Institutet för miljömedicinKarolinska institutet

Stockholm 1998

IMM-rapport 3/98

Health risk assessmentof dichloromethane

Niclas HåkanssonGunnar JohansonFredrik WaernMargareta WarholmKatarina VictorinDan Wikström

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Preface

At the request of the Swedish Environmental Protection Agency, The Institute ofEnvironmental Medicine (IMM) has performed health risk assessments, includingrecommended air quality guidelines for dichloromethane in ambient air, on twoearlier occasions. The first document (Fransson and Ahlborg 1986, referred toin the following text as IMM 1986) was updated some years later (Fransson andAhlborg 1990, referred to in the following text as IMM 1990).

In 1997, the Institute was once again asked to update the toxicology data baseand make a renewed health risk assessment for dichloromethane, this time byAstra AB. The work has been based on the two earlier documents and on amore recent WHO document. All new studies, and those older studies that wereconsidered important for the risk assessment, have been read and cited in ori-ginal. For other older studies, the WHO document or the earlier documents fromIMM have been used as main references.

The authors of this document have co-operated, and all conclusions have beenthoroughly discussed. However, each person has had main responsibility for thefollowing parts:

Niclas Håkansson, B Sc Epidemiology

Gunnar Johanson, Assoc. prof Toxicokinetic modelling, risk assessment

Fredrik Waern, Dr med sci Reproductive toxicity,benchmark modelling

Margareta Warholm, Ph D Metabolism, human case studies

Dan Wikström, B Sc General descriptions, general toxicity,cancer studies

Katarina Victorin, Assoc. prof Genotoxicity, risk assessment

Katarina Victorin was project leader.

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Contents

1. Summary ......................................................................................... 5

2. Sammanfattning ............................................................................... 7

3. Properties, occurrence, exposure levels ...................................... 93.1 General description ........................................................... 93.2 Identity ............................................................................... 93.3 Physical and chemical properties ...................................... 93.4 Conversion factors ............................................................ 103.5 Sources of human and environmental exposure ................103.6 Environmental levels and potential for human exposure ...13

4. Metabolism and toxicokinetics ..................................................... 164.1 Metabolism .........................................................................174.2 Human toxicokinetic data ..................................................274.3 Conclusions ....................................................................... 29

5. Short-term effects in animals ........................................................315.1 Inhalation ......................................................................... 315.2 Oral exposure ................................................................... 345.3 Other exposures ................................................................ 345.4 Effects on the heart .......................................................... 355.5 Conclusions ....................................................................... 36

6. Effects after prolonged exposure in animals ................................376.1 Inhalation ..........................................................................376.2 Oral exposure ....................................................................416.3 Conclusions ....................................................................... 41

7. Chronic toxicity and carcinogenicity ............................................ 437.1 Inhalation ......................................................................... 437.2 Oral exposure ................................................................... 507.3 Conclusions ....................................................................... 53

8. Genotoxicity ................................................................................... 548.1 In vitro .............................................................................. 548.2 In vivo ...............................................................................578.3 Conclusions ....................................................................... 62

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9. Reproductive toxicity .................................................................... 639.1 Conclusions ....................................................................... 64

10. Human data ................................................................................. 6510.1 Accidental and acute exposure ....................................... 6510.2 Controlled exposure ........................................................ 6710.3 Long-term exposure, case studies ....................................6810.4 Epidemiological studies ................................................... 6910.5 Conclusions ..................................................................... 74

11. Quantitative risk estimates ........................................................ 7611.1 Early EPA risk estimates .................................................7611.2 Pharmacokinetic risk estimates ....................................... 7611.3 Benchmark dose models .................................................. 8211.4 Conclusions ..................................................................... 86

12. Summary and health risk assessment ........................................ 8812.1 Metabolism ...................................................................... 8812.2 Non-cancer effects .......................................................... 8912.3 Cancer ............................................................................ 9012.4 Studies important for the interpretation

of animal cancer data ................................................. 9112.5 Epidemiological cancer data .......................................... 9312.6 Quantitative cancer risk assessment ............................... 9412.7 Recommended health-based limit value ........................... 96

13. References ................................................................................... 97

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1. Summary

Dichloromethane (methylene chloride) is a chlorinated solvent with widespreaduse as a cleaning and degreasing agent, for paint removal etc. In Sweden itwas however forbidden in consumer products in 1993, and its professional usewas banned in 1996. The pharmaceutical industry is exempted from the banfor 5 years. In other countries dichloromethane is still in use. The presentliterature survey and risk assessment has been performed by the Institute ofEnvironmental Medicine (IMM) on request by Astra AB. It is an update ofearlier risk assessments performed by IMM on request by the SwedishEnvironmental Protection Agency. The main emphasis is on inhalation exposure.

Dichloromethane is efficiently absorbed by all exposure routes, and evenlydistributed within the body. Dichloromethane is metabolised by two pathways,in which potentially toxic metabolites are formed. In the oxidative P450 2E1pathway, which is the preferred route at low concentrations, carbon monoxideand carbon dioxide are the end products and formyl chloride is formed as anintermediate reaction product. The other metabolic pathway is mediated byglutathione-S-transferase, especially GSTT1, and becomes important at highconcentrations as the P450 pathway becomes saturated at exposure levelsabove approximately 700 mg/m3. In this pathway a reactive conjugate,S-chloromethylglutathione, is formed which decomposes to formaldehyde. Inlaboratory animals, metabolism occurs mainly in the liver, with additional me-tabolism in lung and possibly also in other tissues. The GST activity towardsdichloromethane in liver and lung is considerably higher in mice compared torats, hamsters and humans. Limited data indicate that GSTT1 is expressed inmost human tissues. However, 10% of the Swedish population lack this enzyme,and can thus not metabolise dichloromethane via the GST pathway.

The acute toxicity of dichloromethane is low. As most other solvents,dichloromethane affects the central nervous system at sufficiently highconcentrations. Metabolically formed carbon monoxide binds strongly tohaemoglobin as carboxyhaemoglobin. The formation of carboxyhaemoglobinmost likely produces the cardiotoxic effects that have been seen in somestudies, and appears to be the basis for most occupational limit values fordichloromethane as well as the WHO air quality guidelines for ambient air.

Inhalation exposure of 7000 and 14 000 mg/m3 of dichloromethane has causedliver and lung adenomas and carcinomas in mice and benign mammary tumours

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in rats at similar concentrations, but no increased tumour incidence in Syriangolden hamsters. The liver and lung tumours in mice were not proceeded byovert cytotoxicity or sustained cell proliferation. Slight excess of mortality fromcancer has been found in some occupational studies, including elevated risksof cancer in biliary passages and liver, pancreas and brain. However, thesestudies are not sufficient for any firm conclusions concerning cancer risks.

Dichloromethane is mutagenic in Salmonella bacteria. This effect is probablymainly mediated by the reactive glutathione conjugate, S-chloromethylglutathione.No DNA-binding has been demonstrated. DNA single strand breaks wereformed in liver and lungs of mice, but not in the liver of rats. The DNA damageis also probably mainly GST-mediated. No specific mutations were induced inliver and lung tumours of exposed mice. Taken together, we considerdichloromethane as a genotoxic carcinogen of low potency. Accordingly,quantitative cancer risk estimates can be calculated with extrapolation modelsbased on liver and lung tumours in mice.

Quantitative cancer risk estimates from the literature have either used the so-called linearized multistage model where the dose is recalculated from mouseto man by a species scaling factor (former U.S. EPA risk estimate), or by apharmacokinetic model where the dose is expressed as the amount ofdichloromethane metabolised by GST (Andersen, Reitz and co-workers), orby a modified multistage model partly adopting the pharmakokinetic model(U.S. EPA risk estimate). In the present risk assessment we have chosen touse the so-called benchmark model. The 95% confidence interval of the dose-response curve was used to calculate the lower bound of the dose correspondingto a 10% increased risk for lung tumours in female mice. From this point, alinear extrapolation was done. After recalculation from the experimental protocolto continuous exposure, and compensating with a factor of 4 for non-linearmetabolism, the concentration corresponding to a lifetime risk of 1·10-5 inhumans would be 46 µg/m3. No species-scaling factor was used in thesecalculations. Using the modified multistage model by EPA, the same risk wouldresult from 21 µg/m3.

According to this estimate, we propose a guideline value of 50 µg/m3 as along-term average for dichloromethane in ambient air. The previousrecommendation from IMM in 1990 was 100 ppb (350 µg/m3).

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2. Sammanfattning

Diklormetan (metylenklorid) är ett klorerat lösningsmedel som tidigare använtsi stor utsträckning för sådana ändamål som rengöring, avfettning, färgborttagningetc. I Sverige förbjöds det i konsumentprodukter 1993, och industriell an-vändning är i princip förbjuden sedan 1996, med vissa undantag som t.ex.inom läkemedelsindustrin. I andra länder används det dock fortfarande. Insti-tutet för Miljömedicin (IMM) har gjort denna litteratursammanställning ochriskbedömning på uppdrag av Astra AB. Det är en uppdatering av de risk-bedömningar som IMM gjort åt Naturvårdsverket vid två tidigare tillfällen.Huvudvikten ligger på toxiska effekter vid inandning.

Diklormetan tas upp effektivt och distribueras jämnt till kroppens olika organ.Det metaboliseras via två olika vägar, som bägge bildar toxiska metaboliter.Den oxidativa metabolismen, som dominerar vid låga koncentrationer, sker viacytokrom P450 2E1 och bildar kolmonoxid och koldioxid som slutproduktermed formylklorid som reaktiv mellanprodukt. Den andra metabolismvägen viaglutationtransferas, framför allt GSTT1, är viktig vid höga koncentrationer ef-tersom P450-metabolismen mättas vid exponeringsnivåer över ca 700 mg/m3.Vid denna metabolismväg bildas ett reaktivt glutationkonjugat (S-klorometyl-glutation) som mellanprodukt, vilken bryts ner till formaldehyd. Hos försöks-djur sker metabolismen huvudsakligen i levern, men även i lunga och möjligenäven i andra organ. GST-aktiviteten gentemot diklormetan i lever och lunga ärmycket högre hos mus än hos råtta, hamster eller människa. Hos människatycks GSTT1 uttryckas i de flesta organ. Av den svenska befolkningen saknardock 10% detta enzym och kan alltså inte metabolisera diklormetan via GST.

Den akuta toxiciteten av diklormetan är låg. Liksom andra lösningsmedel på-verkar det dock det centrala nervsystemet om höga koncentrationer inandas.Bildad kolmonoxid binds hårt till blodets hemoglobin och ger upphov till kar-boxyhemoglobin, vilket sannolikt orsakat de effekter på hjärtat som setts ivissa djurförsök. Yrkeshygieniska gränsvärden för diklormetan, liksom ävenWHOs rekommenderade gränsvärde för utomhusluft, baseras på karboxy-hemoglobinbildningen.

Inandning av 7000 och 14 000 mg/m3 har givit upphov till lever- och lungtumörerhos möss samt godartade bröstkörteltumörer hos råttor vid ungefär sammakoncentrationer, men däremot ingen signifikant ökning av tumörer hos hamster.Lever- och lungtumörerna hos möss föregicks inte av uttalad cellprolifiering

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eller cytotoxicitet. I några epidemiologiska studier på yrkesmässigt expone-rade människor har en förhöjd förekomst av cancer i lever och gallgångar,bukspottkörtel och hjärna påvisats, men dessa fynd är inte tillräckliga för attdra några bestämda slutsatser angående cancerrisken hos människor.

Diklormetan är mutagent i Salmonella-bakterier, troligen huvudsakligen medie-rat via det reaktiva glutationkonjugatet S-klormetylglutation. DNA-bindninghar inte påvisats, men däremot enkelsträngsbrott i DNA som också tycks varahuvudsakligen GST-medierat. Enkelsträngsbrott i DNA har påvisats i leveroch lunga hos möss, men ej i råttlever. Inga specifika mutationer kunde på-visas i lever- och lungtumörer från exponerade möss, men vi har ändå bedömtdiklormetan som en lågpotent genotoxisk carcinogen, och menar följaktligenatt kvantitativ cancerriskbedömning utifrån lever- och lungtumörerna hos mösskan göras med hjälp av en extrapoleringsmodell.

De kvantitativa cancerriskuppskattningar som finns beskrivna i litteraturen harantingen utnyttjat den s.k. multistagemodellen med omräkning av dosen frånmus till människa med hjälp av en artomvandlingsfaktor (EPAs tidigare risk-uppskattning), en farmakokinetisk modell där dosen uttrycks som metaboliseradmängd diklormetan via glutationtransferas (Andersen, Reitz och medarbetare),eller en modifierad multistagemodell som delvis utnyttjar den farmakokinetiskamodellen (EPAs nuvarande riskuppskattning). I föreliggande riskbedömninghar vi valt att använda den s.k. benchmarkmodellen. Det 95-procentigakonfidensintervallet för dos-responskurvan har utnyttjats för att beräkna denlägsta dos som gav upphov till 10% ökad risk för lungtumörer hos honmössen,varefter risken har extrapolerats linjärt. Den koncentration som motsvarar enlivstidsrisk för människa på 1·10-5 , efter omräkning till kontinuerlig inhalationoch kompensation med en faktor 4 för icke-linjär metabolism, blir med dettaberäkningssätt 46 µg/m3. Ingen artomvandlingsfaktor har alltså använts. MedEPAs modifierade multistage-modell skulle samma risk uppkomma av 21 µg/m3.

Vi föreslår utifrån detta ett riktvärde för diklormetan i utomhusluft (lågrisknivå)på 50 µg/m3 som långtidsmedelvärde. Den tidigare rekommendationen frånIMM 1990 var 100 ppb (350 µg/m3).

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3.Properties, occurrence, exposure levels

3.1. General description

Dichloromethane is a clear, colourless, highly volatile, non-flammable liquidwith a penetrating ether-like odour (WHO 1996).

3.2. Identity

Formula: CH2Cl2

H

Structure: Cl C ClH

Relative molecular mass: 84.93

Common names: Methylene chloride, dichloromethane

Synonyms: Methane dichloride; methylene bichloride;methylene dichloride

CAS name : Methane, dichloro-

CAS registry number: 75-09-2

3.3. Physical and chemical properties

Boiling temperature: 40ºC

Melting temperature: -95ºC

Solubility in water: 20 (g/kg at 20ºC)

Soluble in: Alcohol, ether, acetone, and benzene

Vapour pressure: 47 (kPa at 20ºC)

Density: 1.33 (g/ml at 20ºC)

Odour threshold: 540-2160 mg/m3 (WHO 1996)743 mg/m3 (WHO 1984)

Conversion products: Phosgene and hydrochloric acid are formed bycontact with hot surfaces or flames.

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3.4. Conversion factors

Conversion factors for dichloromethane concentrations in air, calculated at20ºC and 760 mm Hg are:

1 ppm = 3.53 mg/m3

1 mg/m3 = 0.28 ppm1 mM = 84.93 mg/l

3.5. Sources of human and environmental exposure

3.5.1. ProductionThe world production of dichloromethane was 570 000 tonnes in 1980 (WHO1996). US production of dichloromethane grew steadily through the 1970´sand early 1980´s at about 3% each year, with a peak production of about280 000 tonnes in 1984. By 1988 dichloromethane production had droppedin response to declining demand to about 227 000 tonnes. This decline in thedemand for dichloromethane was expected to continue at a rate of about1-2% per year through 1993 as more manufacturers move towards water-based aerosol systems in anticipation of further regulation of dichloromethane(ATSDR 1993).

The total amount produced in western Europe ranged from 331 500 tonnesin 1986 to 254 200 tonnes in 1991 (WHO 1996).

No manufacture takes place in Sweden (KemI 1991a).

3.5.2. UseThe usage of dichloromethane in western Europe shows a decrease from200 000 tonnes/year in 1975-1985 to 175 000 in 1989 and to 150 000tonnes/year in 1992 (WHO 1996).

Most of the applications of dichloromethane are based on its considerablesolvent capacity. Other important properties are its volatility and stability. It isalso non-flammable (WHO 1996).

In Sweden the use of dichloromethane in consumer products has been bannedsince 1993 and professional use has been banned since 1996. The reasonsgiven for banning were the health hazards, carcinogenicity, high volatility andthe widespread use of dichloromethane (KemI 1991a; 1997). Exceptions fromthe ban are made for use of dichloromethane in research, development, and

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chemical analyses (KemI 1997). The main application areas in Sweden beforethe ban were degreasing and paint removal in the metalworking and enginee-ring industries and cleaning in the electronics industry. Dichloromethane wasalso used in the polyurethane industry and as a solvent in the pharmaceuticaland chemical industries (KemI 1991a).

After the ban, it has been necessary to permit exemptions within certain areasof application. In the pharmaceutical industry it has been difficult to find substitutesfor dichloromethane. Dichloromethane is used both in manufacturing ofpharmaceutical substances and reagents, and in the preparation of medicalarticles. A change in the preparation or formulation of a pharmaceutical demandsa renewed registration and a confirmation from the authorities in all countrieswere the pharmaceutical is sold. For this reason, the pharmaceutical industryis exempted from the ban for five years, i.e. until January 1, 2001 (KemI1997).

The amount of dichloromethane used in Sweden during the years 1988-1996and the number of chemical preparations containing dichloromethane are shownin Table 1. The amount of dichloromethane used in different applications during1988 and 1996 are shown in Table 2.

Table 1. Amounts of dichloromethane used in Sweden (pure substance,tonnes per year) (The Swedish Products Register, 1992-1996. KemI 1996,1998).

Year 1988 1992 1993 1994 1995 1996

Imported pure substance 2500 1290 1050 1230 970 423

Substance imported inchemical preparations 100 50 19 24 17 4

Exported in chemicalpreparations - - - >3 10 2

Exported pure substance - 100 275 202 202 35

Number of productsdeclared to containdichloromethane 450 148 163 137 98 74

Number of consumerproducts among these 100 20 20 16 8 8

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3.5.3. Consumer applicationsIn Sweden the use of dichloromethane in consumer products was banned in1993.

The main use of dichloromethane in other countries in consumer products isin paint strippers, where dichloromethane is the main constituent (70-75%).The second important use is in hair spray aerosols, where dichloromethaneacts as a solvent and a vapour pressure modifier. Other types of dichloromethane-containing products are household cleaning products and lubricating, degrea-sing and automotive products (WHO 1996).

Table 2. Imported amounts of dichloromethane in 1988 and 1996,distributed among different application areas (pure substance, tonnes peryear) (KemI 1991a; 1998).

Function Industry Quantity1988 1996

Degreasing Metal working industry 950 -and engineering

Paint removal Metal working industry, 330 4graffiti removal

Cleaning Electronic industry 270 <1

Solvents Pharmaceuticals industry 380 300

Solvents Chemicals industry 275 -

Cleaning, fermenting Polyurethane industry 270 <0.2agents, glue (polyurethane foam)

Miscellaneous Asphalt, building industry, 50 19laboratories, etc.

Wholesale trading 100

Total 2525 423

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3.6. Environmental levels and potential for human exposure

Dichloromethane is degraded in the atmosphere by reaction with hydroxylradicals and has an atmospheric lifetime of 6 months (WHO 1996). Thecompound is expected to be highly mobile in soil and to evaporate rapidlyfrom surface water to the atmosphere. Biodegradation may be important, butbioconcentration does not appear to be significant (ATSDR 1993).

3.6.1. AirTypical background levels of dichloromethane in rural and remote areas are0.07-0.29 µg/m3 (WHO 1996). The average concentrations in suburban andurban areas are reported to be <2 µg/m3 and <15 µg/m3, respectively. In thevicinity of hazardous waste sites, up to 43 µg/m3 has been found (WHO1996).

The overall mean concentration of dichloromethane was 2.6 µg/m3 in samplesof ambient air taken in 1989 from 17 urban sites in Canada. Mean concentrationsat 16 sites sampled in additional national surveys conducted between 1988and 1990 ranged from 1.0 µg/m3 to 6.2 µg/m3. In general, mean concentrationsof dichloromethane in indoor air are higher than those in ambient air. Basedupon preliminary results, the mean concentration in indoor air in 757 homesacross Canada was 16.3 µg/m3, although complete experimental details werenot provided in the preliminary published account of this survey (Long et al.1994).

Indoor air concentrations resulting from use of dichloromethane-containingproducts have been estimated to range from 0.21 to 19 000 µg/m3 (ATSDR1993).

There are practically no Swedish data on concentrations of dichloromethanein ambient air. According to Astra AB (1993), dispersion modelling of theemissions from a pharmaceuticals industry (total emission of chloromethane15 tons/year) gave a mean value of 0.8 µg/m3 and a 99-percentile value of3.7 µg/m3 for a geographical area 700-800 m away from the plant. Pointmeasurements in the vicinity of the plant revealed a maximum value of33.6 µg/m3.

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3.6.2. WaterAn estimated amount of 0.2% of the total dichloromethane production is releasedin waste water (WHO 1996). In surface water, levels of dichloromethane havebeen reported to vary from not detectable to 10 µg/l. A median concentrationof 0.1 µg/l in the United States was estimated (WHO 1996). The levels ofdichloromethane are considerably lower in open oceans (generally not detected)and a mean concentration of 2.2 ng/l has been reported in the South PacificOcean. Dichloromethane can be found at up to 2.6 µg/l in coastal waters ofthe Baltic sea. Levels up to 0.2 µg/l have been found in North Sea coastalwaters (WHO 1996). Dichloromethane has been detected in both surfacewater and groundwater samples taken at hazardous waste sites. In contaminatedground water in Minnesota USA, up to 250 µg/l has been detected (ATSDR1993). Dichloromethane has been detected in drinking-water supplies in numerouscities in the USA, the mean concentrations reported being generally less than1 µg/l. An average of 3-10 µg/l and a maximum of 50 µg/l were observed ina Canadian study of 30 drinking-water treatment facilities in 1982 (WHO1996), but according to a later survey (Long et al. 1994), the levels ofdichloromethane in municipal drinking water supplies in Canada ranged from0.2 µg/l to 2.6 µg/l.

3.6.3. Soil and sedimentNo data are available on the levels of dichloromethane in soil. The levels ofdichloromethane in sediments have been reported from not detectable to 40 µg/kg.In sediment from one site at the Rhine river, maximum concentrations of 220-2200 µg/kg were found (WHO 1996).

3.6.4. FoodData on levels of dichloromethane in food are very limited. Levels between 1and 300 µg/kg have been reported, with the highest levels found in butter,ready-to-eat cereals and highly processed food (WHO 1996).

Dichloromethane was detected at a mean concentration of 74 µg/l in 17 outof 28 samples of breast milk from women occupationally exposed todichloromethane (WHO 1996). Dichloromethane has been identified, but notquantified, in eight out of eight samples of human breast milk from the USA(KemI 1995).

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3.6.5 . Estimated intake for the general populationThe principal route of exposure to dichloromethane for the general populationis by inhalation. Average daily intake of dichloromethane from urban air hasbeen estimated to range from about 33 to 309 µg (0.5-4.5 µg/kg body weight)based on a daily inhalation of 23 m3 by an adult. Occupational and consumerexposure to dichloromethane in indoor air may be much higher, especially fromspray painting or other aerosol uses (ATSDR 1993). Estimated intakes inCanada are shown in table 3.

Table 3. Estimated mean daily intake of dichloromethane in Canada(Long et al. 1994).

Route of exposure Estimated daily intake (µµµµµg/kg body weight)

Ambient air 0.04 - 0.46

Indoor air 3.9 - 6.0

Drinking water 0 - 0.07

Food 0.03 - 0.11

Total intake 4.0 - 6.6

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4. Metabolism and toxicokinetics

Dichloromethane vapour is rapidly absorbed through the alveoli of the lungsand distributed into the systemic circulation. Dichloromethane can also beabsorbed from the gastrointestinal tract and, at a slower rate, through intactskin. Since dichloromethane is highly soluble in both water and fat, it is rapidlydistributed to all tissues, although at a slower rate to adipose tissue. Tissue:airratios between 4.8 and 9.7 have been reported for most tissues, except foradipose tissue, with a ratio of 85. Dichloromethane can cross the blood-brainbarrier and be transferred across the placenta. Apart from some build up inadipose tissue, dichloromethane and its metabolites do not seem to accumulatein tissues (IMM 1990, Paterson and Mackay 1989, WHO 1996).

The decrease in dichloromethane after exposure follows a multiexponentialpattern. Three phases have been reported for humans with half times in bloodof 8-23 min, 40-80 min and 6-6.5 hours. The final phase reflects washoutfrom adipose tissues. Unmetabolised dichloromethane is primarily excretedfrom the body via the lungs. Small amounts of dichloromethane can be excretedin milk, whereas urinary excretion plays a minor role. In mice, following oraldoses of 1 and 500 mg/kg body weight of dichloromethane in water, 2% and55% of the dose were exhaled unchanged. Thus, at low exposure levels, mostof the absorbed dichloromethane is metabolised, whereas at high doses, mostof the absorbed dichloromethane is exhaled unchanged. Based on comparisonsof the area under the concentration-time curves (AUC) of dichloromethane inplasma, oral doses of 10, 150 and 450 mg/kg body weight correspond to 6 hinhalation exposures to 180, 1800, and 5300 mg/m3, respectively (IMM 1990,Kirschman et al. 1986).

Early toxicokinetic studies have shown that the disposition of dichloromethaneafter oral gavage depends on the vehicle used. Thus dichloromethane administeredin corn oil or olive oil resulted in a slower uptake rate and longer half timesthan when given in water. In addition, a smaller fraction was exhaled unchangedafter oil gavage (IMM 1990).

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4.1. Metabolism

Dichloromethane is metabolised by either or both of two pathways to carbonmonoxide, carbon dioxide and inorganic chloride. The metabolism ofdichloromethane, deduced mainly from studies in rodents, is summarised inFigure 1.

Cytochrome P450 pathway

CH2Cl2 HO - C - Cl

Cl

H

P450

O2, NADPH

HCl

C = O CO + HCl

H

Cl

GSCHO HCOOH

GSH

CO2

CO2

Glutathione S-transferase pathway

CH2Cl2 GSCH2ClGST

GSH

HCl

GSCH2OH HCHO + GSH

HCOOH

CO2

HCl

H2O

HCOOH

GSCHO

C-1 pool

C-1 pool

NAD+

Figure 1. Metabolism of dichloromethane (Adapted from Foster et al.1992).

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The oxidative metabolism of dichloromethane takes place in the endoplasmaticreticulum of the cells. This oxidative dehalogenation reaction is catalysed primarilyby cytochrome P450 2E1 (CYP2E1) (Andersen et al. 1994, Kim and Kim1996, Pankow and Hoffmann 1989, Pankow et al. 1991a). In rodents, someactivity can possibly be attributed to CYP2B1 (Foster et al. 1992, Andersenet al. 1994). The isoenzyme CYP2B1 has not been found in humans (Ingelman-Sundberg and Johansson 1995). The putative formyl chloride intermediate isunstable and reactive, and decomposes to carbon monoxide and hydrogenchloride or reacts with cellular nucleophiles (Gargas et al. 1986). Carbonmonoxide binds to haemoglobin (COHb), but can also be further oxidised tocarbon dioxide. Increased levels of COHb have been demonstrated in humansafter experimental, as well as occupational, exposure to dichloromethane(DiVincenzo and Kaplan 1981, Ghittori et al. 1993, Soden et al. 1996, Åstrandet al. 1975).

The second pathway, which takes place in the cytosol, involves conjugationwith the endogenous tripeptide glutathione (GSH) and is catalysed by glutathioneS-transferases (GST). The conjugate formed, S-chloromethylglutathione, ishighly reactive and unstable and rapidly decomposes to formaldehyde andGSH. Formaldehyde is oxidised to formic acid and ultimately to carbon dioxide.Due to the high reactivity there is only indirect evidence of the intermediaryconjugate (Hashmi et al. 1994).

