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    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),
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    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

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    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
                        2.4.3.1   Blood and tissues
                        2.4.3.2   Urine
                        2.4.3.3   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
                        3.2.1.1   Direct production and procedures
                        3.2.1.2   Indirect production
                        3.2.1.3   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
                        4.2.1.1   Photodegradation
                        4.2.1.2   Photolysis
                        4.2.1.3   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
                        6.1.1.1   Oral
                        6.1.1.2   Dermal
                        6.1.1.3   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
                        6.3.1.1   Dermal
                        6.3.1.2   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
                        7.1.2.1   Oral exposure
                        7.1.2.2   Inhalation exposure
                        7.1.2.3   Subcutaneous and intraperitoneal        
                                  exposure
                        7.1.2.4   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
                        7.5.2.1   Oral exposure
                        7.5.2.2   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
    

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                                 *     *     *

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

         This publication was made possible by grant number 5 U01
    ES02617-15 from the National Institute of Environmental Health
    Sciences, National Institutes of Health, USA, and by financial support
    from the European Commission.

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    FIGURE 1

    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:

    CHEMICAL STRUCTURE 1

    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

    2.4.3.1  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. 

    2.4.3.2  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).

    2.4.3.3  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

    3.2.1.1  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).

    3.2.1.2  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
                                                                            
    
    3.2.1.3  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

    4.2.1.1  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.

    4.2.1.2  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.

    4.2.1.3  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.

    6.1.1.1  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.

    6.1.1.2  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.

    6.1.1.3  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 6.1.1.1). 

         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 6.1.1.3) 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).

    FIGURE 2


         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

    6.3.1.1  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.


    6.3.1.2  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

    7.1.2.1  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.

    7.1.2.2  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.

    7.1.2.3  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.

    7.1.2.4  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
    7.1.2.4).

         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.

    7.5.2.1  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. 

    7.5.2.2  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 6.1.1.1), 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 7.5.2.1.

         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 7.1.2.3) 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.

         The International Agency for Research on Cancer evaluated carbon
    tetrachloride in 1978 (IARC, 1979), and re-evaluated it in 1987 (IARC,
    1987) and 1998 (IARC, in press). The conclusions from the most recent
    evaluation were that there is inadequate evidence for carcinogenicity
    of carbon tetrachloride in humans but sufficient evidence for its
    carcinogenicity in experimental animals. The overall evaluation was
    that carbon tetrachloride is possibly carcinogenic to humans (Group
    2B).

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    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:
       Toxicological Abbreviations
       Carbon Tetrachloride (HSG 108, 1998)
       Carbon tetrachloride (ICSC)
       Carbon tetrachloride (FAO Meeting Report PL/1965/10/2)
       Carbon tetrachloride (FAO/PL:1967/M/11/1)
       Carbon tetrachloride (FAO/PL:1968/M/9/1)
       Carbon tetrachloride (WHO Pesticide Residues Series 1)
       Carbon tetrachloride (Pesticide residues in food: 1979 evaluations)
       Carbon Tetrachloride (IARC Summary & Evaluation, Volume 71, 1999)