayos y pruebas de mutación

 genética in vitro basados en la síntesis no programada de ADN (UDS)

 en células de mamífero fueron siempre negativos. Los ensayos in vitro

 realizados para detectar aberraciones cromosómicas en diferentes tipos

 de células dieron resultados positivos, mientras que en las pruebas de

 inducción de intercambio de cromátides hermanas (SCE) se obtuvieron

 resultados negativos o ambiguos.

 La mayoría de los estudios in vivo publicados no han aportado

 ningún dato indicativo de mutagenicidad del cloruro de metileno

 (determinada por ejemplo, mediante la prueba de aberración

 cromosómica, la prueba de los micronúcleos o el ensayo UDS). Se ha

 notificado un aumento mínimo de la frecuencia de SCE y de micronúcleos

 en el ratón tras la exposición por inhalación a altas concentraciones

 de cloruro de metileno.

 En ratas o ratones a los que se administraron dosis altas de

 cloruro de metileno no se observaron indicios de unión del cloruro de

 metileno al ADN ni de lesiones de éste. Son éstos los estudios

 in vivo potencialmente más sensibles, el mejor de los cuales permite

 detectar una alquilación por cada 106 nucleótidos.

 Dentro de las limitaciones de las pruebas a corto plazo

 actualmente disponibles, no hay pruebas concluyentes de que el cloruro

 de metileno sea genotóxico in vivo.

7.6 Toxicidad crónica y carcinogenicidad

 El cloruro de metileno es carcinógeno en el ratón, en el que la

 exposición a altas concentraciones (7100 y 14 100 mg/m3) es causa de

 tumores tanto pulmonares como hepáticos. La incidencia de esos dos

 tipos de tumores aumentó en ratones expuestos a 7100 mg/m3 durante

 26 semanas y estudiados durante 78 semanas más. No se observaron

 signos claros de toxicidad o hiperplasia asociadas en los órganos

 diana.

 La exposición de hámsters sirios a cloruro de metileno por

 inhalación a concentraciones de hasta 12 400 mg/m3 durante dos años

 no tuvo efectos carcinógenos.

 Se ha observado que las ratas expuestas al cloruro de metileno por

 diversas vías sufren una mayor incidencia de tumores en determinados

 lugares. Se ha notificado un exceso de tumores en la región de las

 glándulas salivales en ratas hembra expuestas a 5300 ó 12 400 mg/m3

 durante dos años. Ese exceso sólo se hizo patente cuando se procedió a

 agrupar los tumores, todos ellos de origen mesenquimatoso, con fines

 estadísticos. El método estadístico utilizado era inapropiado dado que

 los tumores procedían de células de diverso tipo. Además, se señaló

 que las ratas utilizadas se habían visto infectadas al principio del

 estudio por un virus causante de una enfermedad común, la

 sialodacrioadenitis, que afecta sobre todo a la glándula salival.

 Probablemente los tumores no estaban relacionados causalmente con la

 exposición al cloruro de metileno, y la exposición se limitó a

 exacerbar la respuesta a la infección en la región de la glándula

 salival. El efecto no se reprodujo en un segundo estudio realizado con

 ratas expuestas a 3500, 7100 ó 14 100 mg/m3 durante su ciclo de

 vida. Un estudio ulterior realizado con ratas expuestas por inhalación

 a concentraciones de hasta 1770 mg/m3 de cloruro de metileno durante

 todo su ciclo de vida no reveló indicios de carcinogenicidad. En ratas

 expuestas al cloruro de metileno a través del agua que consumían o de

 alimentos administrados con sonda tampoco se observaron indicios

 significativos de carcinogenicidad.

 Tres estudios han puesto de manifiesto un aumento de la incidencia

 de tumores mamarios benignos en ratas expuestas a cloruro de metileno,

 en dos de los casos por inhalación y en el tercero por administración

 forzada. No se ha notificado ningún aumento de la incidencia de

 tumores mamarios en hámsters o en ratones sometidos a dosis

 comparables de cloruro de metileno. La dependencia de los tumores

 mamarios de las hormonas hipofisarias en la rata, tanto macho como

 hembra, es un dato incontrovertible. En la rata, la prolactina actúa

 como iniciador y como promotor de la carcinogénesis mamaria. Hay datos

 convincentes de que el aumento de los niveles de prolactina incrementa

 la incidencia de tumores mamarios (p. ej., el injerto de varias

 hipófisis en ratas Sprague-Dawley aumenta la incidencia de tumores

 mamarios, y se ha observado además una correlación positiva entre la

 existencia de niveles elevados de prolactina en sangre y la incidencia

 de tumores mamarios en ratas hembra R-Amsterdam viejas). En las ratas

 hembra que han recibido carcinógenos, los tratamientos inductores de

 hiperprolactinemia dan lugar a un aumento espectacular de la

 incidencia de tumores. Entre esos tratamientos cabe citar la

 adrenalectomía, los homoinjertos hipofisarios y el consumo de

 alimentos ricos en grasas.

