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   INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY


    ENVIRONMENTAL HEALTH CRITERIA 156





    HEXACHLOROBUTADIENE






    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 Dr. T. Vermeire,
    National Institute of Public Health and
    Environmental Protection, Bilthoven,
    The Netherlands

    Published under the joint sponsorship of
    the United Nations Environment Programme,
    the International Labour Organisation,
    and the World Health Organization

    World Health Orgnization
    Geneva, 1994

          The International Programme on Chemical Safety (IPCS) is a joint
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    Labour Organisation, and the World Health Organization.  The main
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    carried out by the IPCS include the development of know-how for coping
    with chemical accidents, coordination of laboratory testing and
    epidemiological studies, and promotion of research on the mechanisms
    of the biological action of chemicals.

    WHO Library Cataloguing in Publication Data

    Hexachlorobutadiene.

         (Environmental health criteria: 156)

          1. Butadienes - toxicity      2. Environmental exposure
          I.Series

          ISBN 92 4 157126 X         (NLM Classification QV 305.H7)
          ISSN 0250-863X

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    CONTENTS

    ENVIRONMENTAL HEALTH CRITERIA FOR HEXACHLOROBUTADIENE

    1. SUMMARY

         1.1. Identity, physical and chemical properties,
               analytical methods
         1.2. Sources of human and environmental exposure
         1.3. Environmental transport, distribution and
               transformation
         1.4. Environmental levels and human exposure
         1.5. Kinetics and metabolism
         1.6. Effects on organisms in the environment
         1.7. Effects on experimental animals and
                in vitro test systems
               1.7.1. General toxicity
               1.7.2. Reproduction, embryotoxicity and
                       teratogenicity
               1.7.3. Genotoxicity and carcinogenicity
               1.7.4. Mechanisms of toxicity
         1.8. Effects on humans
         1.9. Evaluation of human health risks and
               effects on the environment
               1.9.1. Evaluation of human health risks
               1.9.2. Evaluation of effects on the
                       environment

    2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS

         2.1. Identity
         2.2. Physical and chemical properties
         2.3. Conversion factors
         2.4. Analytical methods

    3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

         3.1. Natural occurrence
         3.2. Anthropogenic sources
               3.2.1. Production levels and processes
               3.2.2. Uses
               3.2.3. Waste disposal

    4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION

         4.1. Transport and distribution between media
         4.2. Abiotic degradation
               4.2.1. Photolysis
               4.2.2. Photooxidation
               4.2.3. Hydrolysis
         4.3. Biodegradation
         4.4. Bioaccumulation

    5. ENVIRONMENTAL LEVELS AND HUMAN 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
         5.3. Occupational exposure

    6. KINETICS AND METABOLISM

         6.1. Absorption and distribution
         6.2. Metabolism
               6.2.1.  In vitro studies
               6.2.2.  In vivo studies
         6.3. Reaction with body components
         6.4. Excretion

    7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT

         7.1. Aquatic organisms
               7.1.1. Short-term toxicity
               7.1.2. Long-term toxicity
         7.2. Terrestrial organisms
               7.2.1. Short-term toxicity

    8. EFFECTS ON EXPERIMENTAL ANIMALS AND  IN VITRO TEST SYSTEMS

         8.1. Single exposure
               8.1.1. Inhalation exposure
                       8.1.1.1  Mortality
                       8.1.1.2  Systemic effects
               8.1.2. Oral exposure
                       8.1.2.1  Mortality
                       8.1.2.2  Systemic effects

               8.1.3. Dermal exposure
                       8.1.3.1  Mortality
                       8.1.3.2  Systemic effects
               8.1.4. Other routes of exposure
         8.2. Short-term exposure
               8.2.1. Inhalation exposure
               8.2.2. Oral exposure
                       8.2.2.1  Rats
                       8.2.2.2  Mice
         8.3. Long-term exposure
         8.4. Skin and eye irritation; sensitization
               8.4.1. Irritation
               8.4.2. Sensitization
         8.5. Reproduction, embryotoxicity and
               teratogenicity
               8.5.1. Reproduction
               8.5.2. Embryotoxicity and teratogenicity
         8.6. Mutagenicity and related end-points
               8.6.1.  In vitro effects
               8.6.2.  In vivo effects
         8.7. Carcinogenicity/long-term toxicity
               8.7.1. Inhalation exposure
               8.7.2. Oral exposure
               8.7.3. Dermal exposure
               8.7.4. Exposure by other routes
         8.8. Other special studies
               8.8.1. Effects on the nervous system
               8.8.2. Effects on the liver
                       8.8.2.1  Acute effects
                       8.8.2.2  Short-term effects
               8.8.3. Effects on the kidneys
                       8.8.3.1  Acute effects
                       8.8.3.2  Short- and long-term effects
         8.9. Factors modifying toxicity; toxicity of
               metabolites
               8.9.1. Factors modifying toxicity
                       8.9.1.1  Surgery
                       8.9.1.2  Inhibitors and inducers of
                                mixed-function oxidases (MFO)
                       8.9.1.3  Inhibitors of gamma-glutamyltrans-
                                 peptidase (EC 2.3.2.2)
                       8.9.1.4  Inhibitors of cysteine conjugate
                                ß-lyase
                       8.9.1.5  Inhibitors of organic anion
                                transport
                       8.9.1.6  Non-protein sylfhydryl scavengers
               8.9.2. Toxicity of metabolites
                       8.9.2.1   In vitro studies
                       8.9.2.2   In vivo studies

         8.10. Mechanisms of toxicity - mode of action
               8.10.1. Mechanisms of toxicity
               8.10.2. Mode of action

    9. EFFECTS ON HUMANS

         9.1. General population exposure
         9.2. Occupational exposure
         9.3.  In vitro metabolism studies
         9.4. Extrapolation of NOAEL from animals to
               humans

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

         10.1. Evaluation of human health risks
               10.1.1. Hazard identification
               10.1.2. Exposure
               10.1.3. Hazard evaluation
         10.2. Evaluation of effects on the environment
               10.2.1. Hazard identification
               10.2.2. Exposure
               10.2.3. Hazard evaluation

    11. FURTHER RESEARCH

    12. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES

    REFERENCES

    RESUME

    RESUMEN
    

    WHO TASK GROUP MEETING ON ENVIRONMENTAL HEALTH CRITERIA FOR
    HEXACHLOROBUTADIENE

     Members

    Dr T.M. Crisp, Reproductive and Development Toxicology Branch, Human
       Health Assessment Group, Office of Health and Environmental
       Assessment, Environmental Protection Agency, Washington, DC, USA
        (Joint Rapporteur)

    Professor W. Dekant, Toxicology Institute, Würzburg University,
       Würzburg, Germany

    Dr I.V. German, Ukrainian Institute for Ecohygiene and Toxicology of
       Chemicals, Kiev, Ukraine

    Dr B. Gilbert, Fundaçao Oswaldo Cruz, Ministry of Health,
       Manguinhos, Rio de Janeiro, Brazil  (Joint Rapporteur)

    Ms E. Kuempel, Document Development Branch, National Institute for
       Occupational Safety and Health, Robert A. Taft  Laboratories,
       Cincinnati, Ohio, USA

    Dr E.A. Lock, Biochemical Toxicology Section, Imperial Chemical
       Industries, Central Toxicological Laboratory, Alderly Park,
       Macclesfield, Cheshire, United Kingdom

    Professor M.H. Noweir, Industrial Engineering Department, College of
       Engineering, King Abdul Aziz University, Jeddah, Saudi Arabia
        (Chairman)

    Dr A. Smith, Toxicology Unit, Health and Safety Executive, Bootle,
       Merseyside, United Kingdom

     Secretariat

    Professor F. Valic, IPCS Consultant, World Health Organization,
       Geneva, Switzerland, also Vice-Rector, University of Zagreb,
       Zagreb, Croatia  (Responsible Officer and Secretary)

    Dr T. Vermeire, National Institute of Public Health and
       Environmental Protection, Toxicology Advisory Centre, Bilthoven,
       The Netherlands

    NOTE TO READERS OF THE CRITERIA MONOGRAPHS

         Every effort has been made to present information in the
    criteria monographs as accurately as possible without unduly
    delaying their publication. In the interest of all users of the
    Environmental Health Criteria monographs, readers are kindly
    requested to communicate any errors that may have occurred to the
    Director of the International Programme on Chemical Safety, World
    Health Organization, Geneva, Switzerland, in order that they may be
    included in corrigenda.

                                  *   *   *

         A detailed data profile and a legal file can be obtained from
    the International Register of Potentially Toxic Chemicals, Case
    postale 356, 1219 Châtelaine, Geneva, Switzerland (Telephone No.
    9799111).

                                  *   *   *

         This publication was made possible by grant number 5 U01
    ES02617-14 from the National Institute of Environmental Health
    Sciences, National Institutes of Health, USA.

    ENVIRONMENTAL HEALTH CRITERIA FOR HEXACHLOROBUTADIENE

         A Task Group on Environmental Health Criteria for
    Hexachlorobutadiene met at the Institute of Hygiene and
    Epidemiology, Brussels, Belgium, from 10 to 15 December 1992. Dr C.
    Vleminckx welcomed the participants on behalf of the host
    institution and Professor F. Valic opened the meeting on behalf 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 and the environment from
    exposure to hexachlorobutadiene.

         The first draft of this monograph was prepared by Dr T.
    Vermeire, National Institute of Public Health and Environmental
    Protection, Bilthoven, The Netherlands.

         Professor F. Valic was responsible for the overall scientific
    content of the monograph and for the organization of the meeting,
    and Dr P.G. Jenkins, IPCS, for the technical editing of the
    monograph.

         The efforts of all who helped in the preparation and
    finalization of the monograph are gratefully acknowledged.

    ABBREVIATIONS

    ACPB        1-( N-acetylcystein- S-yl)-1,2,3,4,4-pentachloro-1,3-
                butadiene

    BCTB        1,4-(bis-cystein- S-yl)-1,2,3,4-tetrachloro-1,3-
                butadiene BGTB 1,4-(bis-glutathion- S-yl)-1,2,3,4-
                tetrachloro-1,3-butadiene

    CMTPB       1-carboxymethylthio-1,2,3,4,4-pentachloro-1,3-
                butadiene

    CPB         1-(cystein- S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene

    GPB         1-(glutathion- S-yl)-1,2,3,4,4-pentachloro-1,3-
                butadiene

    GSH         reduced glutathione

    HCBD        hexachlorobutadiene

    ip          intraperitoneal

    iv          intravenous

    MTPB        1-methylthio-1,2,3,4,4-pentachloro-1,3-butadiene

    NIOSH       National Institute of Occupational Safety and Health

    NOAEL       no-observed-adverse-effect level

    OECD        Organisation for Economic Co-operation and Development

    PATPB       1-(pyruvic acid thiol)-1,2,3,4,4-pentachloro-1,3-
                butadiene

    PBSA        1,2,3,4,4-pentachloro-1,3-butadienyl sulfenic acid

    TBA         2,3,4,4-tetrachloro-1,3-butenoic acid

    TPA         12- o-tetradecanoylphorbol-13-acetate

    TPB         1-thiol-1,2,3,4,4-pentachloro-1,3-butadiene

    UDS         unscheduled DNA synthesis

    1. SUMMARY

    1.1  Identity, physical and chemical properties, analytical methods

         Hexachlorobutadiene is a non-flammable, incombustible, clear,
    oily and colourless liquid at ordinary temperature and pressure. It
    is poorly soluble in water but miscible with ether and ethanol.

         The substance can be detected and determined quantitatively by
    gas chromatographic methods. The detection limits are 0.03 µg/m3
    in air, 0.001 µg/litre in water, 0.7 µg/kg wet weight in soil or
    sediment, and 0.02 µg/litre in blood. A level of 0.47 µg/kg wet
    weight has been determined in tissue.

    1.2  Sources of human and environmental exposure

         Hexachlorobutadiene is not reported to occur as a natural
    product. It is chiefly produced as a by-product of the manufacture
    of chlorinated hydrocarbons where it occurs in the heavy fractions
    (Hex-waste). The world annual production of the compound in heavy
    fractions was estimated in 1982 to be 10 000 tonnes.

         Hexachlorobutadiene can be used for recovery of
    chlorine-containing gas in chlorine plants and as a wash liquor for
    removing certain volatile organic compounds from gas streams. It has
    further been used as a fluid in gyroscopes, as heat transfer,
    transformer, insulating and hydraulic fluids, as a solvent for
    elastomers, and as an intermediate and fumigant.

    1.3  Environmental transport, distribution and transformation

         The main pathways of entry into the environment are emissions
    from waste and dispersive use. Intercompartmental transport will
    chiefly occur by volatilization, adsorption to particulate matter,
    and subsequent deposition or sedimentation. Hexachlorobutadiene does
    not migrate rapidly in soil and accumulates in sediment. In water,
    it is considered persistent unless there is high turbulence.
    Hydrolysis does not occur. The substance seems to be readily
    biodegradable aerobically, though biodegradability has not been
    investigated thoroughly. Hexachlorobutadiene photolyses on surfaces.
    In addition to deposition, reaction with hydroxyl radicals is
    assumed to be an important sink of hexachlorobutadiene in the
    troposphere, and the estimated atmospheric half-life is up to 2.3
    years. The substance has a high bioaccumulating potential as has
    been confirmed by both laboratory and field observations. Average
    steady-state bioconcentration factors of 5800 and 17 000, based on
    wet weight, have been determined experimentally in rainbow trout.
    Biomagnification has not been observed either in the laboratory or
    in the field.

    1.4  Environmental levels and human exposure

         Hexachlorobutadiene has been measured in urban air: in all
    cases levels were below 0.5 µg/m3. Concentrations in remote areas
    are less than 1 pg/m3. In lake and river water in Europe
    concentrations of up to 2 µg/litre have been recorded, but mean
    levels are usually below 100 ng/litre. In the Great Lakes area of
    Canada, much lower levels (around 1 ng/litre) were measured. Bottom
    sediment levels here can be as high as 120 µg/kg dry weight. Older
    sediment layers from around 1960 contained higher concen-trations
    (up to 550 µg/kg wet weight). The sediment concentration was
    demonstrated to increase with particle size in the sediment.

         Concentrations of hexachlorobutadiene in aquatic organisms,
    birds and mammals indicate bioaccumulation but not biomagnification.
    In polluted waters, levels of over 1000 µg/kg wet weight have been
    measured in several species and 120 mg/kg (lipid base) in one
    species. Present levels generally remain below 100 µg/kg wet weight
    away from industrial outflows.

         The compound has been detected in human urine, blood and
    tissues. Certain food items containing a high lipid fraction have
    been found to contain up to about 40 µg/kg and, in one case, over
    1000 µg/kg.

         One study reported occupational exposures of 1.6-12.2 mg/m3
    and urine levels of up to 20 mg/litre.

    1.5  Kinetics and metabolism

         Hexachlorobutadiene is rapidly absorbed following oral
    administration to experimental animals, but the rate of absorption
    following inhalation or dermal exposure has not been investigated.
    In rats and mice, the compound distributes mainly to the liver,
    kidneys and adipose tissue. It is rapidly excreted. Binding to liver
    and kidney protein and nucleic acids has been demonstrated.

         The biotransformation of the compound in experimental animals
    appears to be a saturable process. This process proceeds mainly
    through a glutathione-mediated pathway in which hexachlorobutadiene
    is initially converted to  S-glutathione conjugates. These
    conjugates can be metabolized further, especially in the
    brush-border membrane of renal tubular cells, to a reactive sulfur
    metabolite, which probably accounts for the observed nephrotoxicity,
    genotoxicity and carcinogenicity.

    1.6  Effects on organisms in the environment

         Hexachlorobutadiene is moderately to very toxic to aquatic
    organisms. Fish species and crustaceans were found to be the most
    sensitive, 96-h LC50 values ranging from 0.032 to 1.2 and 0.09 to

    approximately 1.7 mg/litre for crustaceans and fish, respectively.
    The kidney was demonstrated to be an important target organ in fish.

         Based on several long-term tests with algae and fish species, a
    no-observed-effect level (NOEL) of 0.003 mg/litre was established;
    this classifies the compound as very toxic to aquatic species.
    End-points investigated include general toxicity, neurotoxicity,
    biochemistry, haematology, pathology, and reproductive parameters.
    In one 28-day early-lifestage test with fathead minnows,
    reproduction was unaffected at concentrations of up to
    0.017 mg/litre, whereas increased mortality and a decreased body
    weight were observed at 0.013 and 0.017 mg/litre. The NOEL was
    0.0065 mg/litre.

         Only one reliable test with terrestrial organisms has been
    described. In a 90-day test with Japanese quail, receiving a diet
    containing the compound at concentrations from 0.3 to 30 mg/kg diet,
    the survival of chicks was decreased at 10 mg/kg diet only.

    1.7  Effects on experimental animals and in vitro test systems

    1.7.1 General toxicity

         Hexachlorobutadiene is slightly to moderately toxic to adult
    rats, moderately toxic to male weanling rats, and highly toxic to
    female weanling rats following a single oral dose. The major target
    organs are the kidney and, to a much lesser extent, the liver.

         Based on animal data, the vapour of hexachlorobutadiene is
    irritating to mucous membranes and the liquid is corrosive. The
    substance should be regarded as a sensitizing agent.

         In the kidneys of rats, mice and rabbits, hexachlorobutadiene
    causes a dose-dependent necrosis of the renal proximal tubules.
    Adult male rats are less sensitive to renal toxicity than adult
    females or young males. Young mice are more susceptible than adults,
    no sex difference being apparent. In adult female rats the lowest
    single intraperitoneal dose at which renal necrosis was observed was
    25 mg/kg body weight, and in adult male and female mice it was
    6.3 mg/kg body weight. Biochemical changes and distinct functional
    alterations in the kidneys occurred at doses similar to or higher
    than those at which necrosis occurred.

         In six short-term oral tests, two reproductive studies and one
    long-term diet study with rats, the kidney was also the major target
    organ. Dose-related effects included a decreased relative kidney
    weight and tubular epithelial degeneration. The no-observed-
    adverse-effect level (NOAEL) for renal toxicity in rats in a 2-year
    study was 0.2 mg/kg body weight per day. In mice the NOAEL in a
    13-week study was 0.2 mg/kg body weight per day. In both species,
    adult females were more susceptible than adult males.

         In one short-term inhalation test (6 h/day for 12 days),
    similar effects on the kidneys were observed with a nominal vapour
    concentration of 267 mg/m3, at which concentration respiratory
    difficulties and cortical degeneration in the adrenal glands were
    also observed.

