INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY Environmental Health Criteria 208 CARBON TETRACHLORIDE This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the United Nations Environment Programme, the International Labour Organisation, or the World Health Organization. First draft prepared by Ms J. de Fouw, National Institute of Public Health and the Environment, Bilthoven, the Netherlands Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization, and produced within the framework of the Inter-Organization Programme for the Sound Management of Chemicals. World Health Organization Geneva, 1999 The International Programme on Chemical Safety (IPCS), established in 1980, is a joint venture of the United Nations Environment Programme (UNEP), the International Labour Organisation (ILO), and the World Health Organization (WHO). The overall objectives of the IPCS are to establish the scientific basis for assessment of the risk to human health and the environment from exposure to chemicals, through international peer review processes, as a prerequisite for the promotion of chemical safety, and to provide technical assistance in strengthening national capacities for the sound management of chemicals. The Inter-Organization Programme for the Sound Management of Chemicals (IOMC) was established in 1995 by UNEP, ILO, the Food and Agriculture Organization of the United Nations, WHO, the United Nations Industrial Development Organization, the United Nations Institute for Training and Research, and the Organisation for Economic Co-operation and Development (Participating Organizations), following recommendations made by the 1992 UN Conference on Environment and Development to strengthen cooperation and increase coordination in the field of chemical safety. The purpose of the IOMC is to promote coordination of the policies and activities pursued by the Participating Organizations, jointly or separately, to achieve the sound management of chemicals in relation to human health and the environment. WHO Library Cataloguing in Publication Data Carbon tetrachloride. (Environmental health criteria ; 208) 1.Carbon tetrachloride - toxicity 2.Environmental exposure I.International Programme on Chemical Safety II.Series ISBN 92 4 157208 6 (NLM Classification: QD 305.H5) ISSN 0250-863X The World Health Organization welcomes requests for permission to reproduce or translate its publications, in part or in full. Applications and enquiries should be addressed to the Office of Publications, World Health Organization, Geneva, Switzerland, which will be glad to provide the latest information on any changes made to the text, plans for new editions, and reprints and translations already available. (c) World Health Organization 1999 Publications of the World Health Organization enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Convention. All rights reserved. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of any country, territory, city, or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. CONTENTS ENVIRONMENTAL HEALTH CRITERIA FOR CARBON TETRACHLORIDE PREAMBLE ABBREVIATIONS 1. SUMMARY 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS 2.1. Identity 2.2. Physical and chemical properties 2.3. Conversion factors 2.4. Analytical methods 2.4.1. Sampling and analysis in air 2.4.2. Sampling and analysis in water 2.4.3. Sampling and analysis in biological samples 220.127.116.11 Blood and tissues 18.104.22.168 Urine 22.214.171.124 Fish 2.4.4. Sampling and analysis in foodstuffs 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1. Natural occurrence 3.2. Anthropogenic sources 3.2.1. Production 126.96.36.199 Direct production and procedures 188.8.131.52 Indirect production 184.108.40.206 Emissions 3.2.2. Uses 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION 4.1. Transport and distribution between media 4.1.1. Transport 4.1.2. Distribution 4.1.3. Removal from the atmosphere; global warming potential 4.1.4. Removal from water 4.1.5. Removal from soil 4.2. Abiotic degradation 4.2.1. Degradation in atmosphere 220.127.116.11 Photodegradation 18.104.22.168 Photolysis 22.214.171.124 Ozone-depletion potential 4.2.2. Degradation in water 4.2.3. Other degradation processes 4.3. Biotic degradation 4.3.1. Aerobic 4.3.2. Anaerobic 4.4. Bioaccumulation 5. CONCENTRATIONS IN THE ENVIRONMENT AND EXPOSURE 5.1. Environmental levels 5.1.1. Air 5.1.2. Water 5.1.3. Soil and sediment 5.1.4. Biota 5.2. General population exposure 5.2.1. Outdoor air 5.2.2. Indoor air 5.2.3. Drinking-water 5.2.4. Foodstuffs 5.2.5. Intake averages 5.3. Occupational exposure 6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS 6.1. Pharmacokinetics 6.1.1. Absorption 126.96.36.199 Oral 188.8.131.52 Dermal 184.108.40.206 Inhalation 6.1.2. Distribution 6.1.3. Elimination and fate 6.1.4. Physiologically based pharmacokinetic modelling 6.2. Biotransformation and covalent binding of metabolites 6.3. Human studies 6.3.1. Uptake 220.127.116.11 Dermal 18.104.22.168 Inhalation 6.3.2. Elimination 7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS 7.1. Single exposure 7.1.1. Lethality 7.1.2. Non-lethal effects 22.214.171.124 Oral exposure 126.96.36.199 Inhalation exposure 188.8.131.52 Subcutaneous and intraperitoneal exposure 184.108.40.206 Dermal exposure 7.2. Short-term exposure 7.2.1. Oral exposure 7.2.2. Inhalation exposure 7.2.3. Intraperitoneal exposure 7.3. Long-term exposure 7.4. Irritation 7.4.1. Skin irritation 7.4.2. Eye irritation 7.5. Toxicity to the reproductive system, embryotoxicity, teratogenicity 7.5.1. Reproduction 7.5.2. Embryotoxicity and teratogenicity 220.127.116.11 Oral exposure 18.104.22.168 Inhalation exposure 7.6. Mutagenicity 7.7. Carcinogenicity 7.7.1. Mice 7.7.2. Rats 7.8. Special studies 7.8.1. Immunotoxicity 7.8.2. Influence of oxygen levels 7.9. Factors modifying toxicity 7.9.1. Dosing vehicles 7.9.2. Diet 7.9.3. Alcohol 7.9.4. Enhancement of carbon tetrachloride-induced hepatotoxicity by various compounds 7.9.5. Reduction of carbon tetrachloride-induced hepatotoxicity by various compounds 7.10. Mode of action 8. EFFECTS ON HUMANS 8.1. Controlled studies 8.1.1. Inhalation 8.1.2. Dermal 8.2. Case reports 8.3. Epidemiology 8.3.1. Non-cancer epidemiology 8.3.2. Cancer epidemiology 9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD 9.1. Toxicity to microorganisms 9.2. Aquatic toxicity 9.2.1. Algae 9.2.2. Invertebrates 9.2.3. Vertebrates 9.3. Terrestrial toxicity 9.3.1. Earthworms 10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT 10.1. Evaluation of human health risks 10.1.1. Exposure 10.1.2. Health effects 10.1.3. Approaches to health risk assessment 10.1.3.1 Calculation of a TDI based on oral data 10.1.3.2 Calculation of a TC based on inhalation data 10.1.3.3 Summary of the results of risk assessment 10.1.3.4 Conclusions based on exposure and health risk assessment 10.2. Evaluation of effects on the environment 11. FURTHER RESEARCH 12. PREVIOUS EVALUATION BY INTERNATIONAL BODIES REFERENCES RÉSUMÉ RESUMEN NOTE TO READERS OF THE CRITERIA MONOGRAPHS Every effort has been made to present information in the criteria monographs as accurately as possible without unduly delaying their publication. 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Environmental Health Criteria PREAMBLE Objectives In 1973 the WHO Environmental Health Criteria Programme was initiated with the following objectives: (i) to assess information on the relationship between exposure to environmental pollutants and human health, and to provide guidelines for setting exposure limits; (ii) to identify new or potential pollutants; (iii) to identify gaps in knowledge concerning the health effects of pollutants; (iv) to promote the harmonization of toxicological and epidemiological methods in order to have internationally comparable results. The first Environmental Health Criteria (EHC) monograph, on mercury, was published in 1976 and since that time an ever-increasing number of assessments of chemicals and of physical effects have been produced. In addition, many EHC monographs have been devoted to Cevaluating toxicological methodology, e.g., for genetic, neurotoxic, teratogenic and nephrotoxic effects. 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Such meetings have been held in: Ispra, Italy, 1980; Oxford, United Kingdom, 1984; Berlin, Germany, 1987; and North Carolina, USA, 1995. The selection of chemicals has been based on the following criteria: the existence of scientific evidence that the substance presents a hazard to human health and/or the environment; the possible use, persistence, accumulation or degradation of the substance shows that there may be significant human or environmental exposure; the size and nature of populations at risk (both human and other species) and risks for environment; international concern, i.e. the substance is of major interest to several countries; adequate data on the hazards are available. If an EHC monograph is proposed for a chemical not on the priority list, the IPCS Secretariat consults with the Cooperating Organizations and all the Participating Institutions before embarking on the preparation of the monograph. 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It is accepted that the following criteria should initiate the updating of an EHC monograph: new data are available that would substantially change the evaluation; there is public concern for health or environmental effects of the agent because of greater exposure; an appreciable time period has elapsed since the last evaluation. All Participating Institutions are informed, through the EHC progress report, of the authors and institutions proposed for the drafting of the documents. A comprehensive file of all comments received on drafts of each EHC monograph is maintained and is available on request. The Chairpersons of Task Groups are briefed before each meeting on their role and responsibility in ensuring that these rules are followed. WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR CARBON TETRACHLORIDE Members Dr D. Anderson, British Industry Biological Research Association (BIBRA) Toxicology International, Carshalton, Surrey, United Kingdom (Chairperson) Dr E. Elovaara, Finnish Institute for Occupational Health, Helsinki, Finland Dr E. Frantik, National Institute of Public Health, Center of Industrial Hygiene and Occupational Diseases, Prague, Czech Republic Dr B. Gilbert, Ministry of Health, Far-Manguinhas-FIOCRUZ, Rio de Janeiro, Brazil (Co-Rapporteur) Mr M. Greenberg, National Center for Environmental Assessment, Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, North Carolina, USA Mr P. Howe, Institute of Terrestrial Ecology, Monks Wood, Abbots Ripton, Huntingdon, Cambridgeshire, United Kingdom Professor H. Kappus, Virchow Klinikum der Humboldt Universitat, Berlin, Germany Dr D. McGregor, Unit of Carcinogen Identification and Evaluation, International Agency for Research on Cancer, Lyon, France (Co-Rapporteur) Dr P. Parsons, Health and Safety Executive, Bootle, Merseyside, United Kingdom Professor J.A. Sokal, Institute of Occupational Medicine and Environmental Health, Sosnowiec, Poland Secretariat Dr J. de Fouw, Centre for Substances and Risk Assessment, National Institute of Public Health and the Environment, Bilthoven, The Netherlands Professor F. Valic, IPCS Scientific Adviser, Andrija Stampar School of Public Health, Zagreb University, Zagreb, Croatia (Responsible Officer and Secretary of Meeting) ENVIRONMENTAL HEALTH CRITERIA FOR CARBON TETRACHLORIDE A Task Group on Environmental Health Criteria for Carbon Tetrachloride met at the British Industrial and Biological Research Association (BIBRA), Carshalton, United Kingdom, from 2 to 6 March 1998. Dr D. Anderson, welcomed the participants on behalf of the host institution, and Professor F. Valic opened the Meeting on behalf of the heads of the three cooperating organizations of the IPCS (UNEP/ILO/WHO). The Task Group reviewed and revised the draft monograph and made an evaluation of the risks for human health from exposure to carbon tetrachloride. The first draft of this monograph was prepared by Ms J. de Fouw, Centre for Substances and Risk Assessment, National Institute of Public Health and the Environment, Bilthoven, the Netherlands. Professor Valic, Zagreb University, Croatia, was responsible for the overall scientific content of the monograph and for the organization of the Meeting, and Dr P.G. Jenkins, IPCS Central Unit, for the technical editing of the monograph. The efforts of all who helped in the preparation and finalization of the monograph are greatfully acknowledged. ABBREVIATIONS ALAT alanine aminotransferase AP alkaline phosphatase ASAT aspartate aminotransferase ATPase adenosine triphosphatase ATSDR Agency for Toxic Substances and Disease Registry CNS central nervous system CPK creatine phosphokinase CYP cytochrome P-450 Hb haemoglobin Ht haematocrit ip intraperitoneal LDH lactate dehydrogenase LOAEL lowest-observed-adverse-effect level MPV mean packed volume NADPH reduced nicotinamide adenine dinucleotide phosphate NIOSH National Institute for Occupational Safety and Health (USA) NOAEL no-observed-adverse-effect level PBB polybrominated biphenyl PCB polychlorinated biphenyl RBC red blood cell SDH sorbitol dehydrogenase SRBC sheep red blood cells TC tolerable concentration TDI tolerable daily intake 1. SUMMARY Carbon tetrachloride is a clear, colourless, volatile liquid with a characteristic, sweet odour. It is miscible with most aliphatic solvents and is itself a solvent. The solubility in water is low. Carbon tetrachloride is non-flammable and is stable in the presence of air and light. Decomposition may produce phosgene, carbon dioxide and hydrochloric acid. The source of carbon tetrachloride in the environment is likely to be almost exclusively anthropogenic in origin. Most of the carbon tetrachloride produced is used in the production of chlorofluorocarbons (CFCs) and other chlorinated hydrocarbons. The global production of carbon tetrachloride amounted to 960 000 tonnes in 1987. However, since the Montreal Protocol on Substances that Deplete the Ozone Layer (1987) and its amendments (1990 and 1992) have established a timetable for the phase-out of the production and consumption of carbon tetrachloride, manufacture has dropped and will continue to drop. Several sufficiently sensitive and accurate analytical methods for determining carbon tetrachloride in air, water and biological samples have been developed. The majority of these methods are based on direct injection into a gas chromatograph or adsorption on activated charcoal, then desorption or evaporation and subsequent gas chromatographic detection. Nearly all carbon tetrachloride released to the environment will ultimately be present in the atmosphere, owing to its volatility. Since the atmospheric residence time of carbon tetrachloride is long, it is widely distributed. During the period 1980-1990, atmospheric levels were around 0.5-1.0 µg/m3. Estimates of atmospheric lifetime are variable, but 45-50 years is accepted as the most reasonable value. Carbon tetrachloride contributes both to ozone depletion and to global warming. It is in general resistant to aerobic biodegradation but less so to anaerobic. Acclimation increases biodegradation rates. Although the octanol-water partition coefficient indicates moderate potential for bioaccumulation, short tissue lifetime reduces this tendency. In water, reports have indicated levels of less than 10 ng/litre in the ocean and generally less than 1 µg/litre in fresh water, but much higher values close to release sites. Levels of up to 60 µg/kg have been recorded in foods processed with carbon tetrachloride, but this practice has now ceased. The general population is exposed to carbon tetrachloride mainly via air. On the basis of the reported concentrations in ambient air, foodstuffs and drinking-water, a daily carbon tetrachloride intake of around 1 µg/kg body weight has been estimated. This estimate is probably rather high for the present day, because the use of carbon tetrachloride as a fumigant of grain has stopped and the carbon tetrachloride values reported for food and used in the calculation were especially those found in fatty and grain-based foods. Values of 0.1 to 0.27 µg/kg body weight for daily exposure of the general population have been reported elsewhere. Exposure to higher levels of carbon tetrachloride can occur in the workplace as a result of accidental spillage. Carbon tetrachloride is well absorbed from the gastrointestinal and respiratory tract in animals and humans. Dermal absorption of liquid carbon tetrachloride is possible, but dermal absorption of the vapour is slow. Carbon tetrachloride is distributed throughout the whole body, with highest concentrations in liver, brain, kidney, muscle, fat and blood. The parent compound is eliminated primarily in exhaled air, while minimal amounts are excreted in the faeces and urine. The first step in the biotransformation of carbon tetrachloride is catalysed by cytochrome P-450 enzymes, leading to the formation of the reactive trichloromethyl radical. Oxidative biotransformation is the most important pathway in the elimination of the radical, forming the even more reactive trichloromethylperoxyl radical, which can react further to form phosgene. Phosgene may be detoxified by reaction with water to produce carbon dioxide or with glutathione or cysteine. Formation of chloroform and dichlorocarbene occurs under anaerobic conditions. Covalent binding to macromolecules and lipid peroxidation occur via metabolic intermediates of carbon tetrachloride. The liver and kidney are target organs for carbon tetrachloride toxicity. The severity of the effects on the liver depends on a number of factors such as species susceptibility, route and mode of exposure, diet or co-exposure to other compounds, in particular ethanol. Furthermore, it appears that pretreatment with various compounds, such as phenobarbital and vitamin A, enhances hepatotoxicity, while other compounds, such as vitamin E, reduce the hepatotoxic action of carbon tetrachloride. Moderate irritation after dermal application was seen on the skins of rabbits and guinea-pigs, and there was a mild reaction after application into the rabbit eye. The lowest LD50 of 2391 mg/kg body weight (14-day period) was reported in a study on dogs involving intraperitoneal administration. In rats the LD50 values ranged from 2821 to 10 054 mg/kg body weight. In a 12-week oral study on rats (5 days/week), the no-observed-adverse-effect level (NOAEL) was 1 mg/kg body weight. The lowest-observed-adverse-effect level (LOAEL) reported in this study was 10 mg/kg body weight, showing a slight, but significant increase in sorbitol dehydrogenase (SDH) activity and mild hepatic centrilobular vacuolization. A similar NOAEL of 1.2 mg/kg body weight (5 days/ week) was found in a 90-day oral study on mice, with a LOAEL of 12 mg/kg body weight, where hepatotoxicity occurred. When rats were exposed to carbon tetrachloride by inhalation for approximately 6 months, 5 days/week, 7 h/day, a NOAEL of 32 mg/m3 was reported. The LOAEL, based on changes in the liver morphology, was reported to be 63 mg/m3. In another 90-day study on rats, a NOAEL of 6.1 mg/m3 was found after continuous exposure to carbon tetrachloride. The lowest exposure level of 32 mg/m3 (the lowest concentration studied) in a 2-year inhalation study on rats caused marginal effects. The only oral long-term toxicity study available was a 2-year study in rats, which were exposed to 0, 80 or 200 mg carbon tetrachloride/kg feed. Owing to chronic respiratory disease in all animals beginning at 14 months, which resulted in increased mortality, the results reported upon necropsy at 2 years are inadequate for a health risk evaluation. It was concluded that carbon tetrachloride can induce embryotoxic and embryolethal effects, but only at doses that are maternally toxic, as observed in inhalation studies in rats and mice. Carbon tetrachloride is not teratogenic. Many genotoxicity assays have been conducted with carbon tetrachloride. On the basis of available data, carbon tetrachloride can be considered as a non-genotoxic compound. Carbon tetrachloride induces hepatomas and hepatocellular carcinomas in mice and rats. The doses inducing hepatic tumours are higher than those inducing cell toxicity. In humans, acute symptoms after carbon tetrachloride exposure are independent of the route of intake and are characterized by gastrointestinal and neurological symptoms, such as nausea, vomiting, headache, dizziness, dyspnoea and death. Liver damage appears after 24 h or more. Kidney damage is evident often only 2 to 3 weeks following the poisoning. Epidemiological studies have not established an association between carbon tetrachloride exposure and increased risk of mortality, neoplasia or liver disease. Some studies have suggested an association with increased risk of non-Hodgkin's lymphoma and, in one study, with mortality and liver cirrhosis. However, not all of these studies pinpointed specific exposure to carbon tetrachloride, and the statistical associations were not strong. In general carbon tetrachloride appears to be of low toxicity to bacteria, protozoa and algae; the lowest toxic concentration reported was for methanogenic bacteria with an IC50 of 6.4 mg/litre. For aquatic invertebrates acute LC50 values range from 28 to > 770 mg/litre. In freshwater fish the lowest acute LC50 value of 13 mg/litre was found in the golden orfe (Leuciscus idus melanotus), and for marine species an LC50 value of 50 mg/litre was reported for the dab (Limanda limanda). Carbon tetrachloride appears to be more toxic to embryo-larval stages of fish and amphibians than to adults. The common bullfrog (Rana catesbeiara) is the most susceptible species, the LC50 being 0.92 mg/litre (fertilization to 4 days after hatching). The available data indicate that hepatic tumours are induced by a non-genotoxic mechanism, and it therefore seems acceptable to develop a tolerable daily intake (TDI) and a tolerable daily concentration in air (TC) for carbon tetrachloride. On the basis of the study of Bruckner et al. (1986), in which a NOAEL of 1 mg/kg body weight was observed in a 12-week oral study on rats, and incorporating a conversion factor of 5/7 for daily dosing and applying an uncertainty factor of 500 (100 for inter- and intraspecies variation, 10 for duration of the study, and modifying factor 0.5 because it was a bolus study), a TDI of 1.42 µg/kg body weight is obtained. On the basis of a 90-day inhalation study on rats (Prendergast et al., 1967), in which a NOAEL of 6.1 mg/m3 was reported, a TC of 6.1 µg/m3 was calculated using the factors 7/24 and 5/7 to convert to continuous exposure and an uncertainty factor of 1000 (100 for inter- and intraspecies variation and 10 for the duration of the study). This TC corresponds to a TDI of 0.85 µg/kg body weight. Comparing the estimated upper limit of prevailing human daily intake of 0.2 µg/kg body weight with the lowest TDI value (0.85 µg/kg body weight), the conclusion can be drawn that the currently prevailing exposure of the general population to carbon tetrachloride from all sources is unlikely to cause excessive intake of the chemical. In general, the risk to aquatic organisms from carbon tetrachloride is low. However, it may present a risk to embryo-larval stages at, or near, sites of industrial discharges or spills. 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS 2.1 Identity Chemical formula: CCl4 Chemical structure: Common name: carbon tetrachloride Common synonyms: Carbona, carbon chloride, tetrachloromethane, carbon tet, methane tetrachloride, perchloromethane, tetrachlorocarbon Trade names: Benzinoform, Fasciolin, Flukoids, Freon 10, Halon 104, Necatorina, Necatorine, Tetrafinol, Tetraform, Tetrasol, Univerm, Vermoestricid CAS chemical name: tetrachloromethane CAS registry number: 56-23-5 RTECS registry number: FG 4900000 2.2 Physical and chemical properties The most important physical properties of carbon tetrachloride are given in Table 1. Table 1. Physical properties of carbon tetrachloridea Colour colourless Relative molecular mass 153.8 Boiling point at 101.3 kPa, 20°C 76.72 °C Melting point at 101.3 kPa, 20°C -22.92 °C Density (25°C) 1.594 g/ml Table 1. (Continued) Density of solid at - 186 °C 1831 kg/m3 - 80 °C 1809 kg/m3 Refractive index at 20 °C 1.4607 Vapour pressure at 20 °C 91.3 mmHg; 12.2 kPa at 0 °C 32.9 mmHg; 4.4 kPa Autoignition temperature > 1000 °C Critical pressure 4.6 MPa Critical temperature 283.2 °C Solubility in water at 25 °C 785 mg/litre Solubility of water in carbon tetrachloride at 25 °C 0.13 g/kg Henry's law constant at 24.8 °C 2.3 × 10-2 atm-m3/mol Heat of evaporation 194.7 kJ/kg Log Kow 2.64 Log Koc 2.04 a From Kenaga (1980); US EPA (1984b); Huiskamp et al. (1986); ATSDR (1994). Carbon tetrachloride is a volatile colourless clear heavy liquid with a characteristic sweet non-irritant odour. The odour threshold in water is 0.52 mg/litre and in air is > 10 ppm. Carbon tetrachloride is miscible with most aliphatic solvents and it is a solvent for benzyl resins, bitumen, chlorinated rubber, rubber-based gums, oils and fats. The chemical is non-flammable and fairly stable in the presence of air and light. Upon heating by a flame or hot metal surface in air, toxic phosgene is produced. Thermal dissociation in the absence of air proceeds slowly at about 400°C and is extensive at temperatures ranging from 900 to 1300°C with the formation of perchloroethylene, hexachloroethane and some molecular chlorine. A mixture of carbon tetrachloride and excess of water vapour decomposes at 250°C to carbon dioxide and hydrochloric acid. When the amount of water in the mixture is limited, phosgene will be formed too. This decomposition also occurs when moist or wet carbon tetrachloride is exposed to UV radiation (253.7 nm). Like other chloromethanes, carbon tetrachloride reacts (sometimes explosively) with aluminium and its alloys. Similar violent reaction may occur with metals, such as barium, magnesium and zinc, boranes and silanes, and, in the presence of peroxides or light, with unsaturated compounds (such as ethene). Carbon tetrachloride may be reduced to chloroform when treated with zinc and acid, and to methane when treated with potassium amalgam and water (Huiskamp et al., 1986). 2.3 Conversion factors 1 mg carbon tetrachloride/m3 air = 0.156 ppm at 20°C and 101.3 kPa (760 mmHg) 1 ppm = 6.41 mg carbon tetrachloride/m3 2.4 Analytical methods Procedures used for the sampling and determination of carbon tetrachloride in different media are summarized in Table 2. The preferred analytical technique is gas chromatography (GC) using either electron capture detection (ECD), ion trap detection, flame or photo ionisation detection or mass spectrometry. Only one method, reported by Lioy & Lioy (1983), depends on the use of MIRAN-infrared spectrometry, a method of very poor sensitivity. 2.4.1 Sampling and analysis in air Methods reported in Table 2 for detecting carbon tetrachloride in air are of four types. a) Direct measurement These methods are simple, because the air is aspirated or injected directly into the measuring instrument, but they can only be used when carbon tetrachloride is present in the air at relatively high levels. b) Adsorption - liquid desorption In this type of method, air samples are passed through an activated adsorbing agent. The adsorbed carbon tetrachloride is desorbed with an appropriate solvent and then passed through the gas chromatograph. Activated carbon has been described as superior to other adsorbents for adsorption. Elution from the carbon is achieved with carbon disulfide (Morele et al., 1989; ATSDR, 1994). c) Adsorption - thermal desorption After adsorption on an activated adsorbing agent, the carbon tetrachloride is thermally desorbed and driven into the gas chromatograph. Table 2. Sampling and analysis of carbon tetrachloridea Medium Sampling method Analytical method Detection limit Sample size Comments Reference Air aspiration velocity: 28 l/min MIRAN infrared 400 µg/m3 Lioy & Lioy, optical path: 20 m spectrometry 1983 Air direct injection GC with 2 ECD's 0.4 µg/m3 8 ml injected Lillian & in series (estimated) Singh, 1974 Air direct injection GC - ECD 0.2 µg/m3 2 ml injected BIT-SC, 1976 Air direct injection GC - ECD 0.06 µg/m3 5 ml injected Lasa et al., 1979 Air direct injection, methane GC - ECD 0.01 µg/m3 12 ml injected thorough purification of Makide & added carrier gas and apparatus Yokohata, 1983 required Air adsorption on Porapak-N GC - ECD 1 µg/m3 20 litres advantage of using Van Tassel et liquid desorption (methanol) methanol over CS2 is the al., 1981 absence of a background signal in the ECD Air adsorption on activated GC - ECD 0.2 µg up to 30 litres activated charcoal shown Morele et al., charcoal, liquid desorption can be sampled to be more efficient 1989 (ethanol) trichloroethylene trapping material than used as IS XADs, Tenax or Chromosorbs adsorption on activated GC - FID ca. 0.15 mg charcoal liquid desorption (detector (CS2) methylcyclohexane sensitivity) used as IS Table 2. (Continued) Medium Sampling method Analytical method Detection limit Sample size Comments Reference Air adsorption on activated GC - FID 0.01 mg 5-15 litres NIOSH, 1977, charcoal, liquid desorption 1984 (CS2) Air adsorption on Chromosorb GC - ECD 0.003 µg/m3 20 ml Makide et al., 102 or Silicone OV 101 (at 1979 -35 °C), thermal desorption Air adsorption on Porapak-N, GC - ECD 0.005 µg/m3 0.3-3 litres confirmation of results by Russell & thermal desorption at 200 °C use of GC - MS Shadoff, 1977 Air adsorption on Chromosorb GC - ECD 0.01 µg/m3 1 litre Elias, 1977 102, thermal desorption at (collection tube (estimated) 200 °C already connected to GC) Air adsorption on Carbopak-B at GC - ECD 0.01 µg/m3 1 litre calibration with Crescentini et 78 °C, thermal desorption permeation tubes al., 1981 Air adsorption on Chromosorb-102 GC - ECD - FID ca. 0.06 µg/m3 1-3 litres Heil et al., and activated charcoal, (2 detectors in 1979 thermal desorption at 150 °C parallel) Air adsorption on Tenax-GC, GC - MS 0.2 µg/m3 20 litres compounds were Krost et al., thermal desorption at 270 °C cryofocused 1982 Air adsorption on Carbopak-C, GC - MS 0.1 µg/m3 300 ml Crescentini et thermal desorption at 100 °C al., 1983 Air adsorption on activated GC - ECD followed 0.7 µg/m3 24 h sample Coutant & charcoal, liquid desorption by a PID Scott, 1982 (5% CS2 in methanol) Table 2. (Continued) Medium Sampling method Analytical method Detection limit Sample size Comments Reference Air cold trap (liquid oxygen), GC - ECD 0.006 µg/m3 30 ml aliquot measurement of air Harsch & heating in trap samples from the Cronn, 1978 stratosphere Air injection in cold trap, heating GC - MS (SIM) 0.04 µg/m3 100 ml Cronn & Harsch, 1979 Air cold trap (-173 °C), heating to GC - PID - ECD - 0.006 µg/m3 0.5-1.7 litres column is kept at -103 °C Rudolph & 257 °C FID (3 detectors (cryofocusing) Jebsen, 1983 in series) Water dibromomethane used as IS GC - ECD 0.001 µg/litre 500 µl injected suitable for routine Herzfeld et al., analysis of river waters 1989 Water direct aqueous injection GC - MS (SIM) 2 µg/litre 10 µl injected Fujii, 1977 Water direct aqueous injection GC - ECD 0.015 µg/litre 2 µl injected suitable for halocarbons Grob, 1984 in water in the 0.01 to 10 µg/litre range Water direct aqueous injection, GC - ECD 0.05 µg/litre 5-20 µl Simmonds & water removal by injected Kerns, 1979 permeaselective membrane Water liquid-liquid extraction GC - ECD 0.10 µg/litre 10-20 ml Van Rensburg (using hexane) et al., 1978 Water liquid-liquid extraction GC - ECD 0.2 µg/litre Inoko et al., (using xylene) 1984 Water liquid-liquid extraction GC - ECD 0.05 µg/litre Kroneld, 1985 (using pentane) Table 2. (Continued) Medium Sampling method Analytical method Detection limit Sample size Comments Reference Water purge and trap technique, GC - ITD 0.1 µg/litre 5 ml Eichelberger thermal desorption, et al., 1990 fluorobenzene as IS Grain codistillation of carbon GC - ECD 1 µg/kg De Vries et al., tetrachloride in food sample 1985 and mixture of 1,2-dichloropropane and 1,2-dibromopropane as IS in hexane Adipose purge and trap technique GC - MS < 1.3 µg/litre 200-500 mg Peoples et al., tissue (Tenax-silica gel), thermal liquefied fat 1979 desorption samples Blood purge and trap technique GC - MS < 1.3 µg/litre 0.5 ml water- Peoples et al., (Tenax-silica gel), thermal serum sample 1979 desorption Blood warming and passing an inert GC - MS 3 µg/litre 10 ml sample Pellizzari gas, vapours trapped on et al., 1985 Tenax-GC, thermal desorption Urine liquid-liquid extraction using GC - ECD (20% < 1 µg/litre 10 ml sample Youssefi et al., pentane (adding 2.6 g SP-2100/0.1% 1978 ammonium carbonate) Carbowax 1500 column) Fish extraction with pentane and GC - ECD 0.1 µg/kg in Baumann isopropanol, with fresh material Ofstad et al., bromotrichloromethane used as IS 1981 a Abbreviations: GC = gas chromatography; MS = mass spectrometry; ECD = electron capture detector; SIM = single (selected) ion monitoring; FID = flame ionisation detector; ITD = ion trap detector; PID = photo ionisation detector; IS = internal standard. d) Cold trap - heating In this type of procedure, air samples are injected into a cold trap. The trap is then heated and the carbon tetrachloride content transferred into the column of a gas chromatograph. 