Potentially toxic metabolites are thus formed in both pathways; formyl chlorideand carbon monoxide (P450 pathway) and S-chloromethylglutathione andformaldehyde (GST pathway).

Cytochrome P450 has a higher affinity for dichloromethane compared to GST.At lower concentrations of dichloromethane the P450 pathway is thus thepreferred route. In mice dichloromethane is almost exclusively oxidised at lowexposure levels. The P450 pathway is, however, saturated at about 700-1000 mg/m3 dichloromethane. (Andersen and Krishnan 1994, Ottenwälder etal. 1989). In contrast, the GST pathway is not saturated until dichloromethanelevels up to 35 000 mg/m3, and this pathway therefore increases in relativeimportance as exposure levels rise. However, even at high doses, such asexposure to 14 000 mg/m3 in mice, only a part of dichloromethane is meta-bolised by the GST pathway (Ottenwälder et al. 1989).

Studies in mice show that dichloromethane is primarily metabolised in the liver,even after inhalation (Reitz 1991). Certain parts of the lung also metabolise

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dichloromethane, and locally in the lung high concentrations of reactive meta-bolites may be formed. In mouse lung the oxidative metabolism ofdichloromethane takes place in the Clara cells, which comprise approximately5% of the cells in the lung and 70% of the bronchiolar cells (Foster et al.1992).

4.1.1. Glutathione conjugation; polymorphism and localisation ofGSTT1The glutathione transferases are a family of enzymes that catalyse a number ofglutathione dependent reactions. In addition to catalysing the formation ofconjugates, GSTs can also function as peroxidases and isomerases. Basedmainly on their primary structures, GSTs are classified into several differentclasses. In humans GST isoenzymes belonging to class Alpha, Mu, Pi andTheta have been found. Different tissues express different GST isoenzymes.For example, GSTA1/2 (class Alpha) and GSTM1 (class Mu) are commonin the liver, whereas GSTP1(class Pi) is mainly expressed in extrahepaticorgans. As with other drug-metabolising enzymes GST isoenzymes generallyhave broad and overlapping substrate specificities (Hayes and Pulford 1995).

Genetically determined interindividual differences in human GST are common.Such variation is due either to gene deletion, resulting in failure to express theprotein, or to allelic variation, resulting in production of proteins which maydiffer in their catalytic capacity. The gene for GSTM1 has been found to bedeleted in approximately 50% of humans of Caucasian origin, whereas thisdeletion seems to be less common in Africans (Hayes and Pulford 1995).Recently the gene for GSTT1 has also been found to be deleted in someindividuals. There are large differences in the frequency of this deletion indifferent populations. In Sweden approximately 10% of all individuals lackGSTT1 (Warholm et al. 1995), compared to 60% in some Asian populations(Nelson et al. 1995). In most Caucasian populations approximately 15-20%lack GSTT1 (Hayes and Pulford 1995, Nelson et al. 1995). These genedifferences suggest that there may be large interindividual differences insusceptibility to chemicals metabolised by GST.

The conjugation of dichloromethane to glutathione is catalysed by GSTT1(class Theta) (Mainwaring et al. 1996a, Sherratt et al. 1997), although a minorcontribution by GSTM1 cannot be ruled out, as GSTM1 is more abundant,at least in rat liver (Blocki et al. 1994, Casanova et al. 1997). However, the

20

affinity of dichloromethane towards GSTT1 (Km = 0.3 mM) is considerablyhigher compared to GSTM1 (Km = 8 mM) (Blocki et al. 1994). Limited datafrom human liver indicate that GSTM1 levels in individuals possessing thisisoenzyme are 2-10 times higher than GSTT1 levels (Juronen et al. 1996,Ketterer et al. 1991, Meyer et al. 1991). The role of GSTT1 is also supportedby human inhalation experiments with methyl chloride, another substrate forGSTT1. The experiments showed that the metabolism of methyl chloride israpid in individuals possessing GSTT1, absent in GSTT1 deficient individuals,and independent of the GSTM1 genotype (Löf et al. in manuscript).

The cellular localisation of GSTT1 has been studied in liver and lungs using insitu hybridisation for detection of specific mRNA and immunohistochemistryfor the detection of GSTT1 protein. In mouse liver GSTT1 was found in highconcentrations in the centrilobular region, specifically in the cells surroundingthe central vein and bile ducts. In addition to the cytosolic localisation, GSTT1was found in some, but not all, nuclei, and in only one of the two nuclei ofbinucleate cells in mouse liver. In contrast, GSTT1 was only found in thecytosol in rat and human hepatocytes and was considerably less abundant. Inmouse lung, the highest concentration of GSTT1 was found in Clara cells andciliated cells. In rat lung, on the other hand, GSTT1 concentrations weresignificantly lower and confined to Clara cells. In human lung GSTT1 was onlydetected in low levels in a very small number of Clara cells and ciliated cellsat the alveolar/bronchiolar junction (Foster et al. 1992, Green 1997, Mainwaringet al. 1996b, 1998).

Human GSTT1 has been isolated from or detected in a variety of tissues,including liver (Juronen et al. 1996, Meyer et al. 1991), erythrocytes (Schrö-der et al. 1992), lung, kidney, brain, skeletal muscle, heart, small intestine, andspleen (Juronen et al. 1996). When analysing tissues from one male individual,73 years old, the highest concentrations of GSTT1 were found in liver andkidney, with substantial amounts also in the prostate and small intestine. GSTT1was also present in cerebrum, pancreas and skeletal muscle at approximately10% of the level in liver. Lung, spleen, heart and testis expressed GSTT1 atlevels less than 5% of that in liver (Sherratt et al. 1997).

4.1.2. Metabolic data in vitroReitz et al. (1989) determined the enzyme activity in vitro for dichloromethanemetabolism via the GST pathway using the cytosolic fraction from rat, mouse,hamster, and human liver and lung tissue samples. In parallel, enzyme activity

21

of the P450 pathway was determined in microsomal preparations (Table 4).The liver and lung GST activities were comparatively high in the mouse,intermediate in the rat, and low in hamster and man. GST activity was absentin one liver sample and ranged from 2.6 to 3.0 nmol/min/mg protein in theother three human liver samples. The P450 metabolism could be described bysimple Michaelis-Menten kinetics. Overall, the hepatic P450 activity was higherin the mouse and hamster than in the rat and highly variable in man (range 1.5-13 in four subjects). No P450 activity towards dichloromethane could bedetected in human lung microsomes with the analytical method used (detectionlimit 0.1 nmol/min/mg protein). Notably, the Km values determined in vitro inthe study by Reitz et al. (1989) differed by about two orders of magnitudefrom those estimated from in vivo studies by Andersen et al. (1987).

In a review article, Green (1997) reported maximum metabolic rates (Vmax)for P450 dependent metabolism of dichloromethane that are approximately tentimes lower than those of Reitz et al. (1989), although with similar relationshipsbetween the four species. Concerning the GST pathway, Green further reportsactivities of approximately 90, 8, 2, and 1 nmol/min/mg protein for mouse, rat,hamster, and man, respectively (data from Figure 3 in Green 1997). However,the sources of the values given by Green are unclear and are therefore notpresented in Table 4.

GSTT1 purified from mouse and rat have comparable specific activities withdichloromethane as the electrophilic substrate, 5.5 and 11 µmol/min/mg pro-tein, but mouse liver contains at least 10 times more GSTT1 protein (Mainwaringet al. 1996a,b, Meyer et al. 1991), supporting the relative activities in mouseand rat reported by Green (1997).

Metabolism of dichloromethane by the GST pathway can be detected byanalysing DNA-protein crosslinks, which are formed from formaldehyde. Inmice, but not in hamsters, a dose-dependent and non-linear increase in crosslinkswas observed in the liver, but not in the lungs, after exposure to 1800-14 000 mg/m3 dichloromethane, 6 h/day for 3 days (Casanova et al. 1996).When incubating hepatocytes from various species with dichloromethane invitro, crosslink formation was detectable in hepatocytes of male B6C3F1mice, but not in hepatocytes from Fischer 344 rats, Syrian golden hamsters orhumans, with or without functional GSTT1 genes (Casanova et al. 1997). Inthe same study, formation of formaldehyde was also analysed by measuringRNA-formaldehyde adducts. Such adducts were present in hepatocytes from

22

Table 4. Metabolic parameters of dichloromethane in vitro in cytosolic(GST) and microsomal (P450) preparations. Activities are given in nmol/min/mg protein, unless otherwise stated. Values are rounded off. Data frommouse, rat and hamster are mean values. Data from man are groupedaccording to presumed polymorphism.

Substrate Mouse Rat Hamster Man Referencesconcentration(mM)

GST activity, lung homogenate

40 7.3 1.0 0.0 0.4 Reitz et al. 1989

? - - - 0.06-0.23 Mainwaring et al. 1996b

GST activity, liver homogenate

40 25.9 7.1 1.3 0; 2.6-3.0 Reitz et al. 1989

40 - 1.7 - <0.04; Bogaards et al.19930.2-0.4; 0.8-1.4

94 - - - 0.5-0.8; Graves et al. 19952.1-2.4

? male 18 3.7 0.3 0; 0.6±0.3; Thier et al. 1998female 30 1.6±0.5

Vmax 118 - - 7.1; 6.0 Reitz et al. 1989Km (mM) 137 - - 44

GST activity, kidney homogenate

? male 3.2 1.7 0.3 0; 1.4±0.5; Thier et al. 1998female 3.9 3.1±0.7

GST activity, erythrocytes

? - - - 0; 0.9±0.3; Thier et al. 19981.8±0.11

Vmax - - - - - - 0.182 Hallier et al. 1994Km (mM) 60

P450 activity, lung homogenate

5 4.6 0.2 1.0 <0.1 Reitz et al. 1989

P450 activity, liver homogenate

Vmax 15.9 5.4 20.8 1.5-13.0 Reitz et al. 1989Km (mM) 1.8 1.4 2.1 0.9-2.8

1 nmol/min/100 µl erythrocytes2 nmol/min/mg haemoglobin

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all species studied, but in humans only in hepatocytes from individuals posses-sing functional genes for GSTT1 and GSTM1 (Casanova et al. 1997). Yieldsof RNA adducts in hepatocytes from mice were 4-fold higher than those fromrats, 7-fold higher than those of humans, and 14-fold higher than those fromhamsters.

Glutathione transferase activity with dichloromethane as substrate was measuredin vitro in liver and kidney cytosolic fractions from rat, hamster, male andfemale mice and in humans with known GSTT1 genotype (Table 4). Thehighest activity was found in female mouse liver, followed by male mouse liver.The activity in rat liver (sex not specified) was 5-8 times lower compared tomice. The activity in human liver from individuals homozygous for GSTT1(n=12) was twofold higher than in heterozygous individuals (n=11) and absentin GSTT1 null individuals (n=2). Humans had lower liver GSTT1 activity thanrats, but higher than hamsters. In rats and mice the activity in kidney was 2-5 times lower compared to liver, whereas it was equally low in hamster kidneyand liver. In humans, on the other hand, the GSTT1 activity with dichloromethanein kidney was twice that in liver (Thier et al. 1998).

In vitro conjugation of dichloromethane with glutathione by GST was studiedin 22 human liver samples by measuring the formation of formaldehyde. Threeindividuals lacked activity, < 0.04, 11 samples had activities between 0.20 and0.41, and 8 individuals had high activities of between 0.82 and 1.35 nmol/min/mgprotein. The highest activity in human liver was still 1.4 times lower than thatin rat liver (1.7 nmol/min/mg protein) (Bogaards et al. 1993).

In one study, the GST activity in human liver cytosol with dichloromethane assubstrate ranged from 0.50 to 0.84 in four individuals, whereas two individualshad higher activities of 2.1 and 2.4 nmol/min/mg protein, respectively (Graveset al. 1995). In another study, GST activities with dichloromethane in humanlung cytosol were 0.06, 0.21 and 0.23 nmol/min/mg protein in three differentindividuals (Mainwaring et al. 1996b).

Earlier work has shown that monohalogenated methanes, such as methyl chloride,are conjugated by GSTT1 in human erythrocytes. Erythrocytes from rat, mouse,cattle, pig, sheep and rhesus monkey apparently lack this GST activity (Peteret al. 1989). In erythrocytes from one individual, probably homozygous forGSTT1, the maximum metabolic activity with dichloromethane as substratewas estimated as 180 pmol/min/mg Hb (Hallier et al. 1994).

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4.1.3. Repeated exposures, ageingIn mice exposed to 14 000 mg/m3 dichloromethane for 1 or 10 days thecytochrome P450-dependent metabolism of dichloromethane, measured in lungfractions in vitro, was decreased by 50% compared to nonexposed controls,while the GST activity was unaffected (Green 1990a).

In a two-year inhalation study with mice, (7000 mg/m3 dichloromethane, 6 h/day,5 days/week), the animals underwent toxicokinetic studies in a closed chamberat regular intervals. In addition, tissue:air partition coefficients were repeatedlydetermined. The animals were 4-6 weeks of age when the exposure started.Age related changes were seen in muscle:air, lung:air, liver:air, and fat:air, butnot blood:air partition coefficients. Fitting of metabolic parameters of aphysiologically-based pharmacokinetic model to the closed chamber dataindicated that the Vmax value of the P450 pathway was approximately doubledduring the study, and was higher in exposed than in control mice at day oneand at one month, but slightly lower at one and two years. In contrast, themodel suggested that the GST activity decreases with age in control mice aswell as in exposed mice. These age and prior exposure related changes intoxicokinetics will affect target dose estimates, in a complex fashion. Conside-ring only the changes in P450 activity, physiological pharmacokinetic simulationsindicated an age-dependent 18% decrease in GST mediated metabolism (Tho-mas et al. 1996).

4.1.4. Metabolic interactionsEarlier exposure to substances that induce CYP2E1, e.g. benzene (Kim andKim 1996), acetylsalicylic acid (Pankow et al. 1994) or methanol (Pankow etal. 1993) increases the metabolism of dichloromethane to COHb, i.e. by theP450 pathway. Many compounds that induce CYP2E1 are also substrates forthis enzyme. Simultaneous exposure to such substances and dichloromethanemay therefore decrease the metabolism of dichloromethane to carbon mon-oxide, as shown e.g. in rats with aromatic solvents (Pankow et al. 1991b).Rats fed with 10% ethanol via their drinking water (corresponding to ”socialdrinking” according to the authors) expressed approximately two- (using anilineas substrate) to four-fold (using p-nitrophenol as substrate) induction of CYP2E1in the liver. The induction effect developed gradually during 36 weeks. A 50%reduction in dichloromethane blood levels and a doubling of COHb was seenin ethanol treated rats given a single oral dose of dichloromethane in oil. Thiseffect did not parallel the CYP2E1 induction, but was most pronounced after4 weeks of ethanol exposure. Inhalation exposure for 4 h at 350, 1700, or

25

8700 mg/m3 dichloromethane resulted in no or minimal increases in COHb inethanol treated rats as compared to controls (Wirkner et al. 1997).

A physiologically-based pharmacokinetic model was developed by Pelekisand Krishnan (1997) to study the influence of toluene on dichloromethanebiotransformation to COHb. For this purpose, the Andersen model fordichloromethane (Andersen et al. 1991) was linked in series with a conventionalphysiologically-based pharmacokinetic model for toluene, and extended toalso include oral administration of solvents. Metabolism was assumed to occurexclusively in the liver and by the same enzyme, CYP2E1. After validationagainst oral rat data, the model was scaled from oral administration to inha-lation exposure and from rat to man. According to this extrapolation model,an 8 h coexposure to 50 ppm (190 mg/m3) toluene and 50 ppm (180 mg/m3)dichloromethane results in less than 10% reduction in COHb formation, ascompared to 180 mg/m3 dichloromethane alone.

4.1.5. Species scalingAndersen and co-workers (1987) developed a physiologically-basedpharmacokinetic-model for dichloromethane disposition in four species, i.e.mouse, rat, hamster, and man. The model comprised metabolism by two pathways,GST and P450, in the liver and lung compartments. Literature values wereused for physiological parameters. Blood:air partition coefficients were obtainedfor each of the four species, whereas other tissue:air partition coefficients wereextrapolated from rat and hamster experiments. Metabolic parameters wereobtained by fitting the model to closed chamber data for the three animalspecies. The relative contribution from lung and liver metabolism was obtainedusing in vitro metabolic data from Lorentz et al. (1984). The metabolic para-meters of the P450 pathway in humans were taken from unpublished observationsafter exposure to 350 and 1200 mg/m3 dichloromethane for 6 h, whereas therate constant of the GST pathway was obtained by allometric scaling from thethree animal species. There was a high correlation between tumour incidenceand GST metabolism, but not P450 metabolism, in the mouse. The authorsconcluded that the target dose in man expressed as amount dichloromethanemetabolised by GST in liver and lungs was two orders of magnitude lowerthan predicted by linear extrapolation from inhalation or drinking water exposure,as performed by the US EPA (further discussed later, see chapter on Quantitativerisk estimates).

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Reitz et al. (1989) went on to determine enzyme activities in vitro fordichloromethane metabolism in more detail. Using mouse data as a referencepoint for in vitro - in vivo scaling, Reitz et al. obtained a human hepatic GSTactivity in close agreement with that estimated by Andersen et al. (1987) (0.43as compared to 0.53 nmol/min/mg protein). In contrast, the GST activities inrat (0.63 versus 2.2) and hamster (0.16 versus 1.51) liver preparations differedconsiderably between the two studies. These differences illustrate the potentialof introducing errors when scaling metabolism from in vitro data to the wholeorganism and from one species to another.

Andersen et al. (1991) extended their model to include the metabolite CO andCOHb. Metabolic parameters for CO and COHb (diffusion capacity andendogenous formation rate of CO, the Haldane coefficient, and the CO -COHb equilibrium constant) were obtained from experiments where rats wereexposed to 200 ppm CO for 2 h. The extended model could adequatelydescribe the kinetics of dichloromethane and COHb in various rat inhalationexperiments. Furthermore, the model could adequately describe the observedtoxicokinetics in four human inhalation studies, after scaling of physiologicalparameters. The model assumes yield factors of CO of 0.71 and 0.80 forhumans and rats, respectively, i.e. that 0.71 or 0.80 moles CO are formed permole dichloromethane oxidised (see Figure 1).

Later on, Andersen et al. (1994) studied the isotope effect on the toxicokineticsof dichloromethane in mice. The animals inhaled unlabelled or deuterium-labelleddichloromethane in a closed chamber, and the time courses of dichloromethaneand CO concentrations in chamber air were monitored and subsequentlyevaluated by the use of a physiologically-based pharmacokinetic model. Theavailability in the P450 pathway was 0.76 for unlabelled, 0.33 for single-labelled and 0.31 for twin-labelled dichloromethane. The Michaelis constant(Km) increased dramatically with labelling, whereas no effect was seen on themaximum metabolic rate (Vmax).

Hetrick et al. (1991) carried out a sensitivity analysis using a simplephysiologically-based pharmacokinetic model, with metabolism only in the liverand no metabolite kinetics. Dichloromethane levels in blood and amountdichloromethane metabolised was most sensitive to the Vmax, the blood:airpartition coefficient and the Km. Clewell and co-workers (1994) performeda similar sensitivity analysis with a model that also included lung metabolism.Dose surrogates (AUC of dichloromethane and amounts metabolised by P450

27

and GST, respectively) were sensitive to alveolar ventilation, the blood:airpartition coefficient, and metabolic parameters, somewhat sensitive to cardiacoutput and liver blood flow, and insensitive to other parameters.

4.2. Human toxicokinetic data

Three volunteers, who were dermally exposed by immersing one thumb in abeaker with liquid dichloromethane for 30 min, reached average breathconcentrations of 11 mg/m3 at the end of exposure and 2.4 mg/m3 2 h later(Stewart and Dodd 1964). This experiment indicates that spillage may causesubstantial skin penetration of dichloromethane.

DiVincenzo et al. (1972) studied the dichloromethane kinetics after inhalationexposure for 2 h at 350 mg/m3 (4 males) and 700 mg/m3 (7 males). Post-exposure dichloromethane levels in blood and 24 h dichloromethane excretionin urine were approximately 3 times higher after exposure to 700 mg/m3,compared to 350 mg/m3, suggesting partial metabolic saturation.

In a series of experiments, Åstrand et al. (1975) exposed male volunteers todichloromethane vapours during rest and exercise on a bicycle ergometer. Therelative respiratory uptake at rest was about 55% regardless of exposurelevel. Exposure at 880 mg/m3 resulted in average internal levels of 550 mg/m3

dichloromethane in end-tidal air, 5.8 mg/kg dichloromethane in arterial bloodand 3.9 g/l (2.5%) COHb in blood. In comparison, exposure at 1800 mg/m3

resulted in more than two times higher internal levels of dichloromethane,1240 mg/m3 in end-tidal breath and 12.5 mg/kg in arterial blood, whereasCOHb was essentially unaffected (3.4 g/l or 2.2%). These data indicate non-linear kinetics due to metabolic saturation of the P450 pathway resulting inCOHb formation.

The same laboratory went on to analyse uptake and disposition in relation tobody build by exposing 12 males to 2600 mg/m3 dichloromethane for 1 hduring 50W exercise. Obese subjects had a markedly higher uptake and slightlylower levels of dichloromethane in blood than slim subjects during exposure.Dichloromethane in subcutaneous adipose tissue decreased from 6 mg/kg inthe obese and 12 mg/kg in the slim during the first hours after exposure to 1.6-1.7 mg/kg (2 subjects only) 22 h after exposure (Engström and Bjurström1977). This study confirms that fat depots act as reservoirs for dichloromethaneand that body build influences the internal exposure profile.

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DiVincenzo et al. (1972) exposed three male volunteers to 3500 mg/m3 for2 h. Maximum COHb levels of about 7, 9, and 15% were reached one hourafter ending the exposure, indicating interindividual variability in the metaboliccapacity of the P450 pathway.

Eleven male and three female volunteers were experimentally exposed to 170,350, 520 and 700 mg/m3 dichloromethane, with 4-6 subjects per exposurelevel. The exposure lasted for 7.5 h and was interrupted by a 0.5 h lunch. Onaverage, plateau concentrations of dichloromethane of about 0.3, 0.8, 1.2, and1.6 mg/l in blood and 50, 120, 190 and 280 mg/m3 dichloromethane in end-tidal breath samples were reached during the exposure at the four levels. ForCOHb and CO no plateaus were reached. At the end of the exposures COHblevels of about 1.8, 3.2, 5.2 and 6.8% were reached. The correspondingvalues for CO in end-tidal breath were about 6, 12, 24, and 30 ppm. Afterexposure, COHb decreased with a half-time of about 6 h. No saturationeffects were seen in the kinetics at these exposure levels. The average respiratoryuptake was 72% of the inhaled amount. About 5% of the absorbed dose wasexhaled unchanged after exposure and 28% was exhaled as CO (DiVincenzoand Kaplan 1981).

Occupational exposure to dichloromethane was determined in 20 employeesin the pharmaceutical industry. Four hour sampling in the breathing zone withpassive dosimeters revealed exposure levels between 3.5 and 205 mg/m3 withan average of 50 mg/m3. Dichloromethane in urine correlated strongly with airlevels (r=0.90), independent of smoking habits. CO in expired breath correlatedwith dichloromethane in air only in the 8 non-smokers (r=0.87) (Ghittori et al.1993).

Workers in a triacetate fibre production plant were routinely monitored fordichloromethane twice a year by 8 h personal sampling and determination ofCOHb in blood. Exposure levels were between less than 35 and approximately520 mg/m3 dichloromethane (Soden et al. 1996). In non-smokers, there wasa significant correlation between external exposure and COHb levels (r=0.58,n=221). Exposure below 300 mg/m3 in non-smokers gave COHb values ofless than 4.0%. Smokers had higher COHb values (range 4.8-6.4%) whichwere only weakly related to dichloromethane exposure. There were no signsof accumulation of COHb during the work week.

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4.2.1. Influence of work loadExposure to solvent vapours during physical exercise results in increased bloodlevels and target doses of the volatile substance due to increases in alveolarventilation and cardiac output. Experiments with human volunteers exposed to1800 mg/m3 dichloromethane (Åstrand et al. 1975) showed that dichloromethanein blood was approximately doubled during light bicycle exercise (50W) ascompared to rest. Increased work loads gave additional but less markedincreases in blood dichloromethane. Furthermore, the absolute uptake increased,whereas the relative uptake decreased from 55% at rest to 31-44%, 28%,and 23% during exercise levels of 50W, 100W and 150W, respectively.Using a physiologically-based pharmacokinetic model, Johanson and Näslund(1988) showed that these changes could be explained by the shifts in alveolarventilation, cardiac output and cardiac distribution that occur with physicalexercise.

Similar results have been presented in physiological pharmacokinetic simulationsusing metabolic data obtained in vitro with human livers (Dankovic and Bailer1994). Thus, during a simulated exposure to 90 mg/m3 dichloromethane during34W work, the surrogate doses in the liver (amounts metabolised in the liverby P450 and GST, respectively) increased by factors of 1.9 and 2.2, comparedto rest. The corresponding factors for the lungs were 1.5 and 1.8. A small partof the increases is due to differences in exposure times (8 h versus 6 h forworking and resting conditions, respectively).

4.3. Conclusions

Dichloromethane is efficiently absorbed by all exposure routes, and evenlydistributed within the body with an affinity for adipose tissues. In all mammalspecies studied, dichloromethane is metabolised by two pathways, which aremainly mediated by cytochrome P450 2E1 and glutathione-S-transferase (GST)T1, respectively. Potentially toxic metabolites are formed in both pathways,including formyl chloride, carbon monoxide, S-chloromethylglutathione, andformaldehyde.

The P450 pathway becomes saturated at inhalation exposures of about 700-1000 mg/m3, whereas the metabolism via GST is essentially linear at all rele-vant exposure levels. In effect, P450 is the preferred route at low concentrations.However, at high concentrations the GST pathway becomes relatively more

30

important. This is of importance in high dose to low dose extrapolations,especially if toxicity is related to metabolites of the GST pathway only.

In laboratory animals, metabolism occurs mainly in the liver, with additionalmetabolism in lung and possibly also in other tissues. There are considerablespecies differences with respect to tissue specific metabolic capacities of thesepathways. The GST activity in liver and lung is considerably higher in micecompared to other mammals investigated. Except for liver, lung and kidneydata from laboratory animals, there is scarce data on tissue-specific metabo-lism.

Human studies have focused on the uptake and oxidative metabolism ofdichloromethane after inhalation exposure. One reason is probably that at thetime of the experiments there was little awareness of the GST pathway.Dichloromethane levels in blood and exhaled breath rapidly declines whenexposure is ended. The decrease in COHb is slower, and has a half-time ofapproximately 6 h. Experimental exposure to 700 mg/m3 during a work-dayresults in COHb levels of about 7%. In occupational exposures below310 mg/m3, COHb levels are less than 4% in non-smokers. Several in vivostudies confirm metabolic saturation of the P450 pathway in humans.

During physical exercise, increases in uptake, blood levels, and exhalationrates of dichloromethane are seen, but COHb is only slightly affected. Dermalabsorption may be significant upon exposure to liquid dichloromethane.