    1.7.2  Reproduction, embryotoxicity and teratogenicity

         Two reproduction diet studies in rats at doses up to 20 and
    75 mg/kg body weight per day, respectively, revealed reduced birth
    weight and neonatal weight gain at maternally toxic doses of 20 and
    7.5 mg/kg body weight, respectively. The highly toxic dose of
    75 mg/kg body weight per day was sufficient to prevent conception
    and uterine implantation. Skeletal abnormalities were not observed.

         In two teratogenicity tests, where rats were exposed either to
    hexachlorobutadiene vapour at concentrations between 21 and
    160 mg/m3 for 6 h/day (from days 6 to 20 of pregnancy) or
    intraperitoneally to 10 mg/kg body weight per day (from days 1 to 15
    of pregnancy), fetuses demonstrated developmental toxicity,
    including reduced birth weight, delay in heart development and
    dilated ureters, but no gross malformations. The retarded
    development was observed at levels which were also toxic to the
    dams.

    1.7.3  Genotoxicity and carcinogenicity

         Hexachlorobutadiene induces gene mutations in the Ames
    Salmonella test under special conditions favouring the formation of
    glutathione conjugation products. It induced chromosomal aberrations
    in one  in vivo study but not in two  in vitro studies. In one  in
     vitro test the frequency of sister chromatid exchanges was
    increased in Chinese hamster ovary cells. High mutagenic potency by
    sulfur metabolites of hexachlorobutadiene was reported. In  in vitro
    studies, the compound induced unscheduled DNA synthesis in Syrian
    hamster embryo fibroblast cultures but not in rat hepatocyte
    cultures. It induced unscheduled DNA synthesis in rats  in vivo,
    but did not induce sex-linked recessive lethal mutations in
     Drosophila melanogaster.

         In the only long-term (2 years) study, in which rats received a
    diet containing hexachlorobutadiene at doses of 0.2, 2 or 20 mg/kg
    body weight per day, an increased incidence of renal tubular
    neoplasms was observed only at the highest dose level.

    1.7.4  Mechanisms of toxicity

         The nephrotoxicity, mutagenicity and carcinogenicity of
    hexachlorobutadiene is dependent on the biosynthesis of the toxic
    sulfur conjugate 1-(glutathion- S-yl)-1,2,3,4,4-pentachloro-
    1,3-butadiene (GPB). This conjugate is mainly synthesised in the
    liver and is further metabolized in the bile, gut and kidneys to

    1-(cystein- S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene (CPB). The
    activation of CPB, dependent on cysteine conjugate ß-lyase, to a
    reactive thioketene in the proximal tubular cells finally results in
    covalent binding to cellular macromolecules.

    1.8  Effects on humans

         No pathogenic effects in the general population have been
    described.

         There have been two reports of disorders among agricultural
    workers using hexachlorobutadiene as a fumigant, but they were also
    exposed to other substances. An increased frequency of chromosomal
    aberrations was found in the lymphocytes of peripheral blood of
    workers engaged in the production of hexachlorobutadiene and
    reported to be exposed to concentrations of 1.6-12.2 mg/m3.

    1.9  Evaluation of human health risks and effects on the environment

    1.9.1  Evaluation of human health risks

         As there have been very few human studies, the evaluation is
    mainly based on studies in experimental animals. However, limited
    human  in vitro data suggest that the metabolism of
    hexachlorobutadiene in humans is similar to that observed in
    animals.

         Hexachlorobutadiene vapour is considered to be irritating to
    the mucous membranes of humans, and the liquid is corrosive. The
    compound should also be regarded a sensitizing agent.

         The main target organs for toxicity are the kidney and, to a
    much lesser extent, the liver. On the basis of short- and long-term
    oral studies in rats and mice, the NOAEL is 0.2 mg/kg body weight
    per day. In one short-term inhalation study in rats (12 days,
    6 h/day), the NOAEL was 53 mg/m3.

         Reduced birth weight and neonatal weight gain was observed only
    at maternally toxic doses, as was developmental toxicity.

         Hexachlorobutadiene has been found to induce gene mutations,
    chromosomal aberrations, increased sister chromatid exchanges and
    unscheduled DNA synthesis, although some studies have reported
    negative results. There is limited evidence for the genotoxicity of
    hexachlorobutadiene in animals, and insufficient evidence in humans.

         Long-term oral administration of hexachlorobutadiene to rats
    was found to induce an increased frequency of renal tubular
    neoplasms, but only at a high dose level causing marked
    nephrotoxicity. There is limited evidence for carcinogenicity in
    animals and insufficient evidence in humans.

         On the basis of the NOAEL for mice or rats of 0.2 mg/kg body
    weight per day, a NOAEL of 0.03-0.05 mg/kg body weight per day has
    been estimated for humans. There is a margin of safety of 150
    between the estimated NOAEL and the estimated maximum total daily
    intake assuming absorption of the compound via contaminated
    drinking-water and food of high lipid content.

    1.9.2  Evaluation of effects on the environment

         Hexachlorobutadiene is moderately to highly toxic to aquatic
    organisms; crustaceans and fish are the most sensitive species. An
    environmental concern level of 0.1 µg/litre has been established. It
    is estimated that the maximum predicted environmental concentration
    away from point sources is twice the extrapolated environmental
    concern level and, consequently, aquatic organisms may be at risk in
    polluted surface waters. Adverse effects on benthic organisms cannot
    be excluded.

         Considering the toxicity of hexachlorobutadiene to mammals,
    consumption of benthic or aquatic organisms by other species may
    cause concern.

    2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL
        METHODS

    2.1  Identity

    Chemical formula:          C4Cl6

    Chemical structure:

    CHEMICAL STRUCTURE

    Common name:               hexachlorobutadiene

    Common synonyms:           1,3-hexachlorobutadiene, 1,1,2,3,4,4-
                               hexachloro-1,3-butadiene, perchloro-
                               butadiene

    Common trade names:        C-46, Dolen-pur, GP40-66: 120, UN2279

    Common abbreviation:       HCBD

    CAS registry number:       87-68-3

    RTECS registry number:     EJ 0700000

    Relative molecular mass:   260.8

    2.2  Physical and chemical properties

         Hexachlorobutadiene is a non-flammable, incombustible, clear,
    colourless and oily liquid at ordinary temperature and pressure. Its
    odour is described as turpentine-like. The odour threshold for the
    compound in air is reported to be 12 mg/m3 (Ruth, 1986). In water
    an odour threshold of 0.006 mg/litre has been reported (US EPA,
    1980). The compound is poorly soluble in water but is miscible with
    ether and ethanol.

         Hexachlorobutadiene is very stable to acid and alkali in the
    absence of an appropriate solvent and has no tendency to polymerize
    even under high pressure. It reacts with chlorine under severe
    reaction conditions, often with cleavage of the carbon skeleton
    (Ullmann, 1986).

         Some physical and chemical data on hexachlorobutadiene are
    presented in Table 1.

        Table 1.  Some physical and chemical properties of
              hexachlorobutadienea

                                                                       

    Physical state                     liquid

    Colour                             clear, colourless

    Melting point                      -18 °C

    Boiling point                      212 °C at 101.3 kPa

    Water solubility                   3.2 mg/litre at 25 °Cb

    Log  n-octanol-water partition
     coefficient (Kow)                 4.78b, 4.90c

    Density                            1.68 g/cm3 at 20 °C

    Relative vapour density            9.0

    Vapour pressure                    20 Pa (0.15 mmHg) at 20 °Cd

    Autoignition temperature           610 °C
                                                                       

    a  Unless otherwise stated, the data are selected from secondary
       sources.
    b  Experimentally derived by Banerjee  et al. (1980)
    c  Experimentally derived by Chiou (1985)
    d  McConnell  et al. (1975)
    
    2.3  Conversion factors

         1 ppm = 10.67 mg/m3 air at 25 °C and 101.3 kPa (760 mmHg)
         1 mg/m3 air = 0.094 ppm.

    2.4  Analytical methods

         A summary of relevant methods of sampling and gas
    chromatographic analysis is presented in Table 2.

         The analytical method for air, reported by Dillon (1979) and
    Boyd  et al. (1981) has been approved by NIOSH and was published in
    the NIOSH Manual of Analytical Methods (NIOSH, 1979, 1990).


        Table 2.  Sampling, preparation and analysis of hexachlorobutadiene

                                                                                                                                              

    Medium    Sampling method              Analytical method        Detection limit    Sample size      Comments                Reference

                                                                                                                                              

    Air       adsorption on Chromosorb     gas chromatography                          360 litre        developed for personal  Mann et al.
              101; extraction by hexane    with electron capture                                        sampling in industry    (1974)
                                           detection

    Air       adsorption on Amberlite      gas chromatography       10 µg/m3           3 litre          suitable for personal   Boyd et al.
              XAD-2; extraction by         with electron capture                                        and area monitoring;    (1981); Dillon
              hexane                       detection                                                    validation range        (1979)
                                                                                                        10-2000 µg/m3

    Air       adsorption on Tenax-GC;      gas chromatography       11 µg/m3           2 litre          suitable for            Melcher &
              purging of water vapour,     with flame ionization                                        continuous area         Caldecourt
              oxygen, etc., by nitrogen;   detection                                                    monitoring              (1980)
              desorption by heating

    Air       adsorption on Tenax-GC;      gas chromatography       0.03 µg/m3 a                        developed for the       Krost et al.
              desorption by heating        (capillary column)                                           analysis of ambient     (1982); Pellizari
              under a helium flow;         with mass                                                    air                     (1982); Barkley
              cryofocussing                spectro-metric                                                                       et al. (1980)
                                           detection

    Water     extraction by hexane;        gas chromatography       0.05 µg/litre      16 litre         developed for the       Oliver & Nicol
              concentration; drying with   (capillary column)                                           analysis of surface     (1982)
              Na2SO4; clean-up by silica   with electron                                                water
              gel chromatography           capture detection
                                                                                                                                              

    Table 2 (contd).

                                                                                                                                              

    Medium    Sampling method              Analytical method        Detection limit    Sample size      Comments                Reference

                                                                                                                                              

    Water     extraction by                gas chromatography       0.0014 µg/litre    0.8-1 litre      US EPA Method           Lopez-Avila
              dichloro-methane-acetone;    with electron capture                                        8120                    et al. (1989)
              drying; concentration        detection
              by N2 stream

    Water     extraction by                gas chromatography       0.001 µg/litre     12 litre         developed for           Zogorski (1984)
              dichloro-methane;            with electron capture                                        monitoring of
              drying; concentration        detection                                                    domestic and process
                                                                                                        waters

    Water     extraction by                gas chromatography       0.34 µg/litre      1 litre          US EPA Method 612;      US EPA (1984a)
              dichloro-methane;            with electron capture                                        developed for the
              drying; concentration        detection                                                    analysis of municipal
              and exchange to                                                                           and industrial
              hexane; clean-up by                                                                       discharges
              fluorisil chromatography

    Water     extraction by                gas chromatography       0.9 µg/litre       1 litre          US EPA Method 625;      US EPA (1984b)
              dichloro-methane at pH                                                                    developed for the
              >11, then at pH <2;                                                                       analysis of municipal
              drying; concentration                                                                     and industrial
                                                                                                        discharges

    Water     purging by helium;           gas chromatography       0.4 µg/litre       0.1 litre        developed for the       Otson & Chan
              trapping; desorption by      (capillary column)                                           analysis of volatile    (1987);
              heating                      with mass                                                    organics in waters      Eichelberger
                                           spectro-metric                                                                       et al. (1990)
                                           detection
                                                                                                                                              

    Table 2 (contd).

                                                                                                                                              

    Medium    Sampling method              Analytical method        Detection limit    Sample size      Comments                Reference

                                                                                                                                              

    Soil,     extraction by                gas chromatography       0.7 µg/kg                                                   Laseter et
    sediment  acetone-benzene              with electron capture    wet weight                                                  al. (1976)
                                           detection

    Soil      add water; adjust to pH      gas chromatography                                           developed for           Kiang & Grob
              >12; extraction by           (capillary column)                                           screening of soil       (1986)
              dichloromethane;             with flame ionization                                        for priority
              centrifugation; drying;      and mass                                                     pollutants
              concentration                spectro-metric
                                           detection

    Sediment  add water; adjust to pH      gas chromatography                                           developed for           Lopez-Avila et
              > 11; extraction by          (capillary column)                                           screening of            al. (1983)
              dichloromethane;             with flame/electron                                          sediment for
              centrifugation; drying;      capture/mass                                                 priority pollutants
              concentration;               spectro-metric
              clean-up by silica           detection
              gel chromatography

    Sediment  extraction by                gas chromatography       13 µg/kga          10-15 g dry                              Oliver & Nicol
              hexane-acetone; removal      (capillary column)                          weight                                   (1982)
              of acetone by                with electron capture
              water extraction; drying;    detection
              concentration; clean-up
              by silica gel
              chromatography and
              agitation with mercury
                                                                                                                                              

    Table 2 (contd).

                                                                                                                                              

    Medium    Sampling method              Analytical method        Detection limit    Sample size      Comments                Reference

                                                                                                                                              

    Biota    homogenization; filtration;  gas chromatography       0.7 µg/kg                            method applied to       Laseter et
             separation; extraction by    with electron capture                                         analysis of fish        al. (1976)
             hexane; clean-up by          detection
             fluorisil chromatography

    Biota    grind and mix edible         gas chromatography       0.005 mg/kg    25 g (eggs) wet weight                        Yurawecz et
             tissue; extraction;          with electron capture    wet weight     50 g (fish) wet weight                        al. (1976)
             clean-up by fluorisil        detection                or 0.04            3 g (milk fat)
             chromatography                                        mg/kg fat        100 g (vegetables)
                                                                                        wet weight

    Biota    grinding with Na2SO4;        gas chomatography        0.47 µg/kga         15 g             method applied to       Oliver &
    Nicol
             extraction by                (capillary column)                                            analysis of fish        (1982)
             hexane-acetone;              with electron capture
             back-extraction of acetone   detection
             by water; concentration;
             clean-up by silica
             gel chromatography

    Biota    extraction by                gas chromatography       1 µg/kg             2 g              method applied to       Mes et al.
             benzene-acetone;             with electron capture    wet weighta                          analysis of             (1982; 1985;
             filtration; concentration;   detection                                                     chlorinated             1986)
             redissolution in hexane;                                                                   hydrocarbon residues
             clean-up including                                                                         in human adipose
             fluorisil-silicic                                                                          tissue and human milk
             acid chromatography
                                                                                                                                              

    Table 2 (contd).

                                                                                                                                              

    Medium    Sampling method              Analytical method        Detection limit    Sample size      Comments                Reference

                                                                                                                                              

    Biota    extraction by hexane         gas chromatography       0.0182 µg/litre     100 mg           method applied to       Kastl & Hermann
             containing an internal       with electron capture                                         whole (rat) blood       (1983)
             standard; centrifugation;    detection                                                     analysis
             direct injection
                                                                                                                                              

    a lowest reported level measured
    

    The method was validated for the concentration range of
    10-2000µg/m3 in 3 litre air samples. The lowest detectable
    quantity for this method was reported to be 20 ng, the desorption
    efficiency 98%, and the relative standard deviation 9%. Melcher &
    Caldecourt (1980) described a gas chromatographic method for the
    direct determination of organic compounds in air using a collection
    precolumn from which the compounds are directly injected into the
    analytical column by rapid heating of the precolumn. The method was
    reported to be suitable for the analysis of aqueous samples by
    purging the precolumn following injection of the sample
    (0.01-0.2 cm3). The analytical method developed for volatile
    halogenated compounds by Krost  et al. (1982) was applied by
    Pellizari (1982) and Barkley  et al. (1980). Barkley  et al.
    (1980) also described the analysis of volatile halogenated compounds
    in water, blood and urine using a modification of this method: the
    substances are recovered from water by heating and from biological
    matrices by heating and purging and are subsequently trapped on a
    Tenax column.

         A spectrophotometric method for the determination of
    hexachlorobutadiene in blood and urine has been reported. The method
    involves extraction by heptane and determination by either UV
    spectroscopy or colorimetry after derivatization with pyridine.
    Reported detection limits were 0.05 mg/litre for the UV method and
    5 mg/litre for the colorimetric method (Gauntley  et al., 1975).
    Interference by other chlorinated hydrocarbons can be expected.

    3.  SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

    3.1  Natural occurrence

         Hexachlorobutadiene has not been reported to occur as a natural
    product.

    3.2  Anthropogenic sources

    3.2.1  Production levels and processes

         The available data are in general of poor quality and not
    up-to-date. Commercial production of hexachlorobutadiene was
    reported to occur in Germany and Austria (SRI, 1984). In the USA,
    commercial production was apparently terminated around 1970 (Mumma &
    Lawless, 1975). The compound was and is chiefly produced as
    by-product of the manufacture of chlorinated hydrocarbons, often in
    association with hexachlorobenzene. In the USA, the manufacture of
    tetrachloroethene, trichloroethene and carbon tetrachloride
    accounted in 1972 for over 99% of this production of
    hexachlorobutadiene in heavy fractions, the so-called Hex-waste, and
    amounted to 3310-6580 tonnes (Brown  et al., 1975; Mumma & Lawless,
    1975; Yurawecz  et al., 1976; see also section 3.2.3). It was also
    reported to be a by-product of the manufacture of vinyl chloride,
    allyl chloride and epichlorohydrin by chlorinolysis processes (Kusz
     et al., 1984). Hexachlorobutadiene has been identified in the
    effluents of sewage treatment plants (section 5.2) and as a
    by-product of the pyrolysis of trichloro-ethene (Yasuhara & Morita,
    1990) and plastics (Singh  et al., 1982). The annual world
    production of hexachlorobutadiene in heavy fractions was estimated
    in 1982 to be 10 000 tonnes (Hutzinger, 1982). No data have been
    found regarding the amount of hexachlorobutadiene, if any, which is
    now recovered from this waste.

         Apart from the possible commercial production of
    hexachloro-butadiene by recovery from Hex-waste, three pathways for
    chemical synthesis are known: the chlorination and
    dehydro-chlorination of hexachlorobutene; the chlorination of
    polychlorobutanes; and the catalytic chlorination of butadiene
    (Mumma & Lawless, 1975; CESARS, 1981). There is no evidence,
    however, that the latter reactions have ever been used commercially.