2.4.2 Sampling and analysis in water Several methods for sampling analysing the carbon tetrachloride content in water are included in Table 2. Most of these methods are based on direct injection techniques or on liquid-liquid extraction by means of a non-polar non-halogenated solvent. 2.4.3 Sampling and analysis in biological samples 22.214.171.124 Blood and tissues Peoples et al. (1979) developed a method to determine carbon tetrachloride in adipose tissue and blood. In both cases the carbon tetrachloride is purged and trapped on Tenax-silica gel and determined by mass spectrometry after thermal desorption. Pellizzari et al. (1985) similarly passed an inert gas over a warmed plasma sample with adsorption of the vapour on a Tenax-GC cartridge, and then recovered the carbon tetrachloride by thermal desorption. 126.96.36.199 Urine The only method listed in Table 2 for measuring carbon tetra chloride concentrations in urine is based on an extraction technique with pentane and direct gas chromatographic analysis of the pentane extract (Youssefi et al., 1978). 188.8.131.52 Fish Baumann Ofstad et al. (1981) developed a method for the analysis of volatile halogenated hydrocarbons in biological samples and used this method for the analysis of fish samples. It should be noted that the identification and quantification of carbon tetrachloride is especially vulnerable to contamination, so the practical usefulness of this method is very limited. 2.4.4 Sampling and analysis in foodstuffs A method for the determination of 22 compounds (including carbon tetrachloride) in a variety of foods was described by Daft (1988). In this method the samples are extracted with isooctane, and cleaned up according to fat content and food type. Most samples (6-10 µl) are injected for GC with ECD and Hall-electron conductivity detection immediately following the initial extraction or dilution. De Vries et al. (1985) provided a method for analysis of carbon tetrachloride in grain and grain-based products containing 1-2000 µg/kg. A food sample is mixed with water and an internal standard mixture of 1,2-dichloropropane and 1,2-dibromopropane is added. The water is then distilled until 1 ml has been collected under hexane. The hexane is then separated, dried and injected (2 µl) into the GC column. 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1 Natural occurrence It has been suggested that carbon tetrachloride can be formed in the troposphere by the solar-induced photochemical reactions of chlorinated alkenes (Singh et al., 1975). However, so far this reaction has only been demonstrated in the laboratory, and, even if it could happen in nature, it is not certain that it would be a major source of environmental carbon tetrachloride. Carbon tetrachloride has been detected in volcanic emission gases (Isidorov et al., 1990). Several studies have shown that global atmospheric levels of carbon tetrachloride can be explained by anthropogenic sources alone (Singh et al., 1976). 3.2 Anthropogenic sources 3.2.1 Production 184.108.40.206 Direct production and procedures Production of carbon tetrachloride began in about 1907 in the USA. It can be produced by chlorination of methane, methanol, carbon disulfide, propane, 1,2-dichloroethane and higher hydrocarbons. The world production of carbon tetrachloride ranged from 850 to 960 kilotonnes over the years 1980-1988. Table 3 provides some data on past production and production capacities of carbon tetrachloride. These data are based on information in the ECDIN database (ECDIN, 1992) and BUA-Stoffbericht 45 (BUA, 1990). Since 1990 the production of carbon tetrachloride has dropped. The Montreal Protocol of 1990 and its subsequent amendments established the phase-out by 1996 of production and use of carbon tetrachloride and of chlorofluorocarbons (CFCs) by major manufacturing countries. Special conditions were allowed for developing countries, where consumption of controlled substances under Annex B (including carbon tetrachloride) was required to be reduced by 85% of its 1998-2000 average level (or a calculated consumption level of 0.2 kg per capita, whichever is lower) by 2005 and completely stopped by 2010 (UNEP, 1996). 220.127.116.11 Indirect production Carbon tetrachloride can be produced as a by-product during the manufacture of other products and compounds (US EPA, 1984a) and during wood pulp bleaching. Table 3. Past production and production capacity of carbon tetrachloride Country Year Production Capacity (in kilotonnes) (in kilotonnes) France 1988 - 90 Italy 1987 95 - 1988 - 130 Germany (former FRG) 1985 150 - 1987 180 - 1988 170 180 EEC 1985 - 520 1987 480 - 1988 478 540 Japan 1985 - 72 1987 52 - 1988 - 70 United Kingdom 1988 - 75 USA 1986 286 - 1987 340 - 1988 - 281 1991 143 - World 1985 - 1200 1987 960 - 1988 - 1100 18.104.22.168 Emissions According to US EPA (1991), in 1989 approximately 2000 tonnes of carbon tetrachloride were released during manufacturing and processing to the air in the USA. US EPA (1984a) reported emission factors for carbon tetrachloride arising during the chlorination of hydrocarbons ranging from 0.9 kg/tonne of carbon tetrachloride (controlled) to 2.8 kg/tonne of carbon tetrachloride (uncontrolled). Furthermore, emissions may result from industrial water treatment or from old landfill sites. 3.2.2 Uses Most of the carbon tetrachloride produced is used in the production of CFCs, which were primarily used as refrigerants, propellants, foam-blowing agents and solvents and in the production of other chlorinated hydrocarbons. The use of carbon tetrachloride increased in the EEC as well as in the USA during the years 1980-1987. However, this use has decreased in recent years due to the Copenhagen Amendment to the Montreal Protocol (1992) (UNEP, 1996). A survey in Japan could detect no use of carbon tetrachloride in small to medium scale industries in 1996 (Ukai et al., 1997). Carbon tetrachloride has been used as a grain fumigant, pesticide, solvent for oils and fats, metal degreaser, fire extinguisher and flame retardant, and in the production of paint, ink, plastics, semi-conductors and petrol additives. It was previously also widely used as a cleaning agent. All these uses have tended to be phased-out as production has dropped (ECDIN, 1992; ATSDR, 1994). 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION 4.1 Transport and distribution between media 4.1.1 Transport Carbon tetrachloride introduced into water resources is transported by movement of surface water and groundwater. Because of its volatility, evaporation is considered to be the main process for the removal of carbon tetrachloride from aquatic systems. The amount of carbon tetrachloride dissolved in the oceans is reported to be less than 1-3% of that in the atmosphere (Galbally, 1976; Singh et al., 1976). Practically all the carbon tetrachloride released to the environment is thus present in the atmosphere (US EPA, 1991). Because carbon tetrachloride does not degrade readily in the atmosphere, significant global transport is expected. Following releases to soil, most carbon tetrachloride is expected to evaporate rapidly due to its high vapour pressure. A small fraction of carbon tetrachloride may adsorb to organic matter, based on a calculated soil adsorption coefficient of 100 (log Koc = 2.04) (Kenaga, 1980). Walton et al. (1992) studied the adsorption of carbon tetrachloride from solution onto two soils, a silt loam (1.49% organic carbon) and a sandy loam (0.66% organic carbon). The soil was shaken with several concentrations of carbon tetrachloride (100 to 650 mg/kg soil) for 18 h. The Koc values determined were 143.6 for the silt loam and 48.9 for the sandy loam. Duffy et al. (1997) studied the downward movement of carbon tetrachloride in 3 horizons of a fine montmorillonitic soil. Koc values of 55, 77.6 and 269 were calculated for the modern A, buried A and loess C soil horizons. However, the authors point out that Koc values are unreliable in soils with low organic carbon and high clay content. Therefore, the highest Koc value should be treated with some caution. 4.1.2 Distribution The evidence that the residence time of carbon tetrachloride in the atmosphere is long (see section 4.1.3) and that nearly all of the compound is found in this compartment explains the relatively even distribution over the globe as is recorded in Table 4. Only a very small proportion of carbon tetrachloride will remain in water and soil. Table 4. Levels of carbon tetrachloride in air Location Year Mean level (µg/m3) Reference (range in parentheses) Northern 1974 0.71 Cox et al. (1976) hemisphere 1976 0.73 Singh et al. (1977) 1977 0.78 Singh et al. (1979) 1979-1981 0.87 Singh et al. (1983) Southern 1974 0.44 Cox et al. (1976) hemisphere 1977 0.76 Singh et al. (1979) 1979-1981 0.82 Singh et al. (1983) North-West 1975-1980 0.91 Rasmussen et al. Pacific (0.83-0.99) (1981) 1975-1985 0.77 Rasmussen & Khalil (0.67-0.83) (1986) 1976 0.78 Cronn et al. (1977) Antarctica 1975-1980 0.8 Rasmussen et al. (0.77-0.87) (1981) 1975-1985 0.69 Rasmussen & Khalil (0.62-0.76) (1986) Arctic 1982 0.97 Rasmussen & Khalil (1983) North Atlantic 1983 0.56 von Düszeln & Thiemann (1985) North America 1976 0.86 Pierotti et al. (0.33-0.99) (1980) California, USA 1976 0.76-0.86 Singh et al. (0.66-1.85) (1977) Bochum, Germany 1978 0.8 (0.1-1.2) Bauer (1981) Germany (cities) 1980-1981 0.6 von Düszeln & Thiemann (1985) Table 4. (Continued) Location Year Mean level (µg/m3) Reference (range in parentheses) South-West 1986-1988 0.5-0.6 Frank & Frank Germany (1990) The Netherlands 1980 0.83-1.0 Guicherit & (max. 2.2-3.2) Schulting (1985) Turin, Italy 1988 0.96 (0.17-1.94)a Gilli et al. (1990) 1988 0.47 (0.19-1.17)b Gilli et al. (1990) Japan 1979 0.69 (0.62-0.72) Makide et al. (1979) 1994-1995 0.53c Sugama et al. (1995) a cold months b warm months c concentrations were higher in winter than during summer 4.1.3 Removal from the atmosphere; global warming potential The troposphere to stratosphere turnover time has been estimated at around 30 years (Versar Inc., 1979). This is a shorter period of time than is estimated for the degradation processes of carbon tetrachloride in the troposphere (see section 4.2). Therefore tropospheric carbon tetrachloride will attain significant concentration in the stratosphere. Cupitt (1980) calculated that deposition of carbon tetrachloride from the atmosphere will be very slow. Estimates of the atmospheric lifetime (the overall persistence of carbon tetrachloride in the troposphere and the stratosphere combined) are variable, but most values range from 25 to 100 years (Molina & Rowland, 1974; Galbally, 1976; Singh et al., 1979; Edwards et al., 1982a,b; Simmonds et al., 1983, 1988; Rowland, 1985; Huiskamp et al., 1986; Howard, 1990; IPCC, 1990, 1995; WMO, 1991) with 45-50 years generally being accepted as the most reasonable value. The Global Warming Potential (GWP) of carbon tetrachloride, relative to CO2, is estimated (IPCC, 1995) to be 2000, 1400 and 500 at integration time horizons of 20, 100 and 500 years. Its contribution to total warming may be 0.3% as integrated effect over a time horizon of 100 years (IPCC, 1995). Relative to CFC 12, the GWP of carbon tetrachloride has been estimated to be 0.12 (UNEP, 1989). 4.1.4 Removal from water The major removal process from water is volatilization to the atmosphere. This was indicated by laboratory tests performed by Dilling et al. (1975). These tests showed that a 1 ppm concentration of low-molecular-weight chlorinated hydrocarbons will not persist in agitated natural water bodies due to evaporation. In 29 min 50% of the amount of carbon tetrachloride was evaporated, and in 97 min 90% was evaporated. Zoeteman et al. (1980) calculated a half-life of carbon tetrachloride in rivers of 0.3-3 days and in lakes and groundwaters of 30-300 days. 4.1.5 Removal from soil Anderson et al. (1991) studied the loss of carbon tetrachloride from two different soil types, a silt loam (1.49% organic carbon) and a sandy loam (0.66% organic carbon). Carbon tetrachloride was applied to the soil at a concentration of 100 mg/kg (dry weight) and the soil was incubated in the dark at 20°C for 7 days. The mean half-life for disappearance of carbon tetrachloride was 5 days. There was no significant difference between the loss from sterile and non-sterile systems indicating that volatilization was the likely removal process. Jury et al. (1984) predicted that carbon tetrachloride would have a volatilization half-life of 0.2 days at a depth of 1 cm and 0.8 days at a depth of 10 cm in soil, based on volatilization tests and assuming a uniform distribution of the chemical with depth. 4.2 Abiotic degradation 4.2.1 Degradation in atmosphere 22.214.171.124 Photodegradation Carbon tetrachloride is very stable in the troposphere (Lillian et al., 1975; Cox et al., 1976; Singh et al., 1980). This is primarily because carbon tetrachloride, in contrast to most other volatile halocarbons, has low reactivity towards hydroxyl radicals. This is evident from rate constants determined by several authors (Howard Carleton & Evenson, 1976; Cox et al., 1976; Clyne & Holt, 1978). Based on these rate constants, half-lives of > 3.9 to 137 years can be calculated for the decomposition of carbon tetrachloride in the troposphere (Lyman et al., 1982). Cox et al. (1976) found an even higher tropospheric half-life of > 330 years. 126.96.36.199 Photolysis Edwards et al. (1982b) estimated a lifetime in the troposphere due to photolysis of the order of 500 years. The principal degradation process for carbon tetrachloride occurs in the stratosphere, where it is dissociated by short wave length (190- 220 nm) UV radiation to form the trichloromethyl radical and chlorine atoms. Simmonds et al. (1983) estimated a half-life of 18-80 years for this photodissociation process. 188.8.131.52 Ozone-depletion potential The chlorine atoms in carbon tetrachloride interact with oxygen or ozone to produce ClO* groups (Singh et al., 1975). The chlorine atoms and ClO* groups attack the surrounding ozone in a reaction in which they act as catalysts until scavenged by some other chemical reaction (Isaksen & Stordal, 1981; Rowland, 1985; Ember et al., 1986). This effect is reflected in an ODP (ozone depletion potential) of 1.08 (WMO, 1991) and 1.1 (UNEP, 1996), compared with the chlorofluorocarbon CFC-11, and was responsible for the inclusion of carbon tetrachloride in the amended Montreal Protocol of 1990 (UNEP, 1996). Catalytic breakdown of ozone by chloride-containing radicals: CCl4 + h nu -> *CCl3 + *Cl *CCl3 + O2 -> -> COCl2 + ClO* *Cl + O3 -> ClO* + O2 ClO* + O -> *Cl + O2 4.2.2 Degradation in water Carbon tetrachloride dissolved in water does not photodegrade or oxidize in any measurable amount (Howard et al., 1991). The rate of hydrolysis was thought to be second order with respect to carbon tetrachloride with a calculated half-life of 7000 years at a concentration of 1 ppm (Mabey & Mill, 1978). However, Jeffers et al. (1996) found that the rate of hydrolysis for dilute solutions of carbon tetrachloride was first-order and estimated the half-life to be 40 years. The authors reanalysed data previously stated as second-order kinetics and found it to be consistent with a first-order rate of hydrolysis. 4.2.3 Other degradation processes Photodissociation of carbon tetrachloride adsorbed on to silicates has been observed in the laboratory by Ausloos et al. (1977). Gäb et al. (1980) found experimentally that carbon tetrachloride degraded over sand, silica gel and Al2O3. The degradation rate depended, among other factors, on the laboratory conditions. Under the conditions representative for deserts, degradation was about 4.5% after exposure for 115 days. 4.3 Biotic degradation 4.3.1 Aerobic Carbon tetrachloride has been shown to be resistant to aerobic biodegradation by mixed bacterial cultures growing on methane as the carbon source. No degradation of carbon tetrachloride was observed in a mixed culture of methane-utilizing bacteria isolated from soil and incubated in the dark for 6 days (Cochran et al., 1988). Oldenhuis et al. (1989) reported no degradation of carbon tetrachloride by the methanotrophic bacterium Methylosinus trichlosporium in the presence of formate and oxygen. Vannelli et al. (1990) found that carbon tetrachloride was not degraded by the ammonia-oxidizing bacterium Nitrosomonas europea when incubated at 1 mg/litre for 24 h. In contrast, Tabak et al. (1981) found carbon tetrachloride to be significantly degradable under aerobic conditions, with rapid adaptation. Carbon tetrachloride (5 and 10 mg/litre) was incubated at 25°C for 7 days in static culture containing yeast extract inoculated with settled domestic wastewater. Eighty to eighty-seven per cent of the initial concentration disappeared within 7 days in the first culture. An abiotic control showed that 5-23% of this loss could be due to volatilization. In three subsequent cultures, carbon tetrachloride was degraded to concentrations below the detection limit (< 0.1 mg/litre) in the same period. 4.3.2 Anaerobic The biodegradation of carbon tetrachloride has been studied under methanogenic conditions. In batch cultures, carbon tetrachloride at a concentration of 200 µg/litre was incubated in the dark at 35°C with mixed methanogenic bacteria derived from a laboratory-scale digester fed with activated sludge. Carbon tetrachloride was found to be degraded to below the detection limit (< 0.1 µg/litre) within 16 days; carbon dioxide was the only degradation product identified. In a continuous-flow column study, columns were initially seeded with an inoculum of methanogenic bacteria from rum distillery wastewater. Acetate (100 mg/litre) was fed to the column as primary growth substrate and carbon tetrachloride was fed as a secondary substrate. The column had a 2 day retention time, and it was found that carbon tetrachloride was 99% degraded in the column; carbon dioxide being the major degradation product (Bouwer & McCarty, 1983a). Bouwer & McCarty (1983b) studied the biodegradation of carbon tetrachloride under denitrifying conditions. Using batch cultures seeded with primary sewage effluent and containing nitrate as an electron acceptor, carbon tetrachloride (75 µg/litre) was found to be degraded rapidly with no detectable lag period when incubated in the dark at 25°C for 8 weeks. Chloroform and carbon dioxide were the degradation products identified. The biodegradation of carbon tetrachloride using aquifer material has been studied (Parsons et al., 1985). Microcosms were constructed containing groundwater and sediment contaminated with trichloroethene. The concentration of carbon tetrachloride was 4 mg/litre and incubation was carried out in the dark at 25°C. Reductive dehalogenation of carbon tetrachloride to chloroform was found to occur, and 700 µg chloroform/litre was detected after 8 weeks. Egli et al. (1987) observed that pure cultures of Desulfobacterium autotrophicum dechlorinated carbon tetrachloride to trichloromethane and dichloromethane within 6 days. Klecka & Gonsior (1984) provided evidence that reductive dehalogenation of carbon tetrachloride in aqueous solution under anaerobic conditions could be achieved with naturally occurring iron porphyrins and other reducing agents. Carbon tetrachloride (1 mg/litre) was rapidly degraded to chloroform when incubated at 25°C with an iron porphyrin (haematin) and sulfide. Bioremediation studies have shown that anaerobic biodegradation is enhanced by increasing the concentration of primary substrates (such as glucose and acetate) and by lowering the redox potential (providing a relatively higher electron activity which facilitates dechlorination) (Doong & Wu, 1995, 1996; Doong et al., 1996; Jin & Englarde, 1996). 4.4 Bioaccumulation The log octanol-water partition coefficient (Kow) of carbon tetrachloride is 2.64 indicating a moderate potential for bioaccumulation under conditions of constant exposure. However, studies have shown that the compound's short tissue lifetime reduces this tendency. Barrows et al. (1980) reported a bioconcentration factor of 30 for bluegill sunfish (Lepomis macrochirus) with a tissue half-life of less than one day. A similar bioconcentration factor of 30 (whole body; fresh weight) was reported by Veith (1978) in bluegill. Neely et al. (1974) found a bioconcentration factor of 17.7 for muscle tissue of rainbow trout (Oncorhynchus mykiss). A higher bioconcentration factor of 300 (wet weight) has been measured for carbon tetrachloride in the green alga Chlorella fusca exposed to 50 µg/litre for at least 24 h (Geyer, 1984). No significant bioaccumulation in marine food chains was found in an extensive study by Pearson & McConnell (1975) (see Table 6, section 5.1.4). Some plants, due to their lipid content, take up carbon tetrachloride from the air. Thus studies of the equilibrium partitioning of carbon tetrachloride between the gas phase and conifer needles (Pinus sylvestris and Picea abies) on the one hand and hexane-extractable leaf waxes on the other hand showed partition ratios (g/m3 needle; g/m3 air) of 9-17 and 90-400, respectively (Frank & Frank, 1986; Brown et al., 1998). 5. CONCENTRATIONS IN THE ENVIRONMENT AND EXPOSURE 5.1 Environmental levels 5.1.1 Air Reported concentrations of carbon tetrachloride measured in ambient air are presented in Table 4. As seen in Table 4, mean global levels of atmospheric carbon tetrachloride usually lie in the range of 0.5-1.0 µg/m3. Based on an analysis of 4913 ambient air samples (including remote, rural, industrial and source-dominated sites in the USA), the average concentration of carbon tetrachloride was 1.1 µg/m3 (Shah & Heyerdahl, 1988). Urban atmospheric carbon tetrachloride levels and levels in industrial areas can be considerably higher as shown by the measurements by Lillian et al. (1975), Singh et al. (1980, 1982) and Bozzelli & Kebbekus (1982). These authors reported mean levels of 2-3 µg/m3 (several hundred measurements) with maximum levels up to 6 µg/m3. Near a production facility in the United Kingdom, Pearson & McConnell (1975) recorded levels an order of magnitude higher. It has been estimated that concentrations of carbon tetrachloride were increasing worldwide until recently (Simmonds et al., 1988; Howard, 1990). The Intergovernmental Panel on Climate Change (IPCC) has estimated the atmospheric concentration to be about 0.94 µg/m3 and the annual rate of increase to be 1.5% (IPCC, 1990). However, the accumulation of the substance in the atmosphere seems to have stopped (Fraser et al., 1994) and even started to decline (Fraser & Derek, 1994). 5.1.2 Water Some reported aquatic concentrations of carbon tetrachloride are summarized in Table 5. As seen in Table 5, remote oceanic levels of carbon tetrachloride are usually in the range of 0.0005-0.0008 µg/litre. As sites nearer to effluent sources are examined, higher levels are observed. Thus in estuaries, levels from 0.01 to 2.7 µg/litre have been observed, and in remote freshwater sites from 0.0002 to 0.025 µg/litre, while nearer to industrial facilities mean levels in the range of < 0.1-24.2 µg/litre have been recorded. Even higher values, e.g., 160-1500 µg/litre in the River Rhine and a mean of 75 µg/litre in the River Main, recorded in 1976 in Germany, were the result of direct waste release (BUA, 1990). Groundwater levels range from undetectable to a maximum of 80 µg/litre. Table 5. Levels of carbon tetrachloride in surface water Area Mean level (µg/litre) (range in parentheses) Reference Marine East Pacific ocean 0.0005 Su & Goldberg (1976) East Pacific ocean 0.0007 Singh et al. (1983) Arctic ocean 0.0008 Fogelqvist (1985) Estuarine Scheldt Estuary, 0.01-0.02a van Zoest & van The Netherlands (max. 0.29) Eck (1991) Mersey Estuary, UK 2.7 Edwards et al. (1982a) Freshwater Lake Zurich, Switzerland 0.025 (0.02-0.035) Giger et al. (1978) Lake Ontario, Canada (< 0.0002-0.005) Kaiser et al. (1983) Niagara River, Canada 0.0029 (max. 0.018) Kaiser et al. (1983) River Weaver, UK < 0.1 Rogers et al. (1992) River Gowry, UK 0.9 Rogers et al. (1992) River Rhine, Lobith, 1.5 (0.4-2.8) Bauer (1981) Germany Manchester Ship Canal, UK 3.8 Edwards et al. (1982a) Manchester Ship Canal, UK 24.2 Rogers et al. (1992) Groundwater Zurich (industrial area) (< 0.05-3.6) Giger et al. (1978) Birmingham aquifer, UK (0.02-1) Rivett et al. (1990a,b) Coventry aquifer, UK 4.9 (max. 80) Burston et al. (1993) Washington, New Jersey, (ND-34) Suffet et al. (1985) USA Gibbstown, New Jersey, (1.4-1.8) Rosen et al. (1992) USA a range of medians Based on analysis of data from STORET database, carbon tetrachloride was detectable in 1063 of 8858 ambient water samples, with a median concentration of 0.1 µg/litre (Staples et al., 1985). Rain water and snow concentrations of carbon tetrachloride are generally in the range of 0.3 to 2.8 µg/litre (Su & Goldberg, 1976), but a level as high as 300 µg/litre was observed in rainwater collected near a production site in the United Kingdom (Pearson & McConnell, 1975). 5.1.3 Soil and sediment Carbon tetrachloride might occur in soil due to spills, runoff and leaching. However, only 0.8% of 361 measured soil/sediment samples appeared to contain carbon tetrachloride. The concentration was reported to be less than 5.0 mg carbon tetrachloride/kg dry weight soil or sediment (Staples et al., 1985). 5.1.4 Biota Levels of carbon tetrachloride in biota are summarized in Table 6. 5.2 General population exposure The general population can be exposed to carbon tetrachloride through air, foodstuffs and drinking-water. 5.2.1 Outdoor air Levels in ambient air to which the general population may be exposed are recorded in Table 4. 5.2.2 Indoor air Because of its volatility, carbon tetrachloride tends to volatilize from tap water. Although, human exposure by inhalation of carbon tetrachloride transferred to the indoor air from showers and baths, toilets, washing and cooking is conceivable, no experimental data have been reported (McKone, 1987). Several reports on carbon tetrachloride levels in dwellings have been published. Taketomo & Grimsrud (1977) found values ranging between 0.6 and 1.3 µg/m3 for various types of dwellings, which is in agreement with the maximum indoor concentration of 1.2 µg/m3 reported by Clark (1981) and the range of 0.9-1.8 µg/m3 found in a US EPA study. In addition, several measurements have been made in garages, shops, supermarkets, swimming pools, restaurants, etc. (Taketomo & Grimsrud, 1977; Ullrich, 1982). The observed concentrations usually ranged between 0.6 and 2.0 µg/m3. The highest concentration, 10 µg/m3, was found in a dry-cleaning establishment. Wallace (1986) reported typical concentrations in homes in several cities in the USA of about 1 µg/m3; a maximum value of 9 µg/m3 was found. Shah & Heyerdahl (1988) found an average carbon tetrachloride level of 2.6 µg/m3, based on 2120 indoor samples. It should be noted, however, that carbon tetrachloride was not detected in more than half the samples. Table 6. Levels of carbon tetrachloride in biota Organism Location Organ Level (µg/kg) Reference Plankton Liverpool Bay, UK whole body 0.04-0.09 wet weight Pearson & McConnell (1975) Molluscs Firth of Forth, UK whole body 2 wet weight Pearson & McConnell (1975) Liverpool Bay, UK whole body 0.4-1 wet weight Pearson & McConnell (1975) Thames Estuary, UK whole body 0.1-0.9 wet weight Pearson & McConnell (1975) Irish Sea muscle 5-28 dry weight Dickson & Riley (1976) digestive tissue 8-20 dry weight Dickson & Riley (1976) gill 14 dry weight Dickson & Riley (1976) ovary 16 dry weight Dickson & Riley (1976) mantle 2-114 dry weight Dickson & Riley (1976) Crustaceans Firth of Forth, UK whole body 1-3 wet weight Pearson & McConnell (1975) Liverpool Bay, UK whole body 3-5 wet weight Pearson & McConnell (1975) Thames Estuary, UK whole body 0.2 wet weight Pearson & McConnell (1975) Fish Liverpool Bay, UK flesh 2 wet weight Pearson & McConnell (1975) liver ND-0.3 wet weight Pearson & McConnell (1975) Thames Estuary, UK flesh 0.3-6 wet weight Pearson & McConnell (1975) Irish Sea brain 15-191 dry weight Dickson & Riley (1976) gill 3-209 dry weight Dickson & Riley (1976) gut 9-44 dry weight Dickson & Riley (1976) liver 4-51 dry weight Dickson & Riley (1976) muscle 7-83 dry weight Dickson & Riley (1976) skeletal tissue 7-22 dry weight Dickson & Riley (1976) heart 10-40 dry weight Dickson & Riley (1976) ND = not detected. 5.2.3 Drinking-water The National Organics Monitoring Survey (NOMS) in the USA detected carbon tetrachloride (range of 2.4-6.4 ng/litre) in public drinking-water systems in 10% of the 113 samples surveyed (US EPA, 1984b). In 30 out of 954 drinking-water samples from various cities in the USA carbon tetrachloride could be detected. Median concentrations in different groups ranged from 0.3 to 0.7 µg/litre while maximum concentrations reached 16 µg/litre (Westrick et al., 1984). Bauer (1981) reported that drinking-water in Germany contained an average of less than 0.1 µg/litre although a maximum level of 1.4 µg/litre was found (average of 100 towns in 1977). Lahl et al. (1981) reported a carbon tetrachloride concentration less than 0.1 µg/litre in the drinking-water of 50 cities in Germany. In the United Kingdom, < 0.01-2.3 µg/litre was measured in drinking-water (Reynolds et al., 1982; Reynolds & Harrison, 1982). Values as high as a median of 3 µg/litre and a maximum of 39.5 µg/litre were reported in Galicia, Spain (Freiria-Gándara et al., 1992). 5.2.4 Foodstuffs According to investigations carried out in Europe and USA between 1973 and 1989, many foodstuffs contained carbon tetrachloride at concentrations of a few µg/litre or µg/kg. The following concentrations of carbon tetrachloride in foodstuffs in the United Kingdom in 1973 were reported: meat, 7-9 µg/kg; edible oils, 16-18 µg/kg; tea, 4 µg/kg; and fruits and vegetables, 3-8 µg/kg (McConnell et al., 1975). Values in a similar range were found for dairy products, other edible oils, fats, beverages, other fruits and bread, but here carbon tetrachloride and 1,1,1-trichloroethane could not be separated (McConnell et al., 1975). According to a study conducted in Germany, carbon tetrachloride can be present in decaffeinated coffee (4.9-60 µg/kg), milled cereal, flour and starch products (levels in 21 samples ranged from less than 0.1 to 26 µg/kg). The origin in the first case is the caffeine-extraction procedure, and in the second case in all probability fumigation of the raw cereals. The use of carbon tetrachloride for fumigation of stored foodstuffs and decaffeination of coffee appears to have generally ceased and it is unlikely that its occurrence in food stuff will be of significance. Less than 1 µg/kg was found in sugar, fruit, vegetables, beverages, bread, toast, potatoes, olives, oils, milk, butter, eggs, yoghurt, (cream) cheese, meat and fish. In cough mixtures 0.1 to 1.8 µg/kg was found (Bauer, 1981). Entz et al. (1982) and Entz & Hollifield (1982), in analyses of various foods for a series of volatile halogenated hydrocarbons, did not find carbon tetrachloride at a detection limit of 0.5 to 3 µg/kg, depending on the type of product. Decaffeinated coffee and flour products were not included in the studies. Kroneld (1989) detected carbon tetrachloride in meat (0.9 µg/kg), fish (0.6 µg/kg) and juice (0.3 µg/kg) in Finland in 1987. Carbon tetrachloride levels in table-ready foods in the USA were reported by Heikes (1987). He found up to 2.2 µg/kg in four sorts of cheese, 0.10-0.34 µg/kg in cereals, 1.7-5.7 µg/kg in fish sticks and up to 6.0 µg/kg in butter. In a survey by Daft (1989, 1991) carbon tetrachloride was detected in 44 out of 549 food items from the USA, most often in fatty and grain-based foods. The mean level in food items with detectable levels was 31 µg/kg (with a range of 2 to 210 µg/kg). 5.2.5 Intake averages The daily average intake of carbon tetrachloride in Japan by inhalation was calculated to be 7.7 µg/day (based on a daily inhalation volume of 15 m3/day and assuming a 100% absorption) and by ingestion less than 0.1 µg/day (Yoshida, 1993). If adjusted to a daily inhalation volume of 22 m3/day, an absorption of 40% and a body weight of 64 kg, the daily intake would be 11.4 µg/day or 0.18 µg/kg per day. The ATSDR (1994) estimated the daily intake by inhalation to be 0.1 µg/kg body weight based on ambient air level of about 1 µg/m3 (assuming inhalation of 20 m3/day, a body weight of 70 kg and an absorption of 40% based on measurements in monkeys and humans). The daily intake via drinking-water was estimated to be about 0.01 µg/kg body weight based on a typical carbon tetrachloride concentration of 0.5 µg/litre (assuming a water consumption of 2 litres/day and a body weight of 70 kg). In an earlier study of about 500 foodstuffs, an average daily intake via foods and drinks of 8.63 µg/person per day was calculated for inhabitants of Germany (Lahl, 1983). Because the intake by inhalation is expected to be at least as much (BUA, 1990), the total daily average intake would be estimated to be 17.26 µg/person (0.27 µg/kg body weight for a person of 64 kg). This calculation refers to a period when carbon tetrachloride was still used in food processing or in fumigation of grain. 