At occupationally and environmentally realistic exposures, GST metabolismseems to play a quantitatively minor role. Limited qualitative data indicate thatGSTT1 is expressed in most human tissues. However, human quantitative invitro data is limited to a small number of liver, kidney, and lung samples. TheGST isoenzyme of interest, GSTT1, is polymorphic in humans. Thus, between10% (Swedish population) and 60% (Chinese) of the individuals in differentethnic groups lack GSTT1 and can therefore not metabolise dichloromethaneby the GST pathway.

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5. Short-term effects in animals

5.1. Inhalation

The acute toxicity of dichloromethane is low. Reported lethal concentrations(LC50-values) for exposure during 6 h lie between 40 000 and 56 000 mg/m3

for mice, rats and guinea pigs (IMM 1986; WHO 1996).

5.1.1. CNS effectsCNS effects such as changed sleeping patterns appeared in rats down to3500 mg/m3. At higher concentrations (14 000-21 000 mg/m3), decreasedspontaneous activity and reversible narcosis appeared in mice and rats (IMM1986).

Exposure of Fischer 344 rats to 7100 mg/m3 for 2.5 h caused changes insomatosensory evoked responses and electroencephalograph (EEG) pattern.The lack of changes on evoked responses of 157 mg/m3 carbon monoxideindicated that the effects were probably due to dichloromethane itself and notits principal metabolite carbon monoxide (Mattsson et al. 1988, according toWHO 1996).

Alterations in somatosensory evoked potentials were also observed after aone hour exposure of Fischer-344 rats to dose levels of 18 000 mg/m3 ormore (Rebert et al. 1989).

Male NMRI mice were exposed to dichloromethane up to 16 000 mg/m3 ina tolerance study on CNS-effects, (i.e. decreased responsiveness to a chemicalthat arises as a result of a previous exposure to the same chemical). Motoractivity increased on exposure to dichloromethane and decreased on constantexposure. Termination of exposure was followed by hypoactivity (Kjellstrandet al. 1990).

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5.1.2. Cell proliferation in liver and lungGroups of 5 male B6C3F1 mice were exposed to dichloromethane either byoral gavage in corn oil (1000 mg/kg) or by inhalation of 14 000 mg/m3 for 2 h.No evidence of induced DNA synthesis (number of cells in S-phase) in thehepatocytes was seen in the gavage study. The inhalation exposure resulted insome weak, but statistically significant increases in S-phase incidence in theliver, but the biological significance is unclear due to similar increases in somecontrol groups. The strain of mice used is known to be sensitive todichloromethane-induced hepatocarcinogenicity and has also been shown torespond to mitogenic stimuli (Lefevre and Ashby 1989).

Groups of five male B6C3F1 mice were exposed to 0, 440, 880, 1800, 3500,7100 and 14 000 mg/m3 for 6 h. In a separate study 5 animals/group weregiven intraperitoneal injections of a cytochrome P450 inhibitor or a GSHdepletor and then exposed to 7100 mg/m3 dichloromethane for 6 h.Morphological effects in the lung, such as vacuolisation and necrosis of thebronchiolar Clara cells, were seen at 7100 mg/m3 and above. The level ofglutathione (GSH) in cytosol isolates from lung homogenates showed elevatedconcentrations down to 880 mg/m3. An exposure level of 3500 mg/m3 andgreater produced an increased level of DNA synthesis (more cells in S-phase)in Clara cells. This indicates that the Clara cells are primed to divide followingexposure, possibly via a mechanism related to the metabolic activation ofdichloromethane. Mice pre-treated with the P450 inhibitor showed a significantlyreduced number of Clara cells exhibiting vacuolisation following a dose knownto cause lesions in mice without pre-treatment. No effect was seen on thenumber of vacuolised bronchiolar cells following pre-treatment with the GSH-depletor. These findings support that the vacuolisation of the Clara cells is dueto a toxic metabolite produced by the cytochrome P450 pathway (Foster etal. 1994).

B6C3F1 mice exposed to dichloromethane 6 h/day for 3 days, at concentrationsranging from approximately 5300 to 14 000 mg/m3 showed an increased rateof DNA synthesis in the lungs indicating cell proliferation, but increased cellturnover was not detected in mouse lung at exposure concentrations of 530 or1800 mg/m3. Syrian golden hamsters showed no evidence of cell proliferationin the lung at any of the concentrations mentioned above. Cell proliferationwas not apparent in the liver of either mice or hamsters (Casanova et al.1996).

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5.1.3. Other effectsExposure of mice to 18 000 mg/m3 dichloromethane continuously for a weekshowed liver effects such as an increased level of triglycerides and a decrea-sed level of glycogen in the liver. Slight accumulation of fat in the kidneys wasalso seen. (Weinstein 1972, according to IMM 1986). Histopathologicalchanges in the liver were found in guinea pigs at levels above 18 000 mg/m3

and vacuolisation in the liver was found at 38 000 mg/m3 after 6 h of exposure.In addition, lungs showed congestion and haemorrhage. Behavioural changeswere also noted (Balmer et al. 1976, according to IMM 1986).

Cardiac affects such as arrhythmia, tachycardia and hypotension were foundin monkeys, dogs and rabbits exposed for 1-5 min to levels exceeding35 000 mg/m3 (WHO 1996). There are some indication of such effects downto 18 000 mg/m3 (EPA 1985, according to IMM 1986).

Table 5. Acute toxicity by inhalation of dichloromethane. The lowest-observed-effect levels are indicated.

Exposure Duration Species Effect or effected organ Referenceslevel(mg/m3)

38 000 6 h Guinea pig Behavioural effects, Balmer et al. 19761

vacuolisation in liver,haemorrhage in lungs

18 000 6 h Mouse Increased triglyceride and Weinstein et al. 19721

reduced glycogen levels inliver. Fat accumulation inkidneys

18 000 - Not Rat Heart arrhythmia EPA 19851

35 000 stated

18 000 6 h Guinea pig Increase in liver triglycerides Balmer et al. 19761

7100 2.5 h Rat Changes in EEG pattern Mattsson et al. 19882

and somatosensoryevoked response

7100 6 h Mouse Damaged lung Clara cells Foster et al. 1994

3500 6 h Mouse Increased DNA synthesis Foster et al. 1994in lung Clara cells

3500 Not Rat Changed sleeping pattern WHO 1984stated

1) Cited in IMM 1986 2) Cited in WHO 1996

34

5.2. Oral exposure

Histological changes in the liver and the adrenal gland in rats were reportedafter single gavage of dichloromethane (1300 mg/kg). Reversible changes inthe kidney such as decreased volume of urine, hemoglobinuria, increased con-centration of urea in the serum and histological changes were also reportedafter administration of 530 and 800 mg/kg of dichloromethane (Pankow andMarzotko 1987, according to IMM 1990).

CNS effects and evidence of pathological changes in the liver and kidney werefound in Wistar rats receiving 2000 mg/kg (Janssen and Pott 1988, accordingto WHO 1996).

Liver damage was investigated in Sprague-Dawley rats exposed to up to1300 mg/kg given as a single dose by gavage. Serum alanine aminotransferase(ALAT) activity was unaffected, as was liver cytochrome P450 and glutathionecontent. However, increased ornithine decarboxylase activity and DNA damagewere found in the liver (Kitchin and Brown 1989).

5.3. Other exposures

Increased levels of serum aspartate aminotransferase (ASAT) and alanineaminotransferase (ALAT) (enzymes indicating liver damage without histologicalchanges in the liver were found in male rats after intraperitoneal administrationof 0.2 ml 10% dichloromethane in oil (Corsi et al. 1983, according to IMM1990).

Acute CNS-effects (dose-related changes in flash evoked potentials) wereinduced in Long-Evans rats after intraperitoneal administration of 58-460 mg/kgdichloromethane (Herr and Boyes 1997).

Intratracheal administration of dichloromethane in Sprague-Dawley rats showedlethal effects at 350 mg/kg, death occurring in a few seconds. This resultemphasises that aspiration of dichloromethane may present more of a hazardthan oral ingestion (McCarty et al. 1992, according to WHO 1996).

Only slight behavioural effects and macroscopic changes in the liver werefound in Wistar rats receiving 2000 mg/kg by dermal administration under anocclusive dressing for 24 h (Janssen and Pot 1988, according to WHO 1996).

35

5.4. Effects on the heart

Male Wistar rats were used to evaluate the arrhythmogenic properties ofdichloromethane. An ischemia-reperfusion model was chosen becausedichloromethane was shown to produce a transient decrease of coronary bloodflow in dogs. The rats were given dichloromethane by infusion of 3.5 mmol(300 mg) dichloromethane/kg/h. The incidence of atrioventricular block duringthe reperfusion phase was markedly increased by dichloromethane-infusion.Dichloromethane showed a negative dromotropic action (affecting the conductivityof a nerve). According to the authors the results support the view that theinitial coma in dichloromethane poisoning could result not only from theanaesthetic effect, but also from sudden onset of cardiac arrhythmia (Scholzet al. 1991).

Administration by gavage of 3.1, 6.2 and 12 mmol dichloromethane/kg to ratswas found to sensitise the myocardium of rats to arrhythmia development inresponse to catecholamines. Hypertensive adrenaline effects as well as reflexbradycardia after adrenaline and noradrenaline injections were seen indichloromethane-exposed rats. An enhanced negative dromotropic adrenalineaction was also seen (Müller et al. 1991).

Neonatal ventricular myocytes from rats were used to determine if cardiacactions of dichloromethane in vivo correlate with in vitro alterations in Ca2+

dynamics in cardiac myocytes. In cultured myocytes cumulative exposure to0.64-41 mM dichloromethane resulted in a concentration-dependent decreasein the magnitude of intracellular Ca2+ transients with IC10 (inhibitory concentra-tion) and IC50 values of 8.0 and 19 mM, respectively. Total inhibition ofintracellular Ca2+ transients and cessation of beating were observed at 41 mMdichloromethane. Suffusion with dichloromethane for 40 minutes did not causemorphological alterations of the myocytes (Hoffman et al. 1996).

Administration by gavage of 3.1, 6.2 and 12.4 mmol dichloromethane/kg torats resulted in dichloromethane blood concentrations between 1.0 and 1.6 mM,accompanied by a dose dependent decrease in contractile force and heart ratewithout influencing blood pressure and electrocardiograph tracings (Hoffmanet al. 1996).

36

5.5. Conclusions

The acute toxicity of dichloromethane by inhalation and oral administration islow. Toxic effects have been observed in the liver, kidney, lung, heart andCNS. The most common effects are those affecting the liver and CNS.

Since the last report from IMM there are some new studies concerning certainCNS-effects that have not been studied earlier, such as changes in somatosensoryevoked responses and EEG which were seen at levels down to 7100 mg/m3.One of these studies indicated that the effect on the evoked responses probablywas due to dichloromethane itself and not its primary metabolite carbonmonoxide.

Morphological effects such as vacuolisation and necrosis of the Clara cellswere seen in the lungs of mice at 7100 mg/m3. The effect was cytochromeP450-dependent. In the same study increased levels of DNA synthesis inmouse lung was seen at an exposure level of 3500 mg/m3.

One study indicated that the initial coma seen in dichloromethane poisoningcould result from the onset of cardiac arrhythmia as well as from an anaestheticeffect. Dichloromethane exposure was also shown to affect the conductivity ofthe nerves. Another study concluded that dichloromethane could sensitise theheart of rats to arrhythmia development in response to catecholamines.

37

6. Effects after prolonged exposure in animals

6.1. Inhalation

6.1.1. Central nervous systemInhalation exposure of male Sprague-Dawley rats to dichloromethane indicatedbrain effects down to 250 mg/m3 during 6 h/day for 3 days (Fuxe et al. 1984according to IMM 1990). The brain effects were changed levels and turnoverof dopamine and noradrenaline in the forebrain and the hypothalamus,respectively. There was also an increased internal secretion of pituitary glandhormones.

Groups of 5 male and 5 female Fischer 344 rats and B6C3F1 mice wereexposed to dichloromethane at concentrations of 5700, 11 000, 23 000,46 000 or 56 000 mg/m3, 6 h/day for 19 days. Ataxia and hyperactivity wereseen in all the rats exposed to 23 000 mg/m3 or more. Dyspnoea and anaes-thesia were observed at 46 000 mg/m3 or more. Some deaths were alsoobserved at these concentrations. The mice showed a dose-related hyperactivity,but no exposure related pathological findings were observed. Some deathsoccurred in mice exposed to 46 000 mg/m3 or more (NTP 1985, accordingto IMM 1986; WHO 1996).

Exposure of dichloromethane at 35 000 mg/m3, 7 h/day, 5 days/week for6 months resulted in central nervous system (CNS) effects in dogs, monkeys,rats, rabbits and guinea pigs. The initial CNS effect was hyperactivity followedby hypoactivity. No such CNS effects were found at 18 000 mg/m3 (Heppeland Neal 1944, according to IMM 1986).

Mongolian gerbils exposed to 740 mg/m3 for 3 weeks showed brain effectssuch as a statistically significant decrease of glutamate, phosphorus ethanolamineand GABA (gamma- aminobutyric acid) in the cortex of the encephalon. Therewere also significantly increased levels of glutamine and GABA in the cerebellum(Briving et al. 1986, according to IMM 1990).

Another exposure of Mongolian gerbils showed effects on the brain, such asan increased level of two astrogliocyte markers (S-100 and GFA-protein)indicating astroglios (abnormal growth of the astrocytes) in the encephalon. Allthe brain effects occurred after exposure to 1200 mg/m3 for 10 weeks, followedby 4 months of recovery (Rosengren et al. 1986, according to IMM 1990).

38

Male and female Fischer 344 rats were exposed to 180, 7100 or 7100 mg/m3

6 h/day, 5 days/week for 13 weeks. No treatment-related alterations in sensoryevoked potentials or neuropathology were observed (Mattsson et al. 1990).

6.1.2. Liver and kidneysRats (sex and strain unspecified) were continuously exposed to either 90 or350 mg/m3 for 100 days. In both exposure groups fat staining of the liver waspositive and liver cytoplasmic vacuolisation was noted. Rat kidneys from bothexposure groups showed evidence of nonspecific tubular degenerative andregenerative changes. There were no effects on rat organ weights. Mice exposedat the same conditions showed positive fat stains in the liver at the higherexposure level (350 mg/m3). A decrease in the microsomal cytochrome P-450content was found in the liver of mice exposed to 350 mg/m3. No exposurerelated effects were noted in dogs and monkeys after continuous exposure for100 days to either 90 or 350 mg/m3 dichloromethane. Earlier studies hadshown significant toxic lesions, primarily affecting the liver and the kidneys, inall four species after continuous exposure to 3500 mg/m3 dichloromethane for100 days (Haun et al. 1972).

NMRI mice exposed during 30 days showed a significant increase in the levelof buturylcholinesterase in the blood of the males but not in the females at260 mg/m3 dichloromethane and above. According to the authors this effectwas a sign of liver damage. For both sexes there was statistically significantelevated liver weight, starting at 260 mg/m3 for males and 530 mg/m3 forfemales. Both sexes showed an accumulation of fat in the liver starting at260 mg/m3, but the effect was stronger in the females. All the observed effectsseemed to be reversible (Kjellstrand et al. 1986, according to IMM 1990).

Sprague-Dawley rats were exposed to dichloromethane, 3500 mg/m3 2 h/dayfor 15 days. The rats showed decreased body weight and increased hepaticlipid peroxidation after inhalation (Ito et al. 1990, according to WHO 1996).Similar hepatic effects (hypertrophic hepatocytes and increased lipid peroxidation)were reported for male Wistar rats exposed to 3500 mg/m3 2 h/day for20 days (Takashita et al. 1991, according to WHO 1996).

Repeated exposure of guinea pigs for 5 days resulted in a weak increase oftriglyceride concentration in the liver down to 1800 mg/m3 (Balmer et al.1976, according to IMM 1986).

39

6.1.3. LungsExposure of dichloromethane at 18 000 mg/m3, 7 h/day, 5 days/week for6 months showed no organ lesions for dogs, monkeys, rats, rabbits and guineapigs, except for fatty degeneration of the liver for 3 of 14 guinea pigs. Exposureof dichloromethane at 35 000 mg/m3, 4-6 h/day 5 days/week for 8 weeksshowed pulmonary oedema for rabbit, guinea pig and rat (Heppel and Neal1944, according to IMM 1986).

Male B6C3F1 mice were exposed to 14 000 mg/m3 dichloromethane for6 h/day, 5 days/week for up to 13 weeks. Groups of mice were killed atintervals from day 2 to week 13. The time points of examination were selectedso that the effect of dichloromethane exposure on the lungs could be investigatedafter: (a) a single 6 h exposure (day 2), (b) five daily exposures per week, withthe animals being killed immediately after the fifth exposure (Days 5, 40 and89), (c) the 2 days of no exposure, with the animals being killed immediatelyprior to re-exposure (Days 8, 43, 92), and (d) re-exposure following the2 day rest from exposure with the animals being killed 24 h after the initialexposure or re-exposure (days 9, 44 and 93). At each time point, five animalswere taken for pathology, four animals were taken for biochemistry of thelung, and six animals were taken for the isolation of Clara cells. Acute Claracell damage was seen after single exposure to dichloromethane, but the damageresolved after five consecutive days of exposure. After a 2 day interval theClara cell lesion reappeared on subsequent re-exposure to dichloromethane.The severity of the lesions decreased over the duration of the study. Thelesions in the Clara cells correlated well with the activity of cytochrome P450monooxygenase in the whole lung and in the Clara cells and no lesions appearedwhen there was marked decrease in cytochrome P450 activity. The authorssuggested that with time the lung had developed tolerance to dichloromethanepossibly due to the inactivation of a cytochrome P450 isoenzyme. The GSTmetabolism of dichloromethane in the cytosol remained unaltered. Significantincreases of nonprotein sulfhydryl groups in the lungs was observed in all theexposed animals. There was also a transient increase in the number of bronchiolarcells in S-phase when examined at days 5, 8 and 9 after the first exposure todichloromethane (Foster et al. 1992).

6.1.4. Other effectsAn older and briefly reported 90 days study from the mid 70s was cited byLeuschner et al. (1984, according to IMM 1990). In this study, 20 Sprague-Dawley rats and 3 Beagle dogs per sex were exposed. The dogs showed no

40

Table 6. Toxicity after prolonged exposure to dichloromethane by inha-lation. The lowest-observed-effect levels are indicated.

Exposure Duration Species Effect or effected organ Referenceslevel(mg/m3)

46 000 6 h/d; 19 d Mouse, rat Dyspnoea and NTP 19851

anaesthesia, death

35 000 6 h/d; Rat Pulmonary oedema, Heppel and Neal1

5 d/wk; 8 wk CNS-effects

23 000 6 h/d; 19 d Mouse, rat Ataxia and hyperactivity NTP 19851

14 000 6 h/d; Mouse Lesions of lung Clara Foster et al. 19925 d/wk; cells correlating with13 wk cytochrome P450-activity,

increased nonprotein sulfhydryl

3500 2 h/d; 15 d Rat Decreased body weight, Ito et al. 19902

increased lipid peroxidationin liver

3500 2 h/d; 20 d Rat Hypertrophic hepatocytes, Takashita et al. 19912

increased lipid peroxidationin liver

3500 24 h/d; 100 d Mouse, Significant lesions, Haun et al. 1972rat, dog, primarily in liver and kidneymonkey

1800 6 h/d; 5 d Guinea pig Increase in liver Balmer et al. 19761

triglycerides

1200 10 wk; 4 mo Gerbil Indication of astroglios Rosengren et al. 19863

recovery in the brain

740 3 wk Gerbil Changed levels of Briving et al. 19863

glutamate, phospho-etanolamine, glutamine,and GABA in brain

350 24 h/d; 100 d Mouse Decrease in liver P450 Haun et al. 1972content. Positive fatstains in liver

260 24 h/d; 30 d Mouse Increased liver weight, Kjellstrand etal. 19863

fat accumulation inliver, increase inbuturylcholinesterase

250 6 h; 3 d Rat Changed levels and Fuxe et al. 19843

turnover of dopamineand noradrenaline in brain

90 24 h/d; 100 d Rat Renal tubular degenerative Haun et al. 1972and regenerative changes,vacuolisation and positivefat stains in liver

1) Cited in IMM 1986 2) Cited in WHO 1996 3) Cited in IMM 1990

41

effects after exposure to 18 000 mg/m3. The only effect seen in the rats afterexposure to 35 000 mg/m3 was a slight erythema of the conjunctiva in theeyes. The animals seem to have been carefully examined (clinical observations,haematology, clinical and chemical examinations and pathological studies).

6.2. Oral exposure

Rats receiving dichloromethane in the drinking water at a concentration of130 mg/l for 13 weeks did not show any effect on behaviour, body weight,haematology, urine analysis, blood glucose level, plasma free fatty acids, or theoestrous cycle (Bornman and Loeser 1967, according to IMM 1986).

Oral exposure for 90 days of Fischer 344 rats and B6C3F1 mice withdichloromethane administrated via drinking water showed histopathologicalchanges in the liver in both species. Groups of 20 male and 20 female ratswere given 210/170, 610/420, or 1500/1200 (males/females) mg/kg per day.Hepatocellular changes were observed for the high dose rats of both sexesand some mid dose females following treatment for 3 months. Changes observedwere: central lobular necrosis, granulomatous foci, ceroid or lipofuscinaccumulation and cytoplasmatic eosinophilic bodies. A dose-dependent increasein hepatocyte vacuolisation was also observed (Kirschman et al. 1986, accordingto IMM 1990).

Groups of 20 male and 20 female mice were given 230, 590, or 1900/2030(males/females) mg/kg per day for 90 days. Histopathological changes wereobserved in the liver, such as centrilobular fatty changes in the mid-dosefemales and the high-dose males. The authors estimated the no-observed-effect level (NOEL) value to 200 mg/kg/day for rats and to 230-590 mg/kg/dayfor mice (Kirschman et al. 1986, according to IMM 1990).

6.3. Conclusions

Prolonged exposure to dichloromethane primarily affects the liver and theCNS. Effects on the kidney have also been observed. In most cases, significanteffects occur only at inhalation exposures at 3500 mg/m3 and higher. However,slight effects on the liver and kidney were observed in one study where ratswere exposed continuously for 100 days to as low a concentration as 90 mg/m3.

42

Thus, continuous inhalation exposure seems to be more harmful than intermit-tent exposure.

The nonciliated bronchiolar Clara cells in the lungs of mice are initially damagedby inhalation of 14 000 mg/m3, but the damage appears to be resolved after5 consecutive daily exposures. The lesions correlated with the activity ofcytochrome P450 monooxygenase.

43

7. Chronic toxicity and carcinogenicity

7.1. Inhalation

Inhalation studies have been performed with rats, mice and hamsters. Theresults are discussed below, and a summary is presented in Table 7.

7.1.1. Rat studiesGroups of 90 male and female Sprague-Dawley rats were exposed todichloromethane at 180, 700 or 1800 mg/m3, 6 h/day, 5 days/week for 2 years.Two other groups with 30 females in each were also included in the study andone of these groups was exposed to 1800 mg/m3 of dichloromethane duringthe first 12 months of the study. The second group was exposed todichloromethane during the last 12 months. A significant increase in the totalincidence of mammary tumours was seen in the females, but this increase wasonly weakly correlated to the exposure. (Dow Chemical Company 1982,according to IMM 1986).

No dose-related effects were seen on body weight or mortality. There was asignificant increase in local changes such as vacuolisation and multinucleatedliver cells for the females in the high dose group. Males who survived 24 monthshad a significant increase of local changes in the liver at the two highest doselevels. (Dow Chemical Company 1982, according to IMM 1986).

Groups of 95 male and 95 female Sprague-Dawley rats were exposed byinhalation to 0, 1800, 5300 or 12 000 mg/m3 of dichloromethane 6 h/day,5 days per week for two years (Burek et al. 1984). The only reported increasesin tumour incidence occurred in benign mammary gland tumours for bothsexes, and tumours in the salivary gland in males. The increase of animals withmammary tumours was not statistically significant but the number of tumoursper animal with tumours showed an exposure-dependent increase which indicatesa substance-related effect. The increase in incidence of tumours in the salivaryglands was statistically significant only in males at the two highest doses. Itshould be noted that this increase was only evident when the tumours, whichwere all of mesenchymal origin, were grouped together for statistical analysis.Both sexes had a viral disease in the salivary glands during the first two monthsof the study.

44

The female rats exposed to 12 000 mg/m3 had a significantly increased mor-tality from the 18th through the 24th month which appeared to be exposure-related. Pathological effects in the liver, such as increased hepatocellularvacuolisation and fatty change, were seen in both sexes at all dose levels andin a dose-related fashion. The females showed a significant increase inmultinucleated hepatocytes and areas with changed liver cells. In the males, adose-dependent increase in necrosis in the liver was seen which was significantfor the two highest dose levels (Burek et al. 1984).

Groups of 90 male and 108 Sprague-Dawley female rats were exposed to 0,180, 710 or 1800 mg/m3, 6 h/day, 5 days/week for 20 and 24 months,respectively (Nitschke et al. 1988). This study was a follow-up of the studyby Burek et al. (1984) with lower exposure levels. The incidence of tumoursin the mammary glands was not increased but the number of tumours pertumour-bearing high dose females was increased. There was no increasedincidence of tumours in the salivary glands.

The mortality rates for the different groups of female and male rats exposedto dichloromethane were comparable to control values. Due to poor survivalin all groups of male rats, exposure of male rats was terminated after 20 months.No substance-related effects were seen at the two lowest dose levels. Thehigh dose males had a statistically significant increased incidence of hepatocellularvacuolisation and multinucleated hepatocytes.

Groups of 50 male and 50 female Fischer 344 rats were exposed by inhalationto 0, 3500, 7100 or 14 000 mg/m3, 6 h/day, 5 days/week for 102 weeks(NTP 1986; Mennear et al. 1988). There was a statistically significant increasein the incidence of benign tumours in the mammary glands in female rats anda significant positive trend in males (adenomas, fibromas, fibroadenomas, andsarcomas).

Non-carcinogenic treatment-related effects were seen, affecting the incidenceof renal tubular cell degeneration and splenic fibrosis for both sexes. For malesonly, nasal cavity squamous metaplasia was seen. Histopathological changes inthe liver were observed in both sexes. The survival of all the male rat groupswas lower than the historical controls for inhalation studies at the laboratory.Survival at 86 weeks was: control 36/50, low dose 39/50, mid dose 37/50and high dose 33/50. There was a trend toward decreased survival in exposedfemales (30/50, 22/50, 22/50, 15/50). The survival of animal exposed to14 000 mg/m3 was significantly reduced. Survival at 95 weeks was 36/50,33/50, 27/50 and 27/50, respectively.

45

Table 7. Carcinogenicity studies using the inhalation routes (adapted fromWHO 1996). Unless otherwise stated exposures are by inhalation 6 h/day,5 days/week.

Species, Exposure Exposure protocol Observations Referencesstrain levels

(mg/m3)

Rat, 0, 180, 90 males/group, 20 mo; Significant increase in cell Dow ChemicalSprague- 700, 1800 90 females/group, 24 mo changes in the liver in Company 1982,Dawley high dose females and according to

mid- and high dose males. IMM 1986

Rat, 0, 1800 95 animals/sex/group, 2 y Increase in various types Burek et al. 1984Sprague- 5300, of sarcomas in the salivaryDawley 12 000 gland in mid- and high-dose

males (1/92, 0/95, 5/95, 11/97).Slight dose-related increase intotal number of benign mammarytumours in males (8/95, 6/95,11/95, 17/97). Dose -relatedincrease in total number of benignmammary tumours in females(165/96, 218/95, 245/95, 287/97).Increased mortality in high-dose females.

Rat, 0, 180, 90 males/group, 20 mo; Increase in benign Nitschke et al. 1988Sprague- 710, 1800 198 females/group, mammary tumours inDawley 24 mo females at 1800 mg/m3.