         The fraction of hexachlorobutadiene released to the environment
    during its industrial life cycle (not defined) has been estimated to
    be between 1 and 3% (SRI, 1984). The fraction of hexachlorobutadiene
    lost to the environment during its production at a tetrachloroethene
    manufacturing plant in the USA was estimated to be 1.5% (Brown
     et al., 1975). Using a simple model describing the troposphere,
    the global annual emission rate was calculated to be 3000 tonnes of
    hexachlorobutadiene based on air sampling data of 1985 (Class &
    Ballschmiter, 1987; see also section 4.2.2).

    3.2.2  Uses

         Hexachlorobutadiene can be used for the recovery of "snift",
    which is chlorine-containing gas in chlorine plants, and as a wash
    liquor for removing volatile organic compounds from gas streams. It
    can be used as a fluid in gyroscopes, as heat transfer, transformer,
    insulating and hydraulic fluids, and as solvent for elastomers. It
    can be an intermediate in the manufacture of lubricants and rubber
    compounds. In the ex-USSR, the substance was reported to find
    widespread application as a fumigant for treating  Phylloxera on
    grapes, and 600-800 tonnes was used for this purpose in 1975 (Brown
     et al., 1975; Mumma & Lawless, 1975).

    3.2.3  Waste disposal

         Hex-waste containing hexachlorobutadiene may be destroyed by
    incineration, placed in landfill, or simply stored. Another
    procedure involves recycling the compound by catalytic chlorination
    and subsequent high temperature chlorinolysis to carbon
    tetrachloride and tetrachloroethene (Markovec & Magee, 1984).

    4.  ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION

    4.1  Transport and distribution between media

         The main pathways for entry of hexachlorobutadiene into the
    environment are its emission via industrial waste (section 3.2.3)
    and following dispersive use (section 3.2.2). The compound may enter
    surface and ground water, soil and air. In view of its physical
    properties, intercompartmental transport of hexachloro-butadiene is
    expected to occur by volatilization and adsorption to suspended
    particulate matter.

         Considering the vapour pressure of the compound, i.e. 20 Pa at
    20 °C (McConnell  et al., 1975), transfer across soil-air
    boundaries may be significant. Depending on the soil type,
    adsorption will hinder this transport (see below). In a field study
    in the ex-USSR, concentrations of hexachlorobutadiene in air above a
    vineyard were found to be 0.08 and 0.003 mg/m3 at 1 day and 3
    months, respectively, following a spring application of 250 kg/ha.
    The method of analysis was not reported. Volatilization of the
    compound from light soils was more rapid than from heavy soils
    (Litvinov & Gorenshtein, 1982).

         The Henry coefficient of hexachlorobutadiene is 0.43 (1040
    Pa.m3.mol-1) at 25 °C (Shen, 1982) and 0.3 at 22 °C Hellmann,
    1987a). These values are comparable to those of other chlorinated
    aliphatic alkenes. They indicate possible transfer of the compound
    across water-air boundaries leading to a wide distribution, with
    aerial transport playing a major role (McConnell  et al., 1975). In
    a model experiment, hexachlorobutadiene was allowed to evaporate
    from a 20-mg/litre aqueous-methanolic solution, containing 10%
    methanol, in a porcelain basin with slow magnetic stirring at 22 °C.
    UV spectrophotometry recorded a 25% loss within 28 min. It was shown
    that methanol decreased the disappearance time. For the transfer of
    this and other model results to flowing waters, a reduction factor
    of 30 was proposed for the rate of evaporation on the basis of
    limited data for two compounds (Hellmann, 1987a).

         In a model experiment, UV spectrophotometric analysis of
    solutions of hexachlorobutadiene in deionized water to which
    1 g/litre of clay mineral (Fuller's earth) was added revealed a
    clay-water partition coefficient of 500 litre/kg, showing limited
    adsorption to pure clay minerals comparable to that of other
    chlorinated alkenes (Hellmann, 1987b). Based on the log
    octanol-water partition coefficient (log Kow) of 4.78-4.90
    (Table 1), hexa-chlorobutadiene is expected to adsorb strongly to
    organic matter. The organic carbon-water partition coefficient
    (Koc) can be estimated to be 25 120 litre/kg on the basis of a log
    Kow of 4.8 using the semi-empirical equation of Karickhoff (1981).
    Oliver & Charlton (1984) determined a Koc value of 158 500
    litre/kg on the basis of sediment and water concentrations in the
    Niagara River, USA. Partition coefficients of approximately

    200-260 litre/kg were found for two unspecified types of soil in
    model experiments employing gas chromatographic analysis of
    solutions of hexa-chlorobutadiene in water (Leeuwangh  et al.,
    1975; Laseter  et al., 1976). In field experiments conducted along
    the Mississippi river in the USA in 1974-1975, some water samples
    were found to contain 1.0-1.5 µg/litre, whereas levee soil samples
    at the same sites contained 62-1001 µg/kg dry weight. At a more
    polluted site near a Hex-waste landfill, water samples contained
    0.04-4.6 µg/litre and mud samples 270-2370 µg/kg dry weight. These
    studies show that soil-water partition coefficients can range over 2
    to 4 orders of magnitude assuming equilibrium (Laseter  et al.,
    1976). It can be concluded that the compound does not migrate
    rapidly in soils and will accumulate in sediment. It should be noted
    that the micro-particles onto which hexachlorobutadiene is absorbed
    may themselves migrate in the sub-surface resulting in facilitated
    transport. The degree of adsorption to soil is highly dependent on
    the content of organic matter and is less pronounced in sandy soils.

         On the basis of data for Dutch surface waters, the half-lives
    of hexachlorobutadiene were estimated to be 3-30 days in rivers and
    30-300 days in lakes and ground water. This suggests that
    turbulence, and therefore increased aerobic biodegradation,
    volatilization and adsorption, account for the shorter half-lives in
    river water, that the compound is difficult to degrade both
    biologically and chemically (see below), and that, overall, the
    compound is persistent in water (Zoeteman  et al., 1980).

    4.2  Abiotic degradation

    4.2.1  Photolysis

         Hexachlorobutadiene absorbs light within the solar spectrum.
    Irradiation of a solution of hexachlorobutadiene in benzene at
    254 nm for 15 min resulted in the formation of numerous products
    having a relative molecular mass greater than that of
    hexachloro-butadiene itself (Laseter  et al., 1976). The extent of
    mineralization of the compound adsorbed to silica gel and exposed to
    oxygen was examined following irradiation with ultraviolet light
    filtered by quartz (wavelength < 290 nm) or by pyrex (simulating
    tropospheric UV with a wavelength > 290 nm). After 6 days, 50-90%
    mineralization to hydrogen chloride and/or chlorine, and carbon
    dioxide was observed (Gb  et al., 1977). These experiments indicate
    that hexachlorobutadiene present as a virtual monolayer on silica
    gel undergoes quite rapid photolysis.

    4.2.2  Photooxidation

         Using a steady-state mathematical model for the troposphere
    (describing it as 2 boxes one north one south of the equator) and on
    the basis of gas chromatographic analysis of air samples from sites
    far away from anthropogenic sources, the tropospheric lifetime of

    hexachlorobutadiene was estimated to be 2.3 years for the northern
    hemisphere and 0.8 years for the southern hemisphere. It was assumed
    that the reaction with hydroxyl radicals in the troposphere is the
    main sink for hexachloro-butadiene, by analogy with other
    halocarbons. The calculated lifetimes at -8 °C correspond to a
    pseudo-first order rate constant of (2 ± 1) x 10-14
    cm3.molecules-1.sec-1 at estimated hydroxyl radical
    concentrations of 7 x 105 molecules.cm-3 for the northern
    hemisphere and 17 x 105 for the southern hemisphere (Class &
    Ballschmiter, 1987). Experimentally, a half-life of 1 week was
    determined when hexachlorobutadiene was exposed to air in flasks
    outdoors. This relatively short disappearance time was possibly due
    to heterogeneous reactions on the vessel walls, as suggested by the
    authors of the report. Hydrogen chloride was found to be the main
    degradation product after exposure of samples to xenon arc
    radiations (wavelength > 290 nm) (Pearson & McConnell, 1975).

    4.2.3  Hydrolysis

         Hexachlorobutadiene is highly resistant to chemical degradation
    by strong acids and alkalis in the absence of appropriate solvents,
    although it is readily degraded by ethanolic alkali (Roedig &
    Bernemann, 1956). Based on the measured hydrolysis rate of the
    compound in a 1:1 acetone-water mixture, a half-life of over 1800 h
    was calculated (Hermens  et al., 1985).

    4.3  Biodegradation

         Hexachlorobutadiene, at concentrations of 5 or 10 mg/litre, was
    completely degraded by adapted aerobic microorganisms within 7 days
    in a static-culture flask screening procedure at 25 °C, as shown by
    gas chromatography and by determination of total and dissolved
    organic carbon. The inoculum was taken from settled domestic waste
    water (Tabak  et al., 1981). Approximately 70% adsorption to sludge
    and 10% degradation was found to occur within 8 days in a pilot
    low-loaded biological sewage treatment plant (Schröder, 1987).

         Anaerobic degradation of hexachlorobutadiene at 100 mg/litre
    was not observed in 48-h batch assays at 37 °C using an inoculum
    from a laboratory digester (Johnson & Young, 1983).

    4.4  Bioaccumulation

         Considering the low water solubility of 3.2 mg/litre and the
    high log Kow of 4.78-4.90 (Table 1), a strong bioaccumulating
    potential would be expected. Both laboratory and field data support
    this prediction. In flow-through laboratory tests with algae,
    crustaceans, molluscs and fish in fresh or marine waters,
    bioconcentration factors (on a wet weight basis) were between 71 and
    17 000. The results appear to be highly dependent on the exposure
    period and there is great variability between organisms (Leeuwangh

     et al., 1975; Pearson & McConnell, 1975; Laseter  et al., 1976;
    Oliver & Niimi, 1983). Steady state was clearly demonstrated to be
    reached in only one of these tests. Oliver & Niimi (1983) exposed
    rainbow trout  (Salmo gairdnerii) to aqueous solutions of
    hexachlorobutadiene at 0.10 and 3.4 ng/litre and found average
    bioconcentration factors of 5800 and 17 000, steady states having
    been reached after 69 and 7 days, respectively. When Oligochaete
    worms were exposed via spiked Lake Ontario sediments to a pore water
    concentration of 32 ng/litre in a flow-through system, steady state
    was reached within 4 to 11 days and the average bioconcentration
    factor was 29 000, based on dry weight of which about 8% is lipid
    (Oliver, 1987). Biomagnifi-cation, the concentrating of a substance
    through a food chain, was not observed for hexachlorobutadiene in
    two limited laboratory experiments with fish fed contaminated food
    (Pearson & McConnell, 1975; Laseter  et al., 1976).

         The bioaccumulation factors found in plankton, crustaceans,
    molluscs, insects and fish in surface waters are comparable to those
    observed in the laboratory: available bioaccumulation factors based
    on wet weight range between 33 and 11 700 (Goldbach  et al., 1976;
    Laseter  et al., 1976). No biomagnification was observed when
    levels in fish were compared with those of detritus and several
    invertebrates (Goldbach  et al., 1976). The latter was confirmed by
    a trophodynamic analysis in the Lake Ontario ecosystem (Oliver &
    Niimi, 1988).

         Limited bioaccumulation of hexachlorobutadiene was observed in
    the fat of rats following exposure for 4 to 12 weeks to a mixture of
    this compound and 1,2,3,4-tetrachlorobenzene, hexachloroben-zene,
    1,3,5-trichlorobenzene,  o-dichlorobenzene and
    gamma-hexa-chlorocyclohexane in food (each compound at 2 or 4 mg/kg
    body weight per day). Fat concentrations of up to 8 mg/kg were
    observed at the higher dose rates (Jacobs  et al., 1974).

    5.  ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

    5.1  Environmental levels

    5.1.1  Air

         Concentrations of hexachlorobutadiene measured in air at
    different locations are summarized in Table 3.

    5.1.2  Water

         Concentrations of hexachlorobutadiene measured in water at
    different locations are summarized in Table 4.

    5.1.3  Soil and sediment

         Concentrations of hexachlorobutadiene measured in soil and
    sediment at different locations are summarized in Table 5.

    5.1.4  Biota

         Concentrations of hexachlorobutadiene measured in aquatic
    organisms, birds and mammals are summarized in Table 6.

    5.2  General population exposure

         Levels of hexachlorobutadiene encountered in the food and
    drinking-water of the general population are summarized in Table 7.

         Hexachlorobutadiene was not detected in the urine or blood of
    nine individuals living near Old Love Canal, USA, whereas trace
    levels were found in the breath of one of them (Barkley  et al.,
    1980). In another investigation the compound could not be detected
    in the blood of 36 Love Canal area residents (Bristol  et al.,
    1982). Hexachlorobutadiene was found at levels of 0.8-4 µg/kg wet
    weight (fat) and 1.2-13.7 µg/kg wet weight (liver) in postmortem
    tissues from 6 out of 8 United Kingdom residents in 1970 (McConnell
     et al., 1975). In the adipose tissue of accident victims in Canada
    (1976), levels of 1 to 8 µg/kg wet weight were measured in 128 out
    of 135 samples (Mes  et al., 1982, 1985). In Canada (1982),
    hexachlorobutadiene could not be detected in any of 210 samples of
    breast milk (Mes  et al., 1986).

         When 15 samples of hazardous waste from incineration facilities
    in the USA were analysed, 4 sites were found to contain
    hexa-chlorobutadiene, but the levels were reported to be below
    10 mg/kg (Demarini  et al., 1987). In sewage sludge, Alberti &
    Ploger (1986) measured levels of below 1 µg/kg dry weight (3 samples
    of municipal or municipal/industrial sludge), up to 0.6 µg/kg dry
    weight (1 sample of municipal/industrial sludge), and 15 µg/kg dry
    weight (1 sample of industrial sludge).

    5.3  Occupational exposure

         Hexachlorobutadiene levels of 1.6-12.2 mg/m3 air have been
    measured in the workplace, resulting in reported urine levels of up
    to 20 mg/litre in workers at the end of the day (German & Viter,
    1985).


        Table 3.  Levels of hexachlorobutadiene in environmental air

                                                                                                                                              

    Type of       Year       Location                              Detection        Levels determineda      Reference
    air                                                          limit (ng/m3)            (ng/m3)

                                                                                                                                              

    Ambient       1985       Atlantic Ocean, lower                                   0.0001-0.0004 (r)      Class & Ballschmiter
                             troposphere in a south-north                            0.0003 (m, north)      (1987)
                             cross section, 8 sites                                  0.0001 (m, south)

    Urban         1978       USA, Niagara Falls, inside                              nd (n=9)               Barkley et al. (1980)
                             homes near dump site

                             USA, Niagara Falls, outside                             nd (n=6)
                             homes near dump site                                    trace (n=3)

                             USA, Niagara Falls area                                 nd (n=3)
                                                                                     trace (n=1)
                                                                                     50-390 (r, n=2)

    Urban         1980-      USA, 7 cities                                           nd-117 (r,m)           Singh et al. (1982)
                  1981                                                               nd-251 (r)
                                                                                                                                              

    Table 3 (contd).

                                                                                                                                              

    Type of       Year       Location                              Detection        Levels determineda      Reference
    air                                                          limit (ng/m3)            (ng/m3)

                                                                                                                                              

    Polluted      1975       USA, 9 sites with chemical                              nd-460 000 (r)         Li et al. (1976)b
                             industries, on plant property

                             USA, 9 sites with chemical                              nd-22 000 (r)
                             industries, off plant property

    Polluted      1978       USA, Niagara Falls, household                           < 45 (n=1)             Barkley et al.
                             basement near dump site                                                        (1980)

    Polluted      1978       idem                                                    30-410 (r, n=4)        Pellizari (1982)

    Polluted      1982       USA, liquid waste lagoon                  2             nd (n=2)               Guzewich et al.
                                                                                     3-160 (n=4)            (1983)
                                                                                                                                              

    a   nd = not detectable; r = range of individual values; r,m = range of mean values; m = mean; n = number of samples
    b   The highest levels were associated with the production of tetrachloroethene and trichloroethene.  At other plants,
        levels of hexachlorobutadiene remained below 3 ng/m3.  Waste holding areas (especially when involving open storage)
        were often the most significant sources of hexachlorobutadiene, contaminated soil being a secondary source.  The
        total number of samples examined was 405.

    Table 4.  Levels of hexachlorobutadiene in environmental water

                                                                                                                                              

    Type of       Year           Location                       Detection limit     Levels determineda      Reference
    water                                                         (ng/litre)            (ng/litre)

                                                                                                                                              

    Surface                      Canada, Niagara River                50             1.5                    Oliver & Nicol (1982)

    Surface       1982           Canada, Niagara River                               0.82 (m, n=5)          Oliver & Charlton (1984)

    Surface       1981-1983      Canada, Niagara River               0.01            0.78 (m, n=104)        Oliver & Nicol (1984)
                                                                                     0.67 (median)
                                                                                     0.27-3.2 (r)

    Surface       1981           Canada, Niagara River                               nd-0.6 (n=1)           Fox et al. (1983)

    Surface       1972-1973      Netherlands, River IJssel,                          50-130 (r, n=5)        Goldbach et al. (1976)
                                 Ketelmeer, IJsselmeer

    Surface       1976-1978      Netherlands, River Rhine                            1000-2000              Zoeteman et al. (1980)

    Surface       1975           USA, 9 sites with chemical                          nd-240 000 (r)         Li et al. (1976)
                                 industries, on plant property
                                 idem, off plant property                            nd-23 000 (r)
                                                                                                                                              

    Table 4 (contd).

                                                                                                                                              

    Type of       Year           Location                       Detection limit     Levels determineda      Reference
    water                                                         (ng/litre)            (ng/litre)

                                                                                                                                              

    Surface       1976           Germany, River Rhine, 865 km         10             10 (50-percentile)     Alberti (1983)
                                                                                     180 (90-percentile)

                  1978           Germany, idem                        10             20 (50-percentile)
                                                                                     60 (90-percentile)

                  1981           Germany, idem                        10             < 10 (50-percentile)
                                                                                     40 (90-percentile)

                  1980-1981      Germany, 4 River Rhine               10             nd
                                 tributaries
                                 Germany, River Lippe                 10             40-200

    Surface       1979-1981      Germany, River Rhine,                               < 50                   Haberer et al. (1988)
                  1979-1981      Germany, River Main                                 < 1000

    Surface       1983           Netherlands, River Rhine, River Lek                 < 100 (m, n=52)        Meijers (1988)
                                 idem, before dune infiltration                      70 (m, n=13)
                                                                                                                                              

    Table 4 (contd).