5.3 Occupational exposure The most likely route of exposure in the workplace is by inhalation. Workers may be exposed to carbon tetrachloride during, for example, the production of carbon tetrachloride itself, the synthesis of compounds using carbon tetrachloride as a starting material and the use of carbon tetrachloride as a solvent. Furthermore, workers have been exposed to carbon tetrachloride at grain (due to fumigation) and water treatment facilities. The National Institute for Occupational Safety and Health estimated that in the USA around 58 000 workers were potentially exposed to carbon tetrachloride, based on a national survey conducted from 1981 to 1983 (National Library of Medicine, 1992). A few studies on concentrations of carbon tetrachloride in factories, and grain and water treatment facilities have been reported. For water treatment facilities, Lurker et al. (1983) reported exposure concentrations of 0.01-0.23 mg/m3; Clark (1981) reported concen trations ranging from 0 to 1.1 mg/m3. A peak exposure to an inspector during handling of grain at a facility in the USA reached 277 mg/m3. Few employees, however, had a mean exposure above 641 µg/m3 (Deer et al., 1987). Use of carbon tetrachloride in open beakers resulted in exposure levels of 290-620 mg/m3 at a United Kingdom quartz crystal processing plant. Levels were reduced to 50-60 mg/m3 by closing the beakers (Kazantzis & Bomford, 1960). 6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS 6.1 Pharmacokinetics 6.1.1 Absorption Carbon tetrachloride is absorbed readily from the gastrointestinal and respiratory tract. Dermal absorption of carbon tetrachloride, either in vapour or in liquid phase, is possible, but the dermal absorption of the vapour appears to be very low. 184.108.40.206 Oral Carbon tetrachloride is relatively insoluble in water, a source of exposure relevant to environmental scenarios and human health risk. As a result, many studies examining the hepatotoxicity of carbon tetrachloride used corn oil as a dosing vehicle for laboratory animals (Paul & Rubinstein, 1963; Larson & Plaa, 1965; Marchand et al., 1970). Corn oil has been found to delay markedly the absorption of carbon tetrachloride (Kim et al., 1990a) as well as other halocarbons (Withey et al., 1983) from the gastrointestinal tract. In part, because carbon tetrachloride in water is directly relevant to human exposure studies, recent studies in laboratory animals employed Emulphor(R), a polyethoxylated oil, at concentrations up to 10%, as an aqueous vehicle for carbon tetrachloride. Aqueous solutions of carbon tetrachloride in Emulphor(R) were administered to Sprague-Dawley rats both as a bolus and during gastric infusion at a constant rate during a 2-h period (Sanzgiri et al., 1995). Uptake and tissue levels of carbon tetrachloride after gastric infusion were less than after bolus dosing. When the concentration of Emulphor(R) was varied up to 10%, absorption (and distribution) of carbon tetrachloride was not affected (Sanzgiri & Bruckner, 1997). A comparison of the uptake of carbon tetrachloride in corn oil and aqueous emulsions is discussed in section 7.9. Tissue levels of carbon tetrachloride associated with bolus dosing, gastric infusion, and inhalation are discussed in section 6.1.2. The relationship of dosing vehicle, dose rate, and route of exposure to hepatotoxicity is discussed in section 7.9. 220.127.116.11 Dermal Liquid carbon tetrachloride on the intact mouse skin was absorbed at a rate of 8.3 µg/cm2 per minute (Tsuruta, 1975). Jakobson et al. (1982) examined the percutaneous uptake of liquid carbon tetrachloride (1 ml) in guinea-pigs (carbon tetrachloride in a glass depot, covering 3.1 cm2 of clipped skin). A peak blood level of about 1 mg carbon tetrachloride/litre was reached within 1 h. Despite continuation of the exposure the blood levels declined during the following h, possibly due to local vasoconstriction, rapid transport from blood to adipose tissues or biotransformation processes. Wahlberg & Boman (1979) applied 0.5 or 2 ml of carbon tetrachloride in a closed glass container on the skin (3.1 cm2) of guinea-pigs. The deposits were completely absorbed within a few days. McCollister et al. (1951), who exposed the clipped skin of one male and one female monkey to [14C]carbon tetrachloride vapour (whole body exposure), detected radioactivity in the blood and in the expired air. After an exposure of 3 h at 3056 mg/m3, the blood of the female contained a carbon tetrachloride level of 12 µg/100 g and the expired air contained 0.8 µg/litre. After exposure to 7230 mg/m3 for 3.5 h the blood of the male contained a carbon tetrachloride level of 30 µg/100 g and the expired air contained 3 µg/litre. 18.104.22.168 Inhalation In rats exposed by inhalation to carbon tetrachloride concentrations of 100 or 1000 ppm (641 or 6410 mg/m3) for 2 h, the total amounts systemically absorbed were 17.5 and 179 mg/kg body weight. The Cmax values (mg/ml) were approximately 1 and 13, respectively, and the AUC values (mg.min/ml) were approximately 120 and 1900, respectively (Sanzgiri et al., 1995). Steady-state carbon tetrachloride concentrations in the blood of approximately 320 mg/litre were reached within about 5 h when dogs were exposed to a carbon tetrachloride concentration in air of 15 000 ppm (96 150 mg/m3) for several hours (Von Oettingen et al., 1950). McCollister et al. (1951) exposed three female rhesus monkeys to an average [14C]carbon tetrachloride concentration of 46 ppm (295 mg/m3) via air for 139, 244 or 300 min, respectively. Within 300 min, 30% of the inhaled quantity was absorbed but in the blood no steady-state concentration of radioactivity was reached. The radioactivity level in the blood at that moment corresponded to 3 mg carbon tetrachloride/litre blood and was distributed over carbon tetrachloride (56.2%), "acid volatile" carbonates (16.5%) and non-volatile material (27.3%). The US EPA Iris Program uses 40% absorption as a mean for the calculation of human respiratory intake. The determined values ranged from 30% to 65% (US EPA, 1991). 6.1.2 Distribution The tissue distribution of carbon tetrachloride has been investigated in mice after inhalation (Bergman, 1984), in rats after oral administration (Marchand et al., 1970; Teschke et al., 1983; Watanabe et al., 1986) and after inhalation (Paustenbach et al., 1986a), in rabbits after oral administration (Fowler, 1969), in dogs after inhalation (Von Oettingen et al., 1950) and in monkeys after inhalation (McCollister et al., 1951). Bergman (1984) investigated the distribution of [14C]carbon tetrachloride in the mouse after a single inhalation exposure (10 min; 256 000 mg/m3 air). Immediately after the exposure, high levels of radioactivity were found in fat, bone marrow, white matter of the brain, spinal cord and nerves, liver, kidneys, salivary glands and gastrointestinal mucosa. The radioactivity in bronchi, liver, kidneys, salivary glands and the gastrointestinal mucosa (particularly in the mucosa of the glandular part of the stomach and of the colon and rectum) was to a large extent non-volatile. A similar pattern of distribution was observed 30 min after the exposure, except in the liver where a more pronounced accumulation of non-volatile radioactivity was seen than observed immediately after inhalation. A large part of the non-volatile radioactivity in the liver and kidneys appeared to be non-extractable, which may indicate covalent binding to tissue components (see section 6.2). Non-extractable radioactivity was also present in the bronchi and nasal mucosa. Non-volatile and non-extractable radioactivity was present in the vaginal and uterine mucosa and interstitially in the testis. The tissue distribution in rats (in order of decreasing radio-activity), 3 h after oral administration, as reported by Watanabe et al. (1986) was: liver, kidney, brain, muscle and blood. Carbon tetrachloride tends to accumulate in fat. Maximal fat tissue carbon tetrachloride concentrations exceeded the maximal blood levels by a factor of 60 after oral administration to rats (Marchand et al., 1970). Peak levels of carbon tetrachloride were observed 3-6 h following an acute oral carbon tetrachloride dose (1.5 ml/kg body weight administered in olive oil) in the blood (26 mg/litre), liver and fat of female Wistar rats. Subsequently, the carbon tetrachloride levels declined rapidly (Teschke et al., 1983). Peak blood levels of carbon tetrachloride after a 12-h inhalation exposure of rats were 12 mg/litre blood at an airborne concentration of 2 mg/litre (320 ppm), 20 mg/litre blood at 4 mg/litre (640 ppm) and 36 mg/litre blood at 8 mg/litre (1280 ppm). The blood level attained 50% of this value after 60 min. A 4-h exposure to a concentration of 2.6 mg/litre (406 ppm) led to a blood level of 10.5 mg/litre, which dropped to 50% of this peak value within 30 min after exposure (Frantik & Benes, 1984). Paustenbach et al. (1986a) found the highest concentration of carbon tetrachloride equivalents in the fat, liver, lungs and adrenals of male Sprague-Dawley rats repeatedly exposed to 100 ppm (641 mg/m3) of [14C]carbon tetrachloride vapour for 8 or 11.5 h/day for periods of 1 to 10 days. Fowler (1969) administered 1 ml carbon tetrachloride/kg body weight to rabbits by stomach tube as a 20% (v/v) solution in olive oil. Five rabbits were killed 6, 24 and 48 h after receiving carbon tetrachloride and the concentration in fat, liver, kidney and muscle tissue was determined. Two rabbits receiving olive oil were killed as control animals. The highest carbon tetrachloride concentration after 6, 24 and 48 h was found in fat tissue, but the amount found in the fat as well as in the other tissues after 6 h diminished rapidly during the subsequent 42 h. Von Oettingen et al. (1950) studied the distribution of carbon tetrachloride in Beagle dogs after exposure to 15 000 ppm (96 150 mg/m3) and reported a lowest concentration in the blood followed in increasing order by liver, heart and brain. The pattern of distribution immediately after a 5-h inhalation of 46 ppm (295 mg) [14C]carbon tetrachloride/m3 in monkeys (McCollister et al., 1951) was (tissues in order of decreasing concentration of total radioactivity): fat, liver, bone marrow, blood, brain, kidneys, heart, spleen, muscle, lungs, bone. Sanzgiri et al. (1995) demonstrated that the tissue pharmacokinetic profile was influenced by the route and rate of administration of carbon tetrachloride. Inhalation exposure of rats to 1000 ppm (6410 mg/m3) carbon tetrachloride for 2 h resulted in a systemic dose of 179 mg/kg body weight. This dose was subsequently administered as an oral bolus or a constant gastric infusion over 2 h. In all cases tissue levels were highest in fat with levels in all tissues being higher after an oral bolus dose than after inhalation exposure or gastric infusion. For the liver Cmax was higher after an oral bolus dose (58 mg/g) than after inhalation (20 mg/g) or gastric infusion (0.5 µg/g). The authors speculate that the capacity of first-pass metabolism can be exceeded following a large single bolus oral dose, although not during gastric infusion of the same dose over 2 h. 6.1.3 Elimination and fate In a study by Reynolds et al. (1984), all routes of elimination were investigated simultaneously after a single oral administration of [14C]carbon tetrachloride to fasted rats at dose levels ranging from 15.4 to 4004 mg/kg body weight. The exhalation of unchanged carbon tetrachloride increased at higher dose levels (70-90% after administration of 46.2 mg/kg body weight or more). This result might be explained by a saturation of the first pass metabolism, or by an impairment of the overall carbon tetrachloride metabolism due to a breakdown of cytochrome P-450, which is induced by carbon tetrachloride-metabolites (as reported by Noguchi et al., 1982a,b). Both the amount of carbon tetrachloride excreted and the time-course of excretion depended on the dose, tending to become slower as the dose increased. For example, the half-life for exhalation of carbon tetrachloride was 1.3 h at 46.2 mg/kg body weight but was 6.3 h at 4004 mg/kg body weight. Page & Carlson (1994) examined whether faecal excretion, either biliary or by direct exsorption, contributed significantly to carbon tetrachloride elimination from the body of rats. It appeared that biliary and non-biliary mechanisms contributed to the faecal elimination of [14C]carbon tetrachloride, but that this route accounted for less than 1% of the administered dose of 1 mmol/kg body weight in rats. Thus faecal elimination of carbon tetrachloride (as parent compound) does not significantly contribute to the overall elimination of carbon tetrachloride. The carbon tetrachloride levels in blood declined with a half-life of 4 to 5 h during the first 24 h after oral administration of 1.25 ml carbon tetrachloride/kg body weight (Larson & Plaa, 1965) or 2 ml (0.1 mCi) [14C]carbon tetrachloride/kg body weight (Marchand et al., 1970). Carbon tetrachloride levels in the liver declined with a half-life of about 7 h after administration by gastric intubation of 2.5 ml carbon tetrachloride/kg body weight (Dingell & Heimberg, 1968). Kim et al. (1990a) found a half-life for carbon tetrachloride in the blood of 98 min and a whole body clearance of 0.13 ml/min per g when 25 mg carbon tetrachloride/kg body weight was orally administered in four different vehicles to male Sprague-Dawley rats. The elimination appeared to be the same in all the different vehicle groups, whereas the absorption differed (see section 22.214.171.124). According to Paustenbach et al. (1986a) the rate of carbon tetrachloride clearance in rats after inhalation exposure is biphasic, with an initial half-life of 7 to 10 h. Exposure for longer periods of time led to a decreased rate of clearance (and to higher concentrations in the fat) (Paustenbach et al., 1986a,b, 1988). In the study of Sanzgiri et al. (1995) (see section 126.96.36.199) the apparent clearance values after inhalation doses delivering 17.5 or 179 mg/kg body weight, respectively, were 150 and 100 ml/min per kg and the half-life value was about 164 min. Veng-Pedersen et al. (1987) exposed rats repeatedly by inhalation to 100 ppm [14C]carbon tetrachloride (641 mg/m3) for either 8 h/day for 5 days or 11.5 h/day for 4 days. The pulmonary excretion of [14C]activity was clearly biphasic for both dosing regimens, with mean half-lives for the first and second phase being 84 and 400 min for the 8-h exposure and of 91 and 496 min for the 11.5-h exposure, respectively. This indicates that the second phase of the 11.5-h group was longer than the second phase of the 8-h group. This observation suggests that during longer exposure periods a greater fraction of the inhaled carbon tetrachloride is distributed to poorly perfused tissues like fat, thus altering the elimination. McCollister et al. (1951) demonstrated in monkeys that, after an inhalation exposure to [14C]carbon tetrachloride, radioactive material was excreted in faeces, urine and expired air. According to the authors the compounds in the urine consisted of urea, bicarbonate and an acid hydrolysable, non-amino acid substance. 6.1.4 Physiologically based pharmacokinetic modelling A biphasic kinetic in the biotransformation of carbon tetrachloride has been observed in several inhalation studies. The relationship of arterial blood and inhaled carbon tetrachloride concentrations, as found in male Sprague-Dawley rats, suggested that carbon tetrachloride metabolism is limited by blood perfusion of the liver at inhaled concentrations below 100 ppm (641 mg/m3) and that it is saturated at inhaled concentrations above 100 ppm. The estimated rate of reaction (Vmax) measured in the blood was 2.7 mg/kg body weight per hour. This rate gradually decreased during the exposure period of 5 h, which could be due to rapid loss of cytochrome P-450 content. The Vmax in rats pretreated with 100 µl carbon tetrachloride (oral administration, 24 h before inhalation exposure) decreased about 57%, which was in good agreement with the decrease of the cytochrome P-450 content. (Uemitsu, 1986). Gargas et al. (1986) calculated for carbon tetrachloride a Vmax of 0.63 mg/kg body weight per hour in an inhalation study in male Fischer-344 rats. Applications of pharmacokinetic models for the inhalation exposure of rats have provided Vmax and Km estimates in rats of 0.63 mg/h per kg and 0.25 mg/litre (Gargas et al., 1986) and 0.37 mg/h/kg and 1.3 mg/litre (Evans et al., 1994). Paustenbach et al. (1988) constructed a physiologically based pharmacokinetic model (PB-PK) for inhaled carbon tetrachloride and used this model to predict the pharmacokinetics of inhaled [14C]carbon tetrachloride in male Sprague-Dawley rats exposed for 8 or 11.5 h/day for 1 or 2 weeks. The simulations were compared with actual laboratory data (Paustenbach et al., 1986a,b). The model accurately predicted the behaviour of carbon tetrachloride and its metabolites. Metabolites were partitioned in three compartments: the amounts to be excreted in the breath (as [14C]CO2), urine and faeces. Of total carbon tetrachloride metabolites, 6.5, 9.5 and 84% were formed via the pathways leading to CO2, urinary and faecal metabolites, respectively. The PB-PK model suggests that at concentrations up to 100 ppm (641 mg/m3), rats, monkeys and humans metabolize and eliminate carbon tetrachloride in a similar manner. Most species convert 2-5% to CO2, eliminate 4-8% in the urine, and eliminate 40-50% unchanged in the breath. 6.2 Biotransformation and covalent binding of metabolites Metabolism of carbon tetrachloride is initiated by cytochrome P-450-mediated transfer of an electron to the C-Cl bond, forming an anion radical that eliminates chloride, thus forming the trichloromethyl radical. This radical may undergo both oxidative and reductive biotransformation (see Fig. 1). The isoenzymes implicated in this process are CYP2E1 and CYP2B1/2B2 (Raucy et al., 1993; Gruebele et al., 1996). Some isoforms may be preferentially susceptible to degradation by carbon tetrachloride (Tierney et al., 1992). Evidence that carbon tetrachloride inactivates CYP2E1 and reduces total CYP2E1 protein in a cell line that constitutively expresses human CYP2E1 has been obtained by Dai & Cederbaum (1995). When protein synthesis was blocked, inactivation and degradation of CYP2E1 by carbon tetrachloride was more pronounced. Free radical scavengers were unable to prevent CYP2E1 degradation, suggesting that carbon tetrachloride metabolites react at the active site of CYP2E1. Antioxidants prevented carbon tetrachloride-induced lipid peroxidation, but not CYP2E1 degradation, suggesting that these processes are disassociated. The formation of the radical has been demonstrated convincingly in vitro as well as in vivo in electron spin resonance experiments and is mediated by a particular cytochrome P-450, of which the haem moiety is destroyed after carbon tetrachloride exposure (Reiner et al., 1972; Sipes et al., 1977; Poyer et al., 1978, 1980; Lai et al., 1979; Tomasi et al., 1980; Fernández et al., 1982; Noguchi et al., 1982a,b; McCay et al., 1984). The formation of carbon tetrachloride/cytochrome P-450 complexes has been demonstrated by Uehleke et al. (1973), Wolf et al. (1977), Ahr et al. (1980) and Fernández et al. (1982). The most important pathway in the elimination of trichloromethyl radicals is the reaction with molecular oxygen, resulting in the formation of trichloromethylperoxyl radicals (CCl3OO*), as proposed by Packer et al. (1978), Shah et al. (1979), Mico & Pohl (1983), Pohl et al. (1984) and McCay et al. (1984). This intermediate, which is even more reactive than the trichloromethyl radical (Dianzani, 1984), may interact with lipids, causing lipid peroxidation along with the production of 4-hydroxyalkenals (Benedetti et al., 1982; Comporti et al., 1984). Radical-induced lipid peroxidation is also a presumed source of a variety of metabolites, such as acetone, propanal, butanal and malondialdehyde, which appear in rat urine 24 h after exposure to carbon tetrachloride (de Zwart et al., 1997). It is supposed that the trichloromethylperoxyl radical will react further to produce phosgene, which again may interact with tissue macromolecules or with water, finally producing hydrochloric acid and carbon dioxide (Pohl et al., 1984). Carbon tetrachloride has been reported to be metabolised to carbon dioxide in liver homogenates by Rubinstein & Kanics (1964). The biotransformation of carbon tetrachloride to carbon dioxide in vivo has been reported by Reynolds et al. (1984). Condensation of phosgene with cysteine leads to the formation of 2-oxothiazolidine-4-carboxylic acid (Shah et al., 1979; Kubic & Anders, 1980). The condensation of phosgene with glutathione (GSH), resulting in diglutathionyl dithiocarbonate, has been demonstrated by Pohl et al. (1981) in in vivo experiments. Formation of chloroform and CCl2-carbene occur under O2-deficient circumstances (Reiner et al., 1972; Shah et al., 1979; Pohl et al., 1984). Under in vivo conditions CCl2-carbene is of minor importance as an intermediate. Castro et al. (1990) examined the biotransformation of carbon tetrachloride to chloroform by liver nuclear preparations of three different species: C3H mice, Sprague-Dawley rats and Syrian golden hamsters. All species were able to transform carbon tetrachloride to chloroform. This ability was not NADPH dependent and proceeded to an equal extent under nitrogen and air. The relative transforming intensity was mice > hamsters > rats under anaerobic and hamsters >> mice > rats under aerobic conditions, respectively. More detailed experiments with preparations of C3H mice suggested the presence of enzymatic and non-enzymatic pathways of carbon tetrachloride transformation, as revealed by their heat susceptibility and the inhibitory effects of EDTA. Many studies on covalent binding of carbon tetrachloride metabolites to tissue macromolecules have been carried out, but most of them have measured only radioactivity and not identified the adduct formed. Many studies ignored the radioactive impurities present in the carbon tetrachloride used as well as the possible incorporation of 14C-radioactivity via carbon dioxide riginating from carbon tetrachloride. For these reasons, the binding of carbon tetrachloride should be considered as an association of radioactivity, unless further information is provided. According to Shertzer et al. (1988), the active radical metabolic intermediate of carbon tetrachloride may covalently bind to macromolecules, produce lipid peroxidation, and result in the loss of intrahepatic calcium homoeostasis. Cambon-Gros et al. (1986) showed that the fetal rat liver during the last days of pregnancy, as well as the mother liver, can metabolize carbon tetrachloride into a free radical: CCl3*. This radical may bind covalently to the microsomal membranes and cause the destruction of cytochrome P-450 as well as the inhibition of one of the main microsomal activities, the ability to store Ca2+. Contrary to the situation in adults, these radicals do not provoke a membrane phospholipid peroxidation in the fetus. The animals used in the study were nulliparous pregnant female Sprague-Dawley rats (twentieth day of gestation). Tjälve & Löfberg (1983) showed that covalent binding (probably due to metabolic activation) occurred in many tissues of exposed rats (liver, kidney cortex, mucosae of the respiratory tract, the mouth cavity and the oesophagus). The non-extractable part of non-volatile radioactivity in liver and kidney may indicate covalent binding to tissue components (Bergman, 1984). Association of radioactivity to tissue components also appeared to occur in the testis, the uterine and vaginal mucosa and in the nasal mucosa. A similar pattern of association with tissue components to that observed by Bergman (1984) has been found by Tjälve & Löfberg (1983) after intravenous and intraperitoneal administration, indicating that the tissue binding in the nasal mucosa is not specific to the route of administration. Díaz Gómez et al. (1975a) investigated the relations between liver carbon tetrachloride levels, lipid peroxidation, the covalent binding to liver lipids and hepatic centrilobular necrosis after in vivo adminis tration of equimolar doses of carbon tetrachloride in different animal species. The results support the hypothesis that carbon tetrachloride-induced lipid peroxidation is not the only mechanism of its toxic action. In fact, there seems to be a better correlation between irreversible association with tissue components and carbon tetrachloride toxicity than between lipid peroxidation and carbon tetrachloride toxicity. According to Villarruel et al. (1977) association of carbon tetrachloride metabolites with lipids occurs mostly in the liver and kidney cortex and medulla. Ansari et al. (1982) demonstrated the binding of trichloromethyl radicals originating from carbon tetrachloride to cholesterol. Binding to membrane lipids, eventually leading to cross-linking, has been demonstrated by Link et al. (1984). Association of carbon tetrachloride derivatives with macromolecules in vitro has been found mainly in microsomal systems, but binding to lipids and proteins also occurs in purified nuclear preparations (Díaz Gómez & Castro, 1980b). The covalent binding of carbon tetrachloride reactive metabolites to different nuclear and microsomal lipids was studied by Fanelli & Castro (1995) in male Sprague Dawley and Osborne Mendel rats, strains with a marked difference in the carcinogenic response to carbon tetrachloride, the Sprague-Dawley being non-susceptible and the Osborne-Mendel being responsive. The intensity of covalent binding to microsomal lipids in vivo and in vitro was higher in the Osborne Mendel rats. Most of the covalent binding of carbon tetrachloride reactive metabolites in both rat strains occurs in the phospholipid and in the cholesterol/cholesterol ester fractions. The covalent binding to phospholipids is higher in the Sprague Dawley strain, while binding to cholesterol and cholesterol ester is more intense in the Osborne Mendel rat. After administration of [14C]carbon tetrachloride to rats and mice (9 µmol/kg body weight), the quantities of label associated with DNA at 6 h post-dosing were 0.52 and 0.72 pmol/mg DNA, respectively. In this study binding also occurred to nuclear proteins and lipids, especially to phospholipids (diphosphatidylglycerol) and diglycerides (Díaz Gómez & Castro, 1980a). The results of the study of Oraumbo & Van Duuren (1989) indicated that under aerobic incubation conditions, carbon tetrachloride is metabolized to one or more electrophilic metabolites, which bind covalently to chromatin DNA in a dose- and time-dependent manner. In this study chromatin was isolated from male B6C3F1 hybrid mice and incubated with [14C]carbon tetrachloride in the presence of hepatic microsomes from the same animals and a NADPH-regenerating system. The study was carried out with various carbon tetrachloride concentrations and incubation times. 6.3 Human studies 6.3.1 Uptake 188.8.131.52 Dermal Immersion of a thumb in liquid carbon tetrachloride for 30 min produced a maximum alveolar carbon tetrachloride concentration of about 3.8 mg/m3 air 30 min after the end of exposure (Stewart & Dodd, 1964). The concentration declined with a half-life of about 2.5 h. 184.108.40.206 Inhalation Lehmann & Schmidt-Kehl (1936) reported that 60% of the quantity of carbon tetrachloride inhaled was retained in an experiment involving a 30-min exposure of a volunteer to 4200 mg/m3. 6.3.2 Elimination The pulmonary excretion of 33% of the absorbed quantity of [38Cl] carbon tetrachloride occurred during the first hour after a single breath by a volunteer (Morgan et al., 1970). Erickson (1981) found carbon tetrachloride in mother's milk (concentration and exposure not specified). 7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS 7.1 Single exposure 7.1.1 Lethality LD50 values of carbon tetrachloride for various mammalian species are represented in Table 7. For female OF1 mice a LC50 of 7176 ppm (45 998 mg/m3) was reported after a 6-h inhalation exposure to carbon tetrachloride (14 days observation period) (Gradiski et al., 1978). Svirbely et al. (1947) reported a LC50 of 50 000 mg/m3 after a 7-h inhalation exposure (8-h observation period) for male and female Swiss mice. A 100% death rate of Wistar rats due to anaesthesia was observed by Adams et al. (1952) after inhalation exposure to carbon tetrachloride at concentrations of 121 600 mg/m3 for 2.2 h or 46 700 mg/m3 for 8 h. Roudabush et al. (1965) reported that the acute dermal LD50 values of carbon tetrachloride for rabbits and guinea-pigs were in excess of 15 g/kg body weight. Data on mortality and observation period were not given. Wahlberg & Boman (1979) applied carbon tetrachloride in quantities of 800 and 3200 mg (approximately 2100 and 8500 mg/kg body weight) to the skin of guinea-pigs. After 21 days 5/20 and 13/20 of the animals had died in the 800 mg and 3200 mg groups, respectively. 