No increases in benignmammary tumours in malesor in females at 180 or 690 mg/m3.No increase in any malignanttumours. No increased mortality.

Rat, 0, 3500, 50 animals/sex/group, Dose-dependent increase NTP 1986;Fischer 7100, 102 wk in benign mammary Mennear et al. 1988344/N 14 000 tumours, male: 0/50, 0/50,

2/50, 5/50, female, 5/50, 11/50,13/50, 23/50. Increased mortalityin exposed males and females.

Rat, 0, 350 60 females in control No effect on percentage Maltoni et al. 1988Sprague- group, 54 in exposed of animals bearing benignDawley group; exposure during and/or malignant tumours,

and after pregnancy; in maternal or offspring animals.4 h/day for 7 weeks, No statistically significant increasethen 7 h/day for 97 in total malignant tumours.weeks;

46

Species, Exposure Exposure protocol Observations Referencesstrain levels

(mg/m3)

Rat, 0, 210 149-158 offspring/sex As above Maltoni et al. 1988Sprague- in control group, 60-70Dawley in exposed groups;

offspring exposed from12th day in utero; either4 h/day for 15 wk or7 h/day for 104 wk

Mouse, 0, 7100, 50 /sex/group; 102 wk Dose-dependent increases in: NTP 1986;B6C3F1 14 000 - alveolar/bronchiolar Mennear et al. 1988

adenomas (males: 3/50,19/50, 24/50; females: 2/50,23/48, 28/48)- alveolar/bronchiolar carcinomas(males: 2/50, 10/50, 28/50;females: 1/50, 13/48, 29/48)- hepatocellular adenomas andcarcinomas combined (males:22/50, 24/49, 33/49; females:3/50, 16/48, 40/48).Increased mortality during thesecond year in males at all dosesand in females at the high dose.

Mouse, 0, 7100 364 exposed and 364 Mice exposed for more Kari et al. 1993B6C3F1 control females; various than one year showed an

periods up to 104 wk; excess of lung and liveranimals killed at end of tumoursexposure

Mouse, 0, 7100 68 exposed and 68 All exposed groups had Kari et al. 1993B6C3F1 control females; 26, an excess of lung and

52, 78 or 104 wk; liver tumours animals killed at 104 wk

Hamster, 0, 1800, 95 animals/sex/group; No significant increase in Burek et al. 1984Syrian 5300, 2 years tumours. Low survival atgolden 12 000 24 months in all female

dose groups

47

A group of 54 pregnant rats (Sprague-Dawley) was exposed to 350 mg/m3

4 h/day, 5 days/week for 7 weeks and subsequently for 7 h/ day, 5 days/weekfor further 97 weeks (Maltoni et al. 1988). An additional group of 60 ratsserved as controls. Further, groups of 60-70 males or females were exposedto 210 mg/m3 from the 12th day in utero for a total of either 15 or 104 weeks.No effect on percentage of animals bearing tumours, in maternal or offspringanimals was observed. There was a non-significant increase in total malignanttumours. No excess in mortality was seen. There was an absence of relevantdata on the design of the experiment.

7.1.2. Mouse studiesGroups of 50 male and 50 female B6C3F1 mice were exposed by inhalationto 0, 7100 or 14 000 mg/m3, 6 h/day, 5 days/week for 102 weeks (NTP1986; Mennear et al. 1988).

A statistically significant increase in the incidence of tumours in the lungs (adeno-mas and carcinomas) and liver (adenomas and carcinomas combined) wasseen in both sexes.

Histopathological changes in the liver (cell degeneration), testicular atrophy inmales and uterine and ovarian atrophy in females were demonstrated. Theeffects in the gonads may have been secondary toxicity responses to neoplasia.Survival of male and female mice exposed to dichloromethane was reducedduring the second year of the study. Both the dose levels reduced the survivalof the males and the high dose level reduced survival among the females(males: control 39/50, low dose 24/50 and high dose 11/50; females 25/50,25/50 and 8/50). In males, survival at 95 weeks was 82%, 60% and 50%.Survival among the females at week 86 was 72%, 82% and 62%. There wasno increase in hyperplasia in the lungs.

B6C3F1 mice were used to study the time-dependency of exposure todichloromethane leading to tumour formation. Thus, 364 female mice wereexposed to either air or 7100 mg/m3 dichloromethane for 6 h/day, 5 days/week for 104 weeks. At nine different time intervals, 10 exposed and 5 controlmice were randomly selected and examined. Mice exposed for 104 weeks(2 years) showed an increased incidence of adenomas and carcinomas in thelungs (47% versus 7%). There was also an increased incidence of hepatocellularadenomas and carcinomas (67% versus 27%). Mice exposed for less than ayear showed no effects on the lungs and only minimal effect on the liver (5%

48

versus 0%). Mice exposed for longer than one year showed a progressiveincrease in the incidence of lung adenomas and carcinomas. The incidence ofliver tumours in mice exposed for longer than one year showed a time-dependentincrease in the total number of liver tumours per animal. (Kari et al. 1993).

In the second part of the examination a stop-exposure study was conductedwith 8 groups of 68 female B6C3F1 mice per group. The animals were exposedto 7100 mg/ m3, 6 h/day, 5 days/week for either 0, 26, 52, 78 or 104 weeks.The dichloromethane exposure was either preceded or followed by exposureto clean air, in order to study the influence of a latency period. The totalexposure time (dichloromethane + clean air) was 104 weeks (2 years). Alleight treatment groups are shown in Table 8. The incidence of both lung andliver tumours in mice exposed to dichloromethane during the first period(52 weeks) increased with the duration of exposure. In mice exposed todichloromethane during the end of the study, significant increases in the incidenceof lung and liver tumours were only observed following 78 weeks of exposureto dichloromethane. Totally, the study showed an 8-fold increase in the incidenceof animals having a lung adenoma or carcinoma (63 versus 7.5%) and a 13-fold increase in the total number of pulmonary adenomas and carcinomas peranimal (0.97 versus 0.075). The 104 week exposure also caused a 2.5-foldincrease in the incidence of mice having liver tumours (69 versus 27%) and a3-fold increase in the total number of hepatic adenomas and carcinomas peranimal (1.34 versus 0.46). Dichloromethane exposure hastened the firstappearance of lung tumours by one year compared to that observed in controlanimals, whereas dichloromethane-induced and spontaneous liver tumoursoccurred simultaneously. A shorter exposure duration was sufficient to attainmaximal numbers of lung tumours than that needed for a maximal liver tumourburden. Lung tumour multiplicity was substantially increased by having additionaltime after cessation of the chemical treatment. This contrasts the findings inliver, where additional post-exposure latency time did not affect tumour multi-plicity. The results suggest that dichloromethane is a more potent lung carci-nogen than liver carcinogen in this mice strain.

There was an increased rate of hyperplasia in the liver in mice exposed todichloromethane for 78 weeks, followed by air exposure, or mice exposed todichloromethane for 104 weeks. The controls had no signs of hyperplasia upto 26 weeks and only a low incidence was seen during the remaining study.The incidence of lung alveolar hyperplasia in dichloromethane-exposed animalswas very low, even in tumour-bearing animals. The hyperplasias were not seen

49

until at least 13 weeks after the appearance of adenomas and carcinomas(Kari et al. 1993).

Table 8. Incidence of tumours in female mice after different exposureregimens of 7100 mg/m3 of dichloromethane (Kari et al. 1993).

Exposure Percent (%) tumour-bearing animals (adenomas or carcinomas)

Phase 1 Phase 2 Lungs Liver

Air 104 weeks - 7.5 27

Dichloromethane 26 weeks Air 78 weeks 31* 40

Dichloromethane 52 weeks Air 52 weeks 63* 44*

Dichloromethane 78 weeks Air 26 weeks 56* 62*

Dichloromethane 104 weeks - 63* 69*

Air 26 weeks Dichloromethane 78 weeks 19* 48*

Air 52 weeks Dichloromethane 52 weeks 15 31

Air 78 weeks Dichloromethane 26 weeks 4 34

* p< 0.05

In the chronic study described above (Kari et al. 1993), female mice wereexposed to 7100 mg/m3 dichloromethane for up to two years. Groups of10 mice were killed after 13, 26, 52 and 78 weeks of exposure todichloromethane. There was no sustained increase in DNA synthesis in theliver, but a significant decrease was seen after 13 weeks (Foley et al. 1993).

In prechronic studies, female mice (B6C3F1) were exposed to dichloromethaneby inhalation to 3500, 7100, 14 000 or 28 000 mg/m3, 6h/day, 5 days/weekfor up to 4 weeks, followed by a one or two weeks recovery period. Thestudy was designed to investigate if there were dichloromethane-inducedalterations in hepatocellular proliferation that might explain the hepatocarcinogenicresponse. Mice exposed to 7100 mg/m3 and above had sustained increasedliver weight commencing after 1 week of exposure and returning to normalafter the recovery period. The increased liver weight was attributed to

50

hepatocellular hypertrophy secondary to intracellular glycogen accumulation.There was no sustained increase in DNA synthesis in the liver (Foley et al.1993).

The same group of researchers has also examined replicative DNA synthesisin the lungs of a part of the animals in the study by Foley et al (1993). Femalemice were exposed to dichloromethane at 0, 7100 and 28 000 mg/m3 for upto 4 weeks. The labelling index of bronchiolar epithelium was decreasedcompared to controls when measured after 2, 3 and 4 weeks exposure. Micewere also exposed to dichloromethane at 0 and 7100 mg/m3, 6h/day, 5 days/week for up to 78 weeks. This study also failed to detect any increase in thenumber of cells in S-phase, when measured after 13 and 26 weeks of exposure.No obvious treatment-related morphological changes in the lungs were seenup to 26 weeks. (Kanno et al. 1993; see also Kari et al. 1993).

As summarised by Maronpot (1995), these studies have shown that the inductionof liver and lung tumours in B6C3F1 mice is not associated with enhanced cellproliferation, hyperplasia or cytotoxicity.

7.1.3. Hamster studiesGroups of 95 male and 95 female Syrian golden hamsters were exposed byinhalation to 0, 1800, 5300 or 12 000 mg/m3 of dichloromethane, 6 h/day,5 days per week for two years (Burek et al. 1984). No significant increasein tumour incidence resulted from the exposure.

The overall 24 month survival was low in all female groups. In the liver andthe adrenal gland an increased incidence of haemosiderosis was seen.

7.2. Oral exposure

7.2.1. Rat studiesGroups of 85 male and 85 female Fischer 344 rats were administrateddichloromethane by the drinking-water at concentrations equivalent to 0, 5,50, 125 and 250 mg/kg body weight per day for 104 weeks. (Serota et al.1986, according to WHO 1996). Although there was some increase in theincidence of neoplastic nodules and carcinomas in the liver of female mice(IMM 1990), the study showed no increase compared to controls accordingto WHO (1996). Small changes in body weight were seen in rats receiving

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either 125 or 250 mg/kg. Dose related increases were noted in mean haematocrit,haemoglobin levels and number of red blood cells at the three highest doses.Decreases in liver enzymes were found in both sexes at the highest dose.Treatment-related histopathological changes were seen in the liver at the high-est dose.

Groups of 50 male and 50 female Sprague-Dawley rats were given 0, 100,or 500 mg/kg of dichloromethane by gavage in olive oil, 4-5 days/week for64 weeks. Because of a high mortality of the high-dose rats the study wasinterrupted after 64 weeks. The excess (not stated) in mortality was morepronounced in males than in females. There was no statistically significantincreases in any tumours. A slightly higher incidence of adenocarcinomas ofthe mammary glands was observed in females receiving 500 mg/kg (Maltoniet al. 1988).

7.2.2. Mice studies

Groups of male and female B6C3F1 mice received dichloromethane in thedrinking water for 104 weeks at levels of 0, 60, 130, 190 or 250 mg/kg perday. A weak histopathological change (fatty infiltration in liver) was observedfor the two highest dose levels. The study showed no tumour effects (Serotaet al. 1986, according to WHO 1996).

Groups of 50 male and 50 female Swiss mice received either 100 or 500 mg/kgdichloromethane in olive oil by gavage at 4-5 days/week for 64 weeks (Mal-toni et al. 1988, according to IMM 1990; WHO 1996). There was a dose-related increase in lung tumours in male mice.

An excess in mortality was observed in males and females exposed todichloromethane at both the dose levels. The increase in the mortality rate wasmore pronounced in males. The increase in mortality started after 36 weeksand attributed for the interruption of the study after 64 weeks.

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Table 9. Carcinogenicity studies by the oral route (according to WHO1996).

Species, strain Doses (mg/kg/d) Exposure protocol ObservationsReferences

Rat, 0, 5, 50, Drinking water ad No increase in Serota et al.Fischer 130, 250 libitum for 104 wk, incidence of 19861

344 85 animals/sex/dose, neoplasms.scheduled kills: Survival and other5 at 26 wk, 10 at findings not affected.52 wk, 20 at 78 Evidence of liver damagewk, also additional at doses abovegroups of controls 50 mg/kg per dayand 250 mg/kgwhich received DCMfor 78 wk only

Rat, 0, 100, Gavage in olive oil No effect on total Maltoni et al.Sprague- 500 for 64 wk, tumour incidence. 19881

Dawley 50 animals/sex/dose, Higher incidence,additional control not statisticallygroup of 20 males significant, ofand 26 females malignant mammary

tumours in high dosefemales. Survival decreasedin high dose males and females.

Mouse, 0, 60, 130, Drinking water ad No increased Serota et al.B6C3F1 190, 250 libitum for 104 wk, incidence of 19861

50-200 males/females neoplasm,per dose evidence of slight

liver damage at 250 mg/kg

Mouse, 0, 100, Gavage in olive oil Decrease in body Maltoni et al.Swiss 500 for 64 wk; 50-60 weight in exposed 19881

animals/sex/dose males and femalesafter 36-40 weeks.Dose-related increasein pulmonary tumoursin males (only significantwhen considering the mortality rate).No other treatment-related increase inbenign or malignant tumours

1) Cited by WHO 1996

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7.3. Conclusions

Inhalation of high concentrations of dichloromethane (7100 and 14 000 mg/m3)induces lung and liver tumours in B6C3F1 mice and benign mammary tumoursin rats, but there were no tumours in a hamster study. Oral exposure seemsto be less efficient than inhalation in inducing tumours.

IARC (1986) concluded that there is sufficient evidence that dichloromethaneis carcinogenic for experimental animals and dichloromethane is classified aspossibly carcinogenic for humans (group 2B). The National ChemicalsInspectorate has classified dichloromethane as a low potency carcinogenicsubstance (KemI 1995).

The study by Kari et al. (1993) showed clearly that the lungs are moresensitive to dichloromethane exposure compared to the liver in mice. A shorterexposure time was needed to obtain maximum number of tumours in the lungthan in the liver. The data also indicate that early exposure to dichloromethane(first 26 weeks) is more important than exposure later in life with respect tothe development of lung tumours. The induction of liver and lung tumours werenot associated with increased cell proliferation, hyperplasia or cell toxicity.

In the NTP inhalation study with mice (Mennear et al. 1988) there werehistopathological changes in the liver after 2 years of exposure. Liver effects,such as vacuolisation, multinucleated cells and necrosis were also seen in therat studies. Liver vacuolisation was observed in 2-year inhalation studies at700 mg/m3 and higher concentrations for male rats (1700 mg/m3 for females).Other toxic effects seen in rats were those affecting the kidneys, the spleenand the adrenal glands. Increased mortality was seen in several studies.

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8. Genotoxicity

8.1. In vitro

8.1.1. Mutations in bacteria and yeastDichloromethane is mutagenic in Salmonella (strains TA98, TA100, andTA1535) and in different E. coli strains when tested using a vapour phaseexposure, but not in the standard plate incorporation assay or preincubationassay. Metabolic activation by induced rat liver S9 fraction slightly increasedthe mutagenicity in most studies (WHO 1996). When the S9 fraction wasdivided into a microsomal and a cytosolic fraction, the cytosolic fraction wasmore active than the microsomal fraction (tested in the Salmonella strainTA100) (Jongen et al. 1982, Green 1983).

It has been suggested that the bacterial mutagenicity of dichloromethane ismediated by GSH conjugation, catalysed by cytosolic glutathione S-transferase(GST). However, an addition of either GST, GSH or a GSH scavenger onlyslightly attenuated the mutagenic response in Salmonella TA100, probablydue to the bacterial endogenous GSH pool (Jongen et al. 1982; Green 1983).In a comparative study using Salmonella TA100 and the glutathione-deficientTA100 strain NG54;gsh (containing 25% of the normal level of GSH), thelatter strain was slightly less responsive. Various rat liver activation systemsdid not appreciably enhance the mutagenicity in either of the Salmonella strainsor in E. coli WP2. The addition of exogenous GSH had only a small effecton the mutagenic response, although more consistent in the glutathione-deficientstrain (Dillon et al. 1992). Another glutathione-deficient Salmonella TA100strain (NG-11, 10% of the normal GSH content) showed a 2-fold reductionin the number of mutants, which was abolished if glutathione was added to thetest plates (Graves et al. 1994a).

Thier et al. (1993) and Oda et al. (1996) described the development of twoSalmonella TA 1535 strains that contain a plasmid expressing rat theta-classGST with a much higher GST theta activity than the original strain. Theseauthors showed that dichloromethane induced mutations (his+ revertants andumuC gene expression, respectively) more strongly in these strains than in theoriginal strain. These results were confirmed by DeMarini et al. (1997). Theyalso demonstrated that all the induced mutations were GC → AT transitionsin the presence of the rat GSTT1-1 gene, but only 15% of induced mutationswere of this kind in strain TA100 (with no transgenic rat GST gene). According

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to the authors, the findings suggest that dichloromethane may be mutagenic bydifferent pathways, although the GST-mediated pathway may be more significant.Formaldehyde, a metabolite formed by the GSH pathway, is not mutagenic inSalmonella, but formaldehyde as well as dichloromethane activated by mouseliver S9 (but not rat liver S9) was mutagenic in E. coli K12 wild-type cellsand enhanced the cell-killing effect in DNA repair-deficient strains, suggestinga mutagenic role for metabolically derived formaldehyde in E. coli (Graves etal. 1994a).

Dichloromethane has caused gene conversions, mitotic recombinations andreversions at high toxic doses in the yeast Saccaromyces cerevisiae strainD7, but not in the strains D4 and D3. Mitotic segregation was induced in thefungus Aspergillus nidulans (WHO 1996).

8.1.2. DNA binding, DNA damageDichloromethane has not been shown to bind covalently to DNA in experimentswhere DNA was incubated with 14C-labelled dichloromethane in vitro withoutor together with hepatic microsomes. In tests for unscheduled DNA synthesisin cultured cells, negative results were reported, except for one study thatreported a marginally positive result in a primary rat hepatocyte assay (WHO1996).

Dichloromethane has been shown to induce DNA single strand breaks (detectedby alkaline elution) in freshly isolated hepatocytes from mice and rats, incubatedin vitro with dichloromethane for 2 h, although the concentration needed wasmuch higher for rat hepatocytes (30 mM) than for mouse hepatocytes (0.4 mM)(Graves et al. 1994b). When the hepatocytes had been treated with a GSHdepleting agent (buthionine sulfoximine), less DNA damage occurred, whichindicates that the DNA-damaging effect depends on metabolism via the GSTpathway. In parallel experiments, formaldehyde was shown to cause DNAsingle strand breaks in mouse hepatocytes at 0.25 mM and higher concentrations.However, the authors considered it unlikely that such a high concentration offormaldehyde might be formed metabolically, and formaldehyde would thus beof minor importance for the DNA damage.

This conclusion was strengthened by experiments with cell cultures (Chinesehamster ovary cells). It was found that dichloromethane formed both DNAsingle-strand breaks and DNA-protein cross-links, but only after supplementingthe incubation medium with mouse liver S9 fraction. The DNA-protein cross-

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linking effect, but not the DNA single strand breaks, was completely abolishedby an addition of semicarbazide, which binds to aldehydes. Semicarbazideinhibited both types of effects when induced by formaldehyde alone. Thus, theauthor’s conclusion was that GSH-mediated metabolism of dichloromethanecauses DNA single-strand breaks via S-chloromethylglutathione and DNA-protein crosslinks via formaldehyde (Graves et al. 1994b). It was later shownthat only addition of the cytosolic fraction, but not the microsomal fraction ofmouse liver S9 fraction caused DNA single strand breaks in the cells. Further,when GSH was removed from the cytosol the DNA damage was reduced, butit was restored upon addition of exogenous GSH to the incubation medium(Graves et al. 1995).

DNA single strand breaks were not induced by dichloromethane in hamsterhepatocytes in vitro at concentrations from 5 to 90 mM, nor in eight individualsamples of normal human hepatocytes exposed at similar concentrations (Gra-ves et al. 1995).

Bronchiolar Clara cells were isolated from the lungs of B6C3F1 mice andincubated with dichloromethane at 5-60 mM for 2 h. This treatment resultedin a dose-dependent increase in DNA single strand breaks. The DNA damagewas reduced when the glutathione depletor buthionine sulphoximine was addedto the incubation medium (Graves et al. 1995).

In comparative studies on the ability of hepatocytes from different species tometabolise dichloromethane to formaldehyde it was found that only mousehepatocytes, but not hepatocytes from rats, hamsters or humans, formeddetectable amounts of DNA-protein cross-links. RNA-formaldehyde adductswere formed in all rodent species and from humans with functional GSTT1 andGSTM1 genes, but not in human cells lacking these genes. The yield of theseadducts in hepatocytes from different species was in the order: mouse > rat> human > hamster (Casanova et al. 1997).

8.1.3. Mutations in mammalian cellsAccording to WHO (1996) dichloromethane was not mutagenic in mammaliancells with the exception of questionable results reported in one study using themouse lymphoma assay. However, in a more recent study, HPRT-mutationswere induced in CHO cells when activated by mouse liver cytosol (Gravesand Green 1996). Formaldehyde showed only a weak mutagenicity in thesame assay, but induced DNA-protein cross-links. As dichloromethane produced

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only marginal increases in DNA-protein cross-linking, the authors drew theconclusion that formaldehyde does not play a major role in dichloromethanemutagenicity. Instead, the results support the proposal that the mutations arecaused by the glutathione conjugate, S-chloromethylglutathione. This conclusionwas further strengthened in a follow-up study, where the mutations weresequenced and compared with the mutations induced by 1,2-dibromoethaneand formaldehyde. All three compounds induced primarily point mutations.The pattern of mutations from dichloromethane showed greater similarity with1,2-dibromoethane, which is known to act through a glutathione conjugate,rather than with formaldehyde (Graves et al. 1996).

8.1.4. Chromosomal effectsIn one study, chromosome aberrations, but not sister chromatid exchanges,were demonstrated in different cell types. Equivocal results concerning sisterchromatid exchanges were obtained in another study. Negative results for bothchromosome aberrations and sister chromatid exchanges were reported in stillanother study. One test for micronuclei in V79 hamster cells was negative.Two cell transformation assays were also negative (WHO 1996).

In a study by Hallier et al.(1993), the induction of sister chromatid exchanges(SCE) was measured in lymphocytes when whole blood samples from diffe-rent human donors were incubated with dichloromethane. A specific glutathione-S-transferase (GST) activity in the erythrocytes (later identified as GSTT1)was shown to be protective against the genotoxic activity of dichloromethanein the lymphocytes. A marked increase in SCE was only demonstrated inlymphocytes from those donors that lack this GST activity in their erythrocytes.This finding seems contradictory to the enhancing effect of the GST pathwayon the genotoxicity of dichloromethane demonstrated in other studies.

Dichloromethane in water increased the frequency of chromosome aberrationsin root cells of Allium cepa (Rank and Nielsen 1994).

8.2. In vivo

8.2.1. DrosophilaWhen a series of haloalkanes were tested for sex-linked recessive lethals andsomatic mutations and recombinations in Drosophila with inhalation exposure,1,2-dichloroethane and 1,2-dibromoethane were positive, but dichloromethane

58

and dibromomethane were not (Kramers et al. 1991). Out of two other recessivelethal tests using feeding exposure, one was negative and the other showed amarginal increase (WHO 1996).

8.2.2. Chromosomal effectsIntraperitoneal or subcutaneous injections of dichloromethane at doses up to5000 mg/kg did not increase the frequency of micronuclei, sister chromatidexchanges or chromosome aberrations in bone marrow cells of different strainsof mice. Doses of up to 4 g/kg administered by gavage also failed to inducemicronuclei (WHO 1996). On the other hand, inhalation exposure of femaleB6C3F1 mice to 14 000 or 28 000 mg/m3 for 10 days resulted in slightincreases in the frequency of sister chromatid exchanges in lung cells andperipheral blood lymphocytes, in chromosome aberrations in lung and bonemarrow cells, and in micronuclei in peripheral blood erythrocytes. A marginalincrease in lung cell SCEs and micronuclei in erythrocytes was observed followinga 3-month inhalation exposure to 7100 mg/m3 (Allen et al. 1990).

No increase in chromosomal aberrations was observed in bone marrow cellsof Sprague-Dawley rats following inhalation exposure to1800, 3500 or12 000 mg/m3 for 6 months (WHO 1996).

8.2.3. MutationsLung and liver tumours from affected female B6C3F1 mice in the 2-yearinhalation cancer study (7100 mg/m3) by Kari et al. (1993) were examined forthe presence of mutated ras oncogenes. Oncogene mutations in chemicallyinduced and spontaneous tumours were compared, but no significant differencesin the mutation profiles were found, in liver or lung tumours (Devereux et al.1993). Likewise, no dichloromethane-induced inactivation of the p53 tumoursuppressor gene was indicated in studies by Hegi et al. (1993), although theseauthors warned against any conclusions concerning a mutation spectrum fordichloromethane because of the limited number of p53 mutations identified(Hegi et al. 1994).

The dominant lethal test was negative in mice, both after subcutaneous andinhalation exposures (350, 530 or 710 mg/m3 for 6 weeks) (WHO 1996).

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Table 10.In vivo genotoxicity assays. Adapted and extended from WHO (1996)

Assay Strain/type Result Dose, route References

Chromosome Female B6C3F1 mouse; - 2500, 5000 mg/kg; s.c. Allen et al. 1990aberrations bone narrow

Female B6C3F1 mouse; +1 14 000, 28 000 mg/m3, Allen et al. 1990

bone narrow, lung cells 10 d (6 h/d, 5d/wk), inhal.