                                                                                                                                              

    Type of       Year           Location                       Detection limit     Levels determineda      Reference
    water                                                         (ng/litre)            (ng/litre)

                                                                                                                                              

    Surface       1984-1985      Germany, River Rhine                                10-20                  Petersen (1986)
                                 Germany, River Elbe                                 10-150

    Estuarine                    USA, Calcasieu River estuary,                       1298                   Pereira et al. (1988)
                                 vicinity of industrial outfall

    Sea           1972-1973      United Kingdom, Liverpool Bay         1             4 (m, n=150)           Pearson & McConnell
                                                                                     nd-30 (r)              (1975)

    Sea           1977           USA, Gulf of Mexico,                                                       Sauer (1981)
                                  open ocean                           1             nd (n=4)
                                  coast                                1             nd-15 (n=4)

    Ground                       Switzerland, aquifer contaminated                   200-300 (r)            Giger & Schaffner (1981)
    water                        by leachate from a chemical waste
                                 disposal site
                                                                                                                                              

    a   nd = not detectable; r = range of individual values; r,m = range of mean values; m = mean; n = number of samples;
        x percentile = x percent of samples with values up to that given

    Table 5.  Levels of hexachlorobutadiene in soil and sediment

                                                                                                                                              

    Type of soil       Year           Location                             Levels determineda             Reference
    or sediment                                                                  (µg/kg)

                                                                                                                                              

    Soil,                             vineyards infected with Phylloxera     < 7300 (8 mo)                Vorobyeva (1980)
    agricultural                      and treated at 250 kg/ha               < 2990 (32 mo)

    Soil               1975           USA, 9 sites with chemical             nd-980 000 (r)b              Li et al. (1976)
                                      industries, on plant property
                                      idem, off plant property               nd-110 (r)b

    Sediment           1975           idem, on plant property                nd-33 000 (r)b               Li et al. (1976)

                                      idem, off plant property               nd-40 (r)b

    Sediment,                         United Kingdom, Liverpool Bay          < 1 (n=110)                  Pearson & McConnell
    marine                                                                   > 1 (n=30)                   (1975)

    Sediment,                         Canada, Niagara Falls                  18                           Oliver & Nicol (1982)
    river/lake

    Sediment,          1980           Canada, Lake Ontario                   nd (n=9)                     Kaminsky et al. (1983)
    lake                                                                     trace (n=3)
                                                                             8.7 (n=1)
                                                                                                                                              

    Table 5 (contd).

                                                                                                                                              

    Type of soil       Year           Location                             Levels determineda             Reference
    or sediment                                                                  (µg/kg)

                                                                                                                                              

    Sediment,          1981           Canada, Niagara River, downstream      9.6-37 (n=5, dwt)c           Fox et al. (1983)
    river                             idem, upstream                         nd (n=1, dwt)

    Sediment,          1982           Germany, River Rhine, 707 km           0.002 (dwt)                  Alberti (1983)
    river                             idem, 815 km                           0.005 (dwt)

    Sediment,          1981           Canada, Lake Ontario                   12-120 (n=5, dwt)
    lake

    Sediment,          1968-1978      Canada, Niagara Falls sediment         nd                           Durham & Oliver (1983)
    lake               1959-1962      core near Niagara River                550
                       1980-1981                                             18
                       1868-1981                                             nd-550

    Sediment,          1980-1982      Canada, lakes                          0.04-9.3 (r, n=57)           Oliver & Bourbonniere
    lake               1980           Canada, Lake Huron                     0.08 (m, n=9, dwt)           (1985)
                       1982           Canada, Lake St. Clair                 7.3 (m, n=2, dwt)
                       1982           Canada, Lake Erie                      0.2-1.6 (r,m, n=46, dwt)

    Sediment,          1982           Canada, Niagara Falls, settling        nd (n=1)                     Oliver & Charlton (1984)
    lake                              particulates at 20 m depth             2.9-11 (r, n=5), 5.9 (m)

                                      idem, settling particulates at
                                      68 m depth                             7.4 (m)
                                      bottom sediment                        32 (m, n=12)
                                                                                                                                              

    Table 5 (contd).

                                                                                                                                              

    Type of soil       Year           Location                             Levels determineda             Reference
    or sediment                                                                  (µg/kg)

                                                                                                                                              

    Sediment, lake                    Canada, Lake Ontario                   0.1-75 (r, n=3)              Oliver (1984)

    Sediment,                         USA, Eagle Harbour, creosote           < 0.79 (m, n=15, dwt)        Malins et al. (1985)
    sea harbour                       contaminated sediment, 3 sites

    Sediment,                         USA, President Point, 1                < 2.0 (n=1, dwt)
    sea harbour                       reference site

    Sediment                          USA, Calcasieu River estuary,          85 (bottom)                  Pereira et al. (1988)
    estuarine                         vicinity of industrial outfall         1.7 (suspended)
                                                                                                                                              

    a dwt = dry weight; nd = not detectable; r = range of individual values; r,m = range of mean values; m = mean; mo = months after treatment;
      n = number of samples
    b The highest levels were associated with the production of tetrachloroethene and trichloroethene.  Waste holding areas (especially when
      involving open storage) were often the most significant sources of hexachlorobutadiene, contaminated soil being a secondary source.
    c surficial sediment; the sediment concentration increased with fraction size
    d surficial sediment

    Table 6.  Concentrations of hexachlorobutadiene in aquatic organisms, birds and mammals

                                                                                                                                              

    Type of biota                Year           Location                         Levels determineda       Reference
                                                                                     (µg/kg wwt)

                                                                                                                                              

    Detritus (bottom)            1972-1976      Netherlands, surface water               200              Goldbach et al. (1976)
    Detritus (floating)                                                                  220

    Invertebrates

    Plankton                     1972-1973      United Kingdom, sea water              nd-2.0             Pearson & McConnell (1975)
    Ragworm,
     Nereis diversicolor                                                                0.06
    Mussel,
     Mytilus edulis                                                                    nd-3.8
    Crab,
     Cancer pagarus                                                                    nd-1.1
    Others                                                                               nd
     Cerastoderma edule
     Ostrea edulis
     Buccinum undatum
     Crepidula fornicata
     Carcinus maenus
     Eupagurus bernhardus
     Crangon crangon
     Asterias rubens
     Solaster sp.
     Echinus esculentus
                                                                                                                                              

    Table 6 (contd).

                                                                                                                                              

    Type of biota                Year           Location                         Levels determineda       Reference
                                                                                     (µg/kg wwt)

                                                                                                                                              

    Snail                        1972-1976      Netherlands, surface water                                Goldbach et al. (1976)
     Lymnaea peregra                                                                  30, 1670
    Clam,
     Sphaerium sp.                                                                      2410
    Oligochaetes                                                                    0.3 (m, n=3)

    Oligochaetes                 1981           Canada, Lake Ontario                 nd-37 (dwt)          Fox et al. (1983)
    Amphipods                                                                       7.5-62 (dwt)
    Mysids                                                                             6 (dwt)

    Benthic organisms in         1983-1984      USA, sea water                        < 5 (dwt)           Malins et al. (1985)
     stomachs of fish

    Clam,                        1982-1983      Canada, Great Lakes area                                  Kauss & Hamdy (1985)
     E. complanatus                                                                nd-83 (r, n=34)

    Marine algae                 1972-1973      United Kingdom, sea water                                 Pearson & McConnell (1975)
     Enteromorpha compressa                                                              nd
     Ulva lactuca                                                                        nd
     Fucus vesiculosis                                                                   8.9
     Fucus serratus                                                                      0.6
     Fucus spiralis                                                                      0.6
                                                                                                                                              

    Table 6 (contd).

                                                                                                                                              

    Type of biota                Year           Location                         Levels determineda       Reference
                                                                                     (µg/kg wwt)

                                                                                                                                              

    Fish
    Ray,                         1972-1973      United Kingdom, sea water                                 Pearson & McConnell (1975)
     Raja clavata (flesh)                                                              0.1-0.4
     Raja clavata (liver)                                                              0.2-1.5

    Plaice,
     Pleuronectes platessa (flesh)                                                     nd-0.4
     Pleuronectes platessa (liver)                                                     0.2-1.2
    Dab,
     Limanda limanda (flesh)                                                            < 0.1
     Limanda limanda (liver)                                                             nd
    Mackerel,
     Scomber scombrus (flesh)                                                          nd-2.6
    Cod,
     Gadus morrhua (flesh)                                                              < 0.1
     Gadus morrhua (air bladder)                                                        0.35
    Others (liver and/or flesh),                                                         nd
     Platycthus flesus
     Solea solea
     Aspitrigla cuculus
     Trachurus trachurus
     Trisopterus luscus
                                                                                                                                              

    Table 6 (contd).

                                                                                                                                              

    Type of biota                Year           Location                         Levels determineda       Reference
                                                                                     (µg/kg wwt)

                                                                                                                                              

    Trout,                                      USA, Niagara River, Lake                0.47              Oliver & Nicol (1982)
     Salmo gairdneri                            Ontario
    Trout                        1981           Canada, Lake Ontario                  1.3 (dwt)           Fox et al. (1983)

    Catfish (flesh)              1973           USA, surface water near              trace-4600           Yurawecz et al. (1976)
    Gaspergoo (flesh)                           chemical plants manufacturing            200
    Buffalo fish (flesh)                        tetrachloroethene                        100
    Mullet (flesh)                                                                      trace
    Sea trout (flesh)                                                                   trace
    Sheepshead minnow (flesh)                                                           trace

    Catfish                      1973           USA, < 40 km from tetrachloro-         10-1200            Yip (1976)
    Carp                                        ethene or trichloroethene                62
    Gaspergoo                                   manufacturing plants                    12-30
    Buffalo fish                                                                         120
    Whiting                                                                              20
    Drum                                                                                 10
                                                                                                                                              

    Table 6 (contd).

                                                                                                                                              

    Type of biota                Year           Location                         Levels determineda       Reference
                                                                                     (µg/kg wwt)

                                                                                                                                              

    Pike perch,                                                                                           Goldbach et al. (1976)
     Stizostedion lucioperca     1972-1976      Netherlands, Ketelmeer (lake)       440 (m, n=8)
                                                Netherlands, IJsselmeer (lake)       23 (m, n=4)
    Perch,
     Perca fluviatilis                          Netherlands, Ketelmeer             130, 400 (n=2)
    Pike,
     Esox lucius                                Netherlands, Ketelmeer                   260
    Tench,
     Tinca tinca                                Netherlands, Ketelmeer                   950
    Common bream,
     Abramis brama                              Netherlands, Ketelmeer              1520 (m, n=5)
                                                Netherlands, IJsselmeer              33 (m, n=5)
    White bream
     Blicca bjoerkna                            Netherlands, Ketelmeer              360 (m, n=3)
    Roach,
     Rutilis rutilis                            Netherlands, Ketelmeer              910 (m, n=10)
                                                Netherlands, IJsselmeer              61 (m, n=4)
    Eel,
     Anguilla anguilla                          Netherlands, IJsselmeer              33 (m, n=4)

    Smelt
     Osmerus eperlanus                          Netherlands, IJsselmeer              43 (m, n=3)
                                                                                                                                              

    Table 6 (contd).

                                                                                                                                              

    Type of biota                Year           Location                         Levels determineda       Reference
                                                                                     (µg/kg wwt)

                                                                                                                                              

    English sole (liver)         1983-1984      USA, sea water                        < 9 (dwt)           Malins et al. (1985)
    English sole (muscle)                                                            < 0.2 (dwt)

    Catfish                                     USA, vicinity of industrial        46 000-120 000         Pereira et al. (1988)
                                                outfall in Calcasieu River          (lipid base)
                                                estuary
    Atlantic croaker                            idem, in Calcasieu River         41 000 (lipid base)
    Blue crab                                                                    12 000 (lipid base)
    Spotted sea trout                                                            15 000 (lipid base)
    Blue catfish                                                                 46 000 (lipid base)

    Coho salmon                  1980           USA, Great Lakes                      nd (n=31)           Clark et al. (1984)
                                                                                  trace-10 (r, n=5)

    Several species              1983           USA, 14 Lake Michigan                    nd               Camanzo et al. (1987)
                                                tributaries and embayments

    Birds
    Guillemot,                   1972-1973      United Kingdom                                            Pearson & McConnell (1975)
     Uria aalge (eggs)                                                                 1.6-9.9
                                                                                                                                              

    Table 6 (contd).

                                                                                                                                              

    Type of biota                Year           Location                         Levels determineda       Reference
                                                                                     (µg/kg wwt)

                                                                                                                                              

    Swan,                                                                                                 Pearson & McConnell (1975)
     Cygnus olor (liver)                                                                 5.2
     Cygnus olor (kidney)                                                                nd
    Moorhen,                     1972-1973      United Kingdom                                            Pearson & McConnell (1975)
     Gallinula chloropus (liver)                                                         0.8
     Gallinula chloropus (muscle)                                                        2.6
     Gallinula chloropus (eggs)                                                          nd
    Others                                                                               nd
     Sula bassana (liver, eggs)
     Phalacrocerax aristotelis (eggs)
     Alca torda (eggs)
     Rissa tridactyla (eggs)
     Anas platyrhyncos (eggs)

    Mammals                      1972-1973      United Kingdom                                            Pearson & McConnell (1975)
    Grey seal,
     Halichoerus grypus (blubber)                                                      0.4-3.6
     Halichoerus grypus (liver)                                                        nd-0.8
    Common shrew,
     Sorex araneus                                                                       nd
                                                                                                                                              

    a dwt = dry weight; r = range of individual values; m = mean of individueal values; n = number of samples; nd = not detectable; wwt = wet
      weight

    Table 7.  Levels of hexachlorobutadiene in food and drinking-water

                                                                                                                                              

    Type of food                 Year           Location                         Levels determineda       Reference
    or drinking-water                                                          (µg/kg wwt or µg/litre)

                                                                                                                                              

    Tap water                    1978           USA, houses bordering Old         nd-trace (r, n=3)       Barkley et al. (1980)
                                                Love Canal, Niagara Falls        0.06-0.17 (r, n=6)

    Well water                   1978           USA, Tennessee, contaminated      nd-2.53 (r, n=28)       Clark et al. (1982)
                                                by leachate from waste dump        0.15 (m, n=22)

    Fresh milk                                  United Kingdom                          0.08              McConnell et al. (1975)
    Butter                                                                                2
    Cheese, eggs                                                                         nd
    Meat (3 types)                                                                       nd
    Oils/fats (4 out of 5 types)                                                         nd
     Vegetable cooking oil                                                               0.2
    Beverages (5 out of 6 types)                                                         nd
     Light ale                                                                           0.2
    Fruits/vegetables (5 out of 7 types)                                                 nd
     Tomatoes                                   United Kingdom, reclaimed lagoon         0.8
     Black grapes                               United Kingdom, import                   3.7
                                                                                                                                              

    Table 7 (contd).

                                                                                                                                              

    Type of food                 Year           Location                         Levels determineda       Reference
    or drinking-water                                                          (µg/kg wwt or µg/litre)

                                                                                                                                              

    Fresh bread                                 United Kingdom                           nd

    Eggs                         1973           USA, < 40 km from tetrachloro-        nd (n=15)           Yip (1976)
    Milk                                        ethylene or trichloroethylene         nd (n=19)
                                                manufacturing plants, 6-7 sites    1320 (n=1, fat basis)

    Vegetables (7 types)                                                              nd (n=20)
    Condensed milk               1975           Germany, Bonn                             4               Kotzias et al. (1975)
    Milk (products) (2 types)                                                            nd
    Eggs (white)                                                                         nd
    Eggs (yolk)                                                                          42
    Meats (4 types)                                                                      nd
    Tinned fish (2 types)                                                                nd
    Onion bread                                                                          nd
    Chicken feed                                                                         39
    Chicken meal                                                                          2
                                                                                                                                              

    a nd = not detected; m = mean of individual values; n = number of samples; r = range; wwt = wet weight
    

    6.  KINETICS AND METABOLISM

    6.1  Absorption and distribution

         Whole body autoradiography of longitudinal sagittal sections of
    male rats after administration of a single oral dose of 200 mg
    uniformly labelled hexachlorobutadiene/kg body weight in corn oil
    demonstrated that intestinal absorption of the parent compound was
    virtually complete by 16 h. The radioactivity in the
    gastrointestinal tract at this point in time was mainly due to
    water-soluble metabolites, whereas 85% of the radioactivity in the
    small intestine was still present as unchanged hexachlorobutadiene
    4 h after the administration. At all points in time radioactivity
    levels in the stomach were low compared to those in the intestines.
    The autoradiogram showed a specific distribution of radioactivity,
    especially in the outer medulla of the kidney (Nash  et al., 1984).

         Reichert  et al. (1985) orally administered 1 or 50 mg of
    labelled hexachlorobutadiene/kg body weight in tricaprylin to female
    rats and recovered, at 72 h, approximately 7% of the label in
    carcass and tissues, mainly liver, brain and kidneys. Most of the
    label was excreted via urine or faeces within this time period
    (section 6.4). In mice given 30 mg of labelled hexachlorobutadiene
    per kg body weight in corn oil, over 85% of the label was excreted
    within 72 h (section 6.4); 6.7-13.6% was found in the carcass,
    especially in adipose tissue (Dekant  et al., 1988a). This report
    on mice supports the study by Reichert  et al. (1985) on rats with
    respect to the amount of labelled hexachlorobutadiene absorbed.

    6.2  Metabolism

         The extent of metabolic transformation and the identity of
    excretion products found in studies with rodents are summarized in
    Table 8. The available evidence suggests that hexachloro-butadiene
    is metabolized in a glutathione-dependent reaction to toxic sulfur
    metabolites. The glutathione- S-conjugate 1-(glutathion-S-yl)-
    1,2,3,4,4-pentachloro-1,3-butadiene (GPB) is formed in the liver and
    excreted with bile. GPB is reabsorbed from the gut both intact and
    after degradation to 1-(cystein- S-yl)-1,2,3,4,4-pentachloro-
    1,3-butadiene (CPB). Finally, these sulfur conjugates and the
    corresponding mercapturic acid 1-( N-acetylcystein- S-yl)-
    1,2,3,4,4-pentachloro-1,3-butadiene (ACPB) are delivered to the
    kidney. In the kidney, high concentrations of CPB are present due to
    renal accumulation, enzymes with acylase activity and
    gamma-glutamyltranspeptidase. CPB is finally cleaved by renal
    cysteine conjugate ß-lyase to the electrophile
    trichlorovinyl-chlorothioketene. The renal accumulation of sulfur
    conjugates and the location of ß-lyase along the nephron (MacFarlane
     et al., 1989) explain the organ- and site-specific toxicity of
    hexachlorobutadiene (Lock, 1987a,b; Anders  et al., 1987; Dekant
     et al., 1990a,b; Koob & Dekant, 1991).