7.1.2 Non-lethal effects 220.127.116.11 Oral exposure Effects on the liver with changes in several enzyme levels are reported to be the major effects resulting from an acute oral exposure to carbon tetrachloride. In addition to the effects on the liver, effects have also been reported in other organs such as lungs and kidneys. a) Mice Akahori et al. (1983) examined the biochemical alterations in liver and blood, and the histological findings in the liver of female C57BL/6J mice after a single oral administration of carbon tetrachloride in liquid paraffin at doses of 0.02, 0.5 or 1.5 ml/kg body weight (calculated to be 32, 797 or 2391 mg/kg body weight) at various times from 15 to 327 h after administration. The biochemical changes in the liver (decreases in protein, glucose, phospholipids, DNA and RNA concentrations; increases in triglycerides, glycogen, and Table 7. LD50 values (mg/kg body weight) for mammals Species/strain Sex Route Vehicle Observed period LD50a Reference Mice Swiss Webster male intraperitoneal corn oil 24 h 4144 Klaassen & Plaa (1967a) female intraperitoneal corn oil 24 h 4463 Klaassen & Plaa (1967a) Strain and sex unknown oral unknown not reported 12 100-14 400 IARC (1979) Swiss Webster female intraperitoneal corn oil 24 h 4676 Gehring (1968) OF1 (SPF) female intraperitoneal olive oil 14 days 3350 Gradiski et al. (1974) Rats Wistar female oral unknown 14 days 2821 Smyth et al. (1970) Sprague-Dawley male intraperitoneal corn oil 48 h 4463 Klingensmith et al. (1983) Sprague-Dawley male intraperitoneal corn oil 24 h 3029 Klaassen & Plaa (1969) Sprague-Dawley female intraperitoneal peanut oil 24 h 6603 Lundberg et al. (1986) 14 days 2824 Lundberg et al. (1986) Charles River male oral corn oil 14 days 10 054 Kennedy et al. (1986) Dogs Mongrel male/female intraperitoneal corn oil 24 h 2391 Klaassen & Plaa (1967b) a Most of the values are calculated values because they were reported as ml/kg body weight free and esterified cholesterol concentrations) and in blood (an increase in serum aspartate aminotransferase (ASAT) activity and free and esterified cholesterol concentration; a decrease in glucose concentration) were generally dose-related but occurred more slowly in the highest dose groups. The biochemical alterations were reflected in the histological findings in the liver (centrilobular necrosis in the low-dose and a mild midzonal necrosis in the mid- and high-dose groups). These histological findings occurred later in the high-dose group. A dose-related increase in the serum angiotensin converting enzyme level, indicative of pulmonary endothelial cell injury, was reported by Hollinger (1982), who administered carbon tetrachloride in vegetable oil at doses of 0.1 to 2.8 ml/kg body weight (159 to 4463 mg/kg body weight) to male Swiss-Webster mice. Boyd et al. (1980) found morphological effects on the pulmonary Clara cells of mice, including severe dilations of endoplasmic reticulum and occasional cellular necrosis, after administration of 2.5 ml/kg body weight (3985 mg/kg body weight) of carbon tetrachloride in sesame oil. Oral doses of less than 1 ml/kg body weight (1594 mg/kg body weight) failed to produce visible pulmonary lesions. b) Rats Murphy & Malley (1969) reported dose-related increases in liver and serum alanine aminotransferase (ALAT), liver tyrosine transaminase and alkaline phosphatase activities after a single oral dose of 0.5 to 2 ml/kg body weight (797 to 3188 mg/kg body weight) of undiluted carbon tetrachloride in male Holtzman rats. In a study by Korsrud et al. (1972), fasted male Wistar rats received carbon tetrachloride at a dose ranging from 0 to 2.5 ml/kg body weight (0 to 3985 mg/kg body weight) in corn oil. The rats were killed 18 h later. At a dose of 0.0125 ml/kg body weight (19.9 mg/kg), there was histopathological evidence of toxic effects on the liver. At 0.025 ml/kg body weight (39.9 mg/kg), liver fat and weight, serum urea and the activities of sorbitol dehydrogenase, fructose-l-P-aldolase, isocitrate dehydrogenase, ALAT and aspartate aminotransferase (ASAT) were increased. According to Teschke et al. (1984), liver enzymes such as ALAT, ASAT and glutamate dehydrogenase measured in serum reached maximal activities 12-48 h following a single intragastric dose of carbon tetrachloride (1.5 ml/kg body weight) to female Wistar rats. A maximum increase of ASAT and ALAT after 48 h was reported by Nakata et al. (1985) after administration of 5 ml/kg body weight (7970 mg/kg body weight) of carbon tetrachloride in corn oil to male Wistar rats. Regeneration of the liver was characterized by a normalization of the ASAT and ALAT levels and an increase in hepatic thymidylate synthetase and thymidine kinase levels, two enzymes that are believed to be indicative of tissue regeneration. A single oral bolus of carbon tetrachloride (17.5 or 179 mg/kg) to male Sprague-Dawley rats induced a dose-dependent increase in serum sorbitol dehydrogenase and ALAT activities, and a decrease in the hepatic cytochrome P-450 content and glucose-6-phosphatase activity. When the same dose was given as a gastric infusion for 2 h, or by inhalation exposure, the effects were much smaller (Sanzgiri et al., 1995). No statistically significant difference was observed in the toxicity of carbon tetrachloride administered orally in either corn oil, Emulphor, or Tween-85 (Raymond & Plaa, 1997). Significant increases in alpha-GSH, a cytosolic enzyme of short half- life found in high concentration throughout the liver, were detected 2 h after gavage dosing of male Sprague-Dawley rats (Clarke et al., 1997). It was concluded that alpha-GST is a more sensitive and accurate measure of carbon tetrachloride hepatotoxicity than ASAT. Lowrey et al. (1981) reported a dose-dependent decreased capacity of rat liver microsomes to sequester calcium 5 min after the administration of carbon tetrachloride to fasted male Sprague-Dawley rats at doses ranging from 0.025 to 5 ml/kg body weight (40 to 7970 mg/kg body weight). At 10 min after a carbon tetrachloride dose of 2.5 ml/kg body weight (3985 mg/kg body weight), the microsomal calcium uptake was reduced to 15% of the control levels. Chen et al. (1977) observed marked decreases in cytochrome P-450 content and P-450-related N-demethylation of dimethylaniline in the microsomes of the lungs of male Sprague-Dawley rats after an oral carbon tetrachloride dose in mineral oil of 2.5 ml/kg body weight (3985 mg/kg body weight). Shinozuka (1971) found alterations of the rough endoplasmic reticulum membranes of rat hepatic cells, such as detachment of ribosomes, narrowing of cisternal spaces, fusion of membranes and eventual collapse, within 30 min after an administration of 5 ml/kg body weight (7970 mg/kg body weight) of carbon tetrachloride in mineral oil to Wistar rats. Boyd et al. (1980) found lesions in the lungs of male Sprague-Dawley rats that were similar to the lesions found in mice (enlarged pulmonary Clara cells with dilations of the endoplasmic reticulum, occasional cellular necrosis) after carbon tetrachloride administration at doses of 2.4, 3.2 or 4.5 ml/kg body weight (3825, 5100 or 7173 mg/kg body weight) in sesame oil. These lesions, however, were less pronounced and less frequent than in mice. Striker et al. (1968) observed reversible lesions limited to the proximal tubules in the kidneys of male Sprague-Dawley rats after administration of 0.25 ml/kg body weight (400 mg/kg body weight) of carbon tetrachloride in mineral oil. The earliest morphological change was seen in the mitochondria, followed by cellular swelling, loss of basilar interdigitations and swollen microvilli. Proliferation of the smooth endoplasmic reticulum occurred later. Serum parameters, such as creatinine, blood urea nitrogen and bilirubin, temporarily increased. Furthermore, a decrease in the ability to preserve sodium ions and water was observed, accompanied by a reduction of succinate dehydrogenase activity. Rats administered carbon tetrachloride in corn oil as a single intraperitoneal dose had significantly prolonged clotting times that appeared prior to liver necrosis (Pritchard et al., 1987). c) Rabbits Rabbits (strain unspecified), given an intragastric dose of 0.15 ml/kg body weight of a 33% carbon tetrachloride solution in liquid paraffin (equivalent to 239 mg/kg body weight) showed an abnormal electrophoretic lipoprotein pattern. This correlated with the degree of liver injury as measured by ASAT and ALAT activities and plasma lipid levels (Kanaghinis et al., 1982). d) Monkeys Centrilobular hepatocellular necrosis was observed in two out of four monkeys 24 h after administration of a single oral dose of carbon tetrachloride (1590 mg/kg). e) Dogs Dogs were administered single oral doses of 159, 318 and 477 mg/kg. Increased serum ALAT and ASAT activities were observed at 318 mg/kg or more. 18.104.22.168 Inhalation exposure a) Mice Boyd et al. (1980) exposed male Swiss mice to carbon tetrachloride concentrations of 0.46 or 0.92 mmol/litre air for 1 h, 1.84 mmol/litre air for 12 min, and 3.68 mmol/litre air for 2 min (70 750, 141 500, 283 000 and 566 000 mg/m3 air). All exposures produced marked Clara cell lesions, similar to those caused by oral exposure, and hepatic necrosis. b) Rats Brondeau et al. (1983) exposed male Sprague-Dawley rats (IFFA CREDO; 8 males/group) to carbon tetrachloride at concentrations of 259, 531, 967 and 1459 ppm (1660, 3404, 6198 and 9352 mg/m3 air) for 4 h and examined the ASAT, ALAT, SDH and glutamate dehydrogenase activities in the serum 24 h post-exposure. At the lowest exposure level only the glutamate dehydrogenase activity was increased, whereas at the higher exposure levels increases were observed in all enzyme activities. Similar increases in serum activities of liver enzymes have been reported in other rat strains (Magos et al., 1982 (Porton-Wistar and Fischer rats); Jaeger et al., 1975 (Holtzman rats); Siegers et al., 1985 (Wistar rats)). When male Sprague-Dawley rats were exposed to carbon tetrachloride under conditions of various combinations of concentration (; 1350 to 6900 ppm) and exposure time, it appeared that the concentration had more influence on the hepatotoxicity than the exposure time (Uemitsu et al., 1985). Chen et al. (1977) observed a decrease in the cytochrome P-450 content and P-450-related demethylation of dimethylaniline in the microsomes of lungs of male Sprague-Dawley rats exposed for 30 min to air containing 4.38% carbon tetrachloride (280 758 mg/m3). Morphological analysis of the lungs revealed focal changes in pulmonary architecture consisting of alveolar collapse, septal thickening and atypical type II pneumocyte configuration. c) Cats Wong & DiStefano (1966) exposed cats to carbon tetrachloride at a concentration of 10 000 ppm (64 100 mg/m3) for 15, 30, 60 and 240 min. After 15 min the renal lipid content reached maximal levels. An increase of kidney weight occurred within 60 min and was maintained throughout the 24-h observation period. The total lipid content of the liver had increased significantly at 3, 12 and 24 h after the 4-h exposure period. Increased liver weight was observed 24 h after the withdrawal of carbon tetrachloride. According to the authors, the early increases in both the weight and fat content of the kidney suggests that the renal changes precede the liver damage. 22.214.171.124 Subcutaneous and intraperitoneal exposure a) Mice A subcutaneous dose of 28 mg/kg body weight of carbon tetrachloride in olive oil to male Swiss mice (10/group) appeared to be the ED50 for causing prolongation of pentobarbital-induced sleeping time. Histological examination showed changes in the liver after administration of 77 mg/kg body weight. (Kutob & Plaa, 1962). Intraperitoneal administration of carbon tetrachloride in corn oil induced an elevation of the ALAT activity at calculated dose levels of 11.2 to 15.9 mg/kg body weight in female Swiss mice and at 14.4 to 15.9 mg/kg body weight in male Swiss mice (Klaassen & Plaa, 1967a). Bhathal et al. (1983) reported striking differences in the degree of hepatic cell injury among four different strains of mice upon histological examination after subcutaneous injection of 0.3 ml/mouse of olive oil containing 5, 10 and 20% carbon tetrachloride. The SJL/J strain appeared to be the least susceptible and the BALB/c strain the most susceptible one. The hepatic lesions in the C3H and C57BL/6 strains were intermediate. b) Rats The study of Smejkalová et al. (1985) showed the existence of sex differences in the sensitivity of the liver to carbon tetrachloride, including a difference in the rate and quality of liver regeneration. It appeared that in male Wistar rats the biochemical changes occurred earlier (as early as 6 h after intraperitoneal administration of 1200 mg/kg body weight) and persisted longer (reaching a maximum after 12 h and persisting for more than 72 h) than in females. In females these changes reached a maximum after 24 h, and after 72 h the levels were identical to the control values. Whereas in females the liver regeneration started sooner than in males and led to complete healing of the liver tissue, the regeneration in males started more slowly and healing followed a different course, showing the development of fibrosis. Carbon tetrachloride dissolved in olive oil was injected intraperitoneally to male Fischer rats at doses of 30 to 1000 mg/kg body weight. Free and esterified cholesterol, triglycerides, phospholipids and total lipids in plasma were reduced in a dose-dependent manner. Cholesterol, triglyceride, phospholipid and total lipid concentrations in the plasma were significantly lower in rats given 30 mg/kg body weight than in control rats (p < 0.01) (Honma, 1990). When male Wistar rats received carbon tetrachloride as an intraperitoneal injection of 16 or 96 mg/kg body weight in olive oil, the calcium ion (Ca2+) content of liver microsomes was significantly increased by 20% in rats treated with 96 mg/kg body weight. The mitochondrial Ca2+ content was increased in both the 16 and 96 mg/kg body weight group (600% and 1100%, respectively, 3 h after administration) (Yamamoto, 1990). c) Guinea-pigs Divincenzo & Krasavage (1974) administered intraperitoneally 5, 25, 50, 75 or 150 mg/kg body weight to guinea-pigs. At 25 mg/kg or more, increased ornithine decarboxylase activity in the serum was found, an effect that was reflected by histological changes in the liver. d) Hamsters Carbon tetrachloride produced injury to ciliated and non-ciliated tracheal cells (swollen, loss of staining capacity, diluted nuclei) of adult male Syrian golden hamsters that received carbon tetrachloride intraperitoneally at a dose of 2.5 ml/kg body weight (3985 mg/kg body weight). Groups of three hamsters were killed 1, 4, 12 or 24 h after treatment. The number of damaged cells increased markedly after 1 h in the lower trachea, but not until after 4 h in the upper trachea. By 24 h the number of injured cells approached normal values. Effects were consistent within each group (Ahmadizadeh et al., 1990). e) Dogs When five mongrel dogs were given carbon tetrachloride at different intraperitoneal doses, increases in ALAT were observed. The calculated ED50 24-h after exposure was 32 mg/kg (Klaassen & Plaa, 1967b). De Zwart et al. (1997) have identified eight urinary degradation products of carbon tetrachloride-induced lipid peroxidation as potentially useful biomarkers of in vivo hepatocellular damage. Male Wistar rats were injected intraperitoneally with single doses of 38, 77 and 154 mg/kg body weight and the following substances were identified in urine 12 to 48 h later: formaldehyde, acetaldehyde, acetone, propanol, butanol, pentanal, hexanal and malondialdehyde. A dose-dependent increase in histological and clinical chemistry evidence of hepatocellular damage, along with these degradation products, was observed. Increases in urinary concentrations of all eight products were statistically significant at doses of 77 and 154 mg/kg. At 38 mg/kg, acetaldehyde and propanol were the only urinary markers to exhibit a statistically significant increase. 126.96.36.199 Dermal exposure The histopathology of the skin, liver, and kidney in the guinea-pig (weighing 440 to 570 g) was studied by Kronevi et al. (1979) at 15 min and 1, 4 and 16 h after occlusive epicutaneous administration of 1 ml of carbon tetrachloride. After 15 min, some degenerative changes in the epidermis, such a moderate karyopyknosis, marked spongiosis and perinuclear oedema was observed. These changes became more obvious with time, and at 16 h a slight karyolysis also was seen. A junctional separation and cellular infiltration in the dermis was observed after 4 and 16 h. Carbon tetrachloride exposure caused hepatic centrilobular hydropic changes and, in addition, a tendency to necrotic lesions after 16 h. Kidney histology was normal for all exposed guinea-pigs. 7.2 Short-term exposure 7.2.1 Oral exposure a) Mice Hayes et al. (1986) administered carbon tetrachloride in corn oil to CD-1 ICR mice (20/sex/group) for 14 consecutive days at dose levels of 0, 625, 1250 or 2500 mg/kg body weight and for 90 consecutive days at dose levels of 0, 12, 120, 540 or 1200 mg/kg body weight. No compound-related deaths were seen in the 90-day study whereas, in the 14-day study, 6, 8 and 12 males and 0, 1 and 2 females died within 2-4 days in the 625, 1250 and 2500 mg/kg body weight groups, respectively. Dose-dependent effects in the 14-day study consisted of decreased fibrinogen and lymphocyte levels, increased LDH, ALAT and ASAT levels, increased absolute and relative liver weights in both sexes, and decreased lung, thymus and kidney weights in males. In the 90-day study LDH, ASAT, ALAT and AP, cholesterol and bilirubin levels in the blood were increased in a dose-dependent manner while blood glucose levels were decreased at all dose levels. In both sexes and in all 90-day dose groups absolute and relative liver, spleen and thymus weights were increased and liver damage was observed. The results of a 90-day oral study in CD-1 mice by Condie et al. (1986), indicated that the no-observed-adverse-effect level (NOAEL) for hepatotoxic effects after administration of carbon tetrachloride in corn oil was 1.2 mg/kg body weight (see also section 7.9). b) Rats Four groups of weanling rats (six males and six females per group) were fed diet containing 0, 150, 225 or 520 mg/kg; estimated daily doses were 0, 7-13, 13-24, 21-27 mg/kg body weight. The fat content of liver was increased significantly in two higher dose groups in both males (exposed for 6 weeks) and females (exposed for 5 weeks); the difference compared to the control group was 50 to 200% (Alumot et al., 1976). Carbon tetrachloride treatment for 5 consecutive days at a dose level of 400 mg/kg body weight in corn oil to male Fischer-344 rats (CD F/CrlBr) increased the relative liver weight, decreased the CYP enzyme concentration and activity in the liver, and increased the ALAT levels (Dent & Graichen, 1982). Bruckner et al. (1986) administered carbon tetrachloride in corn oil to male Sprague-Dawley rats (5/group) for 5 consecutive days for 12 weeks, then 2 days without dosing followed by another 4 consecutive days of dosing. Groups of rats weighing 300 to 350 g received 0, 20, 40 or 80 mg/kg body weight, whereas groups of rats weighing 200 to 250 g received 0, 20, 80 or 160 mg/kg body weight. In rats weighing 300-350 g, 20 mg/kg body weight caused vacuolization of hepatocytes adjacent to the central vein of most liver lobules. The other dose levels in this group produced comparable increases in SDH and ALAT activities, and vacuolization of 25 to 35% of the hepatocytes in each liver lobule. Carbon tetrachloride appeared to be more toxic to the rats weighing 200-250 g with regard to necrotic cells, which were rarely seen in livers of the 300-350 g rats, while in each 200-250 g rat given 80 mg/kg body weight necrosis was observed. Groups of 15-16 male Sprague-Dawley rats weighing 200 to 250 g were given carbon tetrachloride in corn oil at doses of 0, 1, 10 and 33 mg/kg body weight for 5 days a week. The rats were dosed during the dark part of their light cycle. After 12 weeks, 7 to 9 rats/group were killed. The remaining animals were killed 13 days after exposure. At 10 mg/kg body weight a slightly but significantly increased SDH activity and mild hepatic centrilobular vacuolization was seen. Administration of 33 mg/kg body weight caused elevated serum levels of SDH, ornithine-carbamyl transferase (OCT) and ALAT, which returned to normal during the recovery period except for the OCT. Histopathology of the livers of the 33 mg/kg body weight group revealed cirrhosis, characterized by bile duct proliferation, fibrosis, lobular distortion, parenchymal regeneration, hyperplastic nodules and single-cell necrosis. According to the authors (Bruckner et al., 1986), a NOAEL of 1 mg/kg body weight of carbon tetrachloride could be established. Allis et al. (1990) administered carbon tetrachloride in corn oil to male Fischer-344 rats by gavage at dose levels of 0, 20 or 40 mg/kg body weight for 12 weeks. At both dose levels ALAT, ASAT and LDH levels were elevated and hepatic CYP protein concentrations were reduced. Histopathology showed cirrhotic livers, vacuolar degeneration and hepatocellular necrosis at both dose levels, but this was more severe in the higher dose group. At day 8 and 15 after exposure, all serum indicators and CYP protein concentration had returned to normal levels. In both dose groups, hepatocellular necrosis disappeared by day 8 and vacuolar degeneration decreased in severity but was still present at day 15. Cirrhosis persisted in the high-dose group, although it was less severe. Furthermore the relative liver weight in animals receiving 40 mg/kg body weight remained elevated. Several tests on renal function were conducted on adult male Fischer-344 rats treated for 15 days with carbon tetrachloride in corn oil at doses of 50, 150, 450 or 1350 mg/kg body weight. At 1350 mg/kg body weight, kidney injury could be observed, as indicated by haematuria, enzymeuria and decreases in kidney weight and serum glucose concentration. At 450 mg/kg body weight, a lower body weight and a reduction in the maximum urine-concentrating ability were observed (Kluwe, 1981). c) Dogs Young adult Beagle dogs of the Alderly Park strain (6/sex) that received carbon tetrachloride (in gelatin capsules) as a daily dose of 0.05 ml/kg body weight (80 mg/kg body weight) for 28 days, showed elevated ALAT and ornithine carbamyl transferase activities, whereas no effects were observed on ASAT and alkaline phosphatase. Furthermore there were changes in the livers of all dogs characterized histologically by fatty vacuolation of centrilobular cells. In 3 of the 12 dogs, the vacuolation also occurred mid-zonally and periportally. There was some evidence of individual cell necrosis, and in some cases the sinusoids were mildly congested. No changes in plasma enzyme activities and no histological changes in the liver were observed when three females received a daily dose of 0.02 ml/kg body weight (32 mg/kg body weight) of carbon tetrachloride for 8 weeks. (Litchfield & Gartland, 1974). 7.2.2 Inhalation exposure a) Mice As reported in a translated, extensive summary, BDF1 mice (10/sex/group) were exposed (whole-body) to atmospheres of 0, 10, 30, 90, 270 or 810 ppm carbon tetrachloride (0, 64, 192, 577, 1731 or 5192 mg/m3, respectively) for 6 h a day, 5 days a week for 13 weeks. Mice were observed daily for clinical signs, behavioural changes and mortality and were weighed each week. Urinalysis, haematology, blood chemistry and microscopy were performed at the scheduled end of the experiment. No compound-related deaths occurred. Body weight gain was depressed in males at 30 ppm or more. Slight, but statistically significant, changes in haematology were observed in males at 810 ppm (decreased Hb and increased MPV) and in females at the two highest dose levels (decreased Hb, Ht and RBC). Increased liver enzymes in blood were observed in both sexes at the three highest dose levels. Urinalysis showed a decrease in pH at the highest dose level in females only. Microscopic examination showed slight to moderate dose-related changes in the liver including cytological alterations, even at the lowest dose level in males. At higher dose levels there were more severe changes described as collapse, deposit of ceroid, proliferative ducts, increase in mitosis, pleomorphism and foci (Japan Bioassay Research Centre, 1998). A NOAEL cannot be established on the basis of these results. b) Rats Exposure (whole body) of Sprague-Dawley rats (IFFA CREDO; 8 males/group) to a carbon tetrachloride atmosphere of 3308 mg/m3 (516 ppm) for 6 h a day for 2 or 4 consecutive days resulted in increased serum activities of glutamate dehydrogenase, ASAT, ALAT and SDH after 4 days exposure. After 2 days only the SDH level was significantly increased (Brondeau et al., 1983). When male hooded rats were exposed (whole body) 8 h a day for 12 days to carbon tetrachloride levels of 68 or 680 ppm (436 or 4360 mg/m3) increased ASAT levels were found at low- and high-dose levels after 4 and 2 days, respectively. At both doses the level of liver lipids reached a maximum after 8 days, the level being related to the dose. Additional exposure resulted in a decrease (Kanics & Rubinstein, 1968). An increased liver triglyceride content was also reported by Shimizu et al. (1973) who exposed (whole body) female Sprague-Dawley rats to 10, 50 and 100 ppm of carbon tetrachloride vapour (64, 320 and 641 mg/m3) for 3 h a day for 6 to 8 weeks. Exposures to 320 and 641 mg/m3 resulted in striking increases in the hepatic trigly cerides to a maximum during the first 3 weeks. Afterward this level was nearly maintained in both groups. At 64 mg/m3 the rise in triglycerides was minimal and was maintained for 2 weeks. David et al. (1981) compared the serum enzyme activities and liver lesions in rats exposed to various concentration-time combinations. After four exposures to 50 ppm (320 mg/m3) for 6 h per day, the enzyme activities were significantly increased by 50 to 70%, and steatosis and hydropic changes were found in the liver. The changes were significantly more intensive in rats exposed to 250 ppm (1600 mg/m3) for 72 min per day, not withstanding that the concentration-time product was equal. The same was true for two-fold concentrations and 18 exposures. Bogers et al. (1987) performed 4-week studies in male Wistar rats by exposing (whole body) them to 63 or 80 ppm (404 or 513 mg/m3) carbon tetrachloride in three different concentration profiles: 1) continuous exposure of 6 h/day for 5 days/week; 2) exposure of 2 × 3 h/day (1.5 h interruption) for 5 days/week; and 3) peak loads of 382 ppm (2450 mg/m3) for 5 min (4 peaks for 3 h) with and without the 1.5-h interruption. The interruption of the daily 6-h exposures did not result in less severe but rather in slightly more severe hepatotoxic effects, such as changes in enzyme levels, fat accumulation, increased relative liver weight, lower microsomal protein content and hydropic degeneration of liver cells. Peak loads did not affect the severity of the hepatotoxic effects. Plummer et al. (1990) exposed (whole body) male black-hooded Wistar rats (36/group) for 4 weeks to carbon tetrachloride both continuously (24 h per day, 7 days per week) to 32 ppm (205 mg/m3) or intermittently (6 h per day, 5 days per week) to 176 ppm (1128 mg/m3). The concentration-time products were similar for both groups. The-carbon tetrachloride-induced hepatotoxicity appeared to be similar in the two exposure profiles. However, when rats received the enzyme-inducing agents phenobarbitone or 1,3-butanediol during the study via their drinking-water, the liver injury appeared to be exacerbated in 1,3-butanediol-treated rats, especially in the intermittent exposure profile. Groups of male Sprague Dawley rats were exposed (whole body) to 100 ppm (641 mg/m3) of [14C]carbon tetrachloride for either 8 or 11.5 h/day for periods of 1 to 10 days, and examined with and without recovery in a tissue distribution study (see section 6.1.3 and 6.1.4). The only significant difference between rats exposed to the two different schedules was the serum SDH activity, which was almost always significantly greater for rats exposed to the 11.5 h/day schedule than for the comparable groups exposed to the 8 h/day schedule, except when measured after a recovery period. (Paustenbach et al., 1986b). F-344 rats (10/sex/group) were exposed (whole-body) in a 13-week inhalation study to 10, 30, 90, 270 or 810 ppm carbon tetrachloride (64.1, 192.3, 576.9, 1730.7 or 5192.1 mg/m3, respectively) for 6 h a day, 5 days a week. Control groups were included. Animals were observed for clinical signs, behavioural changes and mortality once a day, and they were weighed once a week. Urinalysis was performed at the end of the dosing period, and haematology, blood biochemistry and microscopy were performed at the scheduled sacrifice. At 810 ppm the body weight gain was depressed in both sexes. Haematological changes were observed at 90 ppm or more in both sexes, and at 30 ppm in females. Increased liver enzymes in blood and urinalysis changes were observed in males at 270 ppm or more and in females at 90 ppm or more. Increased creatine phosphokinase (CPK) was seen at 30 ppm in females. Microscopic examination showed slight to marked changes in the liver described as fatty change, cytological alterations, deposition of ceroid, proliferative ducts, increase in mitosis, pleomorphism, cirrhosis and foci. Furthermore, vacuolic change of tubule, hyaline degeneration of glomerulus and protein cast of the kidney were noted at the two highest dose levels (Japan Bioassay Research Centre, 1998). An NOAEL could not be established on the basis of these results. c) Comparisons between species Adams et al. (1952) exposed (whole body) rats, guinea-pigs, albino rabbits and rhesus monkeys to various concentrations of carbon tetrachloride in air. Histological data were not reported for the control groups; these groups were, however, used for comparison with dose groups. Dose-related effects were seen in Wistar rats (15/sex/group) after exposure to carbon tetrachloride at concentrations of 5, 10, 25, 50, 100, 200 and 400 ppm (32, 63, 160, 320, 630, 1282 and 2520 mg/m3) for 7 h a day, 5 days a week, during approximately 5.5-6.5 months. Increased liver weight, increased liver fat content (especially neutral fat and esterified cholesterol) and fatty degeneration of the liver were observed after 2 to 3 weeks of exposure to concentrations of 63 mg/m3 or more. Cirrhotic livers were found from 630 mg/m3 upwards. At a concentration of 320 mg/m3, kidney tubular epithelium was affected and death rate seemed to be increased, especially in the males. At the two highest exposure levels testicular weights were decreased. At the 32 mg/m3 level, no adverse effects were seen. After exposure for 5 days a week during 13 weeks to 2520 mg/m3 for 3 min a day or to 630 mg/m3 for 18 min a day, no effects could be observed. Similar results were obtained in guinea-pigs from 63 mg/m3 upwards. Rabbits developed slight to moderate fatty degeneration and cirrhosis of the liver at 160 mg/m3 or more; there was no reported effect at 63 mg/m3. Rhesus monkeys developed slight-to-moderate fatty degeneration of the liver 630 mg/m3 (2 monkeys); there was no reported effect at 320 mg/m3 (2 monkeys). Prendergast et al. (1967) studied the effects of continuous and repeated exposure to carbon tetrachloride vapour in rats, guinea-pigs, New Zealand rabbits, beagle dogs and squirrel monkeys (see Table 8). After repeated exposure to 515 mg/m3, all species showed pulmonary interstitial fibrosis or pneumonitis. Mottled livers were seen in all species except in the dog. Histological examination revealed fatty changes in the livers of all species. In addition, fibrosis, bile duct proliferation, hepatocyte degeneration and regeneration, focal inflammatory infiltration and portal cirrhosis were observed in the guinea-pigs. After continuous exposure to 61 mg/m3 all species showed growth retardation and all squirrel monkeys showed alopecia and emaciation. Histopathological examination showed liver changes similar to those reported after repeated exposures. After continuous exposure to 6.1 mg/m3 carbon tetrachloride in 61 mg/m3 n-octane (as a carrier) no visible signs of toxicity were noted in any of the species and no animals died. At termination, all species except the rat showed less body weight gain than the controls. Histopathological examination revealed non-specific inflammatory changes in the lungs of all species and in the liver, kidney and heart of several animals, but no specific pathological changes attributable to the exposure were noted. 7.2.3 Intraperitoneal exposure Biochemical and morphological characterization of carbon-tetrachloride-induced lung fibrosis were investigated in rats after intraperitoneal administration of 1.0 ml/kg body weight (1600 mg/kg in paraffin oil) twice a week for 2 or 5 weeks, and examined after the end of the exposure. The third group (5 rats) was treated for 2 weeks and examined after 3 weeks of recovery. Acute haemorrhagic interstitial pneumonia resulted from the 2 week exposure, while chronic interstitial pneumonia was observed in rats exposed for 5 weeks and in the third group after 3 week of recovery (Pääkkö et al., 1996). 7.3 Long-term exposure In a carcinogenicity study described in section 7.7 (Reuber & Glover, 1970), young male rats were administered 1.3 ml/kg by subcutaneous injection twice a week. Severe cirrhosis was observed in all (16/16) Sprague-Dawley rats by 5 to 16 weeks (the time of death of the animals) and in 13/17 Black rats by 7 to 18 weeks. In Wistar rats, 6/12 rats developed moderate and 6/12 severe cirrhosis by 17-68 weeks, while the cirrhosis was mild in 2/13, moderate in 7 and severe in 4 Osborne Mendel rats by 10-105 weeks; in Japanese rats, the cirrhosis was mild in 9/15, moderate in 5 and severe in one rat by 8 to 78 weeks. Alumot et al. (1976) exposed rats (strain unknown) to carbon tetrachloride in feed at measured levels of 0, 80 and 200 mg/kg feed for 2 years. The highest concentration corresponded to a daily dose of 10 to 18 mg/kg body weight. Because of chronic respiratory disease in all animals beginning at 14 months, which resulted in increased mortality, the results reported upon necropsy at 2 years were inadequate for a health risk evaluation. Muños Torres et al. (1988) administered carbon tetrachloride to female Wistar rats (150 g initial body weight) as weekly intraperitoneal injections at a dose of 0.2 ml in mineral oil for 46 weeks. The hepatic lesions were macroscopically and microscopically evaluated after 8, 16, 22, 30 and 46 injections. After 8 injections, changes in the hepatic architecture due to an increase in the collagenous component accompanied by formation of fibrous bridges were seen. After 46 injections a clearly established cirrhosis with nodules of different sizes was seen. Table 8. Mortality in animals exposed to carbon tetrachloride (from Prendergast at al., 1967). Concentration Type of Ratc Guinea-pig Rabbit Dog Monkey (mg/m3) studyb (Hartley) (New Zealand) (Beagle) (Squirrel) 515 R 0/15 3/15 0/3 0/2 1/3 61 C 0/15 3/15 0/2 0/2 0/3 6.1a C 0/15 0/15 0/3 0/2 0/3 a in 61 mg/m3 n-octane (as a carrier) b R = 30 exposures, 8 h/day, 5 days/week for 6 weeks; C = continuous 90-day exposure. c Long-Evans or Sprague-Dawley rats BDF1 mice (50/sex/group) were exposed (whole-body) in a 2-year inhalation study to 0, 5, 25 or 125 ppm carbon tetrachloride (0, 32.05, 160.25 or 801.25 mg/m3) for 6 h a day, 5 days a week. Animals were observed for clinical signs, behavioural changes and mortality once a day, and they were weighed once a week for the first 13 weeks and every 4 weeks thereafter. Urinalysis was performed at the end of the dosing period, and haematology, blood biochemistry and microscopy were performed at the scheduled sacrifice. No compound-related effects were observed at 5 ppm in female mice. The results from male mice could not be evaluated due to anomalous control group liver enzyme data. A significant decrease in survival was observed at 25 and 125 ppm. Liver tumours were the main cause of death at the highest dose level. Body weight gain was depressed at 25 and 125 ppm. Changes in haematology, blood biochemistry including liver enzymes, and urinalysis were observed at 25 ppm or more. Microscopic examination showed changes of the liver (deposit of ceroid, cyst and degeneration), the kidney (protein cast) and the spleen (increased deposit of haemosiderin at 25 ppm and increased extramedullary haematopoiesis at 125 ppm) at the two highest dose levels in male mice. In female mice changes in the liver included deposit of ceroid, thrombus, necrosis, degeneration and cyst at 25 and 125 ppm. At 25 ppm an increased deposit of haemosiderin of the spleen was observed while at 125 ppm deposit of ceroid of the ovary was seen (Japan Bioassay Research Centre, 1998). A NOAEL could not be established on the basis of these results. F-344 rats (50/sex/group) were exposed (whole-body) in a 2-year inhalation study to 0, 5, 25 or 125 ppm carbon tetrachloride (0, 32.05, 160.25 or 801.25 mg/m3, respectively) for 6 h a day, 5 days a week. Animals were observed for clinical signs, behavioural changes and mortality once a day. They were weighed once a week for the first 13 weeks and every 4 weeks thereafter. Urinalysis was performed at the end of the dosing period, and haematology, blood biochemistry and microscopy were performed at the scheduled sacrifice. A significant decrease in survival was observed at 125 ppm, with liver tumours and/or chronic nephropathy being the main cause of death. Body weight gain was depressed at 25 and 125 ppm. Changes in haematology, blood biochemistry including liver enzymes, and urinalysis were observed at 25 ppm and even at 5 ppm for the nitrate and protein level in the urine of the rats. At the 125 ppm level, only one male and three female rats survived, so no statistical test was performed. Microscopic examination showed changes of the liver (including fatty change, deposit of ceroid, fibrosis, granulation and cirrhosis) at the two highest dose levels in both sexes. An increased deposit of haemosiderin in the spleen was observed in males at all dose levels. An eosinophilic change of the nasal cavity was observed in females at all dose levels and in males at 25 and 125 ppm. A chronic nephropathy (progressive glomerulonephrosis) developed in females at 25 ppm and in both sexes at 125 ppm. At 125 ppm deposit of ceroid and granulation of the lymph node were observed in both sexes (Japan Bioassay Research Centre, 1998). A NOAEL could not be established on the basis of these results. 