Male C57BL/6 mouse; - 0-2000 mg/kg; i.p. Westbrook-bone narrow Collins et al. 1990

Rat; bone narrow - 1800, 3500, Burek et al. 19842

12 000 mg/m3,6 h/day, 5 d/wk,6 mo; inhalation

Sister- Male C57BL/6 mouse; - 0-2000 mg/kg; i.p. Westbrook-chromatid bone narrow Collins et al. 1990exchanges

Female B6C3F1 mouse; - 2500 or 5000 mg/kg; s.c. Allen et al. 1990

bone narrow

Female B6C3F1 mouse; +1 14 000, 28 000 mg/m3, Allen et al. 1990lung cells, peripheral 10 d (6 h/d, 5 d/wk);blood lymphocytes inhalation

Female B6C3F1 mouse; +1 7100 mg/m3; 3 mo; Allen et al. 1990

lung cells inhalation

Micronuclei Female B6C3F1 mouse; +1 14 000, 28 000 mg/m3, Allen et al. 1990peripheral blood 10 d; 7100 mg/m3,erythrocytes 3 mo; inhalation

C57BL/6 mouse; - up to 4 g/kg; p.o. Sheldon et al.bone marrow in corn oil, (1987)2

NMRI mouse; - 10, 20 mM/kg; i.p. Gocke et al. 19812

bone marrow

Unscheduled Male Alpk: AP rat; - 100, 500, 1000 mg/kg; Trueman et al.DNA hepatocytes p.o. 19872

synthesisMale Fischer 344 rat - 7100, 14 000 mg/m3 ; Trueman andand male B6C3F1 2 or 6 h; inhalation Ashby 19872

mouse; hepatocytes

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Assay Strain/type Result Dose, route References

Dominant Male Swiss-Webster - 5 ml/kg, 5% or Raje et al. 19882

lethals mouse 10% v/v; 3 times/wk;4 wk; s.c. in corn oil

Recessive Drosophila - 0-14 260 mg/m3; Kramers et al.lethals melanogaster 6 h/d, 1 or 2 wk; 1991

inhalation

Drosophila +1 130, 630 mM Gocke et al. 19812

melanogaster

DNA binding Male Ficher 344 rat, - 14 000 mg/m3; Green et al. 1988male B6C3F1 mouse; 3 h; inhalationliver and lung

Male B6C3F1 mouse; - 2000 - 2800 mg/m3; Ottenwälder andliver, kidney, lung declining during Peter 1989

experiment; inhalation

DNA - Male B6C3F1 mouse; + 14 000 mg/m3; 6 h/d; Casanova et al.protein liver 2 d; inhalation 1992crosslinks

Male B6C3F1 mouse; - 14 000 mg/m3; 6 h/d; Casanova et al.lung, male hamsters; 2 d; inhalation 1992liver and lung

DNA single B6C3F1 mouse; + 14 0000 mg/m3; 6 h, Graves et al.strand breaks liver and lung 7000 - 21 000 mg/m3; 1994 b; 1995

3 h; inhalation

AP rat - 14 000 mg/m3; 6 h; Graves et al. inhalation 1994b

1) Small, but statistically significant increases, at least at the highest dose tested2) Cited in WHO 1996

8.2.4. DNA binding, DNA damageNo evidence of binding to DNA was found in livers and lungs of mice and ratsexposed to 14 000 or 2000-2800 mg/m3 of 14C-labelled dichloromethane,respectively (Green et al. 1988, Ottenwälder and Peter 1989). DNA-proteincross-links, probably caused by metabolically formed formaldehyde, weredetected in mouse liver, but not in mouse lung or hamster liver or lung afterinhalation exposure to 14 000 mg/m3 for 2 days (Casanova et al. 1992).

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DNA-damage measured as unscheduled DNA synthesis was not increased inmice or rats exposed either by gavage with up to 1000 mg/kg or by inhalationfor 2 or 6 h with up to 14 000 mg/m3. On the other hand, slightly increasedDNA damage in rat hepatocytes, measured by alkaline elution, was reportedafter oral exposure to 2.55 g/kg for 24 h (WHO,1996).

DNA single strand breaks were found in hepatocytes of B6C3F1 mice afterinhalation exposure to 14 000 mg/m3 for 6 h, but not in hepatocytes ofsimilarly exposed rats. By pharmacokinetic modelling this inhalation dose wascalculated to give similar liver concentrations in mice and rats (1.6 and 1.2 mM,respectively), but in parallel experiments it was found that the lowest concent-ration that would induce single strand breaks in vitro was much higher for rathepatocytes than for mouse hepatocytes (30 mM compared to 0.4 mM, alsomentioned above) (Graves et al. 1994b). In later experiments, a dose-dependentincrease in DNA single strand breaks was also detected in the lungs of miceexposed to 7100-21 000 mg/m3 for 3h, although no effects were detected at3500 mg/m3. The DNA damage in liver was undetectable 2 h after exposure,suggesting an active DNA repair mechanism, but in lung the damage waspersistent up to 4 h after exposure. Pre-treatment of mice with the glutathionedepletor buthionine sulphoximine caused a decrease in the amount of DNAdamage detected in both liver and lung, suggesting a GST-mediated mechanism(Graves et al. 1995).

8.2.5. Human dataCytogenetic analyses were performed on a group of 46 styrene-exposed workers,who were also exposed to dichloromethane used to clean tools, machines etc.The time-weighted average during the 8 h working day when samples werecollected was 70 mg/m3 for styrene (range 0-598 by personal sampling for thedifferent individuals) and 108 mg/m3 for dichloromethane (range 0-742). In thelymphocytes of exposed workers, all cytogenetic parameters were significantlyenhanced compared to non-exposed controls (chromosomal aberrations,micronuclei and sister-chromatid exchanges). There was no correlation withthe number of years employed or with styrene exposure during the samplingday, but the dichloromethane concentration was positively correlated with thefrequencies of chromosome aberrations (with gaps) and aberrant cells. Noconclusion could be drawn concerning the causative role of styrene and/ordichloromethane for the induction of these cytogenetic effects (Tates et al.1994).

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8.3. Conclusions

Dichloromethane is mutagenic in Salmonella when tested in a vapour phaseexposure system. The mutagenicity is probably mainly mediated by glutathione(GSH) conjugation, catalysed by cytosolic glutathione S-transferase (GST),since glutathione-deficient strains are less sensitive and strains expressing higherGST activity are more sensitive to dichloromethane-induced mutations. Studieson DNA single-strand breaks in hepatocytes and Chinese hamster ovary (CHO)cells and mutations in CHO cells indicate that these effects are caused by theproposed glutathione conjugate S-chloromethylglutathione. DNA single strandbreaks are formed at much lower concentrations of dichloromethane in mousehepatocytes than in rat hepatocytes. In CHO cells, mutations and DNA damageoccur only in co-incubation with mouse liver cytosolic fraction and not with themicrosomal fraction. This indicates that the microsomal oxidative P450-mediatedmetabolism is of relatively less importance. DNA single strand breaks havealso been induced in mouse bronchiolar Clara cells exposed in vitro, but notin hamster hepatocytes or human hepatocytes. Metabolically formedformaldehyde has been considered to be of little importance for mutations orsingle-strand breaks, but it may form DNA-protein cross-links.

Chromosome aberrations have been demonstrated in vitro whereas negativeor equivocal results were obtained in tests for sister chromatid exchange induction.In one inhalation study with mice in vivo, dichloromethane induced a slightincrease in sister chromatid exchanges in lung cells and in lymphocytes,chromosome aberrations in lung and bone marrow cells, and micronuclei inerythrocytes. However, similar effects did not occur after subcutaneous orintraperitoneal injections. No effects were seen in rats after inhalation exposure.DNA single strand breaks were detected in the hepatocytes of mice, but notin rats, after inhalation of dichloromethane. DNA single-strand breaks werealso detected in the lungs of mice.

Taken together, dichloromethane seems to have genotoxic properties. Thiseffect seems to be mainly related to the GST metabolic pathway.

Cytogenetic effects were demonstrated in workers occupationally exposed tostyrene and dichloromethane. Styrene is known to cause such effects fromother human studies, but it cannot be excluded that dichloromethane contributedto the observed effects.

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9. Reproductive toxicity

A decrease in the number of litters and offspring for Fischer 344 rats exposedto 1700 mg/m3 6 h/day, 5 days/week for 10 weeks and a decreased fertilityfor males at 5300 mg/m3 were observed in a two-generation study by Hammet al. (1985, according to IMM 1986). At 5300 mg/m3 no offspring wereproduced. This level of exposure also showed histopathological changes in thetestes and epididymis. The infertility and pathological changes were not reversibleat 5300 mg/m3. The offspring were exposed to 530 and 1700 mg/m3 withoutany observed toxic effects. No embryo- or fetotoxic effects were reported butthe study indicates a reproduction toxicity potential for dichloromethane exposureto rats.

Another two generation inhalation study with Fischer 344 rats showed noreproduction toxicity after exposure to dichloromethane (Nitschke et al. 1988,according to WHO 1996). Groups of 30 male and female rats were exposedto 0, 350, 1770 or 5300 mg/m3, 6 h/day, 5 days/week for 14-17 weeksbefore mating. During the period of mating, gestation and suckling the ratswere exposed 6 h/day, 7 days/week. The study showed no effect on reproductivecapacity, neonatal growth or neonatal survival of the rats.

Groups of 18 rats were exposed before and/or during 17 days of pregnancyto a concentration of 16 000 mg/m3 of dichloromethane for 6 h/day (Hardinand Manson 1980, according to WHO 1996). Fetal body weight was dec-reased. A slight increase of extra ribs were noted. Carboxyhaemoglobindeterminations ranged from 7.2 to 10.1%. The study showed that behaviourtoxicity (adaptation to new environment) can not be excluded in offspringexposed to dichloromethane during the gestation (Bornschein et al. 1980,according to WHO 1996). The observed behavioural effects might be relatedto elevated maternal carboxyhaemoglobin levels, dichloromethane-inducedchanges in nutrient supply to the foetus, or dichloromethane-induced alterationsin maternal care of the litter.

Exposure of dichloromethane to Wistar rats by drinking water (125 mg/l)during 13 weeks before mating induced no reproduction toxicity (Bornmannand Loeser 1967, according to WHO 1996).

Groups of Sprague-Dawley rats and Swiss-Webster mice were exposed todichloromethane at a concentration of 4400 mg/m3 on days 6-15 of pregnancy

64

for 7 h/day (Schwetz et al. 1975, according to WHO 1996). The offspringshowed an increased incidence of minor skeletal abnormalities such as dilatedrenal pelvis in rats and extra sternebrae in mice. Carboxyhaemoglobin levelsranged from 8.9 to 12.6% in non-pregnant rats and mice exposed at the sametime.

9.1. Conclusions

No new studies have been found since the last update (IMM 1990).

Exposure to dichloromethane does not generally cause any strong embryo- orreproductive toxicity. The noticed changes are light and reversible. One studyshowed decreased fertility and damage of testis in rats exposed to 5300 mg/m3,but no such effects were reported in another similar study. In one study,indications of behavioural effects were noted. Other studies with rats or miceexposed to dichloromethane were without any signs of reproductive toxicity.It should be noted though, that dichloromethane passes the placenta, both inhumans and in experimental animals (KemI 1991b).

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10. Human data

10.1. Accidental and acute exposure

A 56-year-old woman was found deeply unconscious and cyanosed afteringesting approximately 300 ml of a paint remover (Nitromors) containingmainly dichloromethane and methanol. Approximately one hour after ingestionthe COHb level was found to be 9%, and the maximal level was 12.1% andoccurred 36 h after ingestion. The COHb level dropped below 1.5% after60 h. She had metabolic acidosis and haemoglobinuria. During the 3 weeksfollowing ingestion her condition was complicated by progressive renal failure,pneumonia, pancreatitis, ongoing gastrointestinal haemorrhage and sepsis, whicheventually led to death some 25 days following ingestion. It was consideredthat the corrosive properties of the formulation rather than the formation ofCOHb were responsible for the lethal outcome. Post-mortem examinationrevealed acute tubular necrosis, extensive upper oesophageal, gastric, andduodenal ulceration, and necrosis in the pancreas (Hughes and Tracey 1993).

A 67-year-old man who had been using a paint stripper in a poorly ventilatedlocation complained of headache and chest pain. He was also confused,disorientated, had a progressive loss of mental alertness, increased fatigue andlethargy, slurred speech, little recall of either recent or past events, and wasdisorientated to time (WHO 1996).

Chemically induced hepatitis resulting from accidental exposure todichloromethane alone was described by Cordes et al. (cited by WHO 1996).There was exposure to hands, legs and feet and the duration of the exposurewas estimated to be 4 h. The liver was palpable but not enlarged or tender.Initial tests showed elevated serum levels of liver enzymes.

Pulmonary toxicity was reported in a 34-year old man after exposure to apaint remover containing dichloromethane when removing paint with an hot airgun. The patient initially suffered from headache, cough and chest discomfortand subsequently developed non-cardiogenic pulmonary oedema and hyper-reactive airways. There are additional reports suggesting severe pulmonaryinjury and death due to phosgene poisoning caused by the use of dichloromethanenear a heat source. Open flames seem worse than electric heating devices withpotential lethal phosgene concentrations noted within 5 to 10 minutes in a smallenclosure (Snyder et al. 1992).

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A male worker was found unconscious in a tank where he had used a paintstripper with 75% dichloromethane and 8% methanol. Based on the quantityof the paint stripper used and the volume of the tank the authors estimated thatthe concentration of dichloromethane was well above 350 000 mg/m3 whencleaning was carried out. A few hours after admission to a hospital the con-centration of dichloromethane in blood was 281 mg/l and the level of COHbwas < 5%. Post-mortem examination showed cerebral oedema, mucosalhaemorrhages in the larynx, and congestion, haemorrhage and oedema in thelungs. Diffuse fatty change was noted in the liver. There were also skin burns(total estimated area involved was 25-30% of the total body surface area)(Tay et al. 1995).

A 51-year-old man was found dead inside a tank where dichloromethane wasused to remove rust. Internal examination of the brain, kidneys, liver and lungsrevealed prominent organ congestion. Blood samples showed 3% COHb. Theconcentration of dichloromethane was 252 mg/l (blood), 75 mg/kg (brain) and30 mg/kg (heart) (Kim et al. 1996).

An episode of acute exposure to high concentrations of dichloromethane involvingfive victims, including two fatalities, was reported by Leikin et al. (1990). Theauthors concluded that the cause of death was due to solvent-induced narcosisand not carbon monoxide poisoning.

Four cases of serious dichloromethane poisoning, including two fatalities, werereported from furniture-stripping shops in Colorado, USA. In the three patientsdiscovered while still alive, no cardiac irregularities were recorded. Cornealburns with first and/or second degree burns were seen in areas having haddirect contact with the dichloromethane-containing paint-stripper. MeasuredCOHb levels did not exceed 8.6%. In each case, no respiratory protectionwas worn and ventilation was inadequate, but exposure levels were not known.The authors concluded that the toxic effects were due to the anaestheticproperties of dichloromethane (Hall and Rumack 1990).

Two men were found dead in a well, where they had been burying barrelscontaining mixed solvents and other chemicals. Air samples collected a fewhours later contained up to 580 000 mg/m3, which is approximately one-thirdof saturating concentrations of dichloromethane. Blood collected at necropsycontained 571 and 601 mg dichloromethane per litre. Much lower levels ofother solvents were found. Autopsy showed marked congestion and oedemaof the brain and lungs. COHb levels were 30% for both men, accompaniedby severe acidosis (Manno et al. 1992).

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A 35-year old male furniture refinisher had complaints of upper respiratoryirritation, fatigue and light-headedness occurring on a daily basis after using adichloromethane containing paint stripper (80% dichloromethane and 20%methanol). Determinations of blood COHb on three occasions showed anapparently linear correlation of COHb to hours worked on the day of samp-ling, with a maximum COHb level of 10% after working for 6.5 hours (Shustermanet al. 1990).

Miller et al. (cited in WHO 1996) reported a case of acute tubular necrosisin the kidney, as well as hepatocellular injury, in a young male using a tileremover in a poorly ventilated room.

Three myocardial infarctions in one individual were reported to have followedthree exposures to a paint remover containing dichloromethane. Concentrationsof dichloromethane in the breathing zone were up to 4500 mg/m3 (WHO1996).

Dichloromethane has been shown to be irritating to the eyes and skin (WHO1996).

10.2. Controlled exposure

Neurobehavioural changes (vigilance disturbance and impaired combined trackingmonitoring performance) were observed in human volunteers exposed todichloromethane at 690 mg/m3 for 1.5 to 3 h (Putz et al. 1976, cited by WHO1996).

Sixteen healthy male volunteers aged 19-21 were exposed to increasingconcentrations of dichloromethane during 70 min. The concentration ofdichloromethane was increased during 60 minutes in ten geometrical steps upto 2500 mg/m3 and remained at 2500 mg/m3 during the last ten minutes.Another group of 42 persons served as controls in this double-blind study.The exposure to dichloromethane did not impair vigilance performance (Kozenaet al. 1990).

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10.3. Long-term exposure, case studies

During seven winter seasons a young worker was exposed to dichloromethanevapours while cleaning watch parts in a small room that was not ventilatedduring the winter months. He became severely disabled by the development ofswelling and reddening of the skin, blistering and paraesthesia affecting hishands. Treatment with corticosteroids was unsuccessful, but his disorderdisappeared when he changed work and was no longer exposed todichloromethane. COHb levels were 10-13% in different blood samples (Lini-ger and Sigrist 1994).

Thirty-four men with occupational exposure to dichloromethane at the sameplant were self-referred and examined at an Occupational Health Center inCincinnati, Ohio, USA. Twenty-six of these workers were heavily exposed(bonders/prime wipers) with considerable amounts of solvent on their skin andclothing. The average air exposure in this group of workers was reported tobe 240 mg/m3, ranging from 11 to 544 mg/m3. Although the primary complaintof the employees involved problems associated with central nervous dysfunction,8 of the 34 complained of testicular, epididymal or lower abdominal pain, andhad clinical histories relating to infertility. All 8 belonged to the group ofbonders and had carboxyhaemoglobin levels between 1.2 and 17.3% (blooddrawn 4-90 h after exposure). Four out of these 8 workers agreed to providesemen samples and low sperm counts (< 20 million/ml) were found in all thesesamples, compatible with the clinical histories of infertility. The workers in theplant were also exposed to other chemicals, such as styrene and isocyanates.However, the exposure patterns indicate that dichloromethane was the likelycause for the observed inhibitory effect on sperm production. Thus, the 8 affectedworkers were all bonders with high exposure to dichloromethane and lowexposure to styrene (average 7 ppm), whereas higher exposure to styrene(average 41 ppm) in combination with lower exposure to dichloromethane,and exposure to isocyanates and aromatic hydrocarbons were more commonin non-bonders (Kelly et al. 1988).

Irreversible damage to the central nervous system was found in a man whohad been exposed for 5 years to dichloromethane levels between 2300 and12 500 mg/m3. Another man exposed for 3 years to dichloromethane rangingfrom 1700 to 3500 mg/m3 showed bilateral temporal lobe degeneration (WHO1996).

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10.4. Epidemiological studies

10.4.1. Morbidity studiesIn a study by Cherry et al. (1981), an excess of self reported neurologicalsymptoms was found when a group of 46 men exposed to dichloromethane(260-350 mg/m3) was compared with a non-exposed referent group of 12 men.In addition, 29 of the exposed men participated in a follow-up study to seewhether there was any evidence of neuropsychological damage in the exposedmen. Age-matched controls were selected from among men working on asimilar process but with no exposure to solvents. Each man in the study hada clinical examination; motor conduction velocities were measured in the ulnarand median nerves; an ECG was taken and a psychological test battery wasdesigned to detect minimal brain damage. No evidence was found of long-term damage that could be attributed to occupational exposure to dichloro-methane for several years.

Ott et al. (1983) studied 266 workers exposed to dichloromethane (up to1700 mg/m3) and 251 workers who were not exposed to dichloromethane,from two cellulose plants in USA. They observed an increase in red cellcounts, hemoglobin and haematocrit among the women but not among themen. A dose-related increase in serum bilirubin was observed for both menand women, which the authors concluded to be an isolated finding as nocorresponding pattern of dose-related changes consistent with either liver injuryor hemolysis was observed for other serum and blood constituents. In anotherpart of this study, a group of 24 exposed male workers and 26 reference maleworkers from the plants was selected for a 24-hour ECG-monitoring. Underthe conditions of this study, no ECG anomalies were associated with theexposure to dichloromethane that ranged from a time weighted average of 210to 1700 mg/m3.

Taskinen et al. (1986) conducted a register based study on the pregnancyoutcome of female workers in eight Finnish pharmaceutical factories. Informa-tion about all female workers who had been employed in the factories duringthe years 1973 or 1975 to 1980 was obtained from the employers. Theworkers pregnancy data were collected from the nation wide hospital dis-charge register and polyclinic data of hospitals. The total number of 1795pregnancies included 142 spontaneous abortions. A case-control study wascarried out in which the cases were 44 women who had a spontaneous abortionduring employment in the pharmaceutical factory. Three age-matched femalepharmaceutical factory workers who had given birth to a child were chosen as

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controls for every case. The information about occupational exposures wascollected from questionnaires completed by the occupational physician or nurseat the factory. Exposure to chemicals was more common among the casesthan among the controls. For dichloromethane, the odds ratio was 2.3, (95%CI 1.0-5.7, p = 0.06). There was also exposure to other solvents.

Lash et al. (1991) conducted a study to examine the hypothesis that long termexposure to dichloromethane results in lasting effects on the central nervoussystem. A group of 1758 retired airline maintenance workers was surveyed bymail and telephone to identify a cohort of workers with more than 22 yearsof dichloromethane exposure following the stripping of paint from airplanes.Company records revealed exposures between 290 and 830 mg/m3 (time-weighted averages). A cohort of 25 exposed and 21 non-exposed retirees metthe occupational, demographic and medical criteria and were tested extensively.A battery of psychological and physiological tests was performed by profession-als. No statistically significant differences between exposed and control groupswas found at p≤0.05. A subtle decrease in attention was seen in the exposedgroup (p=0.08). Since this is a relatively small study, the results should beinterpreted with caution.

Using a standard battery of medical surveillance questions, a study was under-taken to determine if an increase in reported neurologic symptoms resultedfrom solvent exposure at a pharmaceutical research, development andmanufacturing site (Bukowski et al. 1992). The persons enrolled in exposedsurveillance programs (n=840) were compared with those enrolled in other,non-solvent exposed surveillance (n=1042). The ratio of positive respondersbetween the exposed and unexposed groups was used to generate a relativeprevalence ratio. When adjusting for age, sex, smoking, alcohol use and noiseexposure, there were no significantly elevated relative prevalence ratios. Allsymptoms combined resulted in an odds ratio (OR) of 0.93 (95% CI 0.75-1.17). This study regards solvent exposure in general, and not dichloromethanespecifically, and should therefore be regarded with caution.

Soden (1993) examined a group of active workers (n=150) who had workedfor at least 10 years in an area with relatively high exposures of dichloromethane(the exposure was 1700 mg/m3, 8 h time-weighted average) and comparedthem to an unexposed group of workers (n=260) with regards to symptomsand blood chemistry. Health history and blood samples had been collected aspart of a company sponsored health monitoring program in which both exposedand unexposed workers where participants. No statistically significant differences

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at p≤0.05, were observed in the selected symptoms (including irregularheartbeat, dizziness or loss of memory) or in any of the blood chemistry. Theonly noticeable difference was in ASAT (a liver enzyme test), where the non-exposed group had higher serum levels than the exposed group (means of28.2 versus 25.1, p=0.06).

Bell et al. (1991) examined the relationship between birth weight and exposureto emissions of dichloromethane from manufacturing processes of the Kodakcompany in Monroe County, New York. County census tracts were categorisedas exposed to high, moderate, low or no levels of dichloromethane, withpredicted average exposures of 50, 25, 10, and 0 µg/m3, respectively. Birthweights were obtained from 91 302 birth certificates of white singleton birthsto Monroe county residents from 1976 to 1987. No statistically significanteffect of exposure on birth weight was found. Adjusted birth weight in highexposure census tracts was 18.7 g less than in areas with no exposure (95%confidence interval (CI) -51.6 to + 14.2 g).

10.4.2. Mortality studiesFriedlander et al. (1978) performed a study at a Kodak photographic filmproduction facility in Rochester, New York. Two different approaches tomortality data were followed. The first approach, the proportionate mortalityrate study, included 334 deaths that occurred between 1956 and 1976 amongformer workers who were exposed to dichloromethane at the facility (estimatedtime-weighted average exposure to dichloromethane was 110-440 mg/m3).They were compared with other deceased workers at the facility. No significantlyelevated proportionate mortality rates were noted for any diagnostic group ormalignancy subgroup at p≤0.05. The other approach, the retrospective cohortmortality study, included 751 workers employed during 1964 and involvedfollow-up of this cohort up to 1976. Comparisons were made with mortalityrates from New York state and from an internal unexposed cohort from theKodak facility.

Hearne et al. (1987) extended the follow up of this cohort to 1984 andexpanded the study population to all workers (n=1013) who were exposedfor at least one year between 1964 and 1970. In a subsequent study, Hearneet al. (1990) extended the follow up of this cohort to 1988.

The study by Friedlander et al. (1978) and the subsequent studies by Hearneet al. (1987, 1990) failed to detect a significantly increased risk of ischaemic

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heart disease, lung cancer or liver cancer (which were their diagnoses ofinterest) among dichloromethane exposed workers compared both with theNew York state and the internal unexposed cohort. Hearne et al. (1987)observed an excess of pancreatic cancer mortality with a standardised mor-tality rate (SMR) of 2.58 (compared with the internal unexposed cohort). Thiswas not significant at the 99% confidence level, but became significant afterrecalculation at the 95% confidence level (95% CI 1.11-5.08) which waspointed out by Mirer et al. (1988). No new pancreatic cancer cases wereidentified with additional follow-up, and with the new additional data the ex-cess was no longer statistically significant (SMR=1.90, 95% CI 0.82-3.75)(Hearne et al. 1990). Friedlander et al. (1978) and Hearne et al. (1990) founda decreased risk for the exposed cohort compared with New York state (butnot compared to the internal unexposed cohort), for malignant neoplasms,circulatory diseases and for total mortality (p≤0.05). It could be important tomention that workers at the Kodak facility were not permitted to smoke attheir workstations and that this fact may have induced a negative bias in thesestudies, particularly with respect to lung cancer and cardiovascular disease.

Ott et al. (1983) evaluated a cohort of workers exposed to dichloromethanein the production of triacetate fibre at a manufacturing plant in Rock Hill, SouthCarolina, USA. This cohort included 1271 male and female workers whowere employed for at least three months, between 1954 and 1977. Thedichloromethane exposure was estimated to range from 490-1700 mg/m3, 8 htime-weighted average. Workers from another textile facility that were notexposed to dichloromethane were included for comparison purposes. All workerswere followed up to June 1977. The mortality of the exposed cohort wascompared with the mortality of the USA population. Direct comparisons werealso made between the mortality of the exposed and unexposed cohorts.Mortality from cardiovascular disease or any other cause was not found to besignificantly increased relative to the USA population. However, the authorsdid observe a significant increase in the risk of ischaemic heart disease (relativerisk=3.1, p≤0.05) among white men in the analysis when the mortality rates ofthe exposed and unexposed were compared. A possible confounder in thisstudy could be that the workers also were exposed to methanol and acetone.The follow-up period of the exposed cohort (but not the unexposed one)studied by Ott et al. (1983) was subsequently extended from 1977 to 1986by Lanes et al. (1990). This study also failed to detect an excess of cardiovascularor ischaemic heart disease compared with the USA population. A significantexcess of cancers of the biliary passages and liver (SMR=5.75, 95% CI=1.82-13.78) was observed. Lanes et al. (1993) extended the follow up period of

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the cohort for an additional 4 years. No additional cases of liver or biliarycancer were observed. The excess from the previous study persisted but wasno longer significant (SMR=2.98., 95% CI=0.81-7.63).

Heineman et al. (1994) reported the results of a case-control study of astrocyticbrain cancer and occupational exposure to chlorinated aliphatic hydrocarbons.The study included 300 cases with a hospital diagnosis of astrocytic braincancer and 320 controls matched by age, year of death, and geographicalarea. A job-exposure matrix was used to classify cases and controls in termsof potential exposure to chlorinated aliphatic hydrocarbons, includingdichloromethane. 119 cases and 108 controls were classified as being potentiallyexposed to dichloromethane in this study. The risk was reported to increasewith the probability of exposure (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 dichloromethane after adjustment forother solvent exposures. Combining high probability for exposure and >20 yearsof employment, the odds ratio was higher (OR=8.5, 95% CI 1.3-55.5).