        Table 8.  Tracer studies with [14C] hexachlorobutadiene

                                                                                                                                              

    Species     Route     Dose (mg/kg     Medium                Metabolitea           Fraction of      Time after      Reference
                         body weight)                                                  dose (%)        dosing (h)

                                                                                                                                              

    Rat          ip           0.1         urine                 total                     29               48          Davis et al. (1980)
                                                                water-soluble             25               48
                                          faeces                total                     40               48

                             300.1        urine                 total                      7               48
                                                                water-soluble              6               48
                                          faeces                total                      7               48

    Rat         oral          200         urine                 total                     11               120         Nash et al. (1984)
                                                                PBSA                       1               120
                                                                non-ether soluble          7               120
                                          faeces                total                     39               120

    Rat         oral           1          expired air           total                     8.9              72          Reichert et al. (1985)
                                                                HCBD                      5.3              72
                                                                CO2                       3.6              72
                                          urine                 total                    30.6              72
                                          faeces                total                    42.1              72

                              50          expired air           total                     6.6              72
                                                                HCBD                      5.4              72
                                                                                                                                              

    Table 8 (contd).

                                                                                                                                              

    Species     Route     Dose (mg/kg     Medium                Metabolitea           Fraction of      Time after      Reference
                         body weight)                                                  dose (%)        dosing (h)

                                                                                                                                              

                                                                CO2                       1.2              72
                                          urine                 total                    11.0              72
                                          faeces                total                     69               72

    Rat         oral          100         urine                 total                     5.4              24          Reichert et al. (1985);
                                                                S-containing            ca 4.3             24          Reichert & Schutz (1986)
                                                                ACPB }
                                                                MTPB }                    0.5              24
                                                                CMTPB}
                                          faeces                total                     60               72

                oral           1          expired air           total                    7.45              72
                                                                C2                       2.2
                                          urine                 total                    17.5
                                          faeces & gitb         total                    61.8
                                          carcass               total                    10.5

                              100         expired air           total                    7.57              72
                                                                CO2                       0.7
                                          urine                 total                     9.0
                                          faeces & gitb         total                    72.1
                                          carcass               total                     5.8
                                                                                                                                              

    Table 8 (contd).

                                                                                                                                              

    Species     Route     Dose (mg/kg     Medium                Metabolitea           Fraction of      Time after      Reference
                         body weight)                                                  dose (%)        dosing (h)

                                                                                                                                              

    Rat          iv            1          expired air           total                    8.54              72          Payan et al. (1991)
                                                                CO2                       2.6
                                          urine                 total                    21.1
                                          faeces & gitb         total                    59.3
                                          carcass               total                    12.9

                              100         expired air           total                    8.11              72
                                                                CO2                       0.9
                                          urine                 total                     9.2
                                          faeces & gitb         total                    71.5
                                          carcass               total                    11.1

    Mouse       oral          30          expired air           total = HCBD              4.5              72          Dekant et al. (1988a)
                                          urine                 total                     7.2              72
                                          faeces                total                    72.0              72
                                                                HCBD                     > 57              72
                                                                GPB                       7.2              72
                                                                                                                                              

    a For abbreviations see Fig. 1; "total" indicates that no individual chemicals were specified
    b git = gastrointestinal tract
    

    6.2.1  In vitro studies

         Incubation of hexachlorobutadiene with rat or mouse liver or
    kidney subcellular fractions caused a depletion of non-protein
    sulfhydryl groups, which was not due to oxidation (Kluwe  et al.,
    1981).

         The formation of GPB and of 1,4-(bis-glutathion- S-yl)-
    1,2,3,4-tetrachloro-1,3-butadiene (BGTB) is catalysed by
    glutathione- S-transferase in rat and mouse liver microsomes and
    cytosol (Wolf  et al., 1984; Wallin  et al., 1988; Dekant  et al.,
    1988a,b). GPB formation has also been observed in human liver
    microsomes and those from several other species (Oesch & Wolf, 1989;
    McLellen  et al., 1989). Conjugation in mouse liver microsomes, but
    not in those from rat liver, is significantly faster in females than
    in males (Wolf  et al., 1984; Dekant  et al., 1988a).

         GPB formation has also been demonstrated in the isolated
    perfused rat liver; in this system, GPB formed in the liver was
    almost exclusively excreted with bile by a carrier-mediated active
    transport mechanism; only after infusing very high concentrations of
    hexachlorobutadiene was sinusoidal excretion of GPB into the
    perfusate observed (Gietl & Anders, 1991).

         A large number of studies have used GPB, CPB and ACPB to
    further delineate the fate of hexachlorobutadiene in the organism.
    These studies have demonstrated that CPB is the penultimate
    intermediate in hexachlorobutadiene metabolism. CPB is a substrate
    for renal cysteine conjugate ß-lyase and is metabolized by this
    enzyme to 2,3,4,4-tetrachlorobutenoic acid and
    2,3,4,4-tetrachlorothionobutenoic acid (Dekant  et al., 1988a).
    Trichloro-vinyl-chlorothioketene has been identified as the ultimate
    reactive intermediate in hexachlorobutadiene metabolism catalysed by
    ß-lyase (Dekant  et al., 1991). ACPB accumulated by the renal
    organic anion transporter is cleaved to CPB by renal acylases
    (Vamvakas  et al., 1987; Pratt & Lock, 1988).

    6.2.2 In vivo studies

         In  in vivo studies, hexachlorobutadiene caused a marked,
    dose-related depletion of renal nonprotein sulfhydryl (NP-SH) in
    mice at single intraperitoneal doses of 33-50 mg/kg body weight but
    little or no decrease in hepatic NP-SH (Kluwe  et al., 1981; Lock
     et al., 1984). This pattern was also observed in female rats at
    single intraperitoneal doses from 300 mg/kg body weight (Hook
     et al., 1983). Conversely, the compound caused a marked,
    dose-related depletion of hepatic NP-SH in male rats from 300 mg/kg
    body weight intraperitoneally, but no decrease (or even an increase)
    in renal NP-SH (Kluwe  et al., 1981, 1982; Lock & Ishmael, 1981;
    Baggett & Berndt, 1984).

         When cannulated male rats were given intravenously either a
    tracer dose of 0.071 mg radiolabelled hexachlorobutadiene/kg body
    weight or the same dose at 24 h after an intraperitoneal nephrotoxic
    dose of 300 mg/kg body weight in corn oil, 13 and 10% of the label
    was recovered in the bile, respectively, within the 3 h following
    the tracer dose. The labelled material was completely water soluble
    (Davis  et al., 1980).

         In a study by Payan  et al. (1991), rats with cannulated bile
    ducts received once, either orally or intravenously, 1 or 100 mg of
    radiolabelled hexachlorobutadiene/kg body weight. At 72 h after
    exposure, fractional urinary excretion (7.5% of the dose) was
    independent of the dose and route of administration, in contrast to
    the situation in intact rats (see section 6.4). Fractional biliary
    excretion decreased with increasing dose following oral
    administration (66.8% versus 58%) and intravenous injection (88.7%
    versus 72%). Fractional faecal excretion was minimal following
    intravenous injection (3.1% following the low oral dose and 16.2%
    following the high oral dose). In a group of bile duct-duodenum
    cannula-linked rats given one dose of 100 mg/kg body weight, all
    tissue concentrations (kidney, liver, plasma, carcass) and the
    urinary excretions at 30 h after dosing were higher in bile donor
    rats than in recipient rats. The biliary contribution to both
    urinary and tissue concentrations was calculated to be 40%. Of the
    biliary metabolites entering the recipients, 80% was found to be
    reabsorbed.

         Nash  et al. (1984) administered 200 mg labelled
    hexachloro-butadiene in corn oil/kg body weight to male rats with
    exteriorized bile flow. They recovered 35% of the label in the bile
    during the 48 h following treatment, 40% of which was identified as
    GPB (Fig. 1) and 12% as CPB. In another investigation into the
    identity of biliary excretion products, male rats were given
    intravenously an aqueous suspension of 0.026 mg of labelled
    hexachlorobutadiene. During the next two hours over 30% of the label
    was recovered in bile; 35% of this radioactivity was identified as
    GPB and 6% as BGTB (Fig. 1), but the remaining labelled material was
    not identified. Since some of the unidentified peaks disappeared
    after treatment of bile with inhibitors of
    gamma-glutamyltranspeptidase, they probably represent degradation
    products of GPB and BGTP (Jones  et al., 1985).

    FIGURE 1

         The intestinal absorption of GPB and CPB was studied in rats by
    infusing the compounds into the intestine via a biliary cannula.
    When GPB was infused, both GPB and CPB were found in the blood in
    approximately equal concentrations. Higher blood CPB concentrations
    were found after CPB infusion than after GPB infusion (Gietl
     et al., 1991).

         In studies with radiolabelled hexachlorobutadiene, several
    urinary metabolites were identified. The structure of these
    metabolites further supported the hypothesis that
    hexachloro-butadiene is bioactivated by glutathione conjugation.

         ACPB was found to be the main metabolite (representing
    approximately 80% of the radioactivity present in urine) excreted
    after the administration of [14C] hexachlorobutadiene (200 mg/kg)
    in female rats (Reichert & Schütz, 1986). The same authors also
    identified 1-carboxymethylthion-1,2,3,4,4-pentachlorobuta-1,3-diene
    and 1-methylthio-1,2,3,4,4-pentachloro-1,3-butadiene (MTPB) as
    urinary metabolites (Reichert  et al., 1985). It is the opinion of
    the Task Group that the identification of MTPB by diazomethane
    treatment of the urinary extract is questionable.

         In male rats, 1,2,3,4,4-pentachloro-1,3-butadienyl sulfenic
    acid (PBSA) is the only metabolite excreted in urine that has so far
    been identified. The data presented suggest that ACPB is not a major
    urinary metabolite of hexachlorobutadiene in male rats (Nash
     et al., 1984). In urine of mice exposed to radiolabelled
    hexachlorobutadiene (30 mg/kg), CPB, ACPB and
    2,3,4,4-tetra-chlorobutenoic acid were identified as urinary
    metabolites (Dekant  et al., 1988a).

         It is probable that 2,3,4,4-tetrachlorobutenoic acid is formed
    by reaction of the intermediate thioketene with water and further
    hydrolysis of the thionol acid thus formed (Dekant  et al., 1988a).

         The weight of evidence suggests that oxidative reactions
    involving cytochrome P-450 have little role in the metabolism of
    hexachlorobutadiene (Wolf  et al., 1984; Dekant  et al., 1988a).

    6.3  Reaction with body components

         The covalent binding of [14C]-hexachlorobutadiene-related
    radioactivity to tissue proteins has been shown to be time
    dependent, with the highest level occurring during the first 6 h
    after treatment. The half-life of hexachlorobutadiene binding was
    22 h in both liver and kidney (Reichert, 1983; Reichert  et al.,
    1985).

         In a DNA binding study, rats received a single oral dose of
    20 mg [14C]-hexachlorobutadiene, and DNA was isolated from the
    kidneys of these rats at 6, 18.5 and 30 h after dose administration.
    Although the results have been reported only in summary form,
    various levels of radioactivity were recovered with the DNA, but
    there was a marked variation in the level of radioactivity between
    samples. Furthermore complete analysis of the DNA was not performed
    and protein may have been associated with the DNA (Stott  et al.,
    1981).

         Covalent binding to mouse liver and kidney DNA was demonstrated
    after the oral administration of radiolabelled hexachlorobutadiene
    (30 mg/kg body weight) in corn oil (Schrenk & Dekant, 1989). In the
    liver and kidneys, the binding capacity of mitochondrial DNA was
    significantly higher than that of nuclear DNA. The level of binding
    to nuclear DNA in the liver was indistinguishable from that of
    controls. HPLC separation of the hydrolysed DNA indicated the
    presence of three distinct peaks of radioactivity.

    6.4  Excretion

         Following oral administration in rats and mice of single doses
    of hexachlorobutadiene up to 100 mg/kg body weight, the total
    excretion within 72 h was at least 65% of the dose. In mice, less
    than half of a dose of 30 mg/kg body weight was metabolized (Dekant
     et al., 1988a). In rats, assuming that the faeces mostly contain
    unchanged compound and no non-resorbed conjugates, 44% of an orally
    administered low dose of hexachlorobutadiene (1 mg/kg body weight)
    was metabolized (Reichert  et al., 1985). At higher doses the
    percentage of hexachlorobutadiene metabolized decreased
    dramatically. The biotransformation of hexachloro-butadiene in rats
    appears to be a saturable process considering the reduced excretion
    of carbon dioxide and renal metabolites at increasing doses (Davis
     et al., 1980; Reichert  et al., 1985; Reichert & Scuhtz, 1986;
    Payan  et al., 1991). This could be explained by saturation of the
    gastrointestinal absorption, which was observed by Reichert  et al.
    (1985). It should be noted, however, that the observed increase in
    the amount of unchanged hexachloro-butadiene in faeces with
    increasing dose applies only up to 100 mg/kg body weight. At higher
    dose levels, the amount of unchanged hexachlorobutadiene in faeces
    decreases, probably due to a decrease in faecal output (Davis
     et al., 1980; Nash  et al., 1984).

         The results of studies of Payan  et al. (1991) on bile-duct
    cannulated (see section 6.2.2) and intact (see Table 8) rats show
    that saturation of gastrointestinal absorption indeed occurs
    following oral administration.

         Pharmacokinetic data concerning the fate of
    hexachloro-butadiene in organisms were not available to the Task
    Group.

    7.  EFFECTS ON ORGANISMS IN THE ENVIRONMENT

    7.1  Aquatic organisms

    7.1.1  Short-term toxicity

         A summary of short-term aquatic toxicity data is presented in
    Table 9. In most of these studies the concentration of
    hexachloro-butadiene was not reported. Therefore, the actual effect
    concentrations may be lower than the nominal ones. In several cases
    these nominal values far exceed the solubility limits. Based on
    these data the substance is moderately to highly toxic to aquatic
    organisms (Canton  et al., 1990).

         Adverse effects reported in some of these acute tests included
    loss of equilibrium, erratic swimming (Leeuwangh  et al., 1975;
    Laseter  et al., 1976), decreased activity, increased rate of
    opercular movement, jumping (Leeuwangh  et al., 1975), inverted
    positions, fin fibrillation and muscle tetany (Laseter  et al.,
    1976). In a special investigation into kidney pathology, groups of
    five goldfish  (Carassius auratus) received one intraperitoneal
    injection of hexachlorobutadiene at a dose level of 500 mg/kg body
    weight in corn oil. They were subsequently fasted for up to 6 days
    and sacrificed at different points in time up to day 7. Controls
    received corn oil only. The temperature was kept between 18 and
    21 °C. By 24 h the fish showed decreased activity, swam with dorsal
    fins down, and had difficulty in following food. By day 4
    exophthalmos, distended abdomen and ascites were observed. These
    signs of toxicity were all reversible. Relative kidney weights were
    elevated on day 4 only. From 12 h after exposure, P2 and P3
    renal epithelial cells exhibited marked vacuolation and necrosis,
    which persisted up to day 7. Increased gamma-glutamyl-transferase
    (EC 2.3.2.2) staining was seen in P2 and P3 segments
    (Reimschüssel  et al., 1989).

         In a report of this experiment, the fish were sacrificed at
    different points in time up to day 70 after exposure. In one of the
    two experiments the fish also received 5-bromo-2'-deoxyuridine 4 h
    prior to sacrifice. Morphometric analysis of developing nephrons
    showed an increase in the percentage of volume occupied by
    basophilia clusters and developing nephrons from day 14 onwards. The
    apparent number of basophilic clusters and developing nephrons per
    unit surface area was also increased from day 14 (Reimschüssel
     et al., 1990).


        Table 9.  Short-term aquatic toxicity of hexachlorobutadiene

                                                                                                                                              

    Organisms    Species            Temperature   pH   Dissolved    Hardness    Stat/flowa    Exposure  Parameter  Concentration  Reference
                                       (°C)             oxygen      (mg CaCO3   open/closed    period                (mg/litre)
                                                      (mg/litre)    per litre)                   (h)

                                                                                                                                              

    Fresh water
    Algae        Haematococcus          20                                     stat, closed      24       EC10b       > 2      Knie et al.
                 pluvialis                                                                                                     (1983)

    Bacteria     Pseudomonas putida     25        7.0                          stat, closed      16        TTc        > 25     Bringmann &
                                                                                                                               Kuhn (1977)

    Bacteria     Pseudomonas putida     20        7.2                          stat, open        0.5      EC10b       > 0.9    Knie et al.
                                                                                                                               (1983)

    Protozoans   Chilomonas             20        6.9                          stat, closed      48        TTc        > 10     Bringmann et al.
                 paramecium                                                                                                    (1980)

    Molluscs     great pond snail,      19                                     stat,d closed     24       LC50        0.21     Leeuwangh et
                 Lymnaea stagnalis                                                               96       LC50        0.21     al. (1975)e

    Crustaceans  water flea,            20         7                   250     stat, open        24       EC50        0.5      Knie et al.
                 Daphnia magna                                                                                                 (1983)

                 aquatic sowbug,        19                                     stat,d closed     96       LC50        0.13     Leeuwangh et
                 Asellus aquaticus                                                                                             al. (1975)e
                                                                                                                                              

    Table 9 (contd).

                                                                                                                                              

    Organisms    Species            Temperature   pH   Dissolved    Hardness    Stat/flowa    Exposure  Parameter  Concentration  Reference
                                       (°C)             oxygen      (mg CaCO3   open/closed    period                (mg/litre)
                                                      (mg/litre)    per litre)                   (h)
                                                                                                                                              

    Fish         goldfish,             17.5                                    stat,d open       96       LC50        0.09     Leeuwangh et
                 Carassius auratus                                                                                             al. (1975)e

    Fish         zebrafish,             20        8.0     9.0          180     flow, closed      48       LC50        1        Slooff (1979)
                 Brachydanio rerio

    Fish         rainbow trout,                                                                           LC50        0.320    US EPA (1980)
                 Salmo gairdnerii

    Fish         bluegill sunfish,                                                                        LC50        0.326    US EPA (1980)
                 Lepomis macrochirus

    Fish         sheepshead minnow,                                                                       LC50        0.557    US EPA (1980)
                 Cyprinodon variegatus

    Fish         golden orfe,           20         8                   270     open              48       LC50        3        Knie et al.
                 Leuciscus idus                                                                                                (1983)e

    Fish         fathead minnow,        25      6.7-7.6   8.0          45      flow, open        96       LC50        0.10     Walbridge et
                 Pimephales promelas                                                                                           al. (1983)e
                                                                                                                                              

    Table 9 (contd).