7.4 Irritation 7.4.1 Skin irritation Epicutaneous administration of 1 ml of carbon tetrachloride has been demonstrated to induce degenerative changes in the epidermis 15 min to 16 h after application (Kronevi et al., 1979; see section 188.8.131.52). Moderate dermal irritation was observed after application of 0.5 ml carbon tetrachloride (under occlusion) onto the shaven skin of rabbits (only abraded skin) and male Hartley guinea-pigs (normal and abraded skin) (Roudabush et al., 1965). In a study conducted according to Draize protocol, 0.5 ml carbon tetrachloride was applied under an occlusive dressing for 24 h to the intact and abraded skin of rabbits. Irritation was assessed at 24 and 72 h. Carbon tetrachloride was classified as a "medium" skin irritant. Histopathology of skin samples taken from the application site on day 3 after exposure confirmed the irritant reaction (Duprat et al., 1976). Undiluted carbon tetrachloride (10 µl) was applied to the open skin of guinea-pigs 3 times daily for 3 days. A skin reaction (no further details provided) was observed on day 2 and an average score described as "redness" was seen on day 4 (Anderson et al., 1988). Wahlberg (1984a) rubbed 0.1 ml (159 mg) of carbon tetrachloride into the skin of rabbits and guinea-pigs for ten consecutive days and observed oedema and erythema. 7.4.2 Eye irritation In a study conducted according to Draize protocol, 0.1 ml of carbon tetrachloride caused a mild irritant response in rabbits. The response was evident at 24, 48 and 72 h after exposure and recovery was complete by day 14 (Duprat et al., 1976). 7.5 Toxicity to the reproductive system, embryotoxicity, teratogenicity 7.5.1 Reproduction Groups of six male rats received a single intraperitoneal injection of coconut oil or carbon tetrachloride in coconut oil (1:1 mixture) as 3 ml/kg rat weight (2378 mg/kg body weight) After 15 days, a significant increase in the weight of the pituitary and a decrease in the weights of the testes and seminal vesicles were observed. Histological examination showed testicular atrophy and some abnormality in the process of spermatogenesis in the experimental animals (Chatterjee, 1966). In a study of similar design with female rats, effects on the reproductive system were seen 10 days after dosing. The effects reported were: inhibition of estrous rhythm, reduction in ovarian and uterine weights and vascularization, an increase in adrenal weight and a marked reduction in pituitary gonadotrophin potency (Chatterjee (1968). Kalla & Bansal (1975) injected male rats with a mixture of 3 ml/kg body weight carbon tetrachloride in coconut oil (1:1, v/v) (2378 mg carbon tetrachloride/kg body weight) through an intraperitoneal route for 10, 15 or 20 consecutive days. All dosing periods resulted in decreased weights of testicles and accessory sex organs and impairments in spermatogenesis. Dosing for 20 days resulted in an entire deterioration of testicular tissue accompanied by an absence of spermatids. The study was not reported adequately; number and strain of rats were not reported. 7.5.2 Embryotoxicity and teratogenicity The available data suggest that the fetus is not preferentially sensitive to carbon tetrachloride, and effects of carbon tetrachloride on fetal development and post-natal survival are likely to be secondary to maternal toxicity. 184.108.40.206 Oral exposure When carbon tetrachloride was administered by gavage to F-344 rats on gestation days 6-15 at 0, 25, 50 and 75 mg/kg per day in either corn oil or in an aqueous vehicle containing 10% Emulphor(R), it was more maternally toxic when administered in corn oil, particularly at the highest dose. Full litter resorption (FLR) occurred at 50 and 75 mg/kg with both vehicles. At 75 mg/kg, dams receiving carbon tetrachloride in corn oil had a significantly higher rate of FLR (67%) than those given the aqueous vehicle counterpart (8%) (Narotsky et al., 1997a). Ammonium sulfide staining was used to detect the resorption sites (Narotsky et al., 1997b). Thiersch (1971) dosed pregnant rats with carbon tetrachloride (in corn oil) at a level of 1000 mg/kg body weight on days 7, 7 and 8, 11, or 11 and 12 of gestation. No malformations in the offspring were reported, but the litters of the dams that had received two doses showed more resorptions than the litters of those receiving one dose. Clear information on a control group was not provided. Hamlin et al. (1993) examined the effect on B6D2F1 mice of oral administration of carbon tetrachloride (in corn oil) at concentrations of 82.6 or 826.3 mg/kg body weight for five consecutive days beginning on day 1, 6 or 11 of gestation. No effects were seen on maternal or various neonatal parameters such as weight and crown rump length. No malformations were detected in any pup on day 1 post-partum and the pups developed normally. 220.127.116.11 Inhalation exposure When Schwetz et al. (1974) exposed pregnant Sprague-Dawley rats to measured carbon tetrachloride concentrations of 334 or 1004 ppm (214 or 6435 mg/m3) for 7 h a day on days 6 to 15 of gestation, the dams showed a dose-related decrease in food consumption (and body weight gain). Signs of hepatotoxicity (increased ALAT activity) were observed at both dose levels, but were not dose-related. Fetal body weight and crown-rump length were significantly decreased. No anomalies were seen upon gross examination of the fetuses. In both exposure groups the incidence of fetuses with subcutaneous oedema was increased but was statistically significant only in the lower dose group. The incidence of sternebral anomalies (bipartite and delayed ossification) was significantly increased in the fetuses of rats exposed to the higher dose. In an inhalation study by Gilman (1971), exposure of pregnant rats to carbon tetrachloride at 1575 mg/m3 for 8 h a day on days 10 to 15 of gestation decreased the lactation index (83% compared to 98% in the controls) and the viability index (83% as compared to 99% in the controls). 7.6 Mutagenicity The data from genotoxicity assays conducted with carbon tetrachloride are summarized in Table 9. Since carbon tetrachloride is a volatile compound that partitions preferentially in the hydrophobic phase, the conditions adopted for in vitro experiments are important to the outcome, but these conditions are often not reported in sufficient detail. Carbon tetrachloride was not mutagenic to Salmonella typhimurium in a large number of studies. It did, however, induce DNA damage and mutations in single studies with Escherichia coli. In fungi it induced intrachromosomal and mitotic recombination. However, it did not induce aneuploidy in one study on the yeast Saccharomyces cerevisiae, although aneuploidy was induced in another single study with Aspergillus nidulans. In the only study with Drosophila melanogaster, sex-linked recessive lethal mutations were not induced by carbon tetrachloride. In mammalian in vitro assays, carbon tetrachloride induced cell transformation in a single study with Syrian hamster cells and centromere-positive-staining micronuclei in human cell lines expressing cDNAs for CYP1A2, CYP2A6, CYP3A4, epoxide hydrolase or CYP2E1. The AHH-1 cell line constitutively expressing CYP1A1 showed no increase in either total micronucleus frequency or centromere-staining micronucleus frequency. There is little evidence for the induction in vitro of DNA damage, unscheduled DNA synthesis, sister-chromatid exchange or chromosomal aberrations. In mammalian in vivo tests, carbon tetrachloride did induce DNA strand breakage in one study but not in four others and did not induce: a) unscheduled DNA synthesis in rat hepatocytes; b) micronuclei in mouse hepatocytes, bone marrow cells or peripheral blood erythrocytes; c) chromosomal aberrations in mouse bone marrow; or d) aneuploidy in mouse hepatocytes. Binding of carbon tetra chloride to liver cell DNA has been observed in rats, mice and Syrian hamsters treated in vivo. There has been a report of a reduction in I-compounds (species- and tissue-specific DNA adducts) in mouse liver. The only clear evidence for genotoxicity comes from a number of fungal cell experiments involving mutation and recombinational events. Effects in mammalian cells indicate damage during cytokinesis. This type of damage could result from interactions with proteins, rather than DNA, e.g., of the trichloromethyl radical, and could be induced secondarily to the toxicity of carbon tetrachloride (McGregor & Lang, 1996). Thus, no carbon-tetrachloride-DNA adduct identification has been made, while the polar adducts observed in Syrian hamster liver DNA appear to be derived from lipid peroxidation products (Wang & Liehr, 1995). Consequently, strand-breakage and aneuploidy could arise from the effects of lipid peroxidation products rather than carbon tetrachloride or its metabolites; linoleic acid hydroperoxide, for example, can induce single-strand breaks in DNA of cultured fibroblasts (Nakayama et al., 1986). No resolved DNA damage has been observed in vivo. It is concluded that although carbon tetrachloride has some effects upon genetic material and these could be due to a direct effect of carbon tetrachloride, no supporting evidence is available; the effects are explicable in terms of nuclear protein or DNA damage induced secondarily to carbon tetrachloride toxicity. 7.7 Carcinogenicity 7.7.1 Mice After administration of 0.1 ml of a 40% solution of carbon tetrachloride in olive oil (64 mg/mouse) by stomach tube to male C3H mice, female C mice, and male and female A and Y mice 2 or 3 times a week for 8 to 16 weeks (23 to 58 treatments), hepatomas developed in 126/143 (88%), 34/41 (83%), 63/64 (98%) and 9/15 (60%) of the C3H, C, A and Y mice, respectively. No concurrent control data were reported. Historical control data indicated the following incidence of hepatomas (%) at one year or more: male C3H, 27%; female C, 0%; male and female A, 1.5%; male and female Y, 1.6% (Edwards, 1941; Edwards & Dalton, 1942). In 34 of 73 male and female mice of the L strain that received 0.04 ml carbon tetrachloride per treatment by stomach tube 2 or 3 times a week for 46 treatments and were killed 3 to 3.5 months following treatment, hepatomas were found that were similar to those found in the other strains (Edwards et al., 1942). Table 9. Mutagenicity studies with carbon tetrachloride Speciesa Strain Measured end-pointb Test conditions Activationc Inductiond Resultse Reference Bacterial systems S. typhimurium TA 100 base-pair substitution not reported + rat PCB - McCann et al., TA 1535 1975 S. typhimurium G 46 base-pair substitution not reported + mouse i.n.r. - Kraemer et al., 1974 S. typhimurium TA 1950 base-pair substitution concentrations: unknown - Braun & G 46 10, 20 and 40 mg/ml Schöneich, 1975 S. typhimurium TA 1535 base-pair substitution test performed in + rabbit i.n.r. - Uehleke et al., 1977 TA 1538 frameshift mutation desiccators i.m. S. typhimurium TA 100 base-pair substitution test performed in + rabbit PCB - Simmon et al., 1977 TA 1535 desiccators TA 98 frameshift mutation TA 1537 TA 1538 S. typhimurium TA 1535 base-pair substitution concentration: 8 mM, + mouse PB - Uehleke et al., 1977 TA 1538 frameshift mutation incubation in closed i.m. containers (survival > 90%) S. typhimurium TA 100 base-pair substitution test performed in -/+ i.n.r. - Simmon & Tardiff, TA 1535 desiccators sp. n.r. 1978 S. typhimurium TA 100 base-pair substitution concentrations: 4, 5.7, - - Barber et al., TA 1535 7, 10.2, 12.3, 18.4 1981 TA 98 frameshift mutation µmoles/plate; test for + rat PCB - TA 1537 volatile liquids TA 1538 Table 9. (Continued) Speciesa Strain Measured end-pointb Test conditions Activationc Inductiond Resultse Reference S. typhimurium TA1535/pSK1002 SOS induction open + - Nakamura et al., 1987 S. typhimurium TA1535/pSK 1002 SOS induction open + - Brams et al., 1987 S. typhimurium TA1535/pSK 1002 forward mutation open + - Roldàn-Arjona et al., 1991 S. typhimurium TA1535/pSK 1002 forward mutation open + - Roldàn-Arjona & Pueyo, 1993 S. typhimurium TA100 reverse mutation open + - Zeiger et al., 1988 TA1535 TA1537 TA97 TA98 S. typhimurium TA100 reverse mutation open + - Brams et al., 1987 TA98 TA97 S. typhimurium TA 100 base-pair substitution concentration: below - - De Flora, 1981; TA 1535 toxicity limit De Flora et al., TA 98 frameshift mutation 1984 TA 1537 TA 1538 E. coli K 12 gene mutations not reported + mouse i.n.r. - Kraemer et al., 1974 i.m. E. coli K 12 gene mutations not reported + rabbit i.n.r. - Uehleke et al., 1976 i.m. Table 9. (Continued) Speciesa Strain Measured end-pointb Test conditions Activationc Inductiond Resultse Reference E. coli uvrA back-mutation to trp concentration: + mouse i.n.r. - Norpoth et al., 1980 0.01% v/v E. coli WP2 uvrA reverse mutation 2.5% atmosphere + (+) Norpoth et al., 1980 Non-mammalian eukaryotic systems A. Nidulans unknown somatic segregation spot test technique n.r. - Bignami, 1977 (crossing-over and non-disjunction) A. Nidulans unknown induction of 8-aza- spot test technique n.r. - Bignami, 1977 guanine resistance A. Nidulans 35 (haploid) forward mutation concentration: n.r. (+) Gualandi, 1984 1 (diploid) somatic segregation 0.5% v/v n.r. + A. Nidulans aneuploidy - + Benigni et al., 1993 S. cerevisiae D7 gene conversion at concentration: 21, 28, - + Callen et al., 1980 trps-locus; mitotic 34 mM; test performed recombination at ade-2; in screw-capped glass gene conversion at ilv-1 tubes S. cerevisiae AGY31DEL intrachromosomal - + Schiestl et al., recombination 1989 S. cerevisiae AGY31DEL intrachromosomal - + Galli & Schiestl, recombination 1995 S. cerevisiae D61.M mitotic chromosome - - Whittaker et al., loss 1989 Table 9. (Continued) Speciesa Strain Measured end-pointb Test conditions Activationc Inductiond Resultse Reference S. cerevisiae mitotic recombination - + Galli & Schiestl, 1996 Drosophila SLRL mutation feeding - Foureman et al., melanogaster 1994 Drosophila SLRL mutation injection - Foureman et al., melanogaster 1994 Chinese hamster V79 aneuploidy 2500 µg/ml - + Önfelt, 1987 lung Cell line AHH1 (expressing aneuploidy 10 mM - - Doherty et al., 1996 CYP1A1) (centromere staining) Cell line MCL-5 (cDNAs for aneuploidy 2 mM - + Doherty et al., 1996 CYP1A2, CYP2A6, (centromere staining) CYP3A4, CYP2E1 and epoxide hydrolase) Cell line h2E1 (cDNAs for aneuploidy 2 mM - + Doherty et al., 1996 CYP2E1) (centromere staining) Cell line AHH1 (expressing micronucleus 10 mM - - Doherty et al., 1996 CYP1A1) Cell line MCL-5 (cDNAs for micronucleus 2 mM - + Doherty et al., 1996 CYP1A2, CYP2A6, CYP3A4, CYP2E1 and epoxide hydrolase Table 9. (Continued) Speciesa Strain Measured end-pointb Test conditions Activationc Inductiond Resultse Reference Cell line h2E1 (cDNAs for micronucleus 2 mM - + Doherty et al., 1996 CYP2E1) In vitro mammalian systems Chinese hamster ovary cells anaphase analysis concentration: 5 µl/ml - (+) Coutino, 1979 for chromosomal rearrangements Chinese hamster ovary cells chromosomal highest concentration: + rat PCB - Loveday et al., 1990 aberrations + SCE 3000 µg/ml - - Rat liver epithelium metaphase analysis concentration: 0.005, cells - Dean & for chromosomal 0.01, 0.02 µl/ml in posses Hodson-Walker, abnormalities sealed flasks; toxicity intrinsic 1979 observed activity Human lymphocyte chromosomal concentration: + sp.n.r. i.n.r - Garry et al., 1990 aberrations + SCE 3.8-76 µg/ml Host-mediated assays S. typhimurium TA 1950 base-pair substitution mice NMRI; - Braun & Schöneich, subcutaneous 1975 4 ml/kg body weight S. typhimurium TA 1950 base-pair substitution mice CBA*C57Bl/6; - Shapiro & Fonshtein, subcutaneous 20% 1979 solution in sunflower oil Table 9. (Continued) Speciesa Strain Measured end-pointb Test conditions Activationc Inductiond Resultse Reference Syrian hamster cell transformation 3 µg/ml - (+) Amacher & Zelljadt, embryo cells (clonal assay) 1983 Rat hepatocytes UDS 100 mg/kg × 1 p.o. or - Doolittle et al., in vivo 14 p.o. 1987 Rat hepatocytes SCE and chromosomal 1600 mg/kg × 1 p.o. - Sawada et al., 1991 in vivo aberrations and micronucleus Mouse bone chromosomal 8000 mg/kg × 1 - Lil'p, 1983 marrow in vivo aberrations Mouse bone marrow micronucleus 2000 mg/kg and - Suzuki et al., 1997 and peripheral 3000 mg/kg erythrocytes in vivo Mouse liver binding to DNA + Diaz-Gomez & Castro, in vitro 1980a Mouse, rat, binding to DNA + Castro et al., 1989 Syrian hamster liver in vivo Mouse liver ICR I-compound reduction 1600 mg/kg × 1 p.o. + Nath et al., 1990 in vivo (32P-post-labelling) Syrian hamster binding to DNA + Wang & Liehr, 1995 liver and kidney in vivo a S. typhimurium = Salmonella typhimurium; A. Nidulans = Aspergillus nidulans; S. cerevisiae = Saccharomyces cerevisiae b SCE = sister chromatid exchange; UDS = unscheduled DNA synthesis c + = with metabolic activation; - = without metabolic activation; sp.n.r. = species not reported; n.r. = not reported whether metabolic activation was used d i.n.r. = inducer not reported; i.m. = intact microsomes added; PCB = polychlorinated biphenyls; PB = phenobarbital e - = negative; + = positive; (+) = weakly positive Table 10. Indicator tests with carbon tetrachloride Species Strain Measured end-point Test conditions Activationa Inducationb Resultsc Reference Bacterial systems Escherichia coli WP2 (repair DNA repair variation 1: liquid - + De Flora et al., proficient WP67 micro method 1984 (uvrA polA-) CM871 (uvrAa, + rat PCB + recA-, lexA-) variation 2: 24 h - + preincubation and plating out + rat PCB - In vitro mammalian cells Chinese hamster ovary cells SCE concentration: 0.001, - +d Athanasiou & 0.1, 1 mM; solvent Kyrtopoulos, 1981 DMSO Chinese hamster ovary cells SCE -S9: toxic at 1490 + rat PCB - Loveday et al., µg/ml; +S9: 2930 1990 µg/ml highest dose tested Human lymphocyte SCE concentration: + sp.n.r. i.n.r. - Garry et al., 3.8-76 µg/ml - - 1990 Human lymphocyte UDS concentration: 2.5, - PB - Perocco & 5 µg/ml; solvent + rat - Prodi, 1981 DMSO Rat hepatocyte DNA single strand concentration: 0.03, - + Sina et al., 1983 breaks 0.3, 3 mM Table 10. (Continued) Species Strain Measured end-point Test conditions Activationa Inducationb Resultsc Reference In vivo mammalian systems Mouse NMRI DNA single strand 2.5 mg/kg body - Schwartz et al., breaks weight; single oral 1979 dose, undiluted or in corn oil Mouse CBA*BALB/c sperm head 0.1, 0.25, 0.5, 1.0, - Topham, 1980 abnormalities 1.5 mg/kg body weight (i.p.) for 5 days; solvent corn oil Mouse CDI DNA damage in 3H- 0.02-0.1 ml/kg body + Gans & Korson, 1984 labelled liver weight; single oral cells dose in corn oil Rat Wistar UDS in liver cells 4000 mg/kg body ee Craddock & weight; single oral Henderson, 1978 dose, 2 and 17 h exposure Rat Fischer-344 UDS in liver cells 10 or 100 mg/kg - Mirsalis & body weight; single Butterworth, 1980 oral dose in corn oil, 2 h exposure Rat Wistar DNA damage single oral dose of - Stewart, 1981 (hepatectomized) 200-800 mg/kg body weight in corn oil, 3 weeks after hepatectomy Table 10. (Continued) Species Strain Measured end-point Test conditions Activationa Inducationb Resultsc Reference Rat Fischer-344 DNA single strand 400 mg/kg body - Bermudez et al., breaks weight; single oral 1982 dose in corn oil Rat Fischer-344 UDS in liver cells 40 or 400 mg/kg - Mirsalis et al., body weight; single 1982 oral dose up to 48 h of exposure Rat Sprague-Dawley DNA damage 200 mg/kg body - Brambilla et al., weight; single i.p. 1983 dose a + = with metabolic activation; - = without metabolic activation; sp.n.r.= species not reported b PCB = polychlorinated biphenyls; PB = phenobarbital; i.n.r.; = inducer not reported c - = negative; + = positive; e = equivocal d chromosome aberrations occurred, but no details on this finding were reported e increase in DNA associated with tissue regeneration, but no increase in unscheduled DNA synthesis Eschenbrenner & Miller (1944) administered 30 doses (each 0.005 ml) of 32%, 16%, 8%, 4% and 2% solutions of carbon tetrachloride in olive oil (2540, 1270, 635, 318 or 160 mg/kg body weight) at 1- to 5-day intervals to mice of the A strain. Control group animals were administered olive oil. All animals were examined for hepatomas 150 days after the first dose. Hepatomas were found in 33/60, 32/60, 25/60, 23/60 and 23/60 mice of the 32, 16, 8, 4 and 2% dose groups. A variation in the numerical incidence of hepatomas at a given time was observed to be related both to the total amount administered and to the interval elapsing between successive doses. The incidence of hepatomas increased with the interval between dosing from 1 to 4 days. Weisburger (1977) reported that, in B6C3F1 mice dosed by gavage with carbon tetrachloride in corn oil at levels of 1250 or 2500 mg/kg body weight 5 days/week for 78 weeks and killed at 90 weeks, hepatomas were found in 47/48 males and 43/45 females receiving the higher dose and in 49/49 males and 40/40 females receiving the lower dose. Control incidences of hepatomas were 3/18 in males and 1/18 in females. An increase in adrenal tumours was also reported in the treated mice. Groups of 50 male and 50 female BDF1 mice, 6 weeks of age, were exposed by whole-body inhalation to 0, 5, 25 or 125 ppm (0, 32, 160 or 800 mg/m3) carbon tetrachloride (purity > 99.8%) for 6 h per day on 5 days a week for 104 weeks. Survival of the mid- and high-dose groups in both sexes (35/50, 36/50, 25/50 and 1/50 males; 26/50, 24/49, 10/50 and 1/49 females) was decreased due to liver tumours. Significantly increased incidences of hepatocellular adenomas (9/50, 10/50, 27/50 and 16/50 males; 2/50, 8/49, 17/50 and 5/49 females) were observed in mid- and high-dose males (p < 0.01, Chi-square test) and in low- and mid-dose females (low dose, p < 0.05; mid-dose, p < 0.01), hepatocellular carcinomas (17/50, 12/50, 44/50 and 47/50 males; 2/50, 1/49, 33/50 and 48/49 females) in mid- and high-dose males and females (p < 0.01) and pheochromocytomas of the adrenal gland (0/50, 0/50, 16/50 and 31/50 males; 0/50, 0/49,0/50 and 22/49 females) in mid- and high-dose males (p < 0.01) and high-dose females (p < 0.01) (Nagano et al., 1998). 7.7.2 Rats In a study reported by Reuber & Glover (1970) male rats of the Osborne-Mendel, Japanese, Wistar, Black and Sprague-Dawley strains received twice a week a subcutaneous injection (1.3 ml/kg body weight) of a 50% solution of carbon tetrachloride in corn oil (1036 mg/kg body weight). The Japanese rats survived for 47 weeks and Osborne-Mendel rats for 44 weeks. Wistar rats lived for 33 weeks and Black and Sprague-Dawley rats lived only for 11 and 33 weeks, respectively. Hepatocellular carcinomas developed in 8/13 Osborne-Mendel rats and in 12/15 Japanese rats, and hyperplastic nodules were also found in these strains. The hepatocellular carcinomas found in 4/12 Wistar rats were smaller. Nodules and hyperplasia were also observed in Wistar rats, as well as moderate to severe cirrhosis. The Black rats and Sprague-Dawley rats died with severe cirrhosis before they developed carcinomas. No hepatocellular carcinomas developed in the controls of any strain. Weisburger (1977) reported increases in the incidence of neoplastic nodules and hepatocellular carcinomas after oral administration of carbon tetrachloride in corn oil to Osborne-Mendel rats at doses of 47 and 94 mg/kg body weight (males) and 80 and 160 mg/kg body weight (females) for 78 weeks. The rats were killed at 110 weeks. The incidences of hepatocellular carcinomas in the controls were 0/20 in males and 1/20 in females, and the incidences of liver neoplastic nodules were 0/20 in males and 1/20 in females. Groups of 50 male and 50 female F-344 rats, 6 weeks of age, were exposed by whole-body inhalation to 0, 5, 25 or 125 ppm (0, 32, 160, 800 mg/m3) carbon tetrachloride (purity > 99.8%) for 6 h per day on 5 days a week for 104 weeks. Survival of the high-dose groups in both sexes (22/50, 29/50, 19/50 and 3/50 males; 39/50, 43/50, 39/50 and 1/50 females) was decreased due to liver tumours and chronic nephropathy (progressive glomerulonephrosis). There were significantly increased incidences of hepatocellular adenomas (0/50, 1/50, 1/50 and 21/50 males; 0/50, 0/50, 0/50 and 40/50 females) and hepatocellular carcinomas (1/50, 0/50, 0/50 and 32/50 males; 0/50, 0/50, 3/50 and 15/50 females) in high-dose rats of each sex (p < 0.01, Chi-square test) (Nagano et al., 1998). 7.8 Special studies 7.8.1 Immunotoxicity Carbon tetrachloride treatment of B6C3F1 female mice resulted in marked suppression of both humoral and cell-mediated immune functions. Humoral immunity, as measured by the T-dependent antibody response to sheep red blood cells (SRBC), proved to be the most sensitive indicator of carbon-tetrachloride-induced immunotoxicity. Carbon tetrachloride was immunotoxic in female B6C3F1 mice at all doses tested (500-5000 mg/kg body weight) and there were no significant differences in the magnitude of immunosuppression between oral and intraperitoneal routes of exposure. There was no dose-response relationship with respect to SRBC antibody responses; all dose levels resulted in approximately 50% suppression of the control response. To determine whether a dose-response relationship could be attained, female mice were treated at lower carbon tetrachloride levels (25, 50 and 100 mg/kg body weight) for 30 consecutive days. It appeared that doses as low as 50 mg/kg body weight produced the maximum attainable inhibition of SRBC response (50%); 25 mg/kg body weight caused a 20% reduction (Kaminsky et al., 1989; 1990). Delaney & Kaminski (1994) studied the immunomodulatory activity of serum isolated from carbon tetrachloride-treated B6C3F1 mice on T-cell-independent humoral immune responses. The results of the study suggested that carbon tetrachloride has bifurcating immunological effects. Exposure to carbon tetrachloride appears to suppress T-cell-dependent immune responses but enhance the activity of B-cells. Both effects appear to be mediated by blood-borne factors. Incubation of sera from carbon-tetrachloride-treated mice with neutralizing monoclonal antibodies toward transforming growth factor ß1 reversed the immunosuppression, indicating that TGF ß1 at least in part mediates the immunosuppression induced by carbon tetrachloride. In contrast to the results found in mice by Kaminsky et al. (1989, 1990), Smialowicz et al. (1991) found no consistent alterations in humoral or cell-mediated immune function in male Fischer-344 rats at dosages that clearly resulted in body weight decreases and hepatotoxicity. However, the dose levels used in this study (up to 40 mg/kg body weight) were much lower than those reported by Kaminsky et al. (1989, 1990). Mice (A/PhJ) were administered carbon tetrachloride (about 300 mg/kg per day intraperitoneally in olive oil) for 2, 7, 14 and 23 days. A variety of immunological parameters were evaluated. Morphological examination by light microscopy revealed significant activation of lymphoid tissues in T-cell-dependent areas and only slight activation in B-cell-dependent areas (Jirova et al., 1996). Thymus weights (after exposure for 2, 14 and 23 days) and spleen weights (after exposure for 2 and 23 days) decreased significantly when weights at 23 days were compared to those of controls. The response of SRBC was permanently suppressed from the beginning of exposure. 7.8.2 Influence of oxygen levels It is known that under normal atmospheric conditions carbon tetrachloride initiates lipid peroxidation in mice and rat liver microsomes and in mice and rats in vivo (Sagai & Tappel, 1979; Kornbrust & Mavis, 1980; Gee et al., 1981; Lee et al., 1982). At reduced oxygen concentrations the carbon-tetrachloride-induced lipid peroxidation is greatly enhanced in in vivo experiments and in microsomal preparations, but in these in vitro systems, the process is entirely blocked under anoxic conditions (Kieczka & Kappus, 1980; Noll & De Groot, 1984). Covalent binding to RNA and DNA was enhanced in the absence of oxygen when male rat (Sprague-Dawley) hepatocytes were treated with carbon tetrachloride (Cunningham et al., 1981). DiRenzo et al. (1984) observed that the enhanced covalent binding to protein or lipid, caused by carbon tetrachloride in cultured male rat (Sprague-Dawley) hepatocytes, at decreased oxygen tension was most evident for the binding to lipids. Shen et al. (1982) studied the effect of oxygen concentrations on carbon-tetrachloride-induced hepatotoxicity in male Long-Evans rats exposed to differing oxygen concentrations combined with different carbon tetrachloride concentrations. In this in vivo experiment, carbon tetrachloride appeared to be more toxic when oxygen concentrations were reduced, as shown by increased ALAT activity, more severe centrilobular necrosis, and increased covalent binding to hepatic microsomal lipids and proteins. An increased metabolism of carbon tetrachloride under hypoxic conditions, and consequently an aggravated hepatotoxicity, was reported in male Wistar rats by Siegers et al. (1985). In agreement with the reports of Kieczka & Kappus (1980) and Noll & De Groot (1984), a more pronounced lipid peroxidation was observed, as shown by exhaled ethane, than in animals kept under normal oxygen conditions and treated with the same dose of carbon tetrachloride. Male Sprague-Dawley rats were used in a study by Burk et al. (1988) to examine the effect of hyperbaric oxygen on carbon tetrachloride metabolism by different isoenzymes of cytochrome P-450. The authors concluded that under low oxygen tensions the rate of carbon tetrachloride metabolism depended largely on the amount of cytochrome P-450 present, while under higher oxygen tensions the major determinant was the type of cytochrome P-450. 