 El conocimiento de los mecanismos de inducción de adenomas

 mamarios por el cloruro de metileno en la rata es importante para

 poder evaluar los riesgos para el hombre. Las ratas Sprague-Dawley

 hembras sometidas a cloruro de metileno presentan una elevada

 concentración de prolactina en sangre. Al igual que la respuesta a

 otros agentes cuya acción está mediada por una hiperprolactinemia, la

 respuesta inducida por el cloruro de metileno se limita a la aparición

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 de neoplasias benignas. No hay datos indicativos de una unión del

 cloruro de metileno al ADN de otros tejidos, por lo que parece

 improbable que pueda unirse al tejido mamario, tanto más cuanto que su

 metabolismo se produce fundamentalmente en el hígado. Es más probable,

 por tanto, que el aumento de la incidencia de adenomas mamarios se

 deba a un mecanismo indirecto en el que intervenga la hiperprolac-

 tinemia.

 En cuanto al hombre, hay datos contradictorios respecto a si los

 tumores mamarios son tan sensibles a la prolactina como en la rata.

 Este animal presenta niveles elevados de prolactina cuando es

 alimentado ad libitum en lugar de sometido a una dieta restringida,

 lo cual explica quizá la gran sensibilidad de la incidencia de tumores

 mamarios a diversos efectos ambientales y de otro tipo. En la rata, no

 obstante, la prolactina es luteotrófica. Un aumento de la prolactina

 circulante da lugar a un aumento de los niveles de progesterona y de

 estrógenos exógenos. Es la coincidencia de estos tres factores lo que

 causa el crecimiento túbulo-alveolar de las glándulas mamarias y,

 finalmente, el desarrollo del tumor. La prolactina no es luteotrófica

 en los primates; es improbable, por tanto, que este mecanismo de

 desarrollo tumoral pueda tener importancia en el hombre.

 En la rata, el mecanismo de desarrollo de tumores mamarios mediado

 por la hiperprolactinemia sólo entra en juego a las dosis de cloruro

 de metileno que alteran los niveles de prolactina. No se dispone de

 información directa sobre los niveles de prolactina en ratas sometidas

 a dosis bajas de cloruro de metileno, pero no se ha observado ningún

 aumento de la incidencia de adenomas mamarios tras la administración

 de dosis bajas por inhalación o a través del agua de bebida (p. ej.,

 dosis inferiores a 250 mg/kg peso corporal).

8. Efectos en el hombre

 El cloruro de metileno es irritante para la piel y para los ojos,

 sobre todo cuando se impide su evaporación. En estas condiciones, el

 contacto prolongado puede causar quemaduras químicas. Se ha notificado

 un caso de edema pulmonar grave por inhalación excesiva. Se han

 producido también defunciones en casos de inhalación o contaminación

 cutánea accidentales. Los principales efectos tóxicos del cloruro de

 metileno son la depresión reversible del SNC y la formación de CO-Hb.

 Se ha señalado también la aparición de disfunciones hepáticas y

 renales y de trastornos hematológicos tras la exposición al producto.

 Se han observado problemas neurofisiológicos y neurocomporta-

 mentales en voluntarios humanos expuestos a concentraciones de cloruro

 de metileno de 694 mg/m3 durante 1,5-3,0 horas. No se han observado

 efectos neurológicos en hombres expuestos durante varios años a

 concentraciones del producto comprendidas entre 260 y 347 mg/m3. De

 forma parecida, un grupo de raspadores de pintura de aviones ya

 jubilados con antecedentes de una larga (22 años) exposición a

 concentraciones altas, si bien no especificadas, de cloruro de

 metileno obtuvieron resultados "normales" en una batería de pruebas

 neurofisiológicas y psicológicas en comparación con un grupo testigo

 sin antecedentes de exposición, o en todo caso con antecedentes de una

 baja exposición al compuesto.

 Un aumento de la tasa de abortos espontáneos entre empleadas de la

 industria farmacéutica finlandesa se ha atribuido a la exposición a

 cloruro de metileno. Sin embargo, el diseño incorrecto del estudio ha

 impedido establecer una relación causal.

 Varios estudios de mortalidad realizados en cohortes pertinentes

 muestran resultados dispares en cuanto a las causas de defunción. Se

 ha observado un aumento de la mortalidad por enfermedades especificas

 (como por ejemplo el cáncer pancreático o la cardiopatía isquémica),

 pero de forma irregular y sólo en determinados estudios. Estos efectos

 no se pueden atribuir a la exposición al cloruro de metileno.

 See Also:

Toxicological Abbreviations

Methylene chloride (EHC 32, 1984, 1st edition)

Methylene chloride (HSG 6, 1987)

Methylene chloride (PIM 343)

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