                                                                                                                                              

    Organisms    Species            Temperature   pH   Dissolved    Hardness    Stat/flowa    Exposure  Parameter  Concentration  Reference
                                       (°C)             oxygen      (mg CaCO3   open/closed    period                (mg/litre)
                                                      (mg/litre)    per litre)                   (h)
                                                                                                                                              

    Marine
    Crustaceans  harpacticoid           21        7.9     > 5                  stat, open        96       LC50        1.2      Bengtsson &
                 copepod                                                                                                       Tarkpea (1983)

                 grass shrimp,                                                                            LC50        0.032    US EPA (1980)
                 Palaemonetes pugio

                 Mysid shrimp,                                                                            LC50        0.059    US EPA (1980)
                 Mysidopsis bahia

    Fish         sailfin molly,        22-24    6.6-7.9   8-9                  flow, open        26       LC50        4.2      Laseter et
                 Poecilia latipinna                                                              30       LC50        4.5      al. (1976)e,f
                                                                                                 77       LC50        1.4-1.9
                                                                                                 115      LC50        1.7
                                                                                                 138      LC50        1.2

                 pinfish, Lagodon                                                                         LC50        0.399    US EPA (1980)
                 rhomboides
                                                                                                                                              

    a  static or flow-through test, open or closed system           d  semi-static (daily renewal) test
    b  effect is 10% reduction in oxygen consumption                e  analysis for hexachlorobutadiene was reported
    c  TT = toxic threshold for inhibition of cell multiplication   f  salinity was 0.25%, 96-h LC50 was calculated to be 1.6 mg/litre
    

    7.1.2  Long-term toxicity

         The cell multiplication of green algae  (Scenedesmus
     quadricauda) was not inhibited after 8 days of static exposure to
    a nominal concentration of 25 mg/litre (well above pure water
    solubility) in a closed system at 27 °C and a pH of 7 (Bringmann &
    Kühn, 1977). A 14-day LC50 of 0.4 mg/litre was determined for
    2- to 3-month old guppies  (Poecilia reticulata) in a semi-static
    test using an open system at 22 °C, a water hardness of 25 mg
    CaCO3/litre, and a dissolved oxygen concentration of >
    5 mg/litre. No analysis for hexachlorobutadiene was reported
    (Könemann, 1981). In the same test under the same conditions, but
    with analysis for the compound, the 14-day LC50 was 0.16 mg/litre
    (Hermens  et al., 1985).

         In a study by Leeuwangh  et al. (1975), groups of six goldfish
     (Carassius auratus) were each exposed to hexachlorobutadiene in
    tap water at measured concentrations of 0, 0.0003, 0.003, 0.0096 or
    0.03 mg/litre for 49 and 67 days. The static test in an open system
    was conducted at 19 °C, a pH of 7.6, and a dissolved oxygen
    concentration between 3.2 and 6.3 mg/litre. Body weights were
    decreased after 49 days at 0.03 mg/litre, and body weight gain was
    still reduced at 67 days. Abnormal behaviour, jumping,
    incoordination, increased opercular movement and overall immobility
    were noted at 0.0096 mg/litre. After 49 days at 0.0096 mg/litre (no
    data at 0.03 mg/litre), relative liver weights were increased, and
    the activity of liver glucose-6-phosphatase (EC 3.1.3.9) was
    decreased, whereas the activity of liver glucose-6-phosphate
    dehydrogenase (EC 1.1.1.49) was increased. After 67 days the
    activity of liver phenylalanine hydroxylase (EC 1.14.16.1) showed a
    concentration-related increase. No effects were found on haemoglobin
    concentration and haematocrit after 49 days or on the activity of
    serum alanine aminotransferase (EC 2.6.2.1) and serum alkaline
    phosphatase (EC 3.1.3.1) after 67 days.

         Groups of 12 largemouth bass  (Micropterus salmoides) were
    each exposed to hexachlorobutadiene for 10 days at measured
    concentrations of 0.00343 and 0.03195 mg/litre in filtered tap water
    with a salinity of 0.08-0.1%, at 22-24 °C, a pH of 6.6-7.9 and a
    dissolved oxygen concentration of 7.6-8.5 mg/litre. A control group
    comprised 12 water and 12 vehicle (acetone) controls. Plasma
    cortisol levels were increased at both concentrations, but
    haematocrit values were not affected. At the higher concentration
    there was leukocytic infiltration in the kidneys of one of the fish
    and paleness and accentuated lobulation of parenchyma in the livers
    (Laseter  et al., 1976).

         In an early lifestage test, four replicate groups, each of 30
    fathead minnow  (Pimephales promelas) eggs, 2-4 h after spawning,
    were exposed to measured hexachlorobutadiene concentrations in
    sand-filtered and sterilized lake water of 0.0017, 0.0032, 0.0065,

    0.013 and 0.017 mg/litre in an open system. Following hatching
    (4-5 days after spawning), four replicate groups of 15 larvae
    continued to be exposed for 28 days. Control groups of equal size
    were exposed to slightly contaminated water containing
    0.00008 mg/litre. The temperature was 25 °C, pH was 7.4, dissolved
    oxygen concentration 7 mg/litre, and water hardness 45 mg
    CaCO3/litre. The hatchability of embryos and the percentage of
    normal larvae at hatch were not affected. An increased fish
    mortality and a concentration-related decrease of body weight were
    observed at the two highest concentrations at the end of the
    exposure period (Benoit  et al., 1982).

    7.2  Terrestrial organisms

    7.2.1  Short-term toxicity

         Except for one test with birds, reliable tests with terrestrial
    organisms have not been reported.

         Groups of 12 female and four male Japanese quails  (Coturnix
     coturnix japonica) were exposed to a diet containing
    hexachloro-butadiene at levels of 0, 0.3, 3, 10 or 30 mg/kg diet for
    90 days. Each cage contained three females and one male of the same
    dose group. Feed analysis indicated levels close to the nominal
    values. Adults were all histopathologically examined. Eggs were
    collected on days 37-46, 64-73, and 81-90. Six adults died during
    the study: 4 at 0.3 mg/kg, 1 at 10 mg/kg, and 1 at 30 mg/kg, but
    this was not considered to be related to treatment. The survival of
    chicks from eggs collected on days 81-90 was decreased at 10 mg/kg
    only. Egg production, the percentage of fertile eggs, the percentage
    of hatchable eggs, and eggshell thickness were unaffected compared
    to controls (Schwetz  et al., 1974).

    8.  EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO TEST SYSTEMS

    8.1  Single exposure

         The available mortality data are summarized in Table 10.

    8.1.1  Inhalation exposure

    8.1.1.1  Mortality

         In the only reported study of the mortality of
    hexachloro-butadiene following inhalation, groups of 20 SPF mice of
    the OF1 strain were exposed for 6 h (Gage, 1970). The vapour
    concentrations were measured by gas chromatography, and the
    concentrations were within 10% of the nominal value. Results are
    shown in Table 10.

    8.1.1.2  Systemic effects

         The decrease in respiratory rate (reflex bradypnoea) in groups
    of six male Swiss OF1 mice was measured following exposure for
    15 min to hexachlorobutadiene vapour at concentrations between 886
    and 2625 mg/m3. The vapour concentrations were checked by gas
    chromatography. The mice were restrained in a body plethysmograph,
    while the heads were extended into an inhalation chamber. The 50%
    effect level, calculated from the concentration-effect curve, was
    2250 mg/m3 (De Ceaurriz  et al., 1988).

    8.1.2  Oral exposure

    8.1.2.1  Mortality

         Based on acute mortality data, hexachlorobutadiene is slightly
    to moderately toxic to adult rats, moderately toxic to male weanling
    rats, and highly toxic to female weanling rats following ingestion.
    Young rats are far more sensitive than adult rats (Kociba  et al.,
    1977a). Gradiski  et al. (1975) observed delayed mortality (after
    24 h) in oral LD50 studies on rats and mice.

    8.1.2.2  Systemic effects

         Hexachlorobutadiene mainly affects the kidneys and, to a lesser
    extent, the liver. The effects on these organs and the related
    biochemical findings are discussed extensively in sections 8.8.2
    (liver) and 8.8.3 (kidney). In oral LD50 studies on rats and mice,
    Gradiski  et al. (1975) observed hyper-reactivity just after
    exposure, followed by decreased activity and staggering.


        Table 10. Mortality of hexachlorobutadiene from single exposure

                                                                                                                                              

    Species/strain      Age        Sex         Route of exposure     Observation        LD50 (mg/kg          Reference
                                                                    period (days)      body weight)
                                                                                    or LC50 (mg/m3)a
                                                                                                                                              

    Mouse                                      oral                                   87 (78.1-95.9)      Murzakayev (1963)b
     OF1                adult      female      inhalation (6 h)          14           107 (102-113)       Gage (1970)
     OF1                adult      female      oral                      14           65 (60-70)          Gradiski et al. (1975)c
     OF1                adult      male        oral                      14           80 (75-85)          Gradiski et al. (1975)c
     Alderley Park      adult      male        intraperitoneal           14           67 (53-85)          Lock et al. (1984)e
     Alderley Park      adult      female      intraperitoneal           14           85 (65-111)
     C57BL/10J          adult      male        intraperitoneal           14           57 (41-81)
     C3H                adult      male        intraperitoneal           14           25-75
     BALB/c             adult      male        intraperitoneal           14           32-40
     DBA/2J             adult      male        intraperitoneal           14           53 (36-76)

    Rat                                        oral                                   350 (323-377)       Murzakayev (1963)b
     OF2 rat            adult      female      oral                      14           270 (250-290)       Gradiski et al. (1975)c
     OF2 rat            adult      male        oral                      14           250 (220-280)       Gradiski et al. (1975)c
     Sprague-Dawley     adult      female      oral                                   200-400             Kociba et al. (1977a)d
     Sprague-Dawley     adult      male        oral                                   580 (504-667)       Kociba et al. (1977a)d
     Sprague-Dawley     weanling   female      oral                                   46 (26-81)          Kociba et al. (1977a)d
     Sprague-Dawley     weanling   male        oral                                   65 (46-91)          Kociba et al. (1977a)d
                                                                                                                                              

    Table 10 (contd).

                                                                                                                                              

    Species/strain      Age        Sex         Route of exposure     Observation        LD50 (mg/kg       Reference
                                                                    period (days)      body weight)
                                                                                    or LC50 (mg/m3)a
                                                                                                                                              

     Alderley Park      weanling   male        intraperitoneal            7           57 (38-87)          Hook et al. (1983)e
     Alderley Park      29 days    male        intraperitoneal            7           96 (72-128)
     Alderley Park      adult      male        intraperitoneal            7           360 (325-396)

    Guinea-pig                                 oral                                   90 (81.5-98.5)      Murzakayev (1963)b

    Rabbit
     New Zealand        adult      female      dermal (8 h)              14           1120 (890-1400)     Duprat & Gradiski (1978)f
                                                                                                                                              

    a When available, 95% confidence limits are reported between brackets.
    b Observation period, strain, sex, age (or body weight) and vehicle were not reported.
    c Vehicle was olive oil.
    d Confidence limits not calculable; observation until no toxicity was observed any longer; vehicle was corn oil.
    e Vehicle was corn oil.
    f Application undiluted using glass vials (3.6 cm2).
    

    8.1.3  Dermal exposure

    8.1.3.1  Mortality

         Hexachlorobutadiene was harmful to rabbits following acute
    dermal exposure (LD50 = 1120 mg/kg body weight; (range,
    890-1400 mg/kg; Table 10). After a dose of 780 mg/kg body weight,
    death occurred within 24 h from respiratory and cardiac failure
    (Duprat & Gradiski, 1978).

    8.1.3.2  Systemic effects

         New Zealand rabbits dermally exposed to undiluted
    hexa-chlorobutadiene at doses of 1170 and 1550 mg/kg body weight
    exhibited stupor, dyspnoea and cyanosis (Duprat & Gradiski, 1978).

    8.1.4  Other routes of exposure

         Hexachlorobutadiene has been shown to be toxic to various
    strains of mice after intraperitoneal injection (Lock  et al.,
    1984) and harmful to adult rats (Hook  et al., 1983). The compound
    was considerably more toxic to young and weanling rats (Table 10).

         Rats intraperitoneally exposed to single lethal doses of
    hexachlorobutadiene from 500 to 1000 mg/kg in corn oil exhibited
    piloerection, sedation, hunching, incoordination, loss of muscle
    tone and hypothermia (Lock & Ishmael, 1979). A macroscopic and
    haematological investigation of rats intraperitoneally exposed to
    doses between 121 and 336 mg/kg body weight in olive oil did not
    reveal any damage to the gastrointestinal tract, spleen, heart or
    gonads. In the lungs, congestion, haemorrhage, and oedema were
    observed, but these were attributed by the authors to ether
    anaesthesia. At dose levels of 213 mg/kg body weight or more,
    lymphopenia and related neutrophilia were induced (Gradiski  et al.,
    1975).

    8.2  Short-term exposure

    8.2.1  Inhalation exposure

         In the only inhalation study published, groups of four adult
    Alderley Park SPF rats of each sex were dynamically exposed to
    nominal concentrations of 53, 107 or 267 mg/m3 6 h/day for 15
    days, 1067 mg/m3 6 h/day for 12 days, or 2668 mg/m3 4 h/day for
    2 days. Petroleum ether was used as a solvent for concentrations
    below 1067 mg/m3. Many other chemicals were tested similarly, and
    batches of control rats of unknown size were maintained at intervals
    of 2 months during the whole experimental period. No analysis for
    hexachlorobutadiene was carried out. Two of the four female rats
    exposed to 1067 mg/m3 died, and autopsy revealed pale, enlarged
    kidneys, adrenal regeneration and renal cortical tubular

    degeneration with epithelial regeneration. Rats of both sexes lost
    weight at 1067 mg/m3 and the weight gain of females was reduced at
    107 and 267 mg/m3. Irritation of eyes and nose was observed at the
    two highest levels. At 267 and 1067 mg/m3 rats were in a poor
    condition, females being more affected than males. Respiratory
    difficulties were seen at and above 267 mg/m3. Haematological
    examination at the end of the exposure period showed slight anaemia
    in females at 1067 mg/m3. Urinalysis did not reveal abnormalities
    at any of the exposure levels.  Macroscopically enlarged, pale
    kidneys were found at 267 and 1067 mg/m3 and enlarged adrenals at
    1067 mg/m3. Histopatho-logical investigations revealed proximal
    tubular degeneration in the kidneys and cortical degeneration in
    adrenals at concentrations of 267 mg/m3 or more. No toxic signs
    were observed at the lowest exposure level and autopsy revealed no
    gross abnormalities (Gage, 1970).

    8.2.2  Oral exposure

    8.2.2.1  Rats

         Groups of five adult male Sprague-Dawley rats were exposed to
    daily oral doses of hexachlorobutadiene (0, 0.2 or 20 mg/kg body
    weight) in corn oil for 3 weeks. Only the kidneys were examined
    histologically. At the higher dose level, body weight gain was
    decreased and relative kidney weight increased. Histopathological 
    examination of the kidneys revealed damage in the middle and inner 
    cortical region, including loss of cytoplasm, nuclear pyknosis, 
    increased basophilia and mitotic activity, and increased cellular 
    debris. No toxic signs were observed at a dosage of 0.2 mg/kg per day 
    (Stott  et al., 1981).

         In a study by Kociba  et al. (1971), groups of four female
    Sprague-Dawley rats consumed for 30 days a diet containing
    hexachlorobutadiene, which resulted in ingested nominal daily doses
    of 0, 1, 3, 10, 30, 65 and 100 mg/kg body weight. Analysis of the
    compound in the feed was not reported. Body weights were decreased
    at the two highest dose levels. At 10 mg/kg or more, a dose-related
    decrease in body weight gain was observed along with a decrease in
    food consumption. There was also an increase in haemoglobin
    concentrations, which, although significant, was not clearly dose
    related. There was no effect on serum alanine aminotransferase
    activity (EC 2.6.1.2). A dose-related increase in relative kidney
    weight was observed at dose levels of 3 mg/kg or more.
    Histopathological examination, which was restricted to liver and
    kidneys, showed proximal tubular degeneration, individual cell
    necrosis, and regenerative changes in the kidneys at doses of
    10 mg/kg or more. Hepatocellular swelling was seen at 100 mg/kg. The
    no-observed-adverse-effect level (NOAEL) was 1 mg/kg body weight per
    day.

         In a 2-week experiment, groups of six weanling Wistar-derived
    rats of each sex were exposed to measured dietary
    hexachlorobutadiene levels of 0, 73, 182 or 447 mg/kg (the Task
    Group considered this equivalent to doses of 0, 7.3, 18.2 and
    44.7 mg/kg body weight per day, respectively). At all dose levels,
    body weight and food conversion efficiency (g of weight gain/g of
    food) were decreased in a dose-related manner. Food consumption per
    g of body weight was decreased at 44.7 mg/kg body weight. Relative
    kidney weights were increased at the two highest dose levels. At all
    dose levels a dose-related degeneration of kidney tubular cells was
    observed, especially in the straight limbs of the proximal tubules
    located in the outer medulla. No toxic signs were observed in the
    liver. A NOAEL was not found (Harleman & Seinen, 1979).

         In a follow-up to the dietary study, groups of 10 weanling
    Wistar-derived rats of each sex received daily doses by gavage of 0,
    0.4, 1, 2.5, 6.3 or 15.6 mg/kg body weight in peanut oil for 13
    weeks (Harleman & Seinen, 1979). Body weight gain, food consumption
    and food utilization efficiency were decreased at 6.3 and
    15.6 mg/kg. Polyuria was observed in females at these dose levels
    after week 10, while a dose-related decrease in urine osmolarity
    occurred at dose levels of 2.5 mg/kg or more. In males, the latter
    effect was observed at 15.6 mg/kg. No other changes were observed in
    urinalysis (after week 10) and haematological investigations (after
    week 8). A dose-related increase in relative kidney weight was
    measured in males of all treatment groups but only at 6.3 mg/kg or
    more in females. Dose-related increases in the relative weight of
    liver and spleen were measured at 6.3 mg/kg or more.
    Histopathological examinations revealed changes in liver and
    kidneys. In the livers of males dosed with 6.3 mg/kg or more, an
    increased basophilic, flocky granulation was observed. At dose
    levels of 6.3 mg/kg or more in males and 2.5 mg/kg or more in
    females, there was a dose-related degeneration of the renal proximal
    tubules, as shown by hyperchromatic nuclei, hypercellularity,
    vacuolation and focal necrosis of epithelial cells and a diminished
    brush border. No adverse effects were observed at daily doses of
    1 mg/kg in females or 2.5 mg/kg in males (Harleman & Seinen, 1979).