A cohort study by Gibbs (1996) included 3211 cellulose fibre workers at acellulose acetate fibre plant in Cumberland, Maryland, USA, that were employedfor at least three months in or after 1970 and they were followed until 1989.The cohort was divided into three groups; high (1200-2500 mg/m3), low(180-350 mg/m3) or no exposure to dichloromethane. Comparisons weremade with USA, Maryland and county mortality. There were no elevatedrisks, except for men with 20 or more years of employment who had anincreased mortality from prostate cancer (SMR=2.1, p<0.05), and for womenalso employed for 20 or more years who had an elevated mortality of cervicalcancer (SMR=8.0, p<0.01). Besides dichloromethane, the workers wereexposed to acetone and methanol, and potentially exposed to finishing oilsused as lubricants, and also dyes, pigments and dusts. Furthermore, the biologicalsignificance of these tumour forms is questionable with respect to dichloromethaneexposure.

Tomenson et al. (1997) studied mortality among employees at a factory thatproduced cellulose triacetate film base at Brantham in the United Kingdom.The cohort consisted of 1785 male employees who had worked at the site atany time between 1946 and 1988, of whom 1473 had worked in jobs thatentailed exposure to dichloromethane. The mean duration of exposure todichloromethane was nine years at 67 mg/m3 (8 h time-weighted average).They were followed up to 31 December 1994 and mortality was compared

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with national and local rates. In the cohort of workers exposed todichloromethane, reduced mortality was found for all causes (SMR=0.74,95% CI=0.66-0.83), all cancers (SMR=0.65, 95% CI=0.51-0.82), the can-cer sites of certain interest (liver and biliary tract cancer (0 observed, 1.47expected), lung cancer (SMR=0.48, 95% CI=0.29-0.75) and pancreatic cancer(SMR=0.68, 95% CI=0.14-1.99) and for ischaemic heart disease (SMR=0.92,95% CI=0.76-1.10) compared to national rates.

10.5. Conclusions

10.5.1. Case studiesThe most common acute effects after exposure to high concentrations ofdichloromethane are CNS-effects and elevated COHb levels in the blood.Several other toxic effects due to exposure to dichloromethane have beenreported, including eye- and respiratory irritation, kidney and liver dysfunction,inhibited sperm production, pulmonary oedema and even death. Prolongeddirect contact with liquid dichloromethane to the skin causes burns. Lethaloutcome after ingestion of dichloromethane is believed to be due to the corrosiveproperties of dichloromethane rather than to the formation of COHb. Likewise,cases of death after inhalation of high concentrations of dichloromethane weredue to solvent-induced narcosis and not caused by the formation of COHb.Several studies have found that inhalation of dichloromethane is more harmfuland gives higher levels of COHb than ingestion. Poisoning by phosgene, orig-inating from dichloromethane decomposition after use of electrical heat oropen flames, has been reported. Dichloromethane exposure may possiblyaggravate heart disease.

Limited exposure of dichloromethane during controlled conditions showedneurophysiological and neurobehavioural disturbances at 690 mg/m3 during1.5 - 3 h in one study, but not in another study with increasing concentrationsup to 2500 mg/m3 during 70 min.

IMM has earlier concluded that dichloromethane is not particularly irritating.During controlled conditions no such effects were seen after exposure of humanvolunteers to dichloromethane at concentrations below 3500 mg/m3 (IMM1990).

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10.5.2. Epidemiological studiesThe morbidity studies regarding birth weight (exposure up to 50 mg/m3),neurologic symptoms (260-350 mg/m3), CNS symptoms (irregular heartbeats,dizziness, loss of memory, 290-830 mg/m3), blood chemistry (up to 1700 mg/m3)and spontaneous abortion all failed to show a statistically significant elevatedeffect at p<0.05. In all the morbidity studies the dichloromethane exposurewas occupational exposure, except for the birth weight study where the exposurecame from the outdoor air.

There are only a few mortality studies regarding occupational exposure todichloromethane and they have all been conducted and evaluated with regardto cancer and cardiovascular disease in particular.

The epidemiological studies are inconclusive with respect to mortality fromischaemic heart disease. An excess of cardiovascular disease was reported inone study (490-1700 mg/m3). Further studies did not provide compellingevidence of an increased risk, however, the general population was used asthe referent group in these studies.

Excess of mortality from cancer has been found in some studies, includingelevated risks of cancer in biliary passages and liver (490-1700 mg/m3), pancreas(110-440 mg/m3), and brain. However, there seems to be no consistent patternin tumour appearance. Further, the findings are of borderline significance andhave not been strengthened when cohorts were extended. On the other hand,the use of general mortality rates in occupational cohort mortality studies maybias the results due to the healthy worker effect, thus underestimating the trueeffect.

Another bias may be that in most studies smoking habits were not consideredand in some studies the workers were not permitted to smoke at their workplace. Failure to account for smoking might mask excess risks, in particular oflung cancer and cardiovascular disease.

In conclusion, several epidemiological studies of dichloromethane indicate slightlyelevated risks of cancer. However, these studies are not sufficient for drawingany firm conclusions, neither for cancer risk, nor cardiovascular disease.

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11. Quantitative risk estimates

11.1. Early EPA risk estimates

The U.S. Environmental Protection Agency (EPA) performed a cancer riskestimate of dichloromethane by applying the multistage model to the NTP(1986) study. Combined adenomas and carcinomas in lungs and liver of fem-ale mice were used. Human dose was calculated after body surface areacorrection, i.e. it was assumed that mice and humans exposed to the samedaily dose corrected by body weight elevated by 2/3 encounter the same lifetime cancer risks. The mouse-to-man surface area correction factor used byEPA was 12.7 (12.4 for males, 13.0 for females). In practice, dose con-version was performed by recalculating from ppm to mg/kg/day, from mouseor rat to man, and then back calculating to ppm or µg/m3. The upper boundof the 95% confidence interval of the incremental unit risk for humans inhalingdichloromethane over a life time was estimated to 4.1x10-6 per µg/m3 (EPA1985).

11.2. Pharmacokinetic risk estimates

Andersen et al. (1987) developed a physiologically-based pharmacokinetic(pbpk) model for dichloromethane in four species, namely mouse, rat, ham-ster, and man. The model includes metabolism via the P450 and the GSTpathways in both liver and lung tissues. Tissue volumes and blood flows weretaken from the literature. Blood:air partition coefficients were determined invitro using blood from all four species. Tissue:air coefficients were determinedfor rat and hamster, whereas those of mice and humans were assumed to beequal to those of the rat. Alveolar ventilation rates were partly determined byoptimisation against closed chamber experiments with dichloromethane andpartly obtained by allometric scaling. Metabolic parameters of the two pathwayswere determined for the three animal species by fitting the pharmacokineticdata to closed chamber data for dichloromethane. The relative specific activitiesof the two pathways in lungs and liver, respectively, were estimated from invitro metabolic data. In humans, the metabolic parameters of the P450 pathwaywere estimated from unpublished in vivo exposures at Dow Chemical Co.,whereas those of the GST pathway were obtained by allometric scaling of theanimal parameters.

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Andersen et al. (1987) used their physiological pharmacokinetic model tostudy the correlation between different dichloromethane dose surrogates andliver and lung tumour incidences in mice in the inhalation study by NTP (1986)and the drinking water study by Serota et al. (1984). Based on thesecomparisons, Andersen et al. found that in both tissues the tumour incidencecorrelated well with the area under the concentration-time curve (AUC) ofdichloromethane and the amount metabolised via GST but not with the amountmetabolised via P450 (Table 11). Based on these comparisons, Andersen etal. concluded that the amount metabolised (mg/d/kg tissue) by GST in liverand lung are suitable target doses for dichloromethane. They went on to calculatetarget doses at 3.5 mg/m3 (1 ppm) in man using their pharmacokinetic modelas compared to the approach used by EPA (1985), with the target doses inmouse at inhalation of 14 000 mg/m3 as a reference point. For liver tissue, thetarget dose in man (amount dichloromethane metabolised by GST as predictedby the pharmacokinetic model) was 167 times lower than that obtained usingthe EPA approach for interspecies extrapolation. For lung tissue, thecorresponding ratio was 144. According to Andersen, these ratios may besplit into three factors. Thus, the liver ratio of 167 can be subdivided in afactor of 20.8 due to the smaller relative contribution from GST to overallmetabolism at 3.5 mg/m3 compared to 14 000 mg/m3, a factor of 2.74 toaccount for mouse-man species differences in metabolic activities, and a factorof 2.95 to account for the difference in breathing rates and body surface areasof the two species. The corresponding factors for the lung ratio of 144 are13.1, 3.71, and 2.95, respectively.

Later on more metabolic data obtained in vitro have been presented (Reitzet al. 1988, 1989, review by e.g. Andersen and Krishnan 1994, Clewell1995, Green 1997, see also chapter 3), allowing more reliable target dosecalculations. The new data gives a similar picture; that among the studiedspecies mice have a much higher capacity for dichloromethane metabolism viathe GST pathway. Thus, Reitz et al. (1988) used the model by Andersen etal. (1987) but introduced in vitro metabolic data for GST metabolism inhumans, based on results from two human lung and liver samples. Using themultistage model Reitz et al. estimated an excess lifetime cancer risk of 3.7x10-8

for continuous exposure to 1 µg/m3. This is about two orders of magnitudelower than the EPA (1985) estimate.

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Table 11. Tumour frequencies (% liver adenomas and carcinomas) infemale B6C3F1 mice compared to different dose surrogates ofdichloromethane. Adapted from Andersen et al. (1987). Numbers havebeen rounded off.

Control Inhalation Inhalation Control Drinking7 100 14 000 watermg/m3 mg/m3 250 mg/kg/d

Liver

Tumours (%) 6.0 33 83 6.0 6.0

Delivered dose via P4501 0 3 600 3 700 0 5 200

Delivered dose via GST1 0 850 1 800 0 15

AUC of dichloromethane2 0 360 770 0 6.4

Lung

Tumours (%) 6.0 63 85 8.0

Delivered dose via P4501 0 1 500 1 600 0 1 200

Delivered dose via GST1 0 120 260 0 1.0

AUC of dichloromethane2 0 380 790 0 3.1

1 Expressed in mg dichloromethane per day and kg tissue, corrected for5 d/wk inhalation exposure.

2 Area under the concentration time curve expressed in mg/h/l.

Green and colleagues (ECETOC 1988) introduced saturable enzyme kineticsalso for the GST pathway, used species-specific metabolic data obtained invitro, and incorporated more human experimental data. As GST lung meta-bolism could only be detected in the mouse, the liver:lung metabolic ratio of10 seen in mouse for the GST pathway was assumed to apply also for rat,hamster, and man. The target doses calculated with this model were about 2-4 times higher than those calculated by Andersen et al. (1987). Thus, thedelivered doses via GST in mouse liver were 3800 and 4900 mg/kg/d at 7100and 14 000 mg/m3, respectively. The corresponding values for lung tissuewere 420 and 540 mg/kg/d (see Table 13 for comparison with Andersen´sdata). Applying a quadratic extrapolation model, the predicted human lifetimerisk of cancer (liver and lung tumours combined) was less than 3x10-5 at 1800

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mg/m3 and less than 7x10-9 at 35 mg/m3 (ECETOC 1988). Notably, this isa maximum likelihood estimate for occupational exposure and not the morecommonly given upper bound of the 95% confidence limit of life time risk forcontinuous exposure.

Portier and Kaplan (1989) made an attempt to estimate the individual varia-bility in cancer risk from dichloromethane by combining the Andersenpharmacokinetic model for dichloromethane (Andersen et al. 1987) with aone-stage model for the tumour data obtained in the NTP mouse inhalationstudy (NTP 1986, Mennear et al. 1988). Variability was estimated by repeatedMonte Carlo simulations with random sampling of model parameter valuesfrom different normal and log-normal distributions. The authors demonstratedthat, if no variability is added to the pharmacokinetic parameters, the doseestimated to give a 10-6 added risk in a life time in man varies from 4.7x10-4

(5th percentile) to 7.8x10-4 mg/kg/d (95th percentile) for female mouse livercell adenomas and carcinomas. In contrast, considering only metabolic varia-bility, with log-normal distributions of all metabolic parameters with a standarddeviation of 20% in mouse and 200% in man, the dose giving a 10-6 addedrisk varies from 0.7 to 375x10-4 mg/kg/d, i.e. by almost three orders ofmagnitude. Similar results were obtained for alveolar-bronchial tumours. Thestudy illustrates how inter-individual variability in metabolism, as well asuncertainties in the estimates of metabolic parameters, increase the uncertaintyin the risk estimates. At the time of this study the GSTT1 polymorphism inhumans was not recognised and thus not considered.

Casanova et al. (1992, 1996) analysed DNA-protein crosslinks (DPX) in lungand liver tissues from mouse and hamster. The level of crosslinks in mouseliver following exposure to 7 100 mg/m3 dichloromethane, 6 h/d for 2 dayswas 30-40 pmol/mg DNA, or approximately the same level of crosslinks asseen in the nasal epithelium of rats exposed to 4 ppm formaldehyde. Nocrosslinks were detected in DNA isolated from lung tissue of either mice orhamsters. Although the tissue dose of dichloromethane was approximately thesame in mice and hamsters, no crosslinks could be detected in hamster liver.According to the authors, the crosslinks are secondary to the formation offormaldehyde and the mouse-hamster differences in crosslink levels can beexplained by species differences in formation and elimination rates of thismetabolite. To illustrate this, the authors used the pharmacokinetic model ofAndersen et al. (1987) and included the formation of formaldehyde via theGST pathway and DNA-protein crosslinks formation.

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Using the multistage model Casanova et al. (1996) further showed that byreplacing dichloromethane in ambient air with DNA-protein crosslinks in liveras a dose surrogate, the maximum likelihood of the risk estimate for malemouse liver tumours was marginally increased, whereas the upper confidencelimit of the risk estimate was reduced by two orders of magnitude. Theexplanation for this phenomenon is that dichloromethane in air requires a three-stage model, whereas crosslinks in liver only requires a one-stage model becauseof the nonlinear relationship with dichloromethane in air. Thus, when crosslinksare used as dose surrogates one instead of three parameters are estimated inthe multistage model.

11.2.1. Updated EPA risk estimateThe EPA presented a revised risk estimate in response to the Andersen et al.(1987) pharmacokinetic approach. In this revision the EPA stated that Ander-sen et al. (1987) used ventilation rates that differ from those preferred byEPA. Thus, Andersen used a higher ventilation rate for humans and a lowerrate for mice and, as a consequence, obtained a 3.1-fold higher target dosethan would have been obtained using the EPA ventilation rates. EPA furtherconcluded that, provided that the metabolic rate constants used by Andersenet al. are correct in both species, the target dose is overestimated by a factorof 4.3 in liver and 3.6 in lung by linear extrapolation from high to low doses,i.e. by neglecting the influence of saturation kinetics. A third factor of 12.7relates to the EPA default assumption that the sum of all processes (includingdifferences in pharmacokinetics and life span) leading to cancer are related tobody surface area rather than body mass (EPA 1987).

In view of the pharmacokinetic-based findings of Andersen et al. (1997), andusing the metabolic parameters from the same authors, EPA recalculated theupper bound of the unit risk to 4.7x10-7 per µg/m3, or 8.8 times lower thantheir previous estimate. The new estimate, as well as the previous one, wasbased on lung and liver tumours combined in female mice. The EPA opted tomaintain their own ventilation rates for humans and mice, as well as the bodysurface area scaling factor of 12.7. The lower value of the new risk estimateis largely due to the nonlinearity in the high dose to low dose extrapolation.The EPA pointed out that human in vitro data suggest that the kinetic para-meter for the GST pathway in the Andersen model may be too high. While alower value of GST metabolism would result in a lower risk estimate, the EPAdecided not to use in vitro metabolic data in their quantitative risk assess-ment. The EPA further pointed out that the epidemiology studies, while showing

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no evidence of either liver or lung cancer, are not sufficient to rule out cancerrisk to humans and specifically mentioned the possibility of pancreas cancer(EPA 1987).

11.2.2. Evaluation of the pharmacokinetic approachThere are several assumptions in the pharmacokinetic approach that need tobe examined. The first one is that cancer induced by dichloromethane in miceis indeed related exclusively to the GST pathway. This point is strongly supportedby the close agreement between liver and lung tumour incidences and theamount of dichloromethane metabolised via GST, but not via P450, in themouse at high doses (Table 11). Tumourigenicity is also related to the areaunder the concentration-time curve of unmetabolised dichloromethane in thetwo organs, but it is highly unlikely that dichloromethane itself is a carcinogen.Another assumption is that the pharmacodynamics are linear, i.e. the cancerrisk from dichloromethane is proportional to the target dose. This does nottake into account factors such as the defence mechanisms of the body, includinginduction or inhibition of DNA repair and triggering of the immune system.In the multistage model it is assumed that the relation between dose andcancer risk is species-independent and, thus, quantitatively the same in miceand humans. The same assumption is maintained in the pharmacokinetic ap-proach, except that external exposure is replaced by a presumably moreappropriate target dose. Little data is available to support or invalidate thisassumption.

It is further assumed that life time cancer risk is related to the target doseexpressed as amount of dichloromethane metabolised by the GST pathwayper time unit, e.g. in mg/day/kg tissue. This does not take into account thathumans have larger tissue sizes and a longer life span than mice and thus that,for a given target dose, there is a higher chance of initiating events that mayeventually result in tumour formation. Species differences in size and life span,and not only rates of metabolism and physiological processes, are the reasonsfor the EPA procedure to scale dose by body surface area (EPA 1987,Rhomberg 1995).

Finally, the pharmacokinetic approaches only consider metabolism and tumourrisk in lung and liver tissues. Early human target dose estimates (e.g. Andersenet al. 1987) were made using allometric scaling of GST metabolic rates in lungand liver from rodents to man. Later predictions (ECETOC 1988, Reitz et al.1988) have been improved by using species-specific in vitro data. However,

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they are still based on a limited number of human liver and lung samples.Further, although several studies suggest significant extrahepatic GST activityin various human tissues other than lung, this has so far not been included inthe pharmacokinetic approaches.

In conclusion, some of the uncertainties in the species and dose extrapolationshave been reduced by introducing pharmacokinetic models to calculate targetdoses rather than simply using external doses. However, the greater uncertaintyof not knowing the shape of the target dose-cancer response curve has still notbeen addressed.

11.3. Benchmark dose models

A benchmark dose or exposure concentration is the dose of a substance thatcorresponds to a prescribed increase or decrease in the response of a healtheffect. The statistical lower bound on the benchmark dose can be used as areplacement for the no-observed-effect level (NOEL) in setting acceptabledaily intake levels (Crump 1995). The benchmark method was suggested byCrump (1984) and by Kimmel (1990), as an alternative approach for riskassessment of non-genotoxic substances. The US EPA has in a draft alsosuggested that a variant of the benchmark method could be applied in theassessment of carcinogens (EPA 1996).

The benchmark dose is calculated by fitting a dose-response curve to theexperimental data and calculating the statistical upper bound of the dose-response curve, thus indicating the statistical lower dose corresponding to aspecific response. Commonly, the lower 95% confidence limit of a dose givingrise to a 10% increased risk above the background level is calculated. Thislimit is often denoted as benchmark dose 10% (BMD10) or lower effectivedose 10% (LED10).

Advantages with the benchmark model compared to NOEL are that data fromthe whole dose-response curve are used and that the slope and form of thecurve influence the benchmark dose. Statistical variations in the data alsoinfluence the benchmark dose. The benchmark reflects sample size differentlythan does NOEL. Thus, smaller studies tend to result in smaller benchmarkdoses, whereas the opposite is true for NOEL. An additional advantage is thatthe benchmark dose does not have to be a dose level used in the study(Crump 1984, Kimmel 1990).

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The benchmark dose was calculated from cancer data from the NTP-studieson rat and mouse (NTP 1986, Mennear et al. 1988) using a statistical pro-gram developed at IMM (Kalliomaa et al. 1998). The equation of the doseresponse curve used is similar to the multistage model and is given below. Thesame equation is used by the software THRESH from ICF Kaiser, KS CrumpGroup Inc. (Crump 1984).

P(d) = q0+(1-q0) ⋅ (1-exp(-q1(d-d0)-q2(d-d0)2-…-qk(d-d0)k))

Benchmark doses of dichloromethane were calculated based on extra riskusing tumour data from the NTP (1986) study (Table 12). Calculated versusobserved dose-response curves for lung tumour responses in female mice areshown in Figure 2.

Figure 2. Calculated dose-response curves of pulmonary adenomas,carcinomas and the combined adenomas and carcinomas in female miceexposed to dichloromethane (NTP 1986). Solid lines indicate the maximallikelihood estimate and dotted lines indicate the 95% confidence interval.

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Acceptable intake or exposure limits may be obtained from the benchmarkdose in two ways, either by linear extrapolation to a life-time risk level or byapplying an uncertainty factor. The EPA has, in a draft, suggested the formermodel for handling carcinogens (EPA 1996). For non-genotoxic substances anapproach with uncertainty factors might be more appropriate.

As shown in Table 12, the response giving the smallest BMD10 is the increasein pulmonary adenomas and carcinomas in female mice with a benchmark doseof 640 mg/m3. A linear extrapolation from 10% extra risk down to a life timerisk of 10-5 gives a concentration of 64 µg/m3. This value is based on the NTPstudy with 6 h exposures, 5 days per week. The actual exposure timecorresponds to 18% of the total time in this study. A correction factor of0.18 should therefore be applied on the life time risk above, in order tocorrect for discontinuous exposure in the experiment. To compensate for theinfluence of saturation kinetics when extrapolating from high dose to low dosea correction-factor of 4 should be used (see section 10.2.1). A life time riskof 10-5 for continuous exposure would thus correspond to 46 µg/m3

(64 µg/m3 ⋅ 0.18 ⋅ 4). This approach does not include any species conversionfactors.

Uncertainty factors, if applied, can be further subdivided in factors to accountfor intraspecies variation, interspecies differences in toxicokinetics andtoxicodynamics, and the severity of the effect (Dourson et al. 1996, Haag-Grönlund et al. 1995, Kimmel 1990). In the case of dichloromethane wewould choose factors of 10 for intraspecies variation, 10 for severity, 3 fortoxicodynamics, and 5 to compensate for the relatively high (10%) risk usedfor benchmark dose. A factor of 3 might be considered to compensate formetabolic differences but since there is a metabolic change in the oppositedirection when going from high doses to low doses, such a factor is notincluded. Taken together, this gives an overall uncertainty factor of 1500. Thelowest calculated benchmark dose was 640 mg/m3, hence the ”acceptable”concentration would be 427 µg/m3. Applying a correction factor of 0.18 tocorrect for the exposure regime in the NTP study gives a concentration of77 µg/m3. However, as we regard dichloromethane as a genotoxic compound,we prefer the extrapolation model and not the uncertainly factor model for riskassessment of dichloromethane.

Other endpoints than cancer may also be modelled by the benchmark dosemethod. Clewell et al. (1997) evaluated non-cancer data from a few toxicologicalstudies on dichloromethane, but concluded that the studies were not suited forbenchmark modelling due to inadequate reporting of the data.

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Table 12. Calculated maximal likelihood estimate (MLE) and benchmarkdose at 10% level (BMD10) from the carcinogenicity data in the NTP-studies on mouse and rat. Calculations are based on extra risk and aconfidence interval of 95% was used.

Species, sex MLE BMD10(mg/m3) (mg/m3)

Mouse, male Hepatic adenoma 6200 2700

Hepatic carcinoma 6600 5400

Hepatic adenoma + carcinoma 6100 4800

Mouse, female Hepatic adenoma 6000 5100

Hepatic carcinoma 4400 3800

Hepatic adenoma + carcinoma 3600 3100

Mouse, male Pulmonary adenoma 2300 1700

Pulmonary carcinoma 5100 4400

Pulmonary adenoma + carcinoma 1000 810

Mouse, female Pulmonary adenoma 1500 1200

Pulmonary carcinoma 4700 4100

Pulmonary adenoma + carcinoma 790 640

Rat, male Mammary gland, adenoma 19 400 16 400

Mammary gland, fibroadenoma 16 300 13 200

Mammary gland, adenoma 13 700 10 200+ fibroadenoma

Rat, female Mammary gland, adenoma 19 400 16 400

Mammary gland, fibroadenoma 3500 3000

Mammary gland, adenoma 3600 3000+ fibroadenoma

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11.4. Conclusions

The EPA applied the multistage model on the NTP (1986) data on combinedlung and liver adenomas and carcinomas in female mice. Using body surfacearea correction and the upper bound of the 95% confidence interval, theincremental unit risk for humans inhaling dichloromethane over a life time wasestimated to 4.1x10-6 per µg/m3 (EPA 1985).

Andersen et al. (1987) developed a physiological pharmacokinetic model fordichloromethane based on species-specific metabolic data and compared itwith the same NTP mouse cancer data. Based on predictions of the rate ofmetabolism by the GST pathway in liver and lung tissues, they concluded thatthe EPA procedure overestimates the cancer risk by about two orders ofmagnitude. The pharmacokinetic approach included several assumptions, i.e.that the pharmacodynamics are species-invariant, that risk is related only to theGST pathway, that risk is related to delivered dose per time unit and bodymass (and not body surface area), that metabolic activation takes place andtumours occur only in the liver and lungs, and that the metabolic parametershave been accurately determined. In addition, it did not take into account thedifferences in tissue sizes and life spans between mice and humans. There islimited scientific evidence to support most of these assumptions.

In a subsequent update, the EPA agreed that correction for the high dose tolow dose nonlinearity in the GST pathway should be accounted for, but didnot discontinue the use of surface area correction. Thus, the EPA reducedtheir unit risk estimate to 4.7x10-7 per µg/m3, i.e. by a factor of 8.8 (EPA1987). Linear extrapolation of this estimate gives a concentration of 21µg/m3

for a life time risk of 10-5.

Combining a physiological pharmacokinetic model for dichloromethane withrealistic estimates of metabolic variability and a one-stage model for tumourdata, the variability in cancer risk estimates varied by three orders of magnitude.This illustrates that the pharmacokinetic risk estimates also have a large degreeof uncertainty.

Benchmark dose calculations based on the NTP study give a lowest BMD10of 640 mg/m3 for pulmonary adenomas and carcinomas in female mice. Linearextrapolation to a lifetime risk of 10-5 and correction for continuous exposureand high to low dose extrapolation gives a concentration of 46 µg/m3. Thisapproach does not include species conversion factors.

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Table 13. Summary of published quantitative cancer risk estimates fordichloromethane.

Unit risk The concentration (µµµµµg/m3)corresponding to a lifetime

(lifetime risk at 1 µµµµµg/m3) risk of 1 x 10-5

EPA 1985 4.1 x 10-6 2.4

Reitz et al. 1988 3.7 x 10-8 270

EPA 1987, 1997 4.7 x 10-7 21

IMM 1998 2.2 x 10-7 46

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12. Summary and health risk assessment

This health risk assessment is focused on inhalation exposure, as this is themost important exposure route, and because the aim is to propose a health-based guideline value for ambient air.

12.1. Metabolism

Dichloromethane is efficiently absorbed by all exposure routes, and evenlydistributed within the body with an affinity for adipose tissues. In all mammalspecies studied, dichloromethane is metabolised by two pathways, which aremainly mediated by cytochrome P450 2E1 and glutathione-S-transferase (GST)T1, respectively. Potentially toxic metabolites are formed in both pathways,including formyl chloride and carbon monoxide in the P450 pathway andS-chloromethylglutathione and formaldehyde in the GST pathway.