7.9 Factors modifying toxicity 7.9.1 Dosing vehicles Several studies have demonstrated that carbon-tetrachloride-induced hepatotoxicity, like absorption (see section 18.104.22.168), can be influenced by the dosing vehicle. In order to evaluate the effect of vehicle on the hepatotoxicity in mice, Condie et al. (1986) treated male and female CD-1 mice with oral doses of carbon tetrachloride (0, 1.2, 12 or 120 mg/kg body weight) in either corn oil or 1% Tween-60 vehicle, 5 times/week for 90 days. Differences between the vehicles were observed in mice at the 12 mg/kg dose level. Hepatomegaly and more fat accumulation were observed when carbon tetrachloride was administered in corn oil. At the highest dose level the usage of corn oil as vehicle caused a greater hepatotoxic effect, as shown by necrosis and fatty infiltration in the mice. The data indicated that the NOAEL for hepatotoxic effects after carbon tetrachloride exposure in corn oil was 1.2 mg/kg body weight, while the NOAEL for the Tween-60 groups was 12 mg/kg body weight. When pregnant F-344 rats were dosed by gavage with carbon tetrachloride in corn oil or an aqueous vehicle containing 10% Emulphor during gestation, corn oil was associated with a full litter resorption (FLR) rate of 67%, compared to 8% in those dosed with Emulphor (Narotsky et al., 1997a). Further details regarding this study are described in section 22.214.171.124. Koporec et al. (1995) determined the vehicle effects on the subchronic toxicity of carbon tetrachloride in male Sprague-Dawley rats. Carbon tetrachloride was administered at dose levels of 0, 25 or 100 mg/kg body weight by gavage in either corn oil or a 1% Emulphor aqueous emulsion 5 days/week for 13 weeks. It was concluded that there was no difference in the subchronic hepatotoxicity of carbon tetrachloride in rats when given in corn oil or as an aqueous emulsion. This result contrasts with the result found in mice described by Condie et al. (1986), and with the result of the study in male Sprague-Dawley rats by Kim et al. (1990b), who reported that the hepatotoxicity in male Sprague-Dawley rats was less pronounced at each dose level when corn oil was used as a vehicle. Administration of undiluted carbon tetrachloride or of an aqueous emulsion produced comparable toxicity. Szende et al. (1994) dosed male F-344 rats with carbon tetrachloride (0.2 ml/kg body weight) dissolved in various oils (sunflower, corn, fish or olive oil) by gastric intubation 3 times/week for 8 weeks. The increase of collagen fibres in the liver was only 2-4% when olive oil was used as a vehicle, instead of the 6-8% increase when the other oils were used. 7.9.2 Diet In male NMRI mice, fasted for 24 h before receiving an intraperitoneal injection (0.1 ml/kg body weight) of carbon tetrachloride (159.4 mg/kg body weight) in olive oil, higher hepatic carbon tetrachloride and chloroform levels were found (Pentz & Strubelt, 1983). Fed male Sprague-Dawley rats appeared to be more resistant to the toxic action of carbon tetrachloride than rats starved overnight. Contrary to findings in mice, the carbon tetrachloride concentrations were similar in the livers of fed and fasted rats (Díaz Gómez et al., 1975b). Male Wistar rats were fed various test diets in order to assess the nutritional effects on the liver mixed-function oxidases (MFO) and consequently on the carbon tetrachloride metabolism. The MFO activity increased almost linearly with decreasing food intake. Furthermore it was shown that a diet deficient in carbohydrate enhanced the metabolism and thus the toxic action of carbon tetrachloride, irrespective of the protein or fat content of the diet (Nakajima et al., 1982; Sato & Nakajima, 1985). Cervinková et al. (1987) investigated the effect of long-term intake of high or low protein diet on liver repair processes after the administration of carbon tetrachloride. Rats were fed for 21 days on a low-protein diet (LPD), a standard diet (SD) and a high-protein diet (HPD) and were then given a single intraperitoneal injection (0.75 ml/kg body weight) of carbon tetrachloride (calculated to be 1196 mg/kg body weight). The HPD was found to increase sensitivity to carbon tetrachloride, but it also promoted liver repair processes. The LPD raised liver resistance to carbon tetrachloride, but the development of liver repair activity differed from the process after the SD and HPD, since polyploidy of the hepatocytes predominated and there was also an increase in the number of binuclear hepatocytes. Cell hypertrophy was expressed less in rats fed on the LPD. As far as liver repair was concerned, the HPD showed no explicit advantage over the SD. 7.9.3 Alcohol Several studies have demonstrated that ethanol, methanol and other alcohols potentiate the hepatic toxicity of carbon tetrachloride (Traiger & Plaa, 1971; Cantilena et al., 1979; Harris & Anders, 1980; Ray & Mehendale, 1990; Simko et al., 1992). Dietary ethanol (2 g/80 ml liquid diet for 3 weeks) potentiated carbon tetrachloride (inhalation exposure to 10 ppm (64.1 mg/m3) for 8 h) hepatotoxicity, measured by serum aminotransferases and liver malonaldehyde concentrations, in male Wistar rats. Potentiation did not occur upon exposure to 5 ppm (32 mg/m3) for 8 h (Ikatsu et al., 1991; Ikatsu & Nakajima, 1992). Only a minor potentiating effect on weight gain, but no effect on carbon-tetrachloride-induced hepatotoxicity was observed, when rats were simultaneously treated with ethanol (æ 0.5 ml/kg) and carbon tetrachloride (20 mg/kg) by gavage for 14 days (Berman et al., 1992). Micronodular cirrhosis was observed in all treated rats after 10 weeks of inhalation exposure to carbon tetrachloride (513 mg/m3, 6 h/day, 5 days/week) when the animals were simultaneously given ethanol as a part of a liquid diet, while no animal treated with either ethanol or carbon tetrachloride alone developed cirrhosis (Hall et al., 1991). Similar cirrhosis was also observed in Porton rats treated with carbon tetrachloride and ethanol (Hall et al., 1994). Inhalation exposure to methanol (10 000 ppm for 6 h) increased the liver toxicity of carbon tetrachloride (a single dose of 0.075 ml/kg after 24 h) (Simmons et al., 1995). Similar exposure to methanol also increased the toxicity of inhaled carbon tetrachloride (100, 250 to 1000 ppm (641, 1602 to 6410 mg/m3) for 6 h at 26-27 h after the beginning of the methanol exposure). This potentiation subsided when the interval between methanol and carbon tetrachloride exposures was increased by 24 h (Evans & Simmons, 1996). Malonaldehyde generation induced by carbon tetrachloride in vitro was enhanced by prior exposure of the rats to methanol (10 000 ppm for 6 h); this enhancement coincided with an increased microsomal activity of para-nitrophenol hydroxy lase, used as a marker of cytochrome P-450 2E1; inhibition of CYP 2E1 by allyl sulfone abolished the carbon-tetrachloride-induced lipid peroxidation (Allis et al., 1996). Sato & Nakajima (1985) reported that the metabolism of carbon tetrachloride in the rat was enhanced by pretreatment with ethanol. It was found that the increase in carbon tetrachloride hepatotoxicity was related to the degree of the enhancement. Similar observations were made by Sato et al. (1980), Teschke et al. (1984), Strubelt (1984) and Reinke et al. (1988). In a study by Wang et al. (1997), before exposure to carbon tetrachloride, rats were kept either on an ethanol-containing (2 g/80 ml per rat per day) liquid diet for 3 weeks, to obtain a maximal induction of the alcohol-inducible CYP 2E1 isoenzyme, or on a liquid diet with no alcohol. Both groups were exposed to carbon tetrachloride by inhalation (0, 320 or 3205 mg/m3 for 6 h), or by oral or intraperitoneal administration (0, 0.105 or 1.675 mmol/kg). Ethanol-pretreatment increased significantly the metabolism of carbon tetrachloride as indicated by the carbon tetrachloride concentrations in blood samples. Plasma ALAT and ASAT levels, assayed 24 h after carbon tetrachloride treatment, were highly significantly increased in all carbon tetrachloride-dosed rats pretreated with ethanol, whereas in control diet groups only a slight elevation of transaminases was observed after the high-dose carbon tetrachloride treatment. Shibayama (1988) compared the hepatotoxicity of carbon tetrachloride (intraperitoneal administration in olive oil) in male Wistar rats fed a standard diet and 5 or 20% ethanol solution with the hepatotoxicity of carbon tetrachloride in control rats, which received water instead of ethanol for a period of 1 to 100 weeks. The results indicated that the effect of ethanol on the hepatotoxicity is dependent on the daily amount of alcohol intake and is not affected by the duration of the alcohol consumption. Kniepert et al. (1990), however, reported that an increased duration (30 or 52 weeks instead of 1 or 10) of ethanol pretreatments (10% in drinking water) caused a decrease in ethanol potentiation of carbon-tetrachloride-induced toxicity in male Wistar rats. Ray & Mehendale (1990) studied the effect caused by a single dose of carbon tetrachloride after pretreatment with various homologous alcohols in male Sprague-Dawley rats. A combination of the alcohols methanol, ethanol, isopropanol and decanol with carbon tetrachloride potentiated liver injury but did not affect lethality. A combination of t-butanol, pentanol, hexanol and octanol potentiated liver injury and decreased animal survival significantly. Eicosanol potentiated neither liver injury nor lethality. Hall et al. (1994) observed that chronic administration of alcohol and 'low-dose' carbon tetrachloride vapour caused cirrhosis in all male Porton rats receiving this treatment for 5-7 weeks. A possible mechanism for the interaction between alcohol and carbon tetrachloride is the alcohol-dependent induction of cytochrome P-450 2E1, resulting in enhanced production of toxic metabolites of carbon tetrachloride. These in turn are responsible for the initiation of lipid peroxidation and impaired conjugation of carbon tetrachloride metabolites with glutathione. To determine the dose-response relationships in the production of hepatic fibrosis and cirrhosis, the livers of male Porton rats (4 animals/group) were examined after combined exposure to carbon tetrachloride and alcohol (Plummer et al., 1994). Carbon tetrachloride was administered by inhalation 6 h/night for 5 nights/week at concentrations of 10, 20 or 40 ppm (actual values of 60, 120.1 or 240.1 mg/m3, respectively). Ethanol was administered orally at levels of 75, 150, or 300 kcal/litre liquid diet, leading to mean daily intakes of 2.29, 4.61 and 8.16 g ethanol/kg body weight, respectively. It was proposed to continue administration of alcohol and carbon tetrachloride until animals became cirrhotic, as diagnosed by liver biopsy, or for a maximum of 20 weeks. However, the alcohol consumption declined gradually, approximately to half that at the beginning. Results of the study show that both alcohol and carbon tetrachloride contribute to the liver injury in a dose-related manner. All four rats that received the high dose of both carbon tetrachloride and alcohol, and one of four rats that received the medium alcohol and high-dose carbon tetrachloride treatments, showed liver cirrhosis after 10 weeks of exposure. Two of four rats that received the low alcohol in combination with the high-dose of carbon tetrachloride showed cirrhosis after 20 weeks. Although cirrhosis was observed only at the highest carbon tetrachloride dose, some degree of hepatic fibrosis was observed in all treated rats in a dose-related manner. Daniluk et al. (1994) reported that acute liver injury due to intraperitoneal carbon tetrachloride administration combined with a long-term alcohol consumption may act synergistically in depressing interferon production in C3H/He mice. Strain differences in response to carbon tetrachloride have been described for both mice (see section 126.96.36.199) and rats (see section 7.3). 7.9.4 Enhancement of carbon tetrachloride-induced hepatotoxicity by various compounds According to Klingensmith et al. (1983) the LD50 of carbon tetrachloride after oral administration dropped by a factor of 14 after pretreatment with chlordecone. The potentiation of carbon tetrachloride toxicity by chlordecone appears to be related to a chlordecone-dependent increase in the biotransformation rate of carbon tetrachloride and a chlordecone-dependent reduction of the liver-regenerating capacity (Mehendale, 1984). Mehendale (1989) described a mechanism for the potentiation of carbon tetrachloride hepatotoxicity by chlordecone. The mechanism underlying the highly unusual amplification of carbon tetrachloride toxicity relates to the suppression of the initial hepatocellular regeneration, ordinarily stimulated by low doses of carbon tetrachloride. Pretreatment with phenobarbital enhanced the metabolism of carbon tetrachloride in rats, and consequently the toxicity (Bechtold et al., 1982; Fander et al., 1982; Sato & Nakajima, 1985), whereas pretreatment with PCBs or 3-methylcholanthrene for a few days hardly influenced the carbon tetrachloride metabolism and carbon tetrachloride toxicity (Sato & Nakajima, 1985). Prolonged pretreatment with hexachlorobenzene, PBBs or PCBs, however, made male rats considerably more susceptible to the toxic effects of carbon tetrachloride (Kluwe et al., 1982). The reaction of the liver to carbon tetrachloride was studied in the adaptive stage of organic solvent poisoning. Rats were pretreated daily with benzene, toluene, xylene, phenobarbital or oil for 4 days. On day 4, carbon tetrachloride was given orally and 24 h later the rats were killed. Histological, histochemical and electron microscopic examination revealed a potentiating interaction between the solvents and carbon tetrachloride, similar to the potentiation of carbon tetrachloride toxicity by simultaneous phenobarbital administration. Centrilobular necrosis caused by carbon tetrachloride became confluent and turned submassive in the livers of pretreated animals, and it appeared that in the rats treated with solvent and carbon tetrachloride, the amount of damaged area was twice that induced by carbon tetrachloride alone (Tátrai et al., 1979). Qazi & Alam (1988) reported that phenobarbitone treatment in drinking-water together with carbon tetrachloride (by intraperitoneal injection) induced severe liver cirrhosis with marked proliferation of the bile ducts in female rats. Females receiving only carbon tetrachloride showed moderate cirrhosis whereas the females receiving only phenobarbitone remained healthy, showing only an increased liver weight. ElSisi et al. (1993a,b) studied the effect of retinol (vitamin A) on carbon-tetrachloride-induced hepatoxicity in a time-response and a dose-response study. In the time-response study, male Sprague-Dawley rats were given 75 mg/kg body weight retinol for 1 or 3 days, or 1,2,3 or 5 weeks. In the dose-response study, retinol was given at daily doses of 30 to 75 mg/kg body weight for 3 weeks. At 24 h after the last dose of retinol, 0.15 ml carbon tetrachloride/kg body weight (239 mg/kg body weight) in corn oil was given by intraperitoneal injection and another 24 h later the animals were killed. All treatment durations with retinol, except 1 day, resulted in equivalent potentiation of carbon tetrachloride hepatotoxicity. All rats pretreated with retinol and subsequent administration of carbon tetrachloride had more extensive liver injury than those given carbon tetrachloride alone. As the daily dose of retinol increased, so did the degree of potentiation of carbon tetrachloride hepatotoxicity. Pretreatment with ketonic or ketogenic compounds (e.g., hexane, acetone, isopropanol) potentiated the liver injury in Sprague-Dawley rats produced by an intraperitoneal carbon tetrachloride injection (Charbonneau et al., 1985). The hepatotoxicity of the liver to carbon tetrachloride is considerably enhanced in alloxan-diabetic rats. This potentiation effect can be reversed by an additional insulin treatment before the carbon tetrachloride administration (Villarruel et al., 1982). Intraperitoneal treatment of male Sprague-Dawley rats with pyrazole increased the sensitivity of these rats to carbon tetrachloride hepatotoxicity as assessed by significant loss of cytochrome P-450 and increases in ASAT and ALAT levels. As stated by the author (Ebel, 1989), these observations are consistent with the hypothesis that only certain forms of P-450 (and in this case the 'alcohol-inducible form') are capable of activation of hepatotoxins and potentiate the toxicity. Imidazole and pyrazole, inducers of CYP 2E1, caused 3- to 25-fold enhanced rates of carbon-tetrachloride-induced lipid perioxidation (and chloroform production from carbon tetrachloride); the increase was directly related to the amount of this cytochrome in the microsomes (Johansson & Ingelman-Sundberg, 1985). Acetone, methyl ethylketone and methyl isobutylketone (6.8 mmol/kg body weight for 3 days) increased the hepatotoxicity of carbon tetrachloride (Raymond & Plaa, 1995a); this enhanced toxicity was coincident with an increased microsomal aniline hydroxylase activity (Raymond & Plaa, 1995b). In addition to the effect on cytochrome P-450, acetone, but not the other ketones, increased basal canalicular membrane fluidity, as measured by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene or 1,4-(trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (Raymond & Plaa, 1996). Treatment of male athymic nude rats, male and female Sprague-Dawley rats, and male Fischer-344 rats with retinol (75 mg/kg/day for 7 days) greatly enhanced the hepatotoxicity of carbon tetrachloride in F-344 rats (0.2 or 0.1 mg/kg i.p.), while it protected Balb/C, C3H/HeJ, athymic nude and Swiss-Webster mice against carbon tetrachloride hepatotoxicity (0.0125 to 0.02 ml/kg, respectively) (Hooser et al., 1994). In male Sprague-Dawley rats retinol (> 100 000 IU/kg per day for 3 weeks or 250 000 IU/kg per day for > 1 week) greatly increased the hepatotoxicity of carbon tetrachloride (0.15 ml/kg intraperitoneally) (ElSisi et al., 1993a). There was a simultaneous six- to eight-fold increase in the amount of exhaled ethane and a less than two-fold increase in covalent binding to liver proteins in rats treated with retinol (250 000 IU or 75 mg/kg per day for 1 week) and carbon tetrachloride (0.15 ml/kg), in comparison with rats treated with carbon tetrachloride alone. However, there was no increase in the exhaled 14CO2, exhaled organics or metabolites excreted in the urine, or covalent binding to hepatic lipids from 14C-carbon tetrachloride. Aminobenzotriazole (50 mg/kg intraperitoneally, 2 h before carbon tetrachloride), an inhibitor of cytochrome P-450, blocked the retinol-induced potentiation of the hepatotoxicity of carbon tetrachloride (ElSisi et al., 1993b). A single dose of retinol (> 75 mg/kg orally) 24 h before carbon tetrachloride also very significantly potentiated carbon tetrachloride hepatotoxicity (Badger et al., 1996). While the total cytochrome P-450 content of the liver was not affected by the retinol treatment, the concentration (Western blot analysis) and activity (aniline hydroxylase) of CYP 2E1 were both elevated. Isolated hepatocytes from retinol-treated rats also exhibited enhanced susceptibility to carbon tetrachloride (Badger et al., 1996). Concomitant administration of a single, non-hepatotoxic dose of 6 mmol dichloromethane (DCM)/kg and 308 mg carbon tetrachloride intraperitoneally potentiated carbon tetrachloride-induced hepatotoxicity, as measured by SDH and ALAT. When a radiolabelled tracer dose of carbon tetrachloride was included in the treatment, DCM was found to significantly increase the covalent binding of [14C]-carbon tetrachloride metabolites to microsomal lipids. However, DCM did not affect lipid peroxidation induced by carbon tetrachloride (Kim, 1997). Potentiation of carbon tetrachloride hepatotoxicity was studied after inhalation exposure at concentrations of 0, 5 or 10 ppm (0, 160 or 320 mg/m3) for 8 h and simultaneous exposure to chloroform (0, 25 or 50 ppm for 8 h). While carbon tetrachloride exposure had no effect on plasma ALAT or ASAT levels, co-exposure to chloroform resulted in a slight increase of the transaminase activity in blood (Ikatsu & Nakajima, 1992). 7.9.5 Reduction of carbon tetrachloride-induced hepatotoxicity by various compounds Vitamin E reduces the carbon-tetrachloride-induced lipid peroxidation in rat liver and kidney slices (Gavino et al., 1984), but in an in vivo experiment in rats only limited protection by vitamin E against this process was seen (Gee et al., 1981). A protective action of vitamin E against liver cell membrane damage in Wistar rats was reported in several studies (Ozeki et al., 1982; Martínez-Calva et al., 1984). This protective action could be reinforced by co-administration of vitamin E and riboflavin tetrabutyrate (Miyazawa et al., 1984). When male Wistar rats were given vitamin E 15 h before carbon tetrachloride administration, a partial or complete protection against the necrogenic effect of carbon tetrachloride was induced, depending on the concentration of carbon tetrachloride used. Furthermore, the vitamin supplementation prevented the carbon-tetrachloride-induced increase in total hepatic calcium content (Biasi et al., 1991). Protection, apparent as a decrease in mortality, less pronounced histological damage and lower serum aminotransferase levels was afforded by intravenous administration of alpha-tocopherol as a suspen sion or in liposomes, which are accumulated in Kupffer cells (Yao et al., 1994; Liu et al., 1995). Where incorporated into liposomes, other antioxidants, such as butylated hydroxytoluene and ascorbic acid palmitate, also protected mice against carbon tetrachloride toxicity (Yao et al., 1994). Preventive effects on hepatotoxicity in rats were also reported for vitamin D3 and vitamin C by Fander et al. (1982) and Ademuyiwa et al. (1994), respectively. In contrast to the results of ElSisi et al. (1993a,b), who reported a potentiation in hepatotoxicity when retinol was given to rats prior to carbon tetrachloride administration, Rosengren et al. (1995) observed a protective effect on the liver when retinol was given to mice prior to carbon tetrachloride administration. Studies reported by Bishayee & Chatterjee (1993) and Mandal et al. (1993) indicated a possible hepatoprotective role of carrot (Daucus carota) aqueous extract and Mikania cordata root extract in male Swiss mice. The increased lipid peroxidation and decreased glutathione levels, resulting from carbon tetrachloride treatment, were significantly reversed in a dose-related way after pretreatment of the mice with the carrot extract. According to authors this protective role of carrot extract could be attributed to its antioxidant properties. Pretreatment of rats with SKF-525 A (inhibitor of drug-metabolizing enzymes), cysteine (in particular in combination with tryptophan) or reduced glutathione decreased the carbon-tetrachloride-induced hepatotoxicity (Bechtold et al., 1982; de Ferreyra et al., 1983; Gorla et al., 1983). Administration of 6,6'-methylene-bis(2,2,4-trimethyl-1,2-dihydroquinoline), tert-butyl-4-hydroxyanisole, oltipraz or anethol dithiolthione protects mice against the acute toxic effects of carbon tetrachloride (Fehér et al., 1982; Toncsev et al., 1982; Ansher et al., 1983; Benson, 1993). A preventive effect was also observed when diethyldithiocarbamate and carbon disulfide were administered to mice. Diethyldithiocarbamate was most effective when given orally, while the action of carbon disulfide was less dependent on the route of administration (Masuda & Nakayama, 1982). Rao & Mehendale (1989) reported that administration of fructose 1,6-diphosphate decreased the toxicity of carbon tetrachloride in rats (as shown by a 50-70% decrease in the activity of serum transaminases). This decrease was accompanied by elevated activities of enzymes involved in the polyamine metabolism (important for hepatocellular regeneration and recovery). It appears that nicotinamide administered to male Sprague-Dawley rats at late stages of carbon tetrachloride poisoning (e.g., 6 or 10 h after the hepatotoxin) significantly prevents the liver necrogenic effects of carbon tetrachloride at 24 h. A study by de Ferreyra et al. (1994) did not reveal any relevant effect when nicotinamide was given 30 min before the hepatotoxin. Simultaneous treatment of carbon-tetrachloride-intoxicated rats with zinc (227 mg/litre in drinking-water) resulted in improved serum and liver enzyme levels and attenuated histological abnormalities as well as NADPH-dependent lipid peroxidation (Dhawan & Goel, 1994). Studies of Kaminski et al. (1989, 1990) demonstrated that carbon tetrachloride administration in mice results in a marked suppression of humoral and cell-mediated immune functions. Ahn & Kim (1993) observed that PMC (diphenyl dimethyl dicarboxylate) had a significant preventive effect on carbon-tetrachloride-induced immunotoxic status in ICR mice that were immunized and challenged with sheep red blood cells (SRBC) and were subsequently given PMC (3 or 6 mg/kg body weight; oral administration) once a day for 28 days in combination with carbon tetrachloride (1 ml/kg body weight, 25%, oral administration) twice a week, 2 h after PMC administration. Gadolinium chloride (10 mg/kg), given intravenously 24 h prior to an intragastric dose of carbon tetrachloride (4 g/kg), nearly completely protected rats from hepatic necrosis, as measured by serum ASAT levels and trypan blue exclusion, without an effect on CYP 2E1 (Edwards et al., 1993). This was interpreted to indicate a role of Kupffer cells in carbon-tetrachloride-induced hepatic damage, since gadolinium chloride at this concentration strongly inhibits Kupffer cell phagocytosis (Hustzik et al., 1980). Similar dosage of gadolinium chloride was, however, also reported to decrease the total amount of hepatic cytochrome P-450 in rats, as well as the activity of aniline para-hydroxylase (Badger et al., 1997). In support of the role of Kupffer cells in carbon-tetrachloride-induced hepatic damage, it was reported that gadolinium chloride (10 mg/kg given intravenously 24 h before carbon tetrachloride administration) prevented and methyl palmitate, another Kupffer cell inhibitor, attenuated the periportal oedema observed using proton magnetic imaging 1-2 h after carbon tetrachloride administration (0.8 ml/kg given intraperitoneally) (Towner et al., 1994). In vivo pin trapping using alpha-phenyl- N-tert- butylnitrone and a subsequent electron paramagnetic resonance study of the liver indicated that gadolinium chloride did not affect the generation of trichloromethyl radicals from carbon tetrachloride (Towner et al., 1994). Gadolinium chloride (10 mg/kg given intravenously), methyl palmitate, polyethylene-glycol-coupled superoxide dismutase and polyethylene-glycol-coupled catalase protected Sprague-Dawley rats against retinol-induced potentiation of carbon tetrachloride hepatotoxicity, both after a single dose and daily dosing for seven days of retinol (ElSisi et al., 1993a; Sauer & Sipes, 1995; Badger et al., 1996). Dietary alpha-tocopherol (250 mg/kg diet) partly protected male Wistar rats against carbon-tetrachloride-induced (0.15 ml 3 times a week for 5 weeks) hepatic damage (Parola et al., 1992). In an acute experiment, alpha-tocopheryl hemisuccinate (0.19 mmol, approx. or equivalent 100 mg/kg by gavage) afforded a partial protection against the hepatotoxicity of carbon tetrachloride (1.0 g/kg) administered 18 h later (Tirmenstein et al., 1997). 7.10 Mode of action Recknagel & Glende (1973) suggested that carbon tetrachloride toxicity requires cleavage of the carbon-chlorine bond and that the cleavage takes place after binding of carbon tetrachloride to cytochrome P-450 apoprotein in the mixed-function oxidase system located in the hepatocellular endoplasmatic reticulum. However, cytochrome P-450 is encased in lipid, and peroxidative decomposition of the lipid is initiated by the free radicals formed by the cleavage. Owing to the decomposition of the lipid and the attack on protein functional groups by lipid peroxides, the structure and function of the endoplasmatic reticulum is destroyed. The carbon-tetrachloride-induced destruction of microsomal cytochrome P-450 in vitro (De Groot & Haas, 1980, 1981) and in vivo (Pasquali-Ronchetti et al., 1980; Shen et al., 1982) inhibits the further biotransformation of carbon tetrachloride, the generation of radicals and the concomitant peroxidation of endoplasmic reticular lipids. Burk et al. (1983) proposed a theory in which the toxic action of carbon tetrachloride in vivo is dependent on either trichloromethyl or trichloromethylperoxide radical formation, which is controlled by the oxygen status of the liver cell (peripheral or centrilobular), leading to damage to tissue macromolecules. This theory has been supported by mechanistical arguments as summed up by Slater (1982) and Dianzani (1984), who proposed the trichloromethyl radical as the main covalently binding agent (haloalkylation) and the trichloromethylperoxide radical as the main lipid-peroxidation-inducing agent. Oral dosage of carbon tetrachloride (2.5 ml/kg) decreased the ATP-dependent calcium uptake of liver microsomes within 30 min in Sprague-Dawley rats (Moore et al., 1976). The cytosolic concentration of Ca2+ increased 100-fold in hepatocytes exposed to carbon tetrachloride (about 1 mmol/litre), and this was paralleled by an inhibition of the endoplasmic reticulum Ca-Mg ATPase (Long & Moore, 1986). The inhibition of the ATPase by carbon tetrachloride exposure has been confirmed in several studies (Srivastava et al., 1990), and has led to the hypothesis that this is the specific mechanism by which radical intermediates from carbon tetrachloride lead to cell death. Calcium chelating agents, Calcion and alizarin sodium sulfonate, administered 6 or 10 h after a necrogenic intraperitoneal dose of carbon tetrachloride (1 ml/kg), markedly decreased the necrotizing effect of carbon tetrachloride on the liver, and decreased the hepatic calcium concentration but did not affect carbon-tetrachloride-induced lipid peroxidation in vitro, or lipid accumulation in the liver (de Ferreyra et al., 1989, 1992). Carbon tetrachloride (0.01-0.12 mmol/litre) induced a complete release of calcium from calcium-loaded microsomes in the presence of NADPH; this release was blocked by adding the spin trapping agent, phenyl- tert- butylnitrone (PBN) after a lag period that was dependent on the concentration of carbon tetrachloride. The lag period was shortened using microsomes from pyrazole-treated rats, which showed an elevated activity for para-nitrophenol oxidation, and lengthened in the presence of the CYP-450 2E1 inhibitor, methylpyrazole, or an anti-CYP-450 2E1 antibody. Calcium release was practically complete at concentrations of carbon tetrachloride that had no effect on the Ca-Mg ATPase activity. Ruthenium red, a specific ryanodine receptor inhibitor, completely blocked the carbon-tetrachloride-induced calcium release at a concentration (0.02 mmol/litre) that had no effect on para-nitrophenol hydroxylation or formation of PBN-carbon tetrachloride adducts (Stoyanovsky & Cederbaum, 1996). These results support the notions that the hepatotoxicity of carbon tetrachloride requires metabolism to the *CCl3 radical and is mediated by calcium release from intracellular stores, most likely from the ryanodine-sensitive calcium store. Brattin et al. (1984) stated that the disturbance of the intracellular Ca2+ balance cannot be regarded as an intracellular "toxic messenger". Recknagel (1983), however, suggested that an early disturbance in hepatocellular Ca2+ homoeostasis may be involved in the pathological changes elicited by carbon tetrachloride (Recknagel, 1983). In addition, other authors suggest that the depression of the Ca2+- sequestration capacity of the endoplasmic reticulum (microsomes), resulting in a rise in the concentration of Ca2+ in the cytosol, is an important factor in carbon-tetrachloride-induced hepatotoxicity (Waller et al., 1983; Srivastava et al., 1990; Yamamoto, 1990; Glende & Recknagel, 1991). Mehendale (1990, 1991) showed that hepatic microsomes from carbon-tetrachloride-treated rats accumulated progressively greater concentrations of Ca2+ in response to the rise of Ca2+ levels in cytosol. It is well known that lactic acid plays an important role in hepatic fibrogenesis. Ayub-Ayala et al. (1993) determined the relationship between short-term carbon tetrachloride administration and the rise in lactic acid levels, before the appearance of any signs of hepatic cirrhosis. After intraperitoneal administration of one single and three consecutive carbon tetrachloride doses of 2.0 ml/kg body weight (1:1 dilution in mineral oil) to Sprague-Dawley rats, the blood lactic acid levels were raised, whereas the administration of mineral oil did not increase them. Since carbon tetrachloride increases lactic acid levels prior to cirrhosis development, the authors suggested that chronic presence of lactic acid is one of the factors in hepatic fibrogenesis caused by carbon tetrachloride. Castro et al. (1990) stated that the understanding of the mechanism of the liver carcinogenic effects of carbon tetrachloride might be of relevance because there are reasons to believe that carbon tetrachloride might be one of those carcinogens of a non-genotoxic nature. The authors showed that liver nuclei from three species tested (rat, hamster and mouse) were able to promote a lipid peroxidation process in the presence of carbon tetrachloride, and that NADPH was only required in part for carbon-tetrachloride-induced lipid peroxidation in the case of the mice. There was no correlation between the intensity of carbon-tetrachloride-induced lipid peroxidation, either in liver nuclear or liver slices preparations, in the three species tested and their carcinogenic response to carbon tetrachloride. These results suggest that lipid peroxidation is not determinant or rate-limiting in the process of liver cancer induction by carbon tetrachloride, but does not exclude its participation in given stages of the overall process of cancer development, as is actually believed to occur during chemical carcinogen insult. Carbon tetrachloride induced a hepatic cell proliferation, increasing the frequency of cells in S-phase from < 1% in control animals to around 10% in B6C3F1 mice (100 mg/kg by gavage 48 h before sacrifice) (Mirsalis et al., 1985). In rats a similar increase was observed after a dose of 400 mg/kg (Mirsalis et al., 1985), and the increase was around 30% in CD mice (50 mg/kg) (Doolittle et al., 1987). In male Fischer-344 rats, the frequency of S-phase cells was elevated in one study to 30% 24 h after the only dose tested, 0.4 ml/rat (Cunningham & Matthews, 1991). In another study with 400 mg/kg carbon tetrachloride, the frequency was increased to 3% in fed and to 15% in fasting F-344 rats (Asakura et al., 1994). In yet another study with Fischer rats, an increase to 5% was observed 24 h after an intraperitoneal dose of 400 mg/kg carbon tetrachloride (Mirsalis et al., 1985). An even lower response, approximately 2%, was observed in Tif:RAIf rats (400 mg/kg) (Puri & Müller, 1989). An increase in DNA synthesis was observed 48 h (and the number of ras transcripts was elevated 36-48 h) after an intragastric dose (2.5 ml/kg) of carbon tetrachloride in Sprague-Dawley rats (Goyette et al., 1983). A rapid transient increase in c- fos and c- jun mRNA (1-2 h post-treatment) was also observed in the liver of male Sprague-Dawley rats after a single dose of 160 mg/kg carbon tetrachloride (Zawaski et al., 1993). An increase in the c- fos, c- jun and c- myc nRNA was also observed in male Wistar rats after a single dose of carbon tetrachloride (2 ml/kg intragastrically) (Coni et al., 1990, 1993). In rat liver, ras and myc proteins were observed by immunohistochemical techniques. Their concentrations peaked in periportal areas 32 h after dosing with carbon tetrachloride (2.5 ml/kg), and staining throughout the lobule peaked 96 h after the carbon tetrachloride dose (Richmond et al., 1992). The sequence of fos, myc and Ha- ras mRNA expression, followed by hepatocyte proliferation, was also observed in F-344 rats after a single intraperitoneal dose of 2000 mg/kg carbon tetrachloride by gavage (Goldsworthy et al., 1994). Injection of a polyclonal antiserum to murine tumour necrosis factor alpha one hour before a challenge with carbon tetrachloride (0.1 ml/kg) blocked the increase in c- fos and c- jun mRNA expression and the subsequent increase of S-phase cells, while at the same time prolonging the elevation of serum ALAT, ASAT and sorbitol dehydrogenase (SDH) in female B6C3F1 mice. When recombinant TNF alpha was injected to mice, rapid expression of c- jun and c- fos proto-oncogene mRNA was observed, thus supporting the notion that TNF alpha has a role in the hepatocellular regeneration after carbon tetrachloride administration (Bruccoleri et al., 1997). This idea was originally put forward after the demonstration of increased expression of TNF alpha following an administration of a hepatotoxic dose of carbon tetrachloride (Czaja et al., 1989). On the other hand, injection of soluble TNF alpha receptor preparation to rats had a protective effect against a higher dose of carbon tetrachloride (0.5 ml/kg), reducing the mortality, serum aminotransferase levels and the extent of histological liver damage (Czaja et al., 1995). 