    8.2.2.2  Mice

         In a two-week study, groups of five B6C3F1 mice of each sex
    were fed a diet containing hexachlorobutadiene at nominal doses of
    0, 30, 100, 300, 1000 or 3000 mg/kg feed for 15 days (calculated by
    the Task Group to be equivalent to 0, 4.3, 14.3, 43, 143 and
    430 mg/kg body weight per day, respectively, using standard values
    for average body weight and food consumption in mice). Analysis of
    the feed was carried out by gas chromatography, and no more than 9%
    loss of the chemical was observed in one day (feed was replaced
    every 2 days). All mice given 143 or 430 mg/kg body weight died or
    were sacrificed in a moribund condition within 7 days. A
    dose-related growth retardation was observed. At the two highest

    doses, the observed toxic effects included renal tubular necrosis,
    hepatic cytoplasmic vacuolization, and testicular degeneration
    characterized by the presence of syncytial giant cell formation of
    spermatocytes. At dose levels of 43 mg/kg body weight or more,
    minimal to mild depletion of bone marrow (characterized by a
    decrease in the haematopoietic cells) was seen in two out of five
    mice of both sexes per dose group. At dose levels of 4.3, 14.3 and
    43 mg/kg body weight, at which all animals survived to the end of
    the study, renal tubular cell regeneration was observed (Yang
     et al., 1989; Yang, 1991).

         In a 13-week study, groups of 10 B6C3F1 mice of each sex were
    fed a diet containing hexachlorobutadiene at concentrations of 0, 1,
    3, 10, 30 or 100 mg/kg feed. Using measurements of food consumption
    and body weight, the authors determined doses of 0, 0.1, 0.4, 1.5,
    4.9 or 16.8 mg/kg body weight per day for males, and 0, 0.2, 0.5,
    1.8, 4.5 or 19.2 mg/kg body weight per day for females. Analysis of
    the compound was carried out as in the two-week study. No
    treatment-related clinical signs or deaths were observed. The
    motility of sperm from treated mice was significantly lower than in
    controls, although this effect was not dose related (Yang  et al.,
    1989; Yang, 1991). Body weight gain was reduced at dose levels of
    4.5 and 19.2 mg/kg body weight in females. Reductions in kidney
    weight occurred at dose levels of 1.5 mg/kg or more in males and at
    19.2 mg/kg body weight in females, and reductions in heart weight
    occurred at 19.2 mg/kg body weight in males. Necropsy revealed a
    treatment-related increase in renal tubular regeneration (prominent
    in the outer stripe of the medulla) at dose levels of 0.2 mg/kg body
    weight or more in females (Table 11). Although the author concluded
    that a NOAEL was not observed for females, the Task Group noted that
    the occurrence of renal tubular regeneration in one out of ten
    female mice in the 0.2-mg/kg body weight group is insufficient
    evidence of an adverse effect at this dose level in females.

        Table 11.  Incidences of renal tubular regeneration in 13-week
               feed studies on B6C3F1 micea

                                                                     

    Concentration     Dose (mg/kg body         Number of mice with
    (mg/kg feed)       weight per day)       lesions/number examined

                     Male         Female      Male            Female
                                                                     

          0          0              0         0/10             0/10

          1          0.1            0.2       0/10             1/10

          3          0.4            0.5       0/10             9/10

         10          1.5            1.8        0/9            10/10

         30          4.9            4.5      10/10            10/10

        100         16.8           19.2      10/10            10/10
                                                                     

    a  Modified from: Yang (1991)
    
    8.3  Long-term exposure

         No long-term inhalation studies have been reported. A study
    describing long-term exposure of mice by the dermal route is
    presented in section 8.7.3.

         An oral toxicity/carcinogenicity test in rats has been reported
    (see section 8.7.2).

    8.4  Skin and eye irritation; sensitization

    8.4.1  Irritation

         The vapour of hexachlorobutadiene has been found to be
    irritating to the eyes and nose of rats (Gage, 1970; see section
    8.2.1).

         Groups of six New Zealand rabbits received either 0.78 g
    (0.5 ml) of undiluted hexachlorobutadiene on the intact or abraded
    skin for 24 h, or 0.15 g (0.1 ml) in the conjuctival sac of the left
    eye. Assessment of the degree of irritation was conducted according
    to Draize  et al. (1944) and by calculating the primary irritation
    index. Hexachlorobutadiene was moderately irritating for the skin

    (primary irritation index 4) but not irritating for the eyes
    (primary irritating index 1.5). Moderate conjunctivitis, epithelial
    abrasion and, at day 7, epithelial keratitis were observed in the
    eyes (Duprat  et al., 1976).

         Duprat & Gradiski (1978) applied undiluted hexachloro-butadiene
    to New Zealand rabbits at doses of 0.39, 0.78, 1.17 and 1.55 mg/kg
    body weight (0.25, 0.50, 0.75 and 1.00 ml, respectively) under
    occluded conditions, using glass vials, for 8 h. The observation
    period was 14 days. The skin was histopathologically examined in all
    dead animals, in half the survivors at day 15, and in the remaining
    survivors at day 36. After 12 h of exposure to the two highest
    doses, epidermis and subcutaneous tissue revealed oedema and
    polymorphonuclear leukocyte infiltration. In the epidermal cells,
    degeneration with pyknosis of nuclei and perinuclear oedema, and
    focal separation from the corium with vesicle formation were seen.
    After 3 to 5 days of exposure to the three highest doses, dermal
    necrosis was observed, leading to eschar formation and partial
    destruction of hair follicles. The effects increased with time, not
    with dose. Two to five weeks after application, repair was apparent
    at all dose levels, with scarring and upper dermis fibrosis, and
    epidermal acanthosis with focal dyskeratosis. Diffuse mononuclear
    infiltrate was seen in the dermis.

    8.4.2  Sensitization

         A group of 20 Hartley guinea-pigs were treated according to the
    Magnusson-Kligman protocol by intradermal injections of 5%
    hexachlorobutadiene in peanut oil and, after one week and subsequent
    treatment by sodium lauryl sulfate, by a 48-h dermal application of
    a 25% suspension of the chemical in vaseline. The challenge was
    performed by dermal application of a 20% suspension in vaseline. A
    group of five controls was induced similarly and challenged by
    vaseline only. All exposed animals, but none of the controls, showed
    a positive reaction. The test was repeated in the same fashion
    without adjuvant in five guinea-pigs: all animals showed positive
    reactions (Gradiski  et al., 1975).

    8.5  Reproduction, embryotoxicity and teratogenicity

    8.5.1  Reproduction

         A group of female albino rats was exposed to one dose of
    hexachlorobutadiene (20 mg/kg body weight) administered
    subcutaneously before mating. Within 90 days after exposure, all 86
    newborn rats had died, compared with 13 of the 61 controls. The
    offspring from exposed dams were reported to show excitation,
    disturbances of motor coordination, a decrease in appetite and a
    loss of weight, lymphocytosis, neutropenia, myelocytes, Jolly's and
    Cabeau's bodies, pneumonia, bronchitis, granular dystrophy of renal
    cells, glomerulonephritis, inflammatory destructive lesions of the
    gastrointestinal tract and vascular hyperaemia (Poteryayeva, 1966).

    The Task Group noted major deficiencies and incomplete reporting of
    the experiment, the unusual route of administration, and the high
    percentage of mortality in control rats.

         Groups of 10-12 male and 20-24 female Sprague-Dawley rats
    received a diet containing hexachlorobutadiene at dose levels of
    0.2, 2.0 or 20 mg/kg body weight per day for 90 days prior to
    mating, 15 days during mating, and subsequently throughout gestation
    and lactation. In the mating period, two females were placed with
    one male of the same dose group. The control group consisted of
    17 males and 34 females. The diets were reportedly analysed for the
    test compound. No mortality was observed. At 20 mg/kg, adults showed
    decreased food consumption and body weight gain. Blood urea
    nitrogen, serum alanine aminotransferase (EC 2.6.1.2) and serum
    creatinine were unchanged compared to controls. The dams had an
    increased relative brain weight and the male rats had an increased
    relative liver weight at 20 mg/kg. The relative kidney weights were
    increased in both sexes at 20 mg/kg. At 2 and 20 mg/kg the kidneys
    of adult rats revealed dose-related tubular dilatation and
    hypertrophy with foci of epithelial degeneration and regeneration;
    however, there was no effect at 0.2 mg/kg. The only adverse
    reproductive effect in neonates was a decreased weanling weight at
    20 mg/kg. There was no detectable effect on the percentage
    pregnancy, the period from first cohabitation to delivery, survival
    indices, sex ratio, histopathology of weanlings, and the incidence
    of skeletal alterations and abnormalities in neonates (Schwetz
     et al., 1977).

         In a third reproductive study, groups of six female SPF
    Wistar-derived rats, 10 weeks of age, received a diet containing
    hexa-chlorobutadiene at levels of 0, 150 and 1500 mg/kg diet
    (estimated by the Task Group to be equivalent to 0, 7.5 and 75 mg/kg
    body weight per day) for 3 weeks prior to mating, 3 weeks during
    mating, and subsequently throughout gestation and lactation. In the
    mating period, two untreated males were placed with the females,
    after which the females were housed individually. The 75-mg/kg
    female adults were killed in week 10, while those given 0 or
    7.5 mg/kg were killed in week 18. Food analysis at the low dose
    level revealed hexachlorobutadiene levels within 96% of nominal
    values after 1 week and within 81% of nominal values after 2 weeks.
    Diets were prepared weekly. There was a reduced body weight gain by
    female rats in the two groups receiving hexachlorobutadiene.
    Weakness of hind legs, unsteady gait, incoordination and ataxia were
    seen at 75 mg/kg. The relative kidney weight was increased at both
    dose levels. Histopathological investigations revealed
    hypercellularity of epithelial cells, hydropic degeneration, and
    necrosis of proximal tubules in the kidneys at 7.5 mg/kg. At
    75 mg/kg, slight proliferation of bile duct epithelial cells,
    fragmentation and demyelination of single fibres of the femoral
    nerve, and extensive renal degeneration were observed. Again at
    75 mg/kg, no conceptions occurred, the ovaries showing little

    follicular activity, and there was no uterine implantation sites. At
    7.5 mg/kg fertility and litter size were reduced, but not
    significantly. In both the control and 7.5-mg/kg groups, the
    resorption quotient was low. Compared to controls, pup weights were
    reduced significantly on days 0, 10 and 20 in the 7.5-mg/kg group.
    No gross abnormalities were observed (Harleman & Seinen, 1979).

    8.5.2  Embryotoxicity and teratogenicity

         In a teratology study, groups of 24-25 female rats were exposed
    to hexachlorobutadiene vapour at measured concentrations of 0, 21,
    53, 107 or 160 mg/m3 for 6 h per day from days 6 to 20 of
    pregnancy. The breathing zone atmosphere was analysed by gas
    chromatography. Maternal weight gain decreased at 53 and
    160 mg/m3. At the other two exposure levels, the slight decrease
    in maternal weight was not significant. The mean number of
    implantation sites, total fetal losses, resorptions, live fetuses,
    incidences of pregnancy, and sex ratio were not affected by exposure
    to hexachlorobutadiene, compared to controls. Fetal body weight was
    reduced in both sexes at 160 mg/m3. The incidences of external,
    visceral, and skeletal alterations were not significantly increased
    (Saillenfait  et al., 1989).

         In a study by Hardin  et al. (1981), groups of 10-15 mated
    Sprague-Dawley rats received hexachlorobutadiene in corn oil by
    intraperitoneal injection at a dose level of 10 mg/kg body weight
    per day from days 1 to 15 of gestation. It was reported (without
    further details) that at least two maternal organ weights were
    changed and that pre- or postimplantation survival was reduced.
    Maternal tissues did not reveal histopathological effects. Fetuses
    had a reduced weight or length, a 1-2 day delay in heart
    development, and dilated ureters. No grossly visible external or
    internal malformations were observed (Hardin  et al., 1981).

         It was reported briefly by Badaeva  et al. (1985) that daily
    oral administration of hexachlorobutadiene to pregnant rats at a
    dose level of 8.1 mg/kg body weight per day resulted in
    histopathologi-cal changes of nerve cells and myelin fibres of the
    spinal cord in the dams and their offspring.

    8.6  Mutagenicity and related end-points

    8.6.1  In vitro effects

         Purified hexachlorobutadiene induces gene mutations in the Ames
    Salmonella test when specific incubation conditions are employed.

         In preincubation assays adapted to include rat liver microsomes
    and additional reduced glutathione, hexachlorobutadiene induced
    point mutations in  Salmonella typhimurium TA100 (Vamvakas  et al.,

    1988a). Assays lacking specialized metabolic activation conditions
    have generally yielded negative results (Table 12).

         Data from bacterial mutagenicity assays are consistent with the
    proposed scheme for the biotransformation of hexachlorobutadiene in
    animals (section 6.3; Fig. 1). Activity in  S. typhimurium TA100,
    mediated by subcellular fractions of rat kidney, was inhibited by
    the addition of the ß-lyase inhibitor, AOAA (Vamvakas  et al.,
    1988a, 1989a) and the gamma-glutamyltranspeptidase inhibitor,
    acivicin (Vamvakas  et al., 1989a).

         Several of the proposed metabolites of hexachlorobutadiene have
    been assayed for mutagenic activity in  S. typhimurium TA100
    (Table 13). The mono-glutathione (GPB) and mono-cysteine (CPB)
    conjugates were mutagenic in the presence or absence of rat kidney
    S9. Rat liver microsomes and mitochondria that exhibit high
    gamma-glutamyltranspeptidase activities strongly enhanced the
    mutagenic potency of GPB in the presence of additional glutathione,
    in contrast to liver microsomes that exhibit lower
    gamma-glutamyltranspeptidase activity. Furthermore, AOAA and
    acivicin both inhibit the activation of GPB mediated by kidney
    fractions. The di-glutathione (BGTB) and di-cysteine (BCTB)
    conjugates of hexachlorobutadiene were not mutagenic either in the
    presence or absence of rat kidney S9 (Green & Odum, 1985; Dekant
     et al., 1986; Vamvakas  et al., 1988a, 1989a).

         The mercapturic acid, ACPB, was mutagenic both in the presence
    and absence of rat liver S9. It has been suggested that the
    metabolism of ACPB in animals is catalysed by an N-deacetylase and
    by ß-lyase (section 6.3) (Reichert  et al., 1984). The Task Group
    considered that  S. typhimurium possesses both of these enzymes
    activities. Both MTPB and CMTPB gave negative results in tests with
     S. typhimurium TA100 and are considered to be detoxified
    metabolites of hexachlorobutadiene (Wild  et al., 1986).


        Table 12.  Studies on mutagenicity of hexachlorobutadiene

                                                                                                                                              

    Test description              Species/strain/cell type        Conditionsa                         Resultc   Reference

                                                                                                                                              

    Reverse mutations             Salmonella typhimurium TA98,    +/- rat liver S9, purity 98%,          -      De Meester et al.
                                  TA100, TA1530, TA1535, TA1538   plate incorporation                           (1981)

                                  S. typhimurium TA100            +/- rat liver S9, purity > 99%,        -      Stott et al. (1981)
                                                                  plate incorporation

                                  S. typhimurium TA98, TA100      +/- rat liver S9, purity not           -      Reichert et al. (1983)
                                                                  reported, suspension testb

                                  S. typhimurium TA98, TA100,     +/- rat liver S9, purity not           -      Haworth et al. (1983)
                                  TA1535, TA1537                  reported, preincubation test

                                                                  + rat liver S9*, purity > 99.5%,       +
                                                                  preincubation test

                                  S. typhimurium TA100, TA1535    +/- rat liver S9, purity not           -      Chudin et al. (1985)
                                  TA1538                          reported, plate incorporation

                                  S. typhimurium TA100            +/- rat liver S9,                      -      Reichert et al. (1984)
                                                                  - rat liver S9*                        +

                                  S. typhimurium TA100            + rat liver S9*, purity >99.5%,        +d     Wild et al. (1986)
                                                                  preincubation test

                                  S. typhimurium TA100            no activation, purity 98%              +      Vamvakas et al. (1988a)

                                                                  no activation, purity > 99.5%,         -
                                                                  preincubation test
                                                                                                                                              

    Table 12 (contd).

                                                                                                                                              

    Test description              Species/strain/cell type        Conditionsa                         Resultc   Reference

                                                                                                                                              

                                  S. typhimurium TA100            + rat liver microsomes                 -      Vamvakas et al. (1988a)
                                                                  without additional GSH

                                                                  + rat liver microsomes and             +e
                                                                  additional GSH, purity > 99.5%,
                                                                  plate incorporation

    Sex-linked lethals            Drosophila melanogaster         feeding or injection                   -      Woodruff et al. (1985)

    Chromosome aberrations        CHO cells                       +/- rat liver S9                       -      Galloway et al. (1987)

    Chromosome aberrations        human lymphocytes               - rat liver S9                         -      German (1988)

    Sister chromatid exchanges    CHO cells                       +/- rat liver S9                       +      Galloway et al. (1987)

    Chromosome aberrations        mouse bone marrow cells         inhalation, 4 h                        +      German (1988)

    Chromosome aberrations        mouse bone marrow cells         oral gavage                            +      German (1988)
                                                                                                                                              

    a  S9* = a fortified S9 mix containing 3 times the normal protein concentration; GSH = reduced glutathione
    b  The extreme toxicity of the compound without S9 was supposed to exclude testing in this system
    c  + = > twice the background rate or, in the case of bacterial studies, a reproducible dose-related increase in the number of
       revertants per plate; - = negative
    d  0.23 revertants per nmol
    e  Addition of rat kidney microsomes further increased the number of revertants; positive results were inhibited by the ß-lyase inhibitor
       aminooxyacetic acid
    

         Chinese hamster ovary (CHO) cells were exposed to between 5 and
    24 mg hexachlorobutadiene/litre for 2 h in the presence of rat liver
    S9 and throughout the incubation period (8-26 h, depending on cell
    cycle delay) in the absence of rat liver S9. In comparison with
    concurrent controls, no significant increase in chromosome
    aberration frequency was observed (Galloway  et al., 1987). In a
    further study, in which human lymphocyte cultures were exposed to
    between 0.01 and 0.001 mg hexachlorobutadiene per litre in the
    absence of S9 for 27 h, there was also no clastogenic effect. At the
    highest dose level there was a reduction of approximately 60% in the
    mitotic index of human lymphocyte cultures (German 1988). However,
    hexachlorobutadiene at a dose level of at least 4 mg/litre did cause
    a significant increase in the frequency of sister chromatid exchange
    in CHO cells in both the presence and absence of rat liver S9
    (Galloway  et al., 1987).