The P450 pathway becomes saturated at inhalation exposures of about 700-1000 mg/m3, whereas the metabolism via GST is essentially linear at all rele-vant exposure levels. In effect, P450 is the preferred route at low concentrations.However, at high concentrations the GST pathway becomes relatively moreimportant. This is of relevance in high dose to low dose extrapolations, especiallyif toxicity is related to metabolites of the GST pathway only.

In laboratory animals, metabolism occurs mainly in the liver, with additionalmetabolism in lung and possibly in other tissues as well. There are considerablespecies differences with respect to tissue specific metabolic capacities of thesepathways. However, except for liver and lung data from laboratory animals,there are little data on tissue-specific metabolism.

Human studies have focused on the uptake and oxidative metabolism ofdichloromethane after inhalation exposure. One reason is probably that at thetime of the experiments there was little awareness of the GST pathway. Atenvironmentally realistic exposures, GST metabolism seems to play aquantitatively minor role. However, human quantitative in vitro data is limitedto a small number of liver, lung and kidney samples. Recent data indicate thatthe GST isoenzyme of interest, GSTT1, is expressed in most tissues. GSTT1is polymorphic in humans. Approximately 15-20% of individuals of Caucasianorigin lack functional GSTT1 enzyme, and can therefore not metabolisedichloromethane by the GST pathway.

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12.2. Non-cancer effects

12.2.1. Acute toxicity, CNS-effectsThe acute toxicity of dichloromethane is low. Dichloromethane affects thecentral nervous system, and will cause impairment of behavioural or sensoryresponses at sufficiently high concentrations. CNS effects have been reportedboth in animal experiments and in humans occupationally or accidentally exposedto high levels of dichloromethane. Fatalities have also occurred. The lowestobserved effect level (LOEL) for CNS effects in humans is 690 mg/m3 for1.5 - 3 h (neurobehavioural changes in human volunteers in one out of twostudies). In rats, changes in EEG pattern were detected at 7100 mg/m3, andchanged levels of transmitter substances in the brain at 250 mg/m3.

Some epidemiological studies have investigated neurophysiological andpsychological symptoms in occupationally exposed workers, but no statisticallysignificant increases were demonstrated.

Besides CNS effects, the most important effects seen in animal experimentsare those affecting the liver. In most animal studies toxic effects in the liverbecome evident at levels of 3500 mg/m3 and higher. However, slight alterationswere observed in one study with rats exposed continuously for 100 days toas low a concentration as 90 mg/m3. In a 2 year chronic study, vacuolisationin the liver of rats was observed at 700 mg/m3 in males and 1700 mg/m3 infemales.

12.2.2. Reproductive toxicityDichloromethane passes the placenta both in humans and in experimental animals.Light and reversible effects on the foetus were described in two out of fourinhalation studies. Decreased fertility in male rats was described at 5300 mg/m3

in one out of two studies.

A Finnish study on spontaneous abortions in female workers in pharmaceuticalfactories showed an enhanced risk, although not statistically significant, forthose workers exposed to dichloromethane. There was also exposure to otherchemicals. There is also one case report on heavily exposed male workers,indicating impaired fertility.

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12.2.3. Ischemic heart disease and carboxyhaemoglobinCarbon monoxide (CO) is formed in the oxidative P450-mediated metabolismof dichloromethane. CO binds strongly to haemoglobin as carboxyhaemoglobin(COHb). As this metabolic pathway is saturated at high concentrations, amaximum of < 10% COHb in blood is normally reached, although occasionallystill higher levels have been measured. Human exposure to 170 - 700 mg/m3

for 7.5 h leads to COHb levels of 1.8 - 6.8 %.

The formation of COHb most likely produces the cardiotoxic effects that havebeen seen in some studies. High short-term oral or intravenous doses ofdichloromethane gave rise to heart arrhythmia in rats. Several epidemiologicalstudies have been performed in order to investigate any relationship betweenoccupational exposure to dichloromethane and cardiovascular disease. Thesestudies were inconclusive. An excess of cardiovascular disease was reportedin one study (exposure levels 490 - 1700 mg/m3), but further studies did notprovide compelling evidence of an increased risk.

COHb formation was the basis for the recommendations from WHO Europeon air quality guidelines for ambient air (WHO 1998). A maximum allowableincrease of 0.1% in COHb from dichloromethane led to a 24 h guideline of3 mg/m3, and a weekly average of 0.45 mg/m3. COHb formation also seemsto be the basis for most occupational threshold limit values for dichloromethane.The Swedish occupational exposure limit value (8 h time-weighted average) is120 mg/m3.

12.3. Cancer

Inhalation exposure of 7100 and 14 000 mg/m3 of dichloromethane has givenrise to liver and lung adenomas and carcinomas in B6C3F1 mice and benignmammary tumours in rats at similar concentrations, but no increased tumourincidence in Syrian Golden hamsters. One study with female mice at 7100 mg/m3

demonstrated that the lungs are more sensitive to dichloromethane-inducedtumour induction than the liver. As little as 26 weeks of exposure early in lifewas sufficient to significantly increase the frequency of lung tumours at 2 yearsof age, but the liver tumour frequency continued to increase as exposurecontinued.

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12.4. Studies important for the interpretation of animal cancerdata

The marked species differences called upon mechanistic research in order tobetter understand the process of dichloromethane-induced carcinogenesis, andthereby permit better assessment of human health risks. The main researchgroups include the one initiated by the United States National Institute forEnvironmental Health Sciences (NIEHS) (for review see e.g. Maronpot et al.1995); the one initiated by the manufactures of dichloromethane - ICI/Zeneca(for review see e.g. Green 1997); and the one initiated by the ChemicalIndustry Institute of Toxicology, CIIT (for review see e.g. Casanova andHeck 1996).

12.4.1. Cell proliferationAs chronic inhalation of dichloromethane causes liver and lung tumours inmice, any cell proliferation, hyperplasia or cellular toxicity in these organs is ofinterest for the interpretation of the tumourigenic effects.

A short-term study with mice exposed to dichloromethane at 5300-14 000 mg/m3 for 3 days showed an increased rate of DNA synthesis in thelung indicating cell proliferation, but no such effects were detected at lowerconcentrations (530 or 1800 mg/m3). Hamsters showed no evidence of cellproliferation in the lung at any concentration, and cell proliferation was notapparent in the livers of either mice or hamsters.

In another study with mice exposed to 14 000 mg/m3 there was a transientincrease in the number of cells in S-phase in the lung between days 2 and 9.Further, an acute, selective damage to the bronchiolar Clara cells wasdemonstrated, but the damage disappeared after 5 days of exposure. Therecurring but attenuated Clara cell damage at repeated exposures was correlatedto the amount of cytochrome P450. Thus, the authors speculated that theClara cell damage might be caused by formyl chloride formed in the P450metabolic pathway.

Measurements of replicative DNA synthesis as part of the chronic cancerstudy with female mice at 7100 mg/m3 revealed no sustained increase in DNAsynthesis in liver up to 78 weeks of exposure or in lung up to 26 weeks ofexposure. The only effect on replicative DNA synthesis found in parallelexperiments was a transitory increase in the hepatocellular labelling index at2 weeks in mice exposed to 28 000 mg/m3.

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Thus, these studies indicate that dichloromethane induces liver and lung tumoursin mice that are not preceded by overt cytotoxicity or sustained cell prolife-ration.

12.4.2. GenotoxicityDichloromethane is mutagenic in Salmonella, both with and without additionof rat liver S9. The bacterial mutagenicity of dichloromethane is postulated tobe mainly mediated by GSH conjugation of DCM to S-chloromethylglutathione,catalysed by cytosolic GST. This theory is strengthened by experiments showinga lower mutagenic activity in GSH-deficient strains, and a higher mutagenicactivity in transgenic strains carrying a rat theta-class GST. Mutation tests withmammalian cell cultures have been mainly negative, but in one test with Chinesehamster ovary cells HPRT-mutations were formed if supplemented with thecytosolic fraction from mouse liver. Formaldehyde, the break-down productfrom S-chloromethylglutathione, is not mutagenic in Salmonella. Althoughdichloromethane has not been shown to bind covalently to DNA, DNA single-strand breaks were induced in rat and mouse hepatocytes in vitro, but at amuch higher rate in the mouse hepatocytes. When the cells were treated witha GSH-depleting agent, less DNA damage occurred. In contrast, no DNAsingle-strand breaks were induced in hamster or human hepatocytes in vitro.DNA single-strand breaks were also formed in vitro in bronchiolar Clara cellsisolated from the lungs of mice. The DNA damage was reduced upon theaddition of a glutathione depletor.

In in vivo experiments, DNA single-strand breaks were found in hepatocytesof B6C3F1 mice exposed to 14 000 mg/m3 for 3 h, but not in hepatocytesof similarly exposed rats. DNA single-strand breaks were also detected in thelungs of mice. Pre-treatment of mice with a glutathione depletor caused adecrease in the amount of DNA damage. On an equimolar basis,dichloromethane was as potent as formaldehyde in causing DNA damage,although only a fraction of dichloromethane is converted to formaldehyde.These findings support a genotoxic mechanism for dichloromethane-inducedliver and lung tumours and suggest that the DNA damage is caused byS-chloromethylglutathione rather than by formaldehyde.

In follow-up analyses of the inhalation cancer study by Kari et al.(1993), itwas found that the frequency and pattern of H- and K-ras oncogene mutationswere similar in control tumours and dichloromethane-induced liver and lungtumours, respectively. These findings neither support, nor contradict a genotoxicmechanism for dichloromethane-induced tumours.

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The research group at CIIT has mainly focused on the possible role offormaldehyde, which is formed in the GST metabolic pathway. Only mousehepatocytes, but not hepatocytes from rats, hamsters or humans, formeddetectable amounts of DNA-protein crosslinks in vitro. RNA-formaldehydeadducts, which can be analysed with higher sensitivity than crosslinks, weredetected in hepatocytes from all rodent species and from humans with functionalGSTT1 and GSTM1 genes, but not in human cells lacking these genes. Theyield of these adducts in hepatocytes from different species was in the order:mouse > rat > human > hamster. In vivo, DNA-protein cross-links weredetected in mouse liver, but not in mouse lung or hamster liver or lung afterinhalation exposure to 1800-14 000 mg/m3 for 2 days.

In summary, we consider dichloromethane as a genotoxic substance, althoughit has not been shown to bind covalently to DNA either in vitro or in vivo.DNA single strand breaks were detected in liver and lungs of mice (the organswhere tumours were induced), but not in the liver of rats. DNA-protein crosslinkswere also detected in mouse liver, but not in mouse lung or hamster liver orlung.

12.5. Epidemiological cancer data

Excess of mortality from cancer has been found in some occupational studies,including elevated risks of cancer in biliary passages and liver, pancreas andbrain. However, there seems to be no consistent pattern in tumour appearance.Further, the findings are of borderline significance and have often not beenstrengthened when cohorts were extended. The studies are not sufficient forany firm conclusions.

12.5.1. Comparison between epidemiological and animal cancerstudiesEpidemiology data are rarely capable of refuting or confirming risk predictionsestimated from low-dose extrapolation models based on animal cancer data.In practice, it is almost impossible to show conclusively from epidemiologicdata that an agent does not produce cancer. Tollefson et al. (1990) andStayner and Bailer (1993) have assessed the minimum risk of lung and livertumours detectable in the epidemiological study reported by Hearne et al.(1987) in order to calculate the upper-bound potency of dichloromethane andcompare it to the potency derived from the rodent data. The authors conclude

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that due to the insufficient power the statistically non significant findings in theHearne study are still compatible with the human risk estimate derived fromthe rodent data.

12.6. Quantitative cancer risk assessment

No specific mutations were induced in the ras oncogenes in liver and lungtumours of exposed mice, but on the other hand, the tumours were not associatedwith cytotoxicity, hyperplasia or cell proliferation. Thus, the mode of action oftumour induction and the relative importance of genotoxicity are not known.Yet, we consider dichloromethane as genotoxic and advocate a linear extra-polation model for quantitative cancer risk estimation.

Several findings indicate that humans are less sensitive than mice, for examplethe higher yield of DNA crosslinks in mouse hepatocytes in vitro comparedto human hepatocytes, the detection of GSTT1 in the nucleus of mousehepatocytes but not in human hepatocytes, and a higher content of GSTT1 inmouse lung compared to human lung. It has also been found that ciliated cellsand Clara cells in the lung of mice contain higher levels of GSTT1 than otherlung cell types. The amount of GSTT1 in rat lung was significantly lower andconfined to Clara cells. In human lung, GSTT1 was detected at low levels ina very small number of Clara cells and ciliated cells. Green (1997) has arguedthat some of these conditions are unique for the mouse, and that the mouseis an inappropriate model for human cancer risk assessment. However, thesedifferences in GST metabolism may well be quantitative and not qualitative, aswas also pointed out by Liteplo and Meek (1998).

As the GSTT1 isoenzyme is expressed in several human tissues, it could beargued that dichloromethane-induced tumours in man, if any, does not necessarilyhave to be restricted to the liver and the lung. Also, although the epidemiologicalstudies were not considered sufficient for any firm conclusions, elevated risksof cancer in different organs were found in some studies. Another complicatingfactor is that the P450 metabolic pathway is dominating at low exposurelevels, and that the reactive metabolite in this pathway, formyl chloride, mayalso damage DNA. Still, the only experimental data available for quantitativerisk estimation are the mouse liver and lung tumours found in the NTP (1986)inhalation cancer study. This study has been the basis for all modern humancancer risk estimates.

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In the risk estimate from the US EPA in 1985, the linearised multistage modelwas applied to the increase in liver and lung tumours in female mice. Accordingto the standard procedure of the EPA, the inhalation doses were transformedto intakes in mg/kg body weight as a product of ventilation rate, concentrationand duration of exposure. The animal doses were then decreased by a factorof 12.7 to obtain the equivalent human doses, calculated as the cube root ofthe ratio of human body weight to mouse body weight. This factor, known asthe body surface area correction, is always included in the default EPA app-roach. The estimated unit risk (upper 95% confidence limit) was 4.1⋅10-6

associated with inhalation of 1 µg/m3 for a lifetime. The physiologically basedpharmacokinetic model, developed by Andersen and colleagues, used theamount of dichloromethane metabolised by the GST pathway as target dose.According to this model, estimated target tissue doses in humans exposed tolow concentrations of dichloromethane are 140- to 170-fold lower than wouldbe expected from the linear extrapolation and body surface area factors whichhave been used in conventional risk assessment methods. According to theirmodel, the corresponding unit risk would be two orders of magnitude lower,or 3.7⋅10-8 per µg/m3. The EPA adopted the pharmacokinetic approach, butincluded their standard body surface area correction factor for calculating theequivalent human doses in order to consider potential species differences inpharmacodynamics. The present EPA inhalation unit risk is 4.7⋅10-7 per µg/m3.A lifetime risk of 1⋅10-5 would thus correspond to 21 µg/m3 (IRIS 1997).

The human cancer risk predictions using physiological pharmacokinetic modelsfor dichloromethane suggest that conventional default procedures, such as thoseused by the EPA, overestimate the cancer risk by about two orders of magnitude.However, the model approaches include several assumptions, e.g. that thepharmacodynamics are species-invariant, that risk is related only to the GSTpathway, that risk is related to delivered dose per time unit and body mass(and not body surface area), that metabolic activation takes place only in theliver and lungs, and that the metabolic parameters have been accuratelydetermined. There is limited scientific evidence to support many of theseassumptions. Combining a physiological pharmacokinetic model fordichloromethane with realistic estimates of metabolic variability and a one-stage model for tumour data, the variability in cancer risk estimates varied bythree orders of magnitude. This also illustrates that the pharmacokinetic riskestimates have a large degree of uncertainty. In conclusion, the outcome of thepharmacokinetic exercises should be interpreted with caution.

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An alternative approach is to use the so-called benchmark method that hasalso recently been put forward by EPA in their proposed revised guidelines forcancer risk assessment. As in the multistage model, a dose-response model isfitted to the experimental data. The lower 95% confidence limit on the dosecorresponding to a 10% increased extra risk (BMD10) is calculated, and therisk is then extrapolated linearly towards zero. Benchmark dose calculationsbased on the NTP study gave a lowest BMD10 of 640 mg/m3 for pulmonaryadenomas and carcinomas in female mice. Linear extrapolation to a lifetimerisk of 10-5 leads to a concentration of 64 µg/m3. This concentration wasmultiplied with a factor of 0.18 to correct for continuous exposure and a factorof 4 to compensate for nonlinear metabolism. A lifetime risk of 1⋅10-5 forcontinuous exposure would thus correspond to 46 µg/m3.

12.7. Recommended health-based limit value

In the last update on dichloromethane from IMM (1990), an uncertainty factorapproach was used, as it was considered premature to evaluate thepharmacokinetic approaches. An uncertainty factor of 5000 applied to thelowest concentration of 500 ppm, where benign mammary tumours were observedin rats, led to a guideline value of 100 ppb (350 µg/m3).

In this update we have chosen an alternative to the multistage andpharmacokinetic models, namely the benchmark model, for the quantitativerisk estimate. A lifetime risk of 1⋅10-5 has previously been used by IMM inproposing low-risk levels for other carcinogenic air pollutants. In the benchmarkcalculations, this risk level would correspond to 46 µg/m3. This estimate doesnot account for species differences in metabolic capacity. Humans appear tohave a lower capacity for metabolic activation via the GST pathway than domice. Our estimate may thus be conservative in that it overestimates the truerisk. However, insufficient human data on GST metabolism of dichloromethane,especially in tissues other than liver and lung, prevent a more precise riskestimate. Due to these shortcomings, we only partially adopted thepharmacokinetic approach. This was done by correcting with a factor of 4 fornonlinear metabolism in the high dose to low dose extrapolation.

In conclusion, we propose a guideline value of 50 µg/m3, based on the benchmarkmodel.

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13. References

Allen, J., Kligerman, A., Campbell, J., Westbrook-Collins, B., Erexson, G., Kari, F., andZeiger, E. Cytogenetic analyses of mice exposed to dichloromethane. Environ.Mol.Mutagen.15(4):221-228, 1990.

Andersen, M.E., Clewell, H.J.,3d, Gargas, M.L., Smith, F.A., and Reitz, R.H. Physiologicallybased pharmacokinetics and the risk assessment process for Methylene chloride.Toxicol.Appl.Pharmacol. 87:185-205, 1987.

Andersen, M.E., Clewell, H.J.,3d, Gargas, M.L., MacNaughton, M.G., Reitz, R.H., Nolan,R.J., and McKenna, M.J. Physiologically based pharmacokinetic modeling withdichloromethane, its metabolite, carbon monoxide, and blood carboxyhemoglobin in ratsand humans. Toxicol.Appl.Pharmacol. 108(1):14-27, 1991.

Andersen, M.E., Clewell, H.J.,3rd, Mahle, D.A., and Gearhart, J.M. Gas uptake studies ofdeuterium isotope effects on dichloromethane metabolism in female B6C3F1 mice in vivo.Toxicol.Appl.Pharmacol. 128(1):158-165, 1994.

Andersen, M.E. and Krishnan, K. Physiologically based pharmacokinetics and cancerrisk assessment. Environ.Health Perspect. 102 Suppl 1:103-8:103-108, 1994.

Astra. Miljökonsekvensbedömning för verksamheten vid AB Astras anläggningar iSnäckviken. 1993. (In Swedish)

ATSDR Toxicological profile for methylene chloride. US Department of Health andHuman Services. Agency for Toxic Substances and Disease Registry. 1993.

Bell, B.P., Franks, P., Hildreth, N., and Melius, J. Methylene chloride exposure andbirthweight in Monroe County, New York. Environ.Res. 55(1):31-39, 1991.

Blocki, F.A., Logan, M.S., Baoli, C., and Wackett, L.P. Reaction of rat liver glutathioneS-transferases and bacterial dichloromethane dehalogenase with dihalomethanes.J.Biol.Chem. 269(12):8826-8830, 1994.

Bogaards, J.J., van Ommen, B., and van Bladeren, P.J. Interindividual differences in thein vitro conjugation of methylene chloride with glutathione by cytosolic glutathioneS-transferase in 22 human liver samples. Biochem.Pharmacol. 45(10):2166-2169, 1993.

Bukowski, J.A., Sargent, E.V., and Pena, B.M. Evaluation of the utility of a standardhistory questionnaire in assessing the neurological effects of solvents. Am.J.Ind.Med.22(3):337-345, 1992.

Burek, J.D., Nitschke, K.D., Bell, T.J., Wackerle, D.L., Childs, R.C., Beyer, J.E., Dittenber,D.A., Rampy, L.W., and McKenna, M.J. Methylene chloride: A two-year inhalationtoxicity and oncogenicity study in rats and hamsters. Fundam.Appl.Toxicol. 4:30-47,1984.

Casanova, M., Deyo, D.F., and Heck, H. Dichloromethane (methylene chloride): metabo-lism to formaldehyde and formation of DNA-protein cross-links in B6C3F1 mice andSyrian golden hamsters. Toxicol.Appl.Pharmacol. 114(1):162-165, 1992.

Casanova, M. and Heck, H.,d’A. Of Mice, Men, and Methylene Chloride. CITT Activities16(4):1-10, 1996.

98

Casanova, M., Conolly, R.B., and Heck, H.,d’A. DNA-protein cross-links (DPX) and cellproliferation in B6C3F1 mice but not Syrian golden hamsters exposed to dichloromethane:pharmacokinetics and risk assessment with DPX as dosimeter. Fundam.Appl.Toxicol.31(1):103-116, 1996.

Casanova, M., Bell, D., and Heck, H. Dichloromethane metabolism to formaldehyde andreaction of formaldehyde with nucleic acids in hepatocytes of rodents and humans withand without glutathione S-transferase T1 and M1 genes. Fundam.Appl.Toxicol. 37(2):168-180, 1997.

Cherry, N., Venables, H., Waldron, H.A., and Wells, G.G. Some observations on workersexposed to methylene chloride. Br.J.Ind.Med. 38:351-355, 1981.

Clewell, H.J.,3rd, Lee, T.S., and Carpenter, R.L. Sensitivity of physiologically basedpharmacokinetic models to variation in model parameters: methylene chloride. Risk.Anal.14(4):521-531, 1994.

Clewell, H.J.,3rd Incorporating biological information in quantitative risk assessment: anexample with methylene chloride. Toxicology 102(1-2):83-94, 1995.

Clewell, H.J.,3d, Gentry, P.R., and Gearhart, J.M. Investigation of the potential impact ofbenchmark dose and pharmacokinetic modelling in noncancer risk assessment.J.Toxicol.Environ.Health 52:475-515, 1997.

Crump, K.S. A new method for determining allowable daily intakes. Fundam.Appl.Toxicol.4:854-871, 1984.

Crump, K.S. Calculation of benchmark doses from continous data. Risk.Anal. 15:79-89,1995.

Dankovic, D.A. and Bailer, A.J. The impact of exercise and intersubject variability ondose estimates for dichloromethane derived from a physiologically based pharmacokineticmodel. Fundam.Appl.Toxicol. 22(1):20-25, 1994.

DeMarini, D.M., Shelton, M.L., Warren, S.H., Ross, T.M., Shim, J.Y., Richard, A.M., andPegram, R.A. Glutathione S-Transferase-Mediated Indiction of GC→AT Transitions byHalomethanes in Salmonella. Environ.Mol.Mutagen. 30:440-447, 1997.

Devereux, T.R., Foley, J.F., Maronpot, R.R., Kari, F., and Anderson, M.W. Ras proto-oncogene activation in liver and lung tumors from B6C3F1 mice exposed chronically tomethylene chloride. Carcinogenesis 14(5):795-801, 1993.

Dillon, D., Edwards, I., Combes, R., McConville, M., and Zeiger, E. The role of glutathionein the bacterial mutagenicity of vapour phase dichloromethane. Environ.Mol.Mutagen.20(3):211-217, 1992.

DiVincenzo, G.D., Yanno, F.J., and Astill, B.D. Human and canine exposures to methylenechloride vapor. Am.Ind.Hyg.Assoc.J. 33:125-135, 1972.

DiVincenzo, G.D. and Kaplan, C.J. Uptake, metabolism and elimination of methylenechloride vapor by humans. Toxicol.Appl.Pharmacol. 59:130-140, 1981.

Dourson, M.L., Felter, S.P., and Robinson, D. Evolution of science-based uncertaintyfactors in noncancer risk assessment. Regul.Toxicol.Pharmacol. 24:108-120, 1996.

99

ECETOC Methylene Chloride: Human risk assessment using experimental data. Technicalreport No 32. European Centre for Ecotoxicology and Toxicology of Chemicals, Brussels,1988.

Engström, J. and Bjurström, R. Exposure to methylene chloride. Content in subcutaneousadipose tissue. Scand.J.Work.Environ.Health 3:215, 1977.

EPA Updated Carcinogenicity assessment of dichloromethane. U S Environmental ProtectionAgency, Office of Health and Environmental Assessment. 1985.

EPA Update to the Health assessment document and addendum for Dichloromethane(Methylene Chloride): Pharmacokinetics, Mechanism of Action, and Epidemiology.U S Environmental Protection Agency. 1987.

EPA Proposed Guidelines for carcinogenic risk assessment EPA/600/p-92/003C April 96.US Environment Protection Agency, Washington DC, USA. 1996.

Foley, J.F., Tuck, P.D., Ton, T.V., Frost, M., Kari, F., Anderson, M.W., and Maronpot,R.R. Inhalation exposure to a hepatocarcinogenic concentration of methylene chloridedoes not induce sustained replicative DNA synthesis in hepatocytes of female B6C3F1mice. Carcinogenesis 14(5):811-817, 1993.

Foster, J.R., Green, T., Smith, L.L., Lewis, R.W., Hext, P.M., and Wyatt, I. Methylenechloride—an inhalation study to investigate pathological and biochemical events occurringin the lungs of mice over an exposure period of 90 days. Fundam.Appl.Toxicol. 18(3):376-388, 1992.

Foster, J.R., Green, T., Smith, L.L., Tittensor, S., and Wyatt, I. Methylene chloride: aninhalation study to investigate toxicity in the mouse lung using morphological, biochemicaland Clara cell culture techniques. Toxicology 91(3):221-234, 1994.

Friedlander, B.R., Hearne, T., and Hall, S. Epidemiologic investigation of employeeschronically exposed to Methylene chloride. Mortality analysis. J.Occup.Med. 20(10):657-666, 1978.

Gargas, M.L., Clewell, H.J.,3rd, and Andersen, M.E. Metabolism of inhaled dihalomethanesin vivo: differentiation of kinetic constants for two independent pathways.Toxicol.Appl.Pharmacol. 82(2):211-223, 1986.

Ghittori, S., Marraccini, P., Franco, G., and Imbriani, M. Methylene chloride exposure inindustrial workers. Am.Ind.Hyg.Assoc.J. 54(1):27-31, 1993.

Gibbs, G.W., Amsel, J., and Soden, K. A cohort mortality study of cellulose triacetate-fiber workers exposed to methylene chloride. J.Occup.Environ.Med. 38(7):693-697, 1996.

Graves, R.J., Callander, R.D., and Green, T. The role of formaldehyde and S-chloro-methylglutathione in the bacterial mutagenicity of methylene chloride. Mutat.Res. 320(3):235-243, 1994a.

Graves, R.J., Coutts, C., Eyton-Jones, H., and Green, T. Relationship between hepaticDNA damage and methylene chloride-induced hepatocarcinogenicity in B6C3F1 mice.Carcinogenesis 15(5):991-996, 1994b.