8. EFFECTS ON HUMANS 8.1 Controlled studies 8.1.1 Inhalation Six healthy male volunteers were exposed (three times 4 weeks apart) to carbon tetrachloride vapour at concentrations of 49 ppm (314 mg/m3) for 70 min, 11 ppm (70.5 mg/m3) for 180 min and 10 ppm (64.1 mg/m3) for 180 min. At the high concentration level, all subjects smelled a sweetish odour. None of the volunteers reported irritation, nausea, lightheadedness or disturbance in coordination. No increase in ASAT activity was observed, but a decrease in serum iron concentration was observed in two out of four subjects at the highest concentration. No effects were observed at the lower concentrations. Carbon tetrachloride was detected in exhaled breath at all three exposure levels (Stewart et al., 1961). 8.1.2 Dermal Daily treatments for 10 days with carbon tetrachloride did not cause any increase in skin-fold thickness or erythema on the volar surface of the forearms of a healthy human volunteer. However, no occlusion was used, so it is probable that the chemical evaporated after administration (Wahlberg, 1984a). When an excess of carbon tetrachloride was applied in a glass ring to the volar surface of the forearms of a healthy man for 5 min there was an immediate increase in blood flow. A spontaneous transient whitening of the skin was observed after 5 min and after 10 to 20 min a slight, transient erythema appeared (Wahlberg, 1984b). Immersion of the thumbs of three volunteers for 30 min in carbon tetrachloride caused mild erythema that disappeared in 1 to 2 h after exposure. The volunteers reported a burning sensation in the thumbs, which subsided within 10 min after the immersion (Stewart & Dodd, 1964). 8.2 Case reports Cases of poisoning with carbon tetrachloride have resulted from the accidental or suicidal ingestion of carbon tetrachloride, but the majority resulted from the inhalation of carbon tetrachloride vapour; the concentration of the saturated vapour at ambient temperature can reach 800 000 mg/m3. Carbon tetrachloride appears to be toxic to the liver and kidney. The clinical picture of carbon tetrachloride poisoning is characterized, independent of the route of intake, in the first 24 h with gastrointestinal and neurological symptoms, such as nausea, headache, dizziness, vomiting, diarrhoea and dyspnoea. Liver damage appears, at the earliest, after 24 h. In serious cases, ascites and hepatic coma develop, often accompanied by haemorrhages. Kidney damage is detected later, in 1 to 6 days, but often only 2-3 weeks following the poisoning (Zimmerman, 1978; Kluwe, 1981; Monster & Zielhuis, 1983). Von Oettingen (1964) reviewed the literature on acute carbon tetrachloride intoxication in humans. He concluded that exposure to 10-80 ppm (64.1 to 512.8 mg/m3) for 3-4 h has no adverse effects. At higher concentrations nausea, vomiting, headache, rapid pulse, rapid respiration, sleepiness, dizziness, unconsciousness and immediate death can occur even after only 10-30 min of exposure. Bagnasco et al. (1978) reported a case in which a 22-year-old man ingested 355 ml carbon tetrachloride and an equal amount of water to commit suicide. His liver function deteriorated over the first 24 h, but gradually within the following 3 to 4 days the patient improved. According to the authors fatal cases have been reported with as little as 1.5 ml carbon tetrachloride, whereas some patients have been known to survive after swallowing more than 100 ml. Smetana (1939) described two cases of carbon tetrachloride poisoning. One person, who drank an unknown quantity of carbon tetrachloride, died. Apart from the general symptoms, jaundice, anuria and malaise were observed. Aside from lesions in the liver there was clinical evidence of functional damage of the kidneys, recognized by the presence of albumin in the urine, oliguria, nitrogen retention, oedema and acute hypertension. The patients had a history of alcoholism. Norwood et al. (1950) reported three cases of severe intoxication arising from use of carbon tetrachloride, two by inhalation and one by drinking. The two people who inhaled carbon tetrachloride died, and nephrosis was found to have occurred. All three had a history of heavy drinking. Tracey & Sherlock (1968) described a 59-year-old man, with a history of moderate alcohol consumption, who was exposed to carbon tetrachloride vapour. Five days later, he developed nausea, vomiting and diarrhoea, followed by jaundice and acute renal failure. The liver was found to be enlarged. He recovered uneventfully and liver functions returned to normal. McDermott & Hardy (1963) reported three cases of liver cirrhosis in which repeated exposure to carbon tetrachloride vapour occurred over a number of years. One of the cases involved mixed solvent exposure. There was no evidence of significant alcohol intake for any of the patients. Ruprah et al. (1985) reported details of 19 patients poisoned with carbon tetrachloride during the period 1981-1984. Eight of these patients were known to have ingested other substances, including phenothiazine and benzodiazepine tranquillizers, trichloroethane and trichloroethanol. Carbon tetrachloride exposure was by inhalation (4 cases) or ingestion (15 cases). In each case the diagnosis was confirmed by laboratory analysis of blood specimens. The age of the patients ranged from 3 to 79 years and the whole-blood concentrations at the time of hospital admission varied from 0.1 to 31.5 mg/litre. However, actual doses and exposure concentrations were not known and are difficult to estimate. In none of these cases was the intoxication associated with occupational use or exposure. The commonest symptoms found in these patients were vomiting, abdominal pain, diarrhoea, dizziness, headache and coma. There were no fatalities. Norwood et al. (1950) reported 51 very mild and 4 mild intoxications among industrial workers using carbon tetrachloride. The 4 mildly intoxicated patients showed the general symptoms of carbon tetrachloride intoxication. No data on exposure levels were given. Kazantzis & Bomford (1960) examined a group of 17 factory workers, exposed to carbon tetrachloride atmospheric concentrations of 45 to 100 ppm (288-641 mg/m3). During periods of 24 months to 1 week before the examination, 12 out of 17 workers had experienced one or more of the following symptoms: nausea, anorexia, vomiting, flatulence, epigastric discomfort or distention, depressive symptoms, headache or giddiness. After taking measures to reduce carbon tetrachloride evaporation, these symptoms disappeared and follow-up for 6 months revealed no recurrences. Fourteen workers in an isopropyl alcohol packaging plant became ill after exposure to unspecified carbon tetrachloride levels (Folland et al., 1976). The illness was characterized by the gradual onset of nausea, vomiting, weakness, headache and abdominal pain. In three heavily exposed subjects renal failure developed. Air concentrations of isopropanol in the plant were measured 12 and 9 months before and 2 months after the exposure and were about 400 ppm (2564 mg/m3). Brugnone et al. (1983) investigated 40 workers occupationally exposed to carbon tetrachloride vapour. After several measurements it turned out that the alveolar carbon tetrachloride concentration corresponded to about 53% of the environmental concentration measured in the breathing zone (mean 3.5 ± 5.9 mg/m3). Among the 40 workers, two suffered accidental carbon tetrachloride intoxication with acute renal impairment. Manno et al. (1996) reported that five workers were exposed to carbon tetrachloride vapour for 2 h and two for 6 h following fire accidents. Carbon tetrachloride was present in the fire-extinguishing liquid. Symptoms of carbon tetrachloride poisoning (diarrhoea, nausea, vomiting, fever and liver and kidney impairment) developed only in two heavily drinking workers who consumed, respectively, about 120 and 250 g ethanol daily. The other workers, consuming less than 50 g ethanol per day, did not develop symptoms. Exposure data were not available. 8.3 Epidemiology 8.3.1 Non-cancer epidemiology In a mortality study in a metal fabrication plant (Teta & Ott, 1988) slight increase in mortality from liver cirrhosis was observed. The highest increase (SMR 2.7) was found in workers potentially exposed to carbon tetrachloride before the use of this solvent was discontinued. The authors consider carbon tetrachloride exposure as a possible contributing risk factor for the cirrhosis findings. However, exposure data for carbon tetrachloride, data on other exposures and alcohol consumption were not available, which limit the ability to draw conclusions regarding carbon tetrachloride. Volunteers from three plants were divided into four groups on the basis of estimated exposure to carbon tetrachloride: none (n=262), low (1 ppm (6.4 mg/m3) or less, n=40), medium (1-4 ppm (6.4-25.6 mg/m3), n=54) and high (more than 4 ppm (25.6 mg/m3), n=61). The alcohol consumption was at the same level in all groups. ALAT, ASAT, alkaline phosphatase, gamma-glutamyltransferase, glutamate dehydrogenase, and other biochemical and haematological variables were determined. The percentages of values above the normal range were 2.7% in the non-exposed group and 7.8% in combined exposed groups for ALAT, and 3% and 10.9% for gamma-glutamyltransferase, respectively (both differences statistically significant). The low exposure group did not differ significantly in any enzymatic activity test from the non-exposed group (Tomenson et al., 1995). 8.3.2 Cancer epidemiology A number of epidemiological studies (e.g., cohort mortality, retrospective cohorts, and case-control) have examined potential cause-effect relationships between carbon tetrachloride exposure and incidence of cancer. Because these studies are all characterized by mixed exposures and a lack of carbon tetrachloride exposure data, any contribution from carbon tetrachloride cannot be reliably identified. Thus, information from these studies is not useful for quantitative health risk evaluation. Ott et al. (1985) conducted a cohort mortality study of 1919 men employed for one or more years between 1940 and 1969 at a chemical manufacturing facility in the USA. This cohort included 226 workers assigned to a unit that produced chlorinated methanes (methyl chloride, dichloromethane, chloroform, and carbon tetrachloride) and, recently, perchloroethylene. Exposure levels were not reported. The follow-up period was from 1940 to 1979 and follow-up was 94% complete. Expected numbers of cancer deaths were based on US white male cancer rates for the full cohort; the expected numbers in the full cohort were used for sub-cohort analyses. There were 42 deaths, including nine cancers, three of which were pancreatic cancers. The standardized mortality ratios for all deaths and for all cancers were not elevated. Blair et al. (1990) performed a study to examine the risk of cancer and other causes of death among a cohort of 5365 members of a dry cleaners' union in the USA. The cohort consisted of people who were union members for one or more years before 1978 and had been employed in dry cleaning establishments. Carbon tetrachloride was used extensively in dry cleaning between 1930 and 1960, although other solvents, such as white spirit (Stoddard solvent), were also widely used. The exposure assessment classified members by level of exposure to solvents, but not by type of solvent. The mean year at entry into the cohort was 1956. Follow-up was from 1948 to 1978 and was 88% complete. There were 294 cancer deaths, including a significant excess of oesophageal cancer. Non-significant excesses of several other cancers were found, but only the risk of lymphatic and haematopoietic cancers appeared to be related to the level of solvent exposure. Blair et al. (in press) performed a retrospective cohort mortality study of 14 457 workers employed for at least one year between 1952 and 1956 at an aircraft maintenance facility in the USA. Among this cohort were 6737 workers who had been exposed to carbon tetrachloride (Stewart et al., 1991). Among women, exposure to carbon tetrachloride was associated with an increased risk of non-Hodgkin's lymphoma and multiple myeloma, but among men the corresponding risks were lower. No association was observed with breast cancer and no other site-specific results for carbon tetrachloride were presented. Exposure levels for carbon tetrachloride were not reported, and overlapping exposure to other solvents limits the ability to draw conclusions regarding carbon tetrachloride. A nested case-control study within a cohort of rubber workers in the USA was performed to examine the relationship between exposure to 24 solvents (levels of exposure not reported) and the risk of cancer (Checkoway et al., 1984; Wilcosky et al., 1984). The cohort consisted of 6678 male rubber workers who were either active or retired between 1964 and 1973. The cases comprised all persons with fatal stomach cancer (n=30), respiratory system cancer (n=101), prostrate cancer (n=33), lymphosarcoma (n=9) and lymphocytic leukaemia (n=10). The control group was a 20% age-stratified random sample of the cohort (n=1350). Although an association was observed between exposure for one or more years to carbon tetrachloride and lymphocytic leukaemia and lymphosarcoma after adjusting for year of birth, overlapping exposures limit the ability to draw conclusions regarding carbon tetrachloride. Bond et al. (1986) conducted a nested case-control study of lung cancer among a cohort of 19 608 white male chemical workers in the USA. They were employed for one or more years between 1940 and 1980 at a large facility that produced chlorinated solvents, plastics, chlorine, caustic soda, ethylene, styrene, epoxy, latex, magnesium metal, chlor-nitrogen agricultural chemicals and glycols. The cases were 308 lung cancer deaths that occurred among cohort members between 1940 and 1981. Two control groups, one consisting of other deaths (n=308) and the other a "living" series (n=97), were matched for race, year of birth, and year of hire. No association was observed between having been exposed (levels not reported) to carbon tetrachloride ("ever" versus "never") and lung cancer. Linet et al. (1987) performed an analysis to compare two different methods for determining occupational exposure in a population-based case-control study of chronic lymphocytic leukaemia. No association between chronic lymphocytic leukaemia and carbon tetrachloride was observed in either set of analyses. Heineman et al. (1994) performed a case-control study to examine the relationship between occupational exposure to six chlorinated aliphatic hydrocarbons and risk of astrocytic brain cancer. The study was conducted in three areas of the USA, and 300 cases and 320 controls were included in the analysis. Exposure was assessed using a semi-quantitative job exposure matrix developed for the study (Gómez et al., 1994), and probability of exposure, duration of exposure, average intensity and cumulative exposure were examined. There were 137 cases and 123 controls classified as having been exposed at some time. There was an association between the incidence of astrocytic brain cancer and chlorinated solvent exposure, but not specifically carbon tetrachloride. Cantor et al. (1995) performed a case-control study to examine the relationship between occupational exposure and female breast cancer mortality in 24 states in the USA. Probability and level of workplace exposure to 31 chemical and physical agents were estimated using a job exposure matrix. No association was found with probability of exposure to carbon tetrachloride. After adjustment for age and socioeconomic status, a slightly but significantly elevated risk was observed at the highest exposure level among white women but not among black women. However, the designation of the usual occupation from death certificates in combination with a job-exposure matrix may be a poor indicator of exposure to carbon tetrachloride. Holly et al. (1996) performed a case-control study of intraocular melanoma to examine the role of chemical exposure. Cases were white male patients referred to the Ocular Oncology Unit at the University of California San Francisco between 1978 and 1987. Two white males matched on age and geographic area were selected for each case using random digit dialling. A total of 221 cases and 447 control (93% and 85% participation rates, respectively) were interviewed for the study. Although an association with exposure ("ever" versus "never") to "carbon tetrachloride and other cleaning fluids" was observed, the potential for recall bias for exposure history and the lack of characterization of the exposure atmospheres precludes the ability to draw conclusions regarding carbon tetrachloride alone. In a case-control study carried out in Montreal, the investigators estimated the associations between 293 workplace substances and several types of cancer (Siemiatycki, 1991). Carbon tetrachloride was one of the substances. About 4% of the study subjects had been exposed to carbon tetrachloride at some time. Among the main occupations for which carbon tetrachloride was attributed in this study were fire fighters, machinists and electricians. For most types of cancer examined (oesophagus, stomach, colon, pancreas, prostrate, kidney, skin melanoma, non-Hodgkin's lymphoma), there was no indication of an excess risk. There were, however, elevated risks for rectal cancer and, in the population subgroup of French-Canadians, bladder cancer. 9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD Owing to the volatility of carbon tetrachloride, care must be taken in interpreting test results, particularly those in open static systems where no chemical analysis of the actual concentration was carried out. 9.1 Toxicity to microorganisms Carbon tetrachloride appeared to be of low toxicity to several microorganisms (see Table 11). During studies to determine the toxicity threshold, an initial reduction of cell multiplication or change in culture turbidity was seen at 30 mg/litre in aerobic bacteria, but an IC50 as low as 6.4 mg/litre was found for methanobacteria. The toxicity threshold for protozoa was much higher (> 300 mg/litre). Walton et al. (1989) studied the effect of carbon tetrachloride on the microbial respiration of two slightly acidic soil types, a Captina silt loam (1.49% organic carbon) from Roane County, Tennessee, USA, and a sandy loam (0.66% organic carbon) from Stone County, Mississippi, USA. Carbon tetrachloride was applied at a rate of 1000 µg/g soil (dry weight) and microbial respiration, measured as CO2 efflux, was monitored at 24-h intervals over a 6-day period. Carbon tetrachloride had no effect on the respiration of the silt loam. The CO2 efflux of the sandy loam decreased relative to the control soil but recovered within the 6-day exposure period. 9.2 Aquatic toxicity 9.2.1 Algae Data in Table 11 show the toxicity of carbon tetrachloride to algae to be low. 9.2.2 Invertebrates The acute toxicity values for carbon tetrachloride in Daphnia magna (Table 12) range from 28 to > 770 mg/litre. Carbon tetrachloride had no effect on the embryonic development of sea urchin (Paracentrotus lividus) eggs at concentrations up to the saturated solution concentration (Congiu et al., 1984). 9.2.3 Vertebrates Acute toxicity data for fish are summarized in Table 12. The acute LC50 values for fish range from 13 to 472 mg/litre for the Golden orfe (Leusiscus idus melanotus). Table 11. Carbon tetrachloride toxicity to bacteria, protozoa and algae Organism Test conditions End-point Nominal Reference concentration (mg/litre) Bacteria Pseudomonas fluorescens 16 h, 25 °C, static 3% reduction of turbidity 30 Bringmann, 1973 threshold, log phase Pseudomonas putida 16 h, 25 °C, static 3% reduction of turbidity 30 Bringmann & Kühn, 1977a threshold, log phase Nitromonas sp. 24 h, 25 °C, static IC50, 50% reduction in NH3 51 Blum & Speece, 1991 consumption Methanogens 24 h, 35 °C, static IC50, 50% reduction in gas 6.4 Blum & Speece, 1991 production Aerobic heterotrophs. 24 h, 35 °C, static IC50, 50% reduction in oxygen 130 Blum & Speece, 1991 uptake Protozoa Bacteriovorous flagellate 72 h, 25 °C, static 5% reduction cell count >770 Bringmann, 1978 (Entosiphon sulcatum) Bacteriovorous flagellate 20 h, 25 °C, static 5% reduction in cell count >616 Bringmann & Kühn, 1980 (Uronema parduczi) Saprozoic flagellate 48 h, 20 °C, static 5% reduction in cell count >300 Bringmann et al., 1980 (Chilomonas paramecium) Table 11. (Continued) Organism Test conditions End-point Nominal Reference concentration (mg/litre) Algae Blue-green alga 192 h, 27 °C, static 1% reduction of turbidity 105 Bringmann, 1975 (Microcystis aeroginosa) threshold Green alga 192 h, 27 °C, static 3% reduction of turbidity >600 Bringmann & Kühn, 1977a (Scenedesmus quadricauda) threshold Haematococcus pluvialis 4 h, 20 °C, static EC10, 10% reduction in >136 Knie et al., 1983 oxygen uptake Table 12. Carbon tetrachloride toxicity to invertebrates and fish Organism Test conditions Parameter Concentration Reference (mg/litre) Invertebrates Daphnia magna 21-23 °C reconstituted static 48 h LC50 35 nominal LeBlanc, 1980 well water; pH 7.4-9.4; 24 h LC50 35 nominal hardness 173 mg CaCO3/litre Daphnia magna 20-22 °C dechlorinated static 24 h LC50 >770 Bringmann & Kühn, tap water; pH 7.6; 1977b hardness 173 mg CaCO3 Daphnia magna static 24 h LC50 28 Knie et al., 1983 Fish Freshwater Guppy 22 °C hardness 25 mg static 336 h LC50 67 Könemann, 1981 (Poecilia reticulata) CaCO3/litre renewal Golden orfe 20 °C static 48 h LC50 95 nominala Juhnke & (Leuciscus idus melanotus) 472 nominala Lüdemann, 1978 Golden orfe static 48 h LC50 13 Knie et al., 1983 (Leuciscus idus melanotus) Bluegill sunfish 23 °C well water; static 96 h LC50 125 nominal Dawson et al., (Lepomis macrochirus) pH 7.6-7.9; hardness 1975/77 55 mg CaCO3/litre Table 12. (Continued) Organism Test conditions Parameter Concentration Reference (mg/litre) Bluegill sunfish 21-23 °C pH 6.7-6.8; static 96 h LC50 27 nominal Buccafusco et al., (Lepomis macrochirus) hardness 32-48 1981 mg CaCO3/litre Fathead minnow - - flow - LC50 43.1 measured US EPA, 1984b (Pimephales promelas) Fathead minnow 21.7 °C pH 6.8; hardness flow 96 h LC50 41.4 measured National Library of (Pimephales promelas) 49.2 mg CaCO3/litre Medicine, 1997 Marine Dab - natural seawater flow 96 h LC50 50 measured Pearson & (Limanda limanda) McConnell, 1975 Tidewater silverside 20 °C saltwater; static 96 h LC50 150 nominal Dawson et al., (Menidia beryllina) pH 7.6-7.9; hardness 1975/77 55 mg CaCO3/litre a The authors tested 200 selected chemicals with the golden orfe test under comparable conditions in two different laboratories and found LC50 values of 95 mg/litre (Juhnke) and 472 mg/litre (Lüdemann), respectively, for carbon tetrachloride Rainbow trout (Oncorhynchus mykiss) were exposed to carbon tetrachloride concentrations of between 1 and 80 mg/litre for up to 336 h under semi-static conditions (water was renewed every 48 h). No mortality was observed and no significant changes in enzyme activity were found (Statham et al., 1978). Toxicity data for embryo-larval stages of fish and amphibians are given in Table 13. Carbon tetrachloride is considerably more toxic to the embryo-larval stages of several species of fish and amphibians than it is to the adults (Birge, 1980; Black et al., 1982). The common bullfrog (Rana catesbeiana) was the most susceptible species. At 60 µg/litre the incidence of teratic larvae was 1%, rising to 17% at 7.8 mg/litre. A more striking effect was found in the hatchability of the embryos, which declined from 92% at 60 µg/litre to 23% at 7.8 mg/litre (Birge, 1980). 9.3 Terrestrial toxicity 9.3.1 Earthworms Red earthworms (Eisenia foetida) were exposed to carbon tetrachloride via filter paper in glass vials. An LC50 of 160 µg/cm2 was found (Neuhauser et al., 1985). Table 13. Carbon tetrachloride toxicity to embryo-larval stages of fish and amphibians Organism Test conditions Exposure Parameter Measured Reference period concentration (days) (mg/litre) Fish Rainbow trout 13 °C pH 9.2; hardness flow 27a LC50 1.97 Black et al., 1982 (Oncorhynchus mykiss) 104 mg CaCO3/litre Fathead minnow 20 °C pH 6.4; hardness flow 9b LC50 4.0 Black et al., 1982 (Pimephales promelas) 96 mg CaCO3/litre Amphibians Bullfrog 21 °C pH 8; hardness flow 8b LC50 0.9 Birge, 1980 (Rana catesbeiana) 108 mg CaCO3/litre Pickerel frog 22 °C pH 7.7; hardness flow 8b LC50 2.4 Birge, 1980 (Rana palustris) 104 mg CaCO3/litre Fowler's toad 22 °C pH 7.7; hardness flow 7b LC50 2.8 Birge, 1980 (Bufo fowleri) 104 mg CaCO3/litre European common frog 19 °C pH 7.7; hardness flow 9a LC50 1.2 Black et al., 1982 (Rana temporaria) 96 mg CaCO3/litre Leopard frog 19 °C pH 7.7; hardness flow 9a LC50 1.6 Black et al., 1982 (Rana pipiens) 96 mg CaCO3/litre African clawed toad 19 °C pH 7.7; hardness flow 6a LC50 22.4 Black et al., 1982 (Xenopus laevis) 96 mg CaCO3/litre Northwestern salamander 19 °C pH 7.7; hardness flow 9.5a LC50 1.98 Black et al., 1982 (Ambystoma gracile) 96 mg CaCO3/litre a The organisms were exposed from fertilization until 4 days after hatching b The organisms were exposed from 2-8 h post spawning to 4 days after hatching 10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT 10.1 Evaluation of human health risks 10.1.1 Exposure Carbon tetrachloride can be detected ubiquitously in the environment, mostly in the air due to its volatility and high vapour pressure. Furthermore it is found in foodstuffs and drinking-water. Based on the estimates of mean exposure from various media, as reported in chapter 5, the general population may be exposed via air at a concentration of 0.5-1.0 µg/m3 (retention calculated to be 0.068-0.136 µg/kg body weight per day, assuming 40% retention, ATSDR, 1994), and via drinking-water at levels of 0.1-3 µg/litre (calculated to be 0.003-0.094 µg/kg body weight). Intake via foodstuffs is estimated to be very small (0.13 µg/day) (Yoshida, 1993), but could have been larger in the past in individuals who consumed fumigated or otherwise contaminated foods. These are presumably no longer on the market since this use of carbon tetrachloride has ceased. This means for the general population an estimated maximum daily carbon tetrachloride intake of about 0.23 µg/kg body weight, assuming (IPCS, 1994): - a body weight of 64 kg; - an inhalation volume of 22 m3/day; - a water consumption of 2 litres/day; - a food consumption of 1.536 kg/day. According to estimates made by the ATSDR and in Japan and Germany the general population is considered to be exposed to carbon tetrachloride via ingestion and inhalation leading to an average daily intake of 0.1 to 0.27 µg/kg body weight. Workers involved in the production or use of carbon tetrachloride are likely to be exposed to higher levels than the general population. Based on a national survey conducted from 1981 to 1983, NIOSH estimated that 58 208 workers were potentially exposed to carbon tetrachloride in the USA during that period. Exposure to higher levels of carbon tetrachloride could occur as a result of accidental spillage or near hazardous waste sites contaminated with carbon tetrachloride. 10.1.2 Health effects Acute symptoms after human exposure to carbon tetrachloride are characterized by gastrointestinal and neurological symptoms, such as nausea, vomiting, headache, dizziness and dyspnoea. Liver damage appears after 24 h or more. Kidney damage is evident often only 2 to 3 weeks following the poisoning. Short-term and long-term exposure to low concentrations of carbon tetrachloride can also produce hepatic and renal damage. The toxicity of carbon tetrachloride is associated with the formation of reactive metabolites, the principal enzyme involved being CYP 2E1. The severity of the effects on the liver depends on a number of factors, such as species, susceptibility, route and mode of exposure, diet or co-exposure to other compounds, in particular, ethanol. How these factors affect the CNS and kidney responses is not known. No adequate long-term oral study on laboratory animals, suitable for quantitative health risk evaluation of carbon tetrachloride, was available (section 7.3). In a 12-week oral study on rats (5 days/week), a NOAEL of 1 mg/kg body weight was reported. The LOAEL reported in this study was 10 mg/kg body weight, showing a slight, but significant increase in SDH activity and mild hepatic centrilobular vacuolization (Bruckner et al., 1986). A NOAEL of 1.2 mg/kg body weight was reported in a 90-day oral study on mice (5 days/week). On the basis of hepato toxicity, the LOAEL was 12 mg/kg body weight (Condie et al., 1986). When rats were exposed to carbon tetrachloride by inhalation for approximately 6 months, 5 days/week, 7 h/day, the NOAEL was 32 mg/m3. The LOAEL, based on changes in the liver morphology, was 63 mg/m3 (Adams et al., 1952). In a 90-day study on rats, a NOAEL of 6.1 mg/m3 was found after continuous exposure to carbon tetrachloride (Prendergast, 1967). An exposure level of 32 mg/m3 (the lowest concentration studied) in a 2-year inhalation study on rats caused marginal effects (Japan Bioassay Research Centre, 1998). In experiments with mice and rats, carbon tetrachloride proved to be capable of inducing hepatomas and hepatocellular carcinomas. The doses inducing hepatic tumours were higher than those inducing cell toxicity. It is likely that the carcinogenicity of carbon tetrachloride is secondary to its hepatotoxic effects. There is little evidence to suggest that carbon tetrachloride is genotoxic. Based on the weight of evidence it can be concluded that the hepatic tumours are induced by an indirect mechanism and that a tolerable daily intake or concentration can be derived. The available data suggest that carbon tetrachloride can induce embryotoxic and embryolethal effects, but only at doses that are maternally toxic. Carbon tetrachloride is not teratogenic in rats and mice. 10.1.3 Approaches to health risk assessment There is little evidence to suggest that carbon tetrachloride is genotoxic. A quantitative risk assessment for threshold effects (IPCS, 1994), which includes the effects of non-genotoxic carcinogens, was therefore adopted. 10.1.3.1 Calculation of a TDI based on oral data Calculations of tolerable daily intake (TDI) were based on the 12-week oral study on rats (Bruckner et al., 1986) and the 90-day oral study on mice (Condie et al., 1986), where NOAEL values of 1 mg/kg body weight and 1.2 mg/kg body weight were identified, respectively. a) Rat 1 mg/kg body weight × (5/7) TDI = 500 = 1.42 µg/kg body weight where: * 1 mg/kg body weight is the NOAEL in the 12-week oral study on rats * (5/7) is the conversion from 5 days/week of dosing to 7 days/week * 500 is the uncertainty factor (10 for interspecies variation, 10 for intraspecies variation and 10 for a less-than-long-term study; a modifying factor of 0.5 was applied because this was a bolus study). b) Mouse TDI = 1.2 mg/kg body weight × (5/7) = 1.72 µg/kg body weight 500 where: * 1.2 mg/kg body weight is the NOAEL in the 90-day oral study on mice * (5/7) is the conversion from 5 days/week of dosing to 7 days/week * 500 is the uncertainty factor (10 for interspecies variation, 10 for intraspecies variation and 10 for a less-than-long-term study; a modifying factor of 0.5 was applied because this was a bolus study). 10.1.3.2 Calculation of a tolerable concentration based on inhalation data Calculations of tolerable concentrations (TC) were based on: (a) the 90-day study of Prendegast (1967) where a NOAEL of 6.1 mg/m3 was identified for continuous exposure; (b) the 6-month study of Adams et al. (1952) where a NOAEL of 32 mg/m3 was found; and (c) the 2-year inhalation study by Japan Bioassay Research Centre (1998) where a LOAEL with a marginal adverse effect was 32 mg/m3. a) TC = 6.1 mg/m3 = 6.1 µg/m3 1000 where: * 6.1 mg/m3 is the NOAEL in the 90-day inhalation study on rats * 1000 is the uncertainty factor (10 for interspecies variation, 10 for intraspecies variation; and 10 for a less-than-long-term study) b) TC = 32 mg/m3 × (7/24) × (5/7) = 6.7 µg/m3 1000 where: * 32 mg/m3 is the NOAEL in the 6-month inhalation study on rats * (7/24) × (5/7) is the conversion from 7 h/day and 5 days/week to continuous exposure. * 1000 is the uncertainty factor (10 for interspecies variation, 10 for intraspecies variation and 10 for a less-than-long-term study). c) TC = 32 mg/m3 × (6/24) × (5/7) = 11.4 µg/m3 500 * 32 mg/m3 is the LOAEL in the 2-year inhalation study on rats * (6/24) × (5/7) is the conversion from 6 h/day and 5 days/week to continuous exposure * 500 is the uncertainty factor (10 for interspecies variation, 10 for intraspecies variation and 5 for use of a marginal effect rather than a no-observed-effect level). It is noted that the end-point on which the LOAEL is based in the recent 2-year inhalation bioassay on rats (i.e. proteinuria) was not investigated in the studies of Adams et al. (1952) and Prendergast (1967). 10.1.3.3 Summary of the results of risk assessment TDI Oral studies 1.42 µg/kg body weight 1.72 µg/kg body weight TC TDI (calculated from the TC) Inhalation studies 6.1 µg/m3 0.85 µg/kg body weight 6.7 µg/m3 0.92 µg/kg body weight 11.4 µg/m3 1.56 µg/kg body weight 10.1.3.4 Conclusions based on exposure and health risk assessment On the basis of exposure data presented in chapter 5, an approximate upper-limit estimate of the daily intake of carbon tetrachloride for long-term exposure of the general population can be made for prevailing normal exposure and for a "worst case scenario". The following concentration ranges are considered: * ambient air, 0.5-1.0 µg/m3 (worst case 6 µg/m3 ); * indoor air in dwellings, 0.6-2.0 µg/m3 (worst case 9 µg/m3); * drinking-water, 0.0002-2.3 µg/litre (worst case 16 µg/litre; the abnormally high value of 39.5 µg/litre reported in Spain was not considered); * foodstuffs (particularly table-ready foods) 0.1-6.0 µg/kg (worst case 31 µg/kg). The daily estimates are summarized in Table 14. As is seen from Table 14, the upper limit of human daily intake under prevailing conditions is estimated to be 0.2 µg/kg body weight, well below the lowest tolerable daily intake (0.85 µg/kg body weight) presented in section 10.1.3.3. This leads to the conclusion that the currently prevailing exposure of the general population to carbon tetrachloride from all sources is unlikely to cause excessive intake of the chemical. The hypothetical worst case scenario of exposure may bring about a daily intake of 2.5 µg/kg body weight, more than ten times the daily intake under currently prevailing conditions, and three times the lowest tolerable intake. This would indicate a need for caution. However, conditions similar to those of the worst case scenario are very unlikely to occur in future, due to the expected fall in the use of carbon tetrachloride as a consequence of the Amended Montreal Protocol. 10.2 Evaluation of effects on the environment Carbon tetrachloride may be released into the environment during its production, storage, transport and use. Owing to its volatility, most of the substance emitted into the environment can be found in the air. The residence time of carbon tetrachloride in the atmosphere is long, and it can therefore be transported over long distances from the point of emission. The main degradation site of carbon tetrachloride is the stratosphere where it is photolytically degraded by UV radiation. Carbon tetrachloride contributes both to ozone depletion and to global warming. Carbon tetrachloride is, in general, resistant to aerobic biode gradation, but less so to anaerobic. Acclimation increases biodegradation rates. Although the octanol-water partition coefficient indicates a moderate potential for bioaccumulation, the short lifetime in tissues reduces this tendency. Table 14. Daily intake of carbon tetrachloride for long-term exposure of the general population Prevailing upper limits Worst case scenario Concentration Daily intake Concentration Daily intake Air 1 µg/m3 1 µg/m3 × 22 m3 × 0.4a = 8.8 µg 9 µg/m3 9 µg/m3 × 22 m3 × 0.4a = 79.2 µg Water 0.1 µg/litre 0.1 µg/litre × 2 litres = 0.2 µg 16 µg/litre 16 µg/litres × 2 litres = 32 µg Food 3 µg/kg 3 µg/kg × 1.5 kg = 4.5 µg 31 µg/kg 31 µg/kg × 1.5 kg = 46.5 µg Total daily intake 13.5 µg 157 µg Total daily intake per kg 0.2 µg 2.5 µg body weight a The value of 0.4 derives from the 40% retention reported by ATSDR (1994) Carbon tetrachloride is of low toxicity to the algae and microorganisms tested; the lowest toxic concentration of carbon tetrachloride reported was for methanogenic bacteria (IC50 = 6.4 mg/litre). In aquatic invertebrates, LC50 values range from 28 mg/litre to over 770 mg/litre. The lowest acutely toxic concentration found for freshwater fish was an LC50 of 13 mg/litre for the golden orfe (Leuciscus idus melanotus). The lowest LC50 for a marine species was 50 mg/litre for the dab (Limanda limanda). Carbon tetrachloride is toxic to embryo-larval stages of fish and of amphibians. The most sensitive species tested was the common bullfrog (Rana catesbeiana) with an LC50 of 0.92 mg/litre for the period from fertilization to 4 days post-hatching. Comparing the LC50 value for the most sensitive aquatic species (0.9 mg/litre) with typical levels of carbon tetrachloride in water (< 1.0 µg/litre) gives a ratio of > 900. Therefore, the general risk to aquatic organisms is low. However, carbon tetrachloride may present a risk to embryo-larval stages of aquatic organisms at, or near, sites of industrial discharges or spills, where much higher levels have been reported. 11. FURTHER RESEARCH a) Physiologically based pharmacokinetic models for carbon tetra chloride should be further developed in order to improve their use in defining target organ doses in human exposure conditions. b) Since there is a lack of epidemiological data in those countries where carbon tetrachloride is still used, epidemiological studies of exposed populations would be useful. c) No further research topics are recommended in view of the phase-out of the production and use of carbon tetrachloride as a result of the Montreal Protocol on Substances that Deplete the Ozone Layer. 12. PREVIOUS EVALUATION BY INTERNATIONAL BODIES A drinking-water guideline value of 2 µg/litre has been recommended for carbon tetrachloride by the World Health Organization (WHO, 1993), based on a risk assessment approach for non-genotoxic carcinogens. A NOAEL of 1 mg/kg body weight and an uncertainty factor of 1000 were adopted for calculations. 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Hepatotoxicity -- the adverse effect of drugs and other chemicals on the liver. New York, Appleton-Century-Crofts, pp 297-302. Zoeteman BCJ, Harmsen K, Linders JBHJ, Morra CFH, & Slooff W (1980) Persistent organic pollutants in river water and ground water of the Netherlands. Chemosphere, 9: 231-249. RÉSUMÉ Le tétrachlorure de carbone est un liquide limpide, incolore et volatil qui dégage une odeur douceâtre caractéristique. Il est miscible à la plupart des solvants aliphatiques et il est lui-même un solvant. Il est peu soluble dans l'eau. Le tétrachlorure de carbone n'est pas inflammable et il est stable à l'air et à la lumière. En se décomposant, il peut donner naissance à du phosgène, à du dioxyde de carbone et à de l'acide chlorhydrique. La présence de tétrachlorure de carbone dans l'environnement est vraisemblablement presque exclusivement d'origine humaine. La majeure partie du tétrachlorure de carbone produit sert à la préparation de chlorofluorocarbures (CFC) et autres hydrocarbures chlorés. En 1987, la production mondiale de tétrachlorure de carbone a été de 960 000 tonnes. Toutefois, depuis que le Protocole de Montréal relatif aux substances qui appauvrissent la couche d'ozone (1987) et ses amendements de 1990 et de 1992, a établi un calendrier pour l'abandon progressif de la production et de la consommation de tétrachlorure de carbone, la production a reculé et continuera à le faire. On a mis au point plusieurs méthodes suffisamment sensibles et précises pour la recherche et le dosage du tétrachlorure de carbone dans l'air, l'eau et les milieux biologiques. La plupart d'entre elles sont basées, soit sur l'injection directe de l'échantillon dans un chromatographe en phase gazeuse, soit sur une adsorption sur charbon actif, suivie d'une désorption ou d'une évaporation puis d'une détection par chromatographie en phase gazeuse. La presque totalité du tétrachlorure de carbone libéré dans l'environnement finira tôt ou tard dans l'atmosphère en raison de sa grande volatilité. Comme sa durée de séjour dans l'atmosphère est longue, il est largement distribué. Au cours de la période 1980-1990, la concentration atmosphérique du tétrachlorure de carbone était de l'ordre de 0,5-1,0 µg/m3. Les estimations de sa durée de séjour atmosphériques sont variables mais on pense que la valeur la plus raisonnable est de 45 à 50 ans. Le tétrachlorure de carbone contribue à la fois à la destruction de la couche d'ozone et au réchauffement du climat. Il est généralement résistant à la biodégradation aérobie, mais moins à la biodégradation anaérobie. La vitesse de biodégradation peut s'accroître par suite d'un processus d'acclimatation. Bien que le coefficient de partage octanol-eau indique un potentiel de bioaccumulation moyen, cette tendance est réduite par la brièveté de la demi-vie tissulaire. Dans l'eau, on fait état de concentrations inférieures à 10 ng/litre pour les océans et de moins de 1 µg/litre pour les eaux douces, mais de valeurs beaucoup plus fortes à proximité des sites de décharge. On a mesuré des valeurs allant jusqu'à 60 µg/kg dans des denrées alimentaires qui avaient été traitées avec du tétrachlorure de carbone, mais cette pratique a cessé. C'est essentiellement par l'intermédiaire de l'air que la population dans son ensemble est exposée au tétrachlorure de carbone. Si l'on se base sur les concentrations relevées dans l'air ambiant, les denrées alimentaires et l'eau de boisson, on peut estimer à environ 1 µg/kg de poids corporel la dose de tétrachlorure de carbone absorbée. Cette estimation est probablement un peu forte à l'heure actuelle, du fait qu'on n'utilise plus de tétrachlorure de carbone pour la fumigation des céréales et que les concentrations annoncées pour les aliments et utilisées pour ce calcul, correspondaient tout particulièrement aux matières grasses et aux produits à base de céréales. D'autres sources font état de valeurs comprises entre 0,1 et 0,27 µg/kg p.c. pour l'exposition journalière de la population générale. Une exposition plus importante au tétrachlorure de carbone peut se produire sur le lieu de travail en cas de déversement accidentel. Le tétrachlorure de carbone est bien résorbé au niveau des voies digestives et respiratoires de l'Homme et des animaux. Il peut également y avoir absorption percutanée du produit liquide, mais dans le cas de la vapeur, cette absorption est lente. Le tétrachlorure de carbone se répartit dans tout l'organisme, mais se concentre surtout dans le foie, le cerveau, les reins, les muscles, les tissus adipeux et le sang. Le composé initial s'élimine principalement dans l'air expiré et, en proportion minime, dans l'urine et les matières fécales. La première étape de la biotransformation du tétrachlorure de carbone est catalysée par les enzymes du cytochrome P-450 et aboutit à la formation d'un radical réactif, le radical trichlorométhyl. La voie de biotransformation la plus importante conduisant à l'élimination de ce radical consiste dans une oxydation en un radical encore plus réactif, le radical trichlorométhylperoxyl, qui peut réagir à son tour pour donner du phosgène. Le phosgène peut être détoxifié par réaction avec l'eau pour donner du dioxyde de carbone ou par réaction avec le glutathion ou la cystéine. En anaérobiose, il y a formation de chloroforme et de dichlorocarbène. Les intermédiaires métaboliques du tétrachlorure de carbone peuvent former des liaisons covalentes avec des macromolécules et provoquer la peroxydation des lipides. L'action toxique du tétrachlorure de carbone a pour organes cibles le foie et le rein. La gravité des effets hépatiques dépend d'un certain nombre de facteurs tels que la sensibilité de l'espèce, la voie et le mode d'exposition, le régime alimentaire et une exposition concomitante éventuelle à d'autres substances, notamment l'éthanol. En outre, il semble qu'un traitement préalable par divers composés, comme le phénobarbital ou la vitamine A, accroisse l'hépatotoxicité du tétrachlorure de carbone, alors que d'autres, au contraire, la réduisent, comme la vitamine E. Après application sur l'épiderme de lapins et de cobayes, on a constaté une irritation modérée et une réaction également modérée a été observée après instillation dans l'oeil du lapin. La DL50 la plus faible (2391 mg/kg p.c. sur une période de 14 jours) a été obtenue à l'issue d'une étude sur des chiens qui recevaient le composé par voie intrapéritonéale. Chez le rat, on a obtenu des valeurs comprises entre 2821 et 10 054 mg/kg p.c. Lors d'une étude de 12 semaines sur des rats comportant l'administration du produit par la voie buccale 5 jours par semaine, on a obtenu une dose sans effet nocif observable (NOAEL) de 1 mg/kg p.c. La dose la plus faible produisant un effet nocif observable (LOAEL) était de 10 mg/kg p.c., les effets observés étant une augmentation légère, mais significative, de l'activité de la sorbitol-déshydrogénase et une vacuolisation modérée des hépatocytes centrilobulaires. Une NOAEL similaire de 1,2 mg/kg p.c. (5 jours par semaine) a été obtenue chez des souris lors d'une étude de 90 jours avec administration buccale; dans la même étude, la LOAEL (hépatotoxicité) a été trouvée égale à 12 mg/kg p.c. En exposant des rats à du tétrachlorure de carbone par la voie respiratoire pendant environ 6 mois, 5 jours par semaine, 7 heures par jour, on a obtenu une NOAEL de 32 mg/m3 LOAEL, basée sur des anomalies de la morphologie hépatique, a été trouvée égale à 63 mg/m3. Dans une autre étude de 90 jours sur des rats, on a obtenu une NOAEL de 6,1 mg/m3 après exposition continue à du tétrachlorure de carbone. Lors d'une étude d'inhalation de 2 ans portant également sur des rats, la concentration la plus faible étudiée (32 mg/m3) a provoqué des effets marginaux. La seule étude toxicologique à long terme dont on dispose a consisté à faire ingérer du tétrachlorure de carbone à des rats pendant 2 ans, aux doses respectives de 0, 80 et 200 mg de produit par kg de nourriture. En raison d'une affection respiratoire chronique qui a touché tous les animaux à partir du 14ème mois et a provoqué une augmentation de la mortalité, les résultats de l'autopsie effectuée au bout de deux ans ne peuvent pas être utilisés pour une évaluation du risque sanitaire. Les études d'inhalation effectuées sur des rats et des souris ont permis de conclure que le tétrachlorure de carbone peut avoir des effets embryotoxiques pouvant aller jusqu'à la mort de l'embryon. Toutefois ces effets ne se manifestent qu'aux doses toxiques pour les femelles gravides. Le tétrachlorure de carbone n'est pas tératogène. De nombreuses études de génotoxicité ont été effectuées sur le tétrachlorure de carbone. Sur la base des données disponibles, on peut considérer que ce composé n'est pas génotoxique. Le tétrachlorure de carbone provoque l'apparition d'hépatomes et de carcinomes hépatocellulaires chez le rat et la souris. Les doses qui entraînent la formation de tumeurs hépatiques sont supérieures aux doses cytotoxiques. Chez l'Homme, les manifestations aiguës qui surviennent après exposition au tétrachlorure de carbone sont indépendants du mode d'absorption et se caractérisent par des symptômes gastrointestinaux et neurologiques tels que nausées, vomissements, céphalées, étourdissements, dyspnée qui finissent par aboutir à la mort. Des lésions hépatiques apparaissent au bout de 24 h ou davantage. Les lésions rénales ne se manifestent souvent que 2 ou 3 semaines après l'intoxication. Les études épidémiologiques n'ont pas permis d'établir l'existence d'une association entre l'exposition au tétrachlorure de carbone et un accroissement du risque de mortalité, de cancer ou d'affection hépatique. Certains travaux incitent à penser qu'il pourrait y avoir augmentation du risque de lymphome non Hodgkinien et,selon une étude particulière, du risque de mortalité et de cirrhose du foie. Il faut cependant préciser que toutes ces études ne portaient pas spécifiquement sur l'exposition au tétrachlorure de carbone et qu'il n'y avait pas, en tout cas, de corrélations statistiques fortes. En général, le tétrachlorure de carbone se révèle peu toxique pour les bactéries, les protozoaires et les algues. La concentration toxique la plus faible a été mesurée chez les bactéries méthanogènes (CI50 = 6,4 mg/litre). Pour les invertébrés aquatiques, les valeurs de la Cl50 aiguë varient de 28 à > 770 mg/litre. Dans le cas des poissons d'eau douce, c'est chez l'orfe (Leuciscus idus melanotus) que l'on a trouvé la valeur la plus faible de la CL50 aiguë, avec 13 mg/litre. Chez les espèces marines, c'est la limande (Limanda limanda) qui présente la plus faible valeur de la CL50, avec 50 mg/litre. Chez les poissons et les amphibiens, le tétrachlorure de carbone se révèle plus toxique pour les stades embryo-larvaires que pour les adultes. La grenouille-taureau commune (Rana catesbeiara) est l'espèce la plus sensible, avec une CL50 de 0,92 mg/litre (de la fécondation à 4 jours après l'éclosion). Les données disponibles montrent que le mécanisme de formation des tumeurs hépatiques n'est pas de nature génotoxique et il est donc admissible de fixer une dose journalière tolérable par ingestion (TDI) et une concentration journalière tolérable dans l'air (TC). En s'appuyant sur l'étude de Bruckner et al. (1986) qui ont déterminé une dose sans effet nocif observable (NOAEL) de 1 mg/kg p.c. lors d'une étude de 12 semaines sur des rats auxquels on avait fait ingérer du tétrachlorure de carbone, et en utilisant un facteur de conversion de 5/7 pour la dose journalière et un coefficient d'incertitude de 500 (100 pour les variations inter- et intraspécifiques, 10 pour la durée de l'étude plus un facteur de 0,5 pour tenir compte du fait que l'on avait utilisé des boulettes), on parvient à une TDI de 1,42 µg/kg de poids corporel. En s'appuyant sur une étude d'inhalation de 90 jours pratiquée sur des rats (Prendergast et al., 1967), qui a permis d'aboutir à une NOAEL de 6,1 mg/m3 on a calculé une TC de 6,1 g/m3 en utilisant les coefficients de 7/24 et de 5/7 pour passer à une exposition en continu et un coefficient d'incertitude de 1000 (100 pour les variations inter- et intraspécifiques et 10 pour la durée de l'étude). Cette TC correspond à une TDI de 0,85 µg/kg de poids corporel. En comparant la limite supérieure de la dose journalière absorbée par l'Homme, c'est-à-dire 0,2 µg/kg p.c., à la valeur la plus faible de la TDI, soit 0,85 µg/kg p.c., on peut conclure que l'exposition actuelle de la population générale au tétrachlorure de carbone de toutes origines a peu de chances de causer une absorption excessive de ce composé. En règle générale, les organismes aquatiques ne courent guère de risque imputable au tétrachlorure de carbone. Toutefois, il peut y avoir un danger pour les stades embryo-larvaires sur les sites de décharge ou de déversement de produits industriels ou à proximité de ces sites. RESUMEN El tetracloruro de carbono es un líquido volátil transparente, incoloro, con un olor dulce característico. Es miscible con la mayor parte de los disolventes alifáticos y tiene a su vez propiedades disolventes. La solubilidad en agua es baja. El tetracloruro de carbono no es inflamable y se mantiene estable en presencia del aire y de la luz. Su descomposición puede producir fosgeno, anhídrido carbónico y ácido clorhídrico. La fuente de tetracloruro de carbono en el medio ambiente con toda probabilidad tiene un origen casi exclusivamente antropogénico. La mayor parte del tetracloruro de carbono producido se emplea en la fabricación de clorofluorocarbonos y otros hidrocarburos clorados. La producción mundial ascendió en 1987 a 960 000 toneladas. Sin embargo, desde que en el Protocolo de Montreal relativo a las sustancias que agotan la capa de ozono (1987) y en sus enmiendas (1990 y 1992) se estableció un calendario para la reducción progresiva de la producción y consumo del tetracloruro de carbono, su fabricación ha disminuido y seguirá descendiendo. Se han elaborado varios métodos analíticos suficientemente sensibles y precisos para la determinación del tetracloruro de carbono en muestras de aire, agua y biológicas. La mayoría de estos métodos se basan en la inyección directa en un cromatógrafo de gases o la adsorción en carbón activado, seguida de la desorción o la evaporación y la posterior detección por cromatografía de gases. Casi todo el tetracloruro de carbono que se libera en el medio ambiente estará en último término presente en la atmósfera, debido a su volatilidad. Dado que su tiempo de permanencia en la atmósfera es prolongado, tiene una distribución muy amplia. Durante el período 1980-1990, las concentraciones atmosféricas fueron de alrededor de 0,5-1 µg/m3. Las estimaciones de su permanencia en la atmósfera son variables, pero se aceptan como los valores más razonables los 45-50 años. El tetracloruro de carbono contribuye tanto a la reducción del ozono como al calentamiento mundial. Es en general resistente a la biodegradación aerobia, pero menos a la anaerobia. La aclimatación aumenta la velocidad de biodegradación. Aunque el coeficiente de reparto octanol/agua indica un potencial de bioacumulación moderado, el breve período de permanencia en los tejidos reduce esta tendencia. En el agua, se han notificado concentraciones inferiores a 10 ng/litro en los océanos y generalmente inferiores a 1 µg/litro en el agua dulce, pero con valores mucho más elevados cerca de los lugares de liberación. Se han registrado concentraciones de hasta 60 µg/kg en alimentos elaborados con tetracloruro de carbono, pero esta práctica se ha suprimido. La población general está expuesta al tetracloruro de carbono fundamentalmente a través del aire. A partir de las concentraciones notificadas en el aire ambiente, los productos alimenticios y el agua potable, se ha estimado que la ingesta diaria de tetracloruro de carbono es de alrededor de 1 µg/kg de peso corporal. En la actualidad esta estimación es probablemente demasiado elevada, porque se ha suprimido el uso del tetracloruro de carbono como fumigante de los cereales y los valores notificados para los alimentos y utilizados en el cálculo fueron fundamentalmente los obtenidos en alimentos a base de grasas y cereales. En otras partes se han descrito valores de 0,1 a 0,27 µg/kg de peso corporal para la exposición diaria de la población general. Se puede producir una exposición a concentraciones más elevadas de tetracloruro de carbono en el lugar de trabajo debido a un derrame accidental. El tetracloruro de carbono se absorbe bien de los tractos gastrointestinal y respiratorio en los animales y en el ser humano. Es posible la absorción cutánea de tetracloruro de carbono líquido, pero la absorción cutánea del vapor es lenta. El tetracloruro de carbono se distribuye por todo el organismo, alcanzando las concentraciones más altas en el hígado, el cerebro, el riñón, los músculos, la grasa y la sangre. El compuesto original se elimina fundamentalmente en el aire exhalado, mientras que se excretan cantidades mínimas en las heces y la orina. En la biotransformación del tetracloruro de carbono, el primer paso es la catálisis por enzimas del citocromo P-450 para formar un radical reactivo, el triclorometilo. La biotransformación oxidativa es la ruta más importante en la eliminación del radical, produciendo otro radical incluso más reactivo, el triclorometilperoxilo, que puede reaccionar de nuevo para formar fosgeno. Éste se puede destoxificar mediante la reacción con el agua para producir anhídrido carbónico, o con el glutatión o la cisteína. En condiciones anaerobias se forma cloroformo y diclorocarbeno. Se produce la unión a macromoléculas mediante enlaces covalentes y la peroxidación de lípidos a través de intermediarios metabólicos del tetracloruro de carbono. El hígado y el riñón son los órganos destinatarios de la toxicidad de este compuesto. La gravedad de los efectos hepáticos depende de diversos factores, como la susceptibilidad de la especie, la ruta y el modo de exposición, la alimentación o la exposición simultánea a otros compuestos, en particular el etanol. Además, parece que el tratamiento previo con diversos compuestos, como el fenobarbital y la vitamina A, aumenta la hepatotoxicidad, mientras que otros compuestos, como la vitamina E, reducen la acción hepatotóxica del tetracloruro de carbono. Tras la aplicación cutánea se ha observado una irritación moderada en la piel de conejos y cobayas, y se puso de manifiesto una reacción leve después de aplicar el compuesto en el ojo del conejo. La DL50 más baja, de 2391 mg/kg de peso corporal (período de 14 días), se notificó en un estudio realizado con perros mediante administración intraperitoneal. En ratas, los valores de la DL50 oscilaron entre 2821 y 10 054 mg/kg de peso corporal. En un estudio de administración por vía oral a ratas durante 12 semanas (5 días/semana), la concentración sin efectos adversos observados (NOAEL) fue de 1 mg/kg de peso corporal. La concentración más baja sin efectos adversos observados (LOAEL) notificada en este estudio fue de 10 mg/kg de peso corporal, registrándose un aumento ligero, pero significativo, de la actividad de la sorbitol deshidrogenasa y una ligera vacuolación centrilobular hepática. En un estudio de 90 días por vía oral realizado en ratones, se encontró una NOAEL semejante de 1,2 mg/kg de peso corporal (5 días/semana) con una LOAEL de 12 mg/kg de peso corporal cuando se produjo hepatotoxicidad. Cuando se expusieron ratas a tetracloruro de carbono mediante inhalación durante unos seis meses, cinco días a la semana, siete horas diarias, se notificó una NOAEL de 32 mg/m3. Se informó de una LOAEL, basada en cambios en la morfología del hígado, de 63 mg/m3. En otro estudio de 90 días en ratas se encontró una NOAEL de 6,1 mg/m3 tras una exposición continua a tetracloruro de carbono. El nivel de exposición más bajo, de 32 mg/m3 (la concentración más baja estudiada), en un estudio de inhalación en ratas de dos año produjo efectos marginales. El único estudio de toxicidad prolongada por vía oral fue uno de dos años realizado en ratas expuestas a 0, 80 y 200 mg de tetracloruro de carbono/kg de alimentos. Debido a una enfermedad respiratoria crónica que contrajeron todos los animales a partir del 14° mes y que provocó un aumento de la mortalidad, los resultados notificados de la necropsia a los dos años son insuficientes para evaluar el riesgo para la salud. Se llegó a la conclusión de que el tetracloruro de carbono puede inducir efectos embriotóxicos y embrioletales, pero sólo a dosis tóxicas para la madre, como se observó en los estudios de inhalación realizados en ratas y ratones. El tetracloruro de carbono no es teratogénico. Se han realizado numerosas valoraciones de la genotoxicidad del tetracloruro de carbono. Tomando como base los datos disponibles, se puede considerar que este producto es un compuesto no genotóxico. El tetracloruro de carbono induce la formación de hepatomas y carcinomas hepatocelulares en ratones y ratas. Las dosis que inducen la formación de tumores hepáticos son más elevadas que las que producen toxicidad celular. En el ser humano, los síntomas agudos tras la exposición a tetracloruro de carbono son independientes de la ruta de ingestión y se caracterizan por síntomas gastrointestinales y neurológicos, como náuseas, vómitos, dolor de cabeza, desvanecimiento, disnea y la muerte. Después de las 24 horas o más aparecen lesiones hepáticas. Son evidentes los trastornos renales con frecuencia sólo dos o tres semanas después de la intoxicación. Los estudios epidemiológicos no han establecido una asociación entre la exposición al tetracloruro de carbono y el aumento del riesgo de mortalidad, neoplasia o enfermedad hepática. Algunos estudios han indicado una asociación con un aumento del riesgo de linfoma no-Hodgkin y, en un estudio, con la mortalidad y la cirrosis hepática. Sin embargo, no en todos estos estudios se señaló la exposición específica al tetracloruro de carbono y las asociaciones estadísticas no fueron convincentes. En general, el tetracloruro de carbono parece tener una toxicidad baja para las bacterias, los protozoos y las algas; la concentración tóxica más baja notificada para las bacterias metanogénicas correspondió a una CI50 de 6,4 mg/litro. Para los invertebrados acuáticos, los valores de la CL50 aguda fueron de 28 a >770 mg/litro. En los peces de agua dulce el valor más bajos de la CL50 aguda, de 13 g/litro, se encontró en el cachuelo dorado (Leuciscus idus melanotus), y para las especies marinas se notificó un valor de la CL50 de 50 mg/litro para la limanda (Limanda limanda). El tetracloruro de carbono parece ser más tóxico para las fases embrionaria y larvaria de los peces y anfibios que para los adultos. La rana toro común (Rana catesbeiara) es la especie más susceptible, con una CL50 de 0,92 mg/litro (desde la fecundación hasta los cuatro días después de la eclosión). Los datos disponibles indican que la inducción de tumores hepáticos se debe a un mecanismo no genotóxico, por lo que parece aceptable el establecimiento de una ingesta diaria tolerable (IDT) y de una concentración diaria tolerable (CDT) en el aire para el tetracloruro de carbono. Tomando como base el estudio de Bruckner et al. (1986), en el cual se observó una NOAEL de 1 mg/kg de peso corporal en un estudio de 12 semanas con administración por vía oral a ratas, e incorporando un factor de conversión de 5/7 para la dosificación diaria y aplicando un factor de incertidumbre de 500 (100 por la variación interespecífica e intraespecífica, 10 por la duración del estudio y un factor de modificación de 0,5 porque se trataba de un estudio de bolo), se obtiene una IDT de 1,42 µg/kg de peso corporal. Al comparar el límite superior estimado de la ingesta diaria predominante de 0,2 µg/kg de peso corporal con el valor más bajo de la IDT (0,85 µg/kg de peso corporal), se puede llegar a la conclusión de que la exposición predominante en la actualidad de la población general al tetracloruro de carbono procedente de todas las fuentes es poco probable que dé lugar a una ingesta excesiva de la sustancia química. En general, el riesgo del tetracloruro de carbono para los organismos acuáticos es bajo. Sin embargo, puede presentar un riesgo para las fases embrionaria y larvaria en lugares de vertidos o escapes industriales o en zonas próximas a ellos.
See Also: Carbon tetrachloride (CHEMINFO) Carbon tetrachloride (IARC Summary & Evaluation, Volume 71, 1999) Carbon tetrachloride (ICSC)