Graves, R.J., Coutts, C., and Green, T. Methylene chloride-induced DNA damage: aninterspecies comparison. Carcinogenesis 16(8):1919-1926, 1995.

100

Graves, R.J., Trueman, P., Jones, S., and Green, T. DNA sequence analysis of methylenechloride-induced HPRT mutations in Chinese hamster ovary cells: comparison with themutation spectrum obtained for 1,2-dibromoethane and formaldehyde. Mutagenesis11(3):229-233, 1996.

Graves, R.J. and Green, T. Mouse liver glutathione S-transferase mediated metabolism ofmethylene chloride to a mutagen in the CHO/HPRT assay. Mutat.Res. 367(3):143-150,1996.

Green, T. The metabolic activation of dichloromethane and chlorofluoromethane in abacterial mutation assay using Salmonella typhimurium. Mutation Research 118:277-288,1983.

Green, T. Changes in metabolism during toxicity tests. Xenobiotica 20(11):1233-1240,1990a.

Green, T. Species differences in carcinogenicity: the role of metabolism in human riskevaluation. Teratog.Carcinog.Mutagen. 10(2):103-113, 1990b.

Green, T. Methylene chloride induced mouse liver and lung tumours: an overview of therole of mechanistic studies in human safety assessment. Hum.Exp.Toxicol. 16(1):3-13,1997.

Green, T., Provan, W.M., Collinge, D.C., and Guest, A.E. Molecular interactions ofinhalated Methylene chloride in rats and mice. Toxicol.Appl.Pharmacol. 93:1-10, 1988.

Haag-Grönlund, M., Fransson-Steen, R., and Victorin, K. Application of the benchmarkmethod to risk assessment of trichloroethene. Regul.Toxicol.Pharmacol. 21:261-269,1995.

Hall, A.H. and Rumack, B.H. Methylene chloride exposure in furniture-stripping shops:ventilation and respirator use practices. J.Occup.Med. 32(1):33-37, 1990.

Hallier, E., Langhof, T., Dannappel, D., Leutbecher, M., Schröder, K., Goergens, H.W.,Müller, A., and Bolt, H.M. Polymorphism of glutathione conjugation of methyl bromide,ethylene oxide and dichloromethane in human blood: influence on the induction of sisterchromatid exchanges (SCE) in lymphocytes. Arch.Toxicol. 67(3):173-178, 1993.

Hallier, E., Schröder, K.R., Asmuth, K., Dommermuth, A., Aust, B., and Goergens, H.W.Metabolism of dichloromethane (methylene chloride) to formaldehyde in humanerythrocytes: influence of polymorphism of glutathione transferase theta (GST T1-1).Arch.Toxicol. 68(7):423-427, 1994.

Hashmi, M., Dechert, S., Dekant, W., and Anders, M.W. Bioactivation of:13C:dichloromethane in mouse, rat, and human liver cytosol: 13C nuclear magneticresonance spectroscopic studies. Chem.Res.Toxicol. 7(3):291-296, 1994.

Haun, C.C., Vernot, E.H., and Darmer, K.I.J. Continous animal exposure to low levels ofdochloromethane.Proceedings, 3rd Annual Conference on Environmental Toxicology.Dayton, Ohio, Wright-Patterson Air Force Base. AMRL-TR-72-130 Paper No 12 AD 773766:199-208, 1972.

Hayes, J. and Pulford, D. The glutathione S-transferase supergene family: Regulation ofGST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance.Crit. Rev. Biochem. Mol. Biol. 30(6):445-600, 1995.

101

Hearne, F.T., Grose, F., Pifer, J.W., Friedlander, B.R., and Raleigh, R.L. Methylene chloridemortality study: Dose-response characterization and animal model comparison.J.Occup.Med. 29(3):217-228, 1987.

Hearne, F.T., Pifer, J.W., and Grose, F. Absence of adverse mortality effects in workersexposed to methylene chloride: an update. J.Occup.Med. 32(3):234-240, 1990.

Hegi, M.E., Söderkvist, P., Foley, J.F., Schoonhoven, R., Swenberg, J.A., Kari, F., Maronpot,R., Anderson, M.W., and Wiseman, R.W. Characterization of p53 mutations in methylenechloride-induced lung tumors from B6C3F1 mice. Carcinogenesis 14(5):803-810, 1993.

Hegi, M.E., Devereux, T.R., Dietrich, W.F., Cochran, C.J., Lander, E.S., Foley, J.F., Maronpot,R.R., Anderson, M.W., and Wiseman, R.W. Allelotype analysis of mouse lung carcinomasreveals frequent allelic losses on chromosome 4 and an association between allelicimbalances on chromosome 6 and K-ras activation. Cancer Res. 54(23):6257-6264, 1994.

Heineman, E.F., Cocco, P., Gömez, M.R., Dosemeci, M., Stewart, P.A., Hayes, R.B., Zahm,S.H., Thomas, T.L., and Blair, A. Occupational exposure to chlorinated aliphatichydrocarbons and risk of astrocytic brain cancer. Am.J.Ind.Med. 26(2):155-169, 1994.

Herr, D.W. and Boyes, W.K. A comparison of the acute neuroactive effects ofdichloromethane, 1,3-dichloropropane, and 1,2-dichlorobenzene on rat flash evokedpotentials (FEPs). Fundam.Appl.Toxicol. 35(1):31-48, 1997.

Hetrick, D.M., Jarabek, A.M., and Travis, C.C. Sensitivity analysis for physiologicallybased pharmacokinetic models. J.Pharmacokinet.Biopharm. 19(1):1-20, 1991.

Hoffmann, P., Müller, S.P., Heinroth, K., Büchner, E., Richards, D., and Toraason, M.Calcium dynamics in cardiac myocytes as a target of dichloromethane cardiotoxicity.Arch.Toxicol. 70(3-4):158-163, 1996.

Hughes, N.J. and Tracey, J.A. A case of methylene chloride (nitromors) poisoning,effects on carboxyhaemoglobin levels. Hum.Exp.Toxicol. 12(2):159-160, 1993.

IARC. Some halogenated hydrocarbons and pesticide exposure. IARC Monograph onthe Evaluation of Carcinogenic Risk of Chemicals to Humans. Lyon:International Agencyfor Research on Cancer. Volume 41:43-85, 1986.

IMM. Fransson, R., and Ahlborg, U.G. Medicinska och hygieniska effekter av metylen-klorid i omgivningsluft. (In Swedish). Stockholm:The Swedish Environmental ProtectionBoard. Report No. 3199, 1986.

IMM. Fransson, R., and Ahlborg, U.G. Medicinska och hygieniska effekter av diklormetan,trikloreten och tetrakloreten i omgivningsluft (In Swedish with English summary).Stockholm:National Institute of Environmental Medicine. Report No. 5/90, 1990.

Ingelman-Sundberg, M. and Johansson, I. The molecular genetics of the human drugmetabolizing cytochrome P450s. In: Advances in Drug Metabolism in Man (G. Pacificiand G.N.Fracchia,eds), EUR 15439 EN, EC, Luxembourg, Office for Official Publicationsof the European Communities. :543-585, 1995.

IRIS. Integrated risk information system. Dichloromethane. U.S. Environmental ProtectionAgency. 1997.

Johanson, G. and Näslund, P.H. Spreadsheet programming - a new approach inphysiologically based modeling of solvent toxicokinetics. Toxicol.Lett. 41:115-127, 1988.

102

Jongen, W.M.F., Harmsen, E.G.M., Alink, G.M., and Koeman, J.H. The effect of glutathioneconjugation and microsomal oxidation on the mutagenicity of dichloromethane inS. typhimurium. Mutat.Res. 95:183-189, 1982.

Juronen, E., Tasa, G., Uusküla, M., Pooga, M., and Mikelsaar, A.-V. Purification,characterization and tissue distribution of human class Theta glutathione S-transferaseT1-1. Biochem. Mol. Biol. Internat. 39(1):21-29, 1996.

Kalliomaa, K., Haag-Grönlund, M., and Victorin, K. A new model function for continousdata sets in health risk assessment of chemicals using the benchmark dose concept.Regul. Toxicol. Pharmacol. 27:98-107, 1998.

Kanno, J., Foley, J.F., Kari, F., Anderson, M.W., and Maronpot, R.R. Effect of methylenechloride inhalation on replicative DNA synthesis in the lungs of female B6C3F1 mice.Environ.Health Perspect. 101 Suppl 5:271-6:271-276, 1993.

Kari, F.W., Foley, J.F., Seilkop, S.K., Maronpot, R.R., and Anderson, M.W. Effect ofvarying exposure regimens on methylene chloride-induced lung and liver tumors infemale B6C3F1 mice. Carcinogenesis 14(5):819-826, 1993.

Kelly, M. Case reports of individuals with oligospermia and Methylene chloride exposures.Reproductive Toxicology 2:13-17, 1988.

KemI. Risk reduction of chemicals. A government commission report. Report No 1/91.The Swedish National Chemicals Inspectorate, 1991a.

KemI. Effects on Reproduction of Dichloromethane, n-hexane and 1,1,1-trichloroethane.Report No 10/91. The Swedish National Chemicals Inspectorate, 1991b.

KemI. Chlorine and chlorinated compounds in Sweden. Report on governmental assignment.Report No 5/95. The Swedish National Chemicals Inspectorate, 1995.

KemI.The Swedish Product Register Flödesanalyser över kemiska ämnen; (Flow analysesof chemical substances), 1992; 1993; 1994; 1995; 1996. The Swedish National ChemicalsInspectorate, 1996. (In Swedish)

KemI. Tillsyn med stöd av LKP, Lagen (1985:426) om kemiska produkter (Supervisionaccording to the Act (1985:426) on Chemical Products). The Swedish National ChemicalsInspectorate, 1997. (In Swedish)

KemI. Information from the Swedish Product Register (personal communication). TheSwedish National Chemicals Inspectorate, 1998.

Ketterer, B., Meyer, D.J., Lalor, E., Johnson, P., Guengerich, F.P., Distlerath, L.M., Reilly,P.E.B., Kadlubar, F.F., Flammang, T.J., Yamazoe, Y., and Beaune, P.H. A comparison oflevels of glutathione transferases, cytochromes P450 and acetyltransferases in humanlivers. Biochem. Pharmacol. 41(4):635-638, 1991.

Kim, N.Y., Park, S.W., and Suh, J.K. Two fatal cases of dichloromethane or chloroformpoisoning. J.Forensic Sci. 41(3):527-529, 1996.

Kim, S.K. and Kim, Y.C. Effect of a single administration of benzene, toluene or m-xyleneon carboxyhaemoglobin elevation and metabolism of dichloromethane in rats.J.Appl.Toxicol. 16(5):437-444, 1996.

103

Kimmel, C.A. Quantitative approaches to human risk assessment for noncancer healtheffects. Neurotoxicology 11:189-198, 1990.

Kirschman, J.C., Brown, N.M., Coots, R.H., and Morgareidge, K. Review of investigationsof dichloromethane metabolism and subchronic oral toxicity as the basis for the designof chronic oral studies in rats and mice. Food Chem. Toxicol. 24(9):943-949, 1986.

Kitchin, K.T. and Brown, J.L. Biochemical effects of three carcinogenic chlorinatedmethanes in rat liver. Teratog.Carcinog.Mutagen. 9(1):61-69, 1989.

Kjellstrand, P., Månsson, L., Holmquist, B., and Jonsson, I. Tolerance during inhalationof organic solvents. Pharmacol.Toxicol. 66(5):409-414, 1990.

Koñená, L., Frantík, E., and VodiHková, A. Methylene chloride does not impair vigilanceperformance at blood levels simulating limit exposure. Act.Nerv.Super.(Praha) 32(1):35-37, 1990.

Kramers, P.G., Mout, H.C., Bissumbhar, B., and Mulder, C.R. Inhalation exposure inDrosophila mutagenesis assays: experiments with aliphatic halogenated hydrocarbons,with emphasis on the genetic activity profile of 1,2-dichloroethane. Mutat.Res. 252(1):17-33, 1991.

Lanes, S.F., Cohen, A., Rothman, K.J., Dreyer, N.A., and Soden, K.J. Mortality of cellulosefiber production workers. Scand.J.Work.Environ.Health 16(4):247-251, 1990.

Lanes, S.F., Rothman, K.J., Dreyer, N.A., and Soden, K.J. Mortality update of cellulosefiber production workers. Scand.J.Work.Environ.Health 19(6):426-428, 1993.

Lash, A.A., Becker, C.E., So, Y., and Shore, M. Neurotoxic effects of methylene chloride:are they long lasting in humans? Br.J.Ind.Med. 48(6):418-426, 1991.

Lefevre, P.A. and Ashby, J. Evaluation of dichloromethane as an inducer of DNAsynthesis in the B6C3F1 mouse liver. Carcinogenesis 10(6):1067-1072, 1989.

Leikin, J.B., Kaufman, D., Lipscomb, J.W., Burda, A.M., and Hryhorczuk, D.O. Methylenechloride: report of five exposures and two deaths. Am.J.Emerg.Med. 8(6):534-537, 1990.

Liniger, B. and Sigrist, T. Carboxyhemoglobinemia caused by dichloromethane exposurewith dermatologic effects: Hautarzt. 45(1):8-11, 1994.

Liteplo, R.G., Long, G.W., and Meek, M.E. Relevance of carcinogenecity bioassays inmice in assesing potential health risks associated with exposure to methylene chloride.Human & Experimental Toxicology 17:84-87, 1998.

Long, G., Meek, M.E., Caldwell, I., Bartlett, S., and Savard, S. Dichloromethane: Evaluationof risks to health from environmental exposure in Canada. Environ. Carcino. & Ecotox.Revs. C12(2):305-318, 1994.

Lorenz, J., Glatt, H.R., Fleischmann, R., Ferlinz, R., and Oesch, F. Drug metabolism in manand its relationship to that in three rodent species: Monooxygenase, epoxide hydroxylase,and glutathione S-transferase activities in subcellular fractions of lung and liver. Biochem.Med. 32:43-56, 1984.

Löf, A., Rannug, A., Warholm, M., and Johanson, G. Toxicokinetics of methyl chloridein relation to polymorphic distribution of glutathione S-transferase in volunteers. 1998.(manuscript)

104

Mainwaring, G.W., Nash, J., Davidson, M., and Green, T. Isolation of a mouse thetaglutathione S-transferase active with methylene chloride. Biochem.J. 314(Pt 2):445-448,1996a.

Mainwaring, G.W., Williams, S.M., Foster, J.R., Tugwood, J., and Green, T. The distri-bution of theta-class glutathione S-transferases in the liver and lung of mouse, rat andhuman. Biochem.J. 318(Pt 1):297-303, 1996b.

Mainwaring, G.W., Foster, J.R., and Green, T. Nuclear and cellular immunolocalization ofThetaclass glutathione S-transferase GSTT1-1 in the liver and lung of the mouse. Biochem.J.329:431-432, 1998.

Maltoni, C., Cotti, G., and Perino, G. Long-term carcinogenicity bioassays on methylenechloride administered by ingestion to Sprague-Dawley rats and Swiss mice and byinhalation to Sprague-Dawley Rats. Annals New York Academy of Sciences 534:352-366,1988.

Manno, M., Rugge, M., and Cocheo, V. Double fatal inhalation of dichloromethane.Hum.Exp.Toxicol. 11(6):540-545, 1992.

Maronpot, R.R., Devereux, T.R., Hegi, M., Foley, J.F., Kanno, J., Wiseman, R., andAnderson, M.W. Hepatic and pulmonary carcinogenicity of methylene chloride in mice:a search for mechanisms. Toxicology 102(1-2):73-81, 1995.

Mattsson, J.L., Albee, R.R., and Eisenbrandt, D.L. Neurotoxicologic evaluation of ratsafter 13 weeks of inhalation exposure to dichloromethane or carbon monoxide.Pharmacol.Biochem.Behav. 36(3):671-681, 1990.

Mennear, J.H., McConnell, E.E., Huff, J.E., Renne, R.A., and Giddens, E. Inhalationtoxicology and carcinogenesis studies of methylene chloride (Dichloromethane) in F344/NRats and B6C3F1 Mice. Annals New York Academy of Sciences :343-351, 1988.

Meyer, D.J., Coles, B., Pemble, S.E., Gilmore, K.S., Fraser, G.M., and Ketterer, B. Theta,a new class of glutathione transferases purified from rat and man. Biochem.J. 274:409-414, 1991.

Mirer, F.E., Silverstein, M., and Park, R. Methylene Chloride and cancer of the pancreas[letter]. J.Occup.Med. 30:475-476, 1988.

Müller, S., Weise, M., Krug, T., and Hoffmann, P. Adrenergic cardiovascular actions inrats as affected by dichloromethane exposure. Biomed.Biochim.Acta 50(3):307-311, 1991.

Nelson, H.H., Wiencke, J.K., Christiani, D.C., Cheng, T.J., Zuo, Z.-F., Schwartz, B.S., Lee,B.K., Spitz, M.R., Wang, M., Xu, X.P., and Kelsey, K.T. Ethnic differences in theprevalence of the homozygous deleted genetype of glutathione S-transferase theta.Carcinogenesis 16(5):1243-1245, 1995.

Nitschke, K.D., Burek, J.D., Bell, T.J., Rampy, L.W., and McKenna, M.J. Methylenechloride: A 2-year inhalation toxicity and oncogenicity study in rats. Fundam.Appl.Toxicol.11:48-59, 1988.

NTP. Toxicology and carcinogenesis studies of dichloromethane (methylene chloride)in F344/N rats and B6C3F1 mice (Inhalation studies). NTP Technical report TR306.National Toxicology Program, Research Triangle Park, NC, USA. 1986.

105

Oda, Y., Yamazaki, H., Thier, R., Ketterer, B., Guengerich, F.P., and Shimada, T. A newSalmonella typhimurium NM5004 strain expressing rat glutathione S-transferase 5-5: usein detection of genotoxicity of dihaloalkanes using an SOS/umu test system. Carcinogene-sis 17(2):297-302, 1996.

Ott, M.G., Skory, L.K., Holder, B.B., Bronson, J.M., and Williams, P.R. Health evaluationof employees occupationally exposed to methylene chloride. Scand.J.Work.Environ.Health9(Suppl 1):1-38, 1983.

Ottenwälder, H. and Peter, H. DNA binding assay of methylene chloride in rats and mice.Arch.Toxicol. 63(2):162-163, 1989.

Ottenwälder, H., Jäger, R., Thier, R., and Bolt, H.M. Influence of cytochrome P-450inhibitors on the inhalative uptake of methyl chloride and methylene chloride in maleB6C3F1 mice. Arch.Toxicol.Suppl. 13:258-61:258-261, 1989.

Pankow, D. and Hoffmann, P. Dichloromethane metabolism to carbon monoxide can beinduced by isoniazid, acetone and fasting. Arch.Toxicol.Suppl. 13:302-3:302-303, 1989.

Pankow, D., Kretschmer, S., and Weise, M. Effect of pyrazole on dichloromethanemetabolism to carbon monoxide. Arch.Toxicol.Suppl. 14:246-8:246-248, 1991a.

Pankow, D., Matschiner, F., and Weigmann, H.J. Influence of aromatic hydrocarbons onthe metabolism of dichloromethane to carbon monoxide in rats. Toxicology 68(1):89-100,1991b.

Pankow, D. and Jagielki, S. Effect of methanol or modifications of the hepatic glutathioneconcentration on the metabolism of dichloromethane to carbon monoxide in rats.Hum.Exp.Toxicol. 12(3):227-231, 1993.

Pankow, D., Damme, B., and Schrör, K. Acetylsalicylic acid—inducer of cytochromeP-450 2E1? Arch.Toxicol. 68(4):261-265, 1994.

Paterson, S. and Mackay, D. Correlation of tissue, blood, and air partition coefficientsof volatile organic chemicals. Br.J.Ind.Med. 46(5):321-328, 1989.

Pelekis, M. and Krishnan, K. Assessing the relevance of rodent data on chemicalinteractions for health risk assessment purposes: a case study with dichloromethane-toluene mixture. Regul.Toxicol.Pharmacol. 25(1):79-86, 1997.

Peter, H., Deutschmann, S., Reichel, S., and Hallier, E. Metabolism of methyl chloride byhuman erythocytes. Arch.Toxicol. 63:351-355, 1989.

Portier, C.J. and Kaplan, N.L. Variability of safe dose estimates when using complicatedmodels of the carcinogenic process. A case study: methylene chloride. Fundam.Appl.Toxicol. 13(3):533-544, 1989.

Rank, J. and Nielsen, M.H. Evaluation of the Allium anaphase-telophase test in relationto genotoxicity screening of industrial wastewater. Mutat.Res. 312(1):17-24, 1994.

Rebert, C.S., Matteucci, M.J., and Pryor, G.T. Acute effects of inhaled dichloromethaneon the EEG and sensory-evoked potentials of Fischer-344 rats. Pharmacol.Biochem.Behav.34(3):619-629, 1989.

106

Reitz, R.H., Mendrala, A.L., Park, C.N., Andersen, M.E., and Guengerich, F.P. Incorporationof in vitro enzyme data into the physiologically-based pharmacokinetic (PB-PK) modelfor methylene chloride: implications for risk assesment. Toxicol.Lett. 43:97-116, 1988.

Reitz, R.H., Mendrala, A.L., and Guengerich, F.P. In vitro metabolism of methylenechloride in human and animal tissues: use in physiologically based pharmacokineticmodels. Toxicol.Appl.Pharmacol. 97(2):230-246, 1989.

Reitz, R.H. Estimating the risk of human cancer associated with exposure to methylenechloride. Ann.Ist.Super.Sanita. 27(4):609-614, 1991.

Rhomberg, L. Use of quantitative modelling in methylene chloride risk assessment.Toxicology 102(1-2):95-114, 1995.

Scholz, J., Klapperstück, M., Weise, M., and Hoffmann, P. Acute effects of dichloromethaneon arrhythmia development during the early phase of myocardial ischemia and reperfusionin the rat. Arch.Toxicol.Suppl. 14:128-31:128-131, 1991.

Schröder, K.R., Hallier, E., Peter, H., and Bolt, H.M. Dissociation of a new glutathioneS-transferase acticity in human erythrocytes. Biochem.Pharmacol. 43(8):1671-1674, 1992.

Serota, D.G., Thakur, A.K., Ulland, B.M., Kirschman, J.C., Brown, M.M., and Coots, R.H.A Two-year drinking-water study of Dichloromethane in rodents. II. mice. Fd ChemToxic 24(9):959-963, 1986.

Sherratt, P., Pulford, D., Harrison, D., Green, T., and Hayes, J. Evidence that human classTheta glutathione S-transferase T1-1 can catalyse the activation of dichloromethane, aliver and lung carcinogen in the mouse. Comparison of the tissue distribution of GSTT1-1 with that of classes Alpha, Mu and Pi GST in human. Biochem.J. 326 (Pt 3):837-846, 1997.

Shusterman, D., Quinlan, P., Lowengart, R., and Cone, J. Methylene chloride intoxicationin a furniture refinisher. A comparison of exposure estimates utilizing workplace airsampling and blood carboxyhemoglobin measurements. J.Occup.Med. 32(5):451-454, 1990.

Snyder, R.W., Mishel, H.S., and Christensen, G.C.,3d Pulmonary toxicity following exposureto methylene chloride and its combustion product, phosgene. Chest 101(3):860-861, 1992.

Soden, K.J. An evaluation of chronic methylene chloride exposure. J.Occup.Med. 35(3):282-286, 1993.

Soden, K.J., Marras, G., and Amsel, J. Carboxyhemoglobin levels in methylene chloride-exposed employees :see comments: J.Occup.Environ.Med. 38(4):367-371, 1996.

Stayner, L.T. and Bailer, A.J. Comparing toxicologic and epidemiologic studies: methylenechloride—a case study. Risk.Anal. 13(6):667-673, 1993.

Stewart, R.D. and Dodd, H.C. Absorption of carbon tetrachloride, trichloroethylene,tetrachloroethylene, methylene chloride, and 1,1,1-trichloroethane through the humanskin. Am.Ind.Hyg.Assoc.J. 25:439-446, 1964.

Taskinen, H., Lindbohm, M.-L., and Hemminki, K. Spontaneous abortion among womenworking in the pharmaceutical industry. Br.J.Ind.Med. 43:199-205, 1986.

107

Tates, A.D., Grummt, T., van Dam, F.J., de Zwart, F., Kasper, F.J., Rothe, R., Stirn, H.,Zwinderman, A.H., and Natarajan, A.T. Measurement of frequencies of HPRT mutants,chromosomal aberrations, micronuclei, sister-chromatid exchanges and cells with highfrequencies of SCEs in styrene/dichloromethane-exposed workers. Mutat.Res. 313(2-3):249-262, 1994.

Tay, P., Tan, K.T., and Sam, C.T. Fatal gassing due to methylene chloride - a case report.Singapore.Med.J. 36(4):444-445, 1995.

Thier, R., Taylor, J.B., Pemble, S.E., Humphries, W.G., Persmark, M., Ketterer, B., andGuengerich, F.P. Expression of mammalian glutathione S-transferase 5-5 in Salmonellatyphimurium TA535 leads to base-pair mutations upon exposure to dihalomethanes.Proc. Natl. Acad. Sci. USA 90:8576-8580, 1993.

Thier, R., Wiebel, F.A., Schulz, T.G., Hinke, A., Brüning, T., and Bolt, H.M. Comparisonof GST Theta activity in liver and kidney of four species. Arch.Toxicol.Suppl. 20:471-474, 1998.

Thomas, R.S., Yang, R.S., Morgan, D.G., Moorman, M.P., Kermani, H.R., Sloane, R.A.,O’Connor, R.W., Adkins, B.,Jr., Gargas, M.L., and Andersen, M.E. PBPK modeling/Monte Carlo simulation of methylene chloride kinetic changes in mice in relation to ageand acute, subchronic, and chronic inhalation exposure. Environ.Health Perspect.104(8):858-865, 1996.

Tollefson, L., Lorentzen, R.J., Brown, R.N., and Springer, J.A. Comparison of the cancerrisk of methylene chloride predicted from animal bioassay data with the epidemiologicevidence: see comments: Risk.Anal. 10(3):429-435, 1990.

Tomenson, J., Bonner, S., Heijne, C., Farrar, D., and Cummings, T. Mortality of workersexposed to methylene chloride employed at a plant producing cellulose triacetate filmbase. Occupational & Environmental Medicine 54(7):470-476, 1997.

Warholm, M., Rane, A., Alexandrie, A.-K., Monaghan, G., and Rannug, A. Genotypic andphenotypic determination of polymorphic glutathione transferase T1 in a Swedish popu-lation. Pharmacogenetics 5:252-254, 1995.

Westbrook-Collins, B., Allen, J.W., Sharief, Y., and Campbell, J. Further evidence thatdichloromethane does not induce chromosome damage. J.Appl.Toxicol. 10(2):79-81, 1990.

WHO Methylene Chloride. Environmental Health Criteria 32. WHO/IPCS Geneva. 1984.

WHO Methylene Chloride (Second Edition); Environmental Health Criteria 164, WHO/IPCS Geneva. 1996.

WHO Air Quality Guidelines for Europe. Copenhagen:World Health Organization, 1998.(in press)

Wirkner, K., Damme, B., Poelchen, W., and Pankow, D. Effect of long-term ethanolpretreatment on the metabolism of dichloromethane to carbon monoxide in rats.Toxicol.Appl.Pharmacol. 143(1):83-88, 1997.

Åstrand, I., Övrum, P., and Carlsson, A. Exposure to methylene chloride. I. Its concent-ration in alveolar air and blood during rest and exercise and its metabolism. Scand.J.Work.Environ.Health 1:78, 1975.

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