         Hexachlorobutadiene was found to induce unscheduled DNA
    synthesis (UDS) in Syrian hamster embryo fibroblast cultures.
    Moreover, the magnitude of the response was increased when a
    preincubation period with rat liver S15 was employed (Schiffman  et
     al., 1984). However, there was no induction of UDS in a study
    using rat hepatocyte cultures (Stott  et al., 1981).

         In summary, the Task Group concluded that hexachloro-butadiene
    was genotoxic  in vitro and that the negative results reported in
    some studies may have resulted from the use of inappropriate
    conditions for metabolic activation.

    8.6.2  In vivo effects

         Hexachlorobutadiene induced a significant increase in the
    frequency of chromosomal aberrations in mouse bone marrow cells
    following the administration of acute oral doses of 2 or 10 mg/kg
    body weight or acute inhalation exposure to 10 mg/m3 for 4 h. Both
    experiments used six mice per dose group, and the animals were
    sacrificed after 24 h (German, 1988).

         Six hours after the administration of a single oral dose of
    20 mg hexachlorobutadiene/kg body weight to two groups of five male
    Sprague-Dawley rats, there were statistically significant increases
    in kidney UDS of 27% and 54% above concurrent control levels.
    Administration of a positive control substance,
    dimethyl-nitrosamine, resulted in an increase of 187% over controls
    (Stott  et al., 1981).

         As described in section 6.3, radiolabelled nucleotides were
    recovered from the kidneys of rats and mice administered
    14C-labelled hexachlorobutadiene by gavage (Stott  et al., 1981;
    Schrenk & Dekant, 1989). The Task Group concluded that these studies
    indicated covalent binding of hexachlorobutadiene or its metabolites


        Table 13.  Tests for reverse mutations in  Salmonella typhimurium TA100 by proposed metabolites of hexachlorobutadiene

                                                                                                                                              

    Metabolite and abbreviationa                                         Conditionsb                  Resultc   Reference

                                                                                                                                              

    1-(glutathion- S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene (GPB)        no activation                   -      Green & Odum (1985)
                                                                          + rat kidney S9                 +
                                                                          +/- rat kidney fractions        +d     Vamvakas et al. (1988a)

    1,4-(bis-glutathion- S-yl)-1,2,3,4-tetrachloro-1,3-butadiene          +/- rat kidney fractions        -      Vamvakas et al. (1988a)
        (BGTB)

    1,4-(bis-cystein- S-yl)-1,2,3,4-tetrachloro-1,3-butadiene             +/- rat kidney fractions        -      Vamvakas et al. (1988a)
        (BCTB)

    1-(cystein- S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene (CPB)           +/- rat kidney S9               +e     Green & Odum (1985)
                                                                          no activation                   +f     Dekant et al. (1986)

    1-( N-acetylcystein- S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene         - rat liver S9                 -      Wild et al. (1986)
      (ACPB)                                                              + rat liver S9                  +g

    1-carboxymethylthio-1,2,3,4,4-pentachloro-1,3-butadiene               + rat liver S9*                -      Wild et al. (1986)
      (CMTPB)
                                                                                                                                              

    Table 13 (contd).

                                                                                                                                              

    Metabolite and abbreviationa                                         Conditionsb                  Resultc   Reference

                                                                                                                                              

    1-methylthio-1,2,3,4,4-pentachloro-1,3-butadiene (MTPB)              + rat liver S9                  -      Wild et al. (1986)

    2,2,3,4,4-pentachloro-3-butenoic acid (PBA)                          +/- rat liver S9                +      Reichert et al. (1984)

    2,2,3,4,4-pentachloro-3-butenoic acid chloride (PBAC)                +/- rat liver S9                +      Reichert et al. (1984)
                                                                                                                                              

    a  See also Figure 1
    b  Plate incorporation assays with, except in the case of the tests by Green & Odum, preincubation; S9* = a fortified S9 mix containing
       3 times the normal protein concentration
    c  + = > twice background rate; - = negative
    d  Mutagenic potency enhanced by rat kidney microsomes or mitochondria and less so by cytosol; positive results were inhibited by the
       ß-lyase inhibitor aminooxyacetic acid
    e  Mutagenic potency enhanced by rat kidney S9
    f  Positive results were inhibited by the ß-lyase inhibitor aminooxyacetic acid
    g  18.7 revertants per nmol; mutagenic potency decreased by addition of pyridoxal phospate; activation by cytosol, with and without cofactors,
       had the same results as S9 mix; microsome mix was inactive
    

    to kidney DNA  in vivo. The study with mice showed that the level
    of binding to mitochondrial DNA was greater than that to nuclear
    DNA. In addition, radioactivity was recovered in mitochondrial DNA,
    but not nuclear DNA, from mouse liver (Schrenk & Dekant, 1989).

         Hexachlorobutadiene did not induce sex-linked recessive lethal
    mutations in  Drosophila melanogaster following treatment of adults
    either via the diet or by injection (Woodruff  et al., 1985).

    8.7  Carcinogenicity/long-term toxicity

    8.7.1  Inhalation exposure

         No long-term carcinogenicity studies, where inhalation was the
    route of exposure, have been reported.

    8.7.2  Oral exposure

         In a study by Kociba  et al. (1977a,b), groups of 39-40
    adult Sprague-Dawley rats of each sex received a diet containing
    hexachlorobutadiene at 0.2, 2 or 20 mg/kg body weight per day for 22
    (males) or 24 (females) months. Control groups comprised 90 rats of
    each sex. Analysis for the compound was not reported. An increased
    mortality was observed in males at 20 mg/kg. Hexachlorobutadiene
    caused a depression of the body weight gain in both sexes at the
    highest dose level without any effect on food consumption.
    Haematological investigations performed at 12-14 and 22-24 months,
    revealed a slight, but statistically significant, depression in the
    red blood cell count of males at 20 mg/kg (22 months). Urinalysis at
    12-14 months and 22-24 months did not reveal effects except for a
    small increase in coproporphyrin excretion. The analysis of the
    clinical chemistry parameters of blood urea nitrogen, serum alanine
    aminotransferase (EC 2.6.1.2) and serum alkaline phosphatase
    (EC 3.1.3.1) at 12 months revealed no treatment-related effects,
    except for statistically significant decreases in serum alanine
    aminotransferase in males of the 20-mg/kg dose group and females of
    the 0.2- or 20-mg/kg dose groups. These changes were considered by
    the authors to be of questionable toxicological significance. The
    relative kidney weights were elevated at 20 mg/kg for both sexes, as
    were the relative weights of the brain in females and of the testes
    in males. In both sexes, an extensive histopathological examination
    revealed tubular epithelial hyperplasia at 2 and 20 mg/kg, but not
    at 0.2 mg/kg, and an increased incidence of renal tubular neoplasms
    at 20 mg/kg (see Table 14) (Kociba  et al., 1977a,b).

    8.7.3  Dermal exposure

         In a study by Van Duuren  et al. (1979), groups of 30 female
    Ha:ICR Swiss mice received 6.0 mg hexachlorobutadiene in acetone
    applied 3 times per week to the shaven dorsal skin for between 144
    and 594 days. A group of 100 untreated females were included in the

    study, together with 30 controls which received acetone only. The
    study duration was described as being between 440 and 594 days.
    Sections of skin, liver, stomach and kidney were sampled at autopsy,
    but no increase in the number of distant tumours was observed.

         In a two-stage initiation-promotion experiment, each of
    20 female Swiss mice received one application of 15.0 mg
    hexa-chlorobutadiene in acetone to the dorsal skin. After 14 days,
    the mice similarly received 5 µg of the tumour promoter
    12- o-tetra-decanoylphorbol-13-acetate (TPA) three times weekly for
    between 428 and 576 days. Hexachlorobutadiene administration did not
    induce a significant increase in the fraction of mice developing
    skin papillomas in this study (Van Duuren  et al., 1979).

        Table 14. Renal tubular neoplasms in rats after long-term
              exposure to hexachlorobutadienea

                                                                       

    Dose (mg/kg   Sex    Incidence of renal tubular neoplasms
    body weight
    per day)               adenoma   adenocarcinoma    total

                                                                      

        0         males     1/90          0/90         1/90

        0.2                 0/40          0/40         0/40

        2.0                 0/40          0/40         0/40

       20                   2/39          7/39         9/39 (P < 0.05)

        0         females   0/90          0/90         0/90

        0.2                 0/40          0/40         0/40

        2.0                 0/40          0/40         0/40

       20                   3/40          3/40b        6/40 (P < 0.05)
                                                                       

    a  From: Kociba  et al. (1977a)
    b  One of these was an undifferentiated carcinoma
    
    8.7.4  Exposure by other routes

         In a study of repeated exposure to hexachlorobutadiene by ip
    injection, groups of 20 A/St strain male mice (from 6 to 8 weeks of
    age) received 12 or 13 ip injections of hexachlorobutadiene (4 or
    8 mg/kg body weight) in tricaprylin, respectively. The purity of the
    hexachlorobutadiene was stated to exceed 99.9%. Urethane was used as
    a positive control for carcinogenesis, and a negative control group
    of 50 mice receiving tricaprylin only. Survival was 95% for mice
    receiving 4 mg/kg and 70% for mice receiving 8 mg/kg, compared to
    92% for controls. Mice were sacrificed at 24 weeks after the first
    injection, and the number of surface adenomas in the lungs was
    counted. No significant increase in adenomas, compared to the
    vehicle-treated control, was observed (Theiss  et al., 1977). The
    Task Group noted major deficiencies of this study; including the
    choice of a sensitive strain of mice, the short duration of both the
    exposure period (4 weeks) and the follow-up period (24 weeks), the
    small group sizes of the experimental animals, the unusual route of
    administration, and the limited histopathology. The strain A mice
    used in this study are highly predisposed to spontaneous lung
    cancer, which is likely to have further compromised the value of the
    study.

    8.8  Other special studies

    8.8.1  Effects on the nervous system

         Acute high exposure to hexachlorobutadiene has a depressant
    effect on the central nervous system (see sections 8.1.1.2 and
    8.1.2).

         Subchronic exposure of rats at high dose levels (1500 mg/kg
    diet for 13 weeks) also produced some signs of neurotoxicity, which
    was associated with demyelinization and fragmentation of femoral
    nerve fibres (Harleman & Seinén, 1979; see also Badaeva  et al.,
    1985, section 8.5.2).

    8.8.2  Effects on the liver

    8.8.2.1  Acute effects

         Hexachlorobutadiene causes hydropic changes in the liver of
    rats (Gradiski  et al., 1975; Lock & Ishmael, 1981; Lock  et al.,
    1982), mice (Lock  et al., 1985), and rabbits (Duprat & Gradiski,
    1978), sometimes accompanied by fat accumulation (Duprat & Gradiski,
    1978; Lock & Ishmael, 1981; Lock  et al., 1982).

         Male rats, exposed to a single intraperitoneal dose of
    hexachlorobutadiene (200 or 300 mg/kg body weight) in corn oil
    showed increased relative liver weights, mitochondrial swelling in
    liver and bile duct, proliferation of smooth endoplasmic reticulum,

    lipid accumulation, and increased water content in the liver.
    Biochemical changes in the liver were a decrease, followed after 1
    day by an increase, in non-protein sulfhydryl (NP-SH) concentration,
    and an increase in potassium content. All effects were reversible
    within 10 days. Increases in plasma urea and alkaline phosphatase
    (EC 3.1.3.1.) were also reported. In a separate experiment, the
    highest dose administered by ip injection which did not cause an
    increased water content in the liver was 25 mg/kg body weight (Lock
     et al., 1982).

         Male rats exposed to single intraperitoneal doses up to
    100 mg/kg body weight showed an increase in serum bile acids and
    bilirubin (Bai  et al., 1992).

         Male mice, exposed to single intraperitoneal doses of 50, 100
    and 200 mg/kg body weight in corn oil, showed a dose-related
    increase in relative liver weight at 100 and 200 mg/kg, and, at all
    dose levels, dose-related, reversible changes in the liver
    (mitochondrial swelling, proliferation of smooth endoplasmic
    reticulum, and an increased water content). Reversible biochemical
    changes included increases in sodium and potassium content, NP-SH
    concentration in the liver, and serum alanine aminotransferase
    activity (EC 2.6.1.2) at 50 mg/kg (Lock  et al., 1985).

    8.8.2.2  Short-term effects

         As discussed in section 8.2.2.1, slight hepatotoxic effects
    have been observed following oral exposure of rats (Kociba
     et al., 1971; Harleman & Seinen, 1979).

    8.8.3  Effects on the kidneys

         This section will describe the main features of the renal
    toxicity induced by hexachlorobutadiene. For more detail the reader
    is referred to the reviews of Rush  et al. (1984), Lock (1988),
    Yang (1988) and Dekant  et al. (1990a).

    8.8.3.1  Acute effects

         Inhalation exposure of rats produces renal tubular necrosis
    (Gage, 1970; see section 8.2.1). Enzyme histochemical
    investi-gations were performed on groups of 10 male Swiss OF1 mice
    24 h after whole-body inhalation exposure for 4 h to
    hexachloro-butadiene at measured concentrations of 29.3, 53.4, 106.7
    or 266.8 mg/m3. A concentration-related increase in the percentage
    of damaged kidney tubules, which had been stained for alkaline
    phosphatase (EC 3.1.3.1), was observed at all exposure levels. The
    EC50 was calculated to be 76.8 mg/m3 (De Ceaurriz  et al.,
    1988).

         A single oral dose of hexachlorobutadiene (200 mg/kg body
    weight) in polyethylene glycol caused an increase in plasma urea
    concentration, a decrease in plasma alanine aminotransferase
    activity, and, in urine, increases in the levels of glucose,
    protein, alanine aminotransferase,  N-acetyl-ß-D-glucosaminidase,
    gamma-glutamyltranspeptidase (EC 2.3.2.2) and alanine aminopeptidase
    (EC 3.4.11.12) (Nash  et al., 1984).

         Following  in vivo administration, hexachlorobutadiene caused
    dose-dependent necrosis of the renal proximal tubules in rats
    (Gradiski  et al., 1975; Lock & Ishmael, 1979, 1981; Kluwe  et al.,
    1982; Hook  et al., 1982, 1983; Ishmael  et al., 1982; Ishmael &
    Lock, 1986), mice (Ishmael  et al., 1984) and rabbits (Duprat &
    Gradiski, 1978). In rats, the lesions were restricted to the pars
    recta (S3-segment) and were macroscopically observed as a distinct
    band of damage in the outer stripe of the medulla (Lock & Ishmael,
    1979; Ishmael  et al., 1982). In mice and rabbits both the pars
    recta and the pars convoluta of the proximal convoluted tubules were
    damaged (Duprat & Gradiski, 1978; Ishmael  et al., 1984). The
    lesion is characterized microscopically by necrotic epithelial
    cells, most of which are devoid of nuclei. The few remaining nuclei
    show karyorrhexis, and the cytoplasm is strongly eosinophilic. Many
    renal tubules contained cellular debris (Duprat & Gradiski, 1978;
    Lock & Ishmael, 1979; Lock  et al., 1984; Ishmael  et al., 1984).
    Vacuolation of the pars convoluta was observed (Duprat & Gradiski,
    1978; Ishmael  et al., 1982, 1984). Mitochondrial swelling and loss
    of brush-borders were prominent ultrastructural findings (Ishmael
     et al., 1982, 1984).

         Adult male rats have been found to be less sensitive to the
    renal toxicity induced by hexachlorobutadiene than adult females and
    young males (Hook  et al., 1983; Kuo & Hook, 1983). When male rats
    were dosed intraperitoneally with a single dose of 300 mg/kg in corn
    oil, the earliest pathological change was mitochondrial swelling in
    proximal tubular cells observed after 1-2 h. Extensive necrosis was
    evident between days 1 and 4, and active regeneration by day 5
    (Ishmael  et al., 1982). Similar renal toxicity was seen at a dose
    level of 25 or 50 mg/kg body weight in female rats and young males,
    respectively. A similar pattern of pathological changes with
    comparable intensity was observed in mice at an intraperitoneal dose
    of 50 mg/kg body weight (Ishmael  et al., 1984). In a study by Lock
     et al. (1984), young mice were found to be more susceptible than
    adults, but no sex difference was apparent. In both rats and mice,
    differences in strain susceptibility were observed (Hook  et al.,
    1983; Lock  et al., 1984). The lowest intraperitoneal dose at which
    renal necrosis was observed in adult female rats was 25 mg/kg body
    weight (Lock & Ishmael, 1985) and in adult male and female mice was
    6.3 mg/kg body weight (Lock  et al., 1984).

         Biochemical changes found following intraperitoneal exposure in
    both rats and mice were increases in renal water content (Kluwe  et
     al., 1982; Ishmael  et al., 1982, 1984; Gartland  et al., 1989),
    plasma urea (Lock & Ishmael, 1979, 1981; Ishmael  et al., 1982,
    1984; Hook  et al., 1983; Lock  et al., 1984; Ishmael & Lock,
    1986; Stonard  et al., 1987; Gartland  et al., 1989), plasma
    alkaline phosphatase (EC 3.1.3.1.) (Lock & Ishmael, 1981), serum
    alanine aminotransferase (EC 2.6.1.2) (Gradiski  et al., 1975; Kuo
    & Hook, 1983) and serum aspartate aminotransferase (Gradiski  et
     al., 1975; Davis  et al., 1980). In the urine of rats, increases
    in urinary protein, glucose and ketones have been measured (Lock &
    Ishmael, 1979; Berndt & Mehendale, 1979; Davis  et al., 1980;
    Stonard  et al., 1987), as well as increases in the activities of
    alkaline phosphatase and  N-acetyl-ß-D-glucosaminidase (EC
    3.2.1.50) (Lock & Ishmael, 1979; Stonard  et al., 1987) and in
    lactic acid level (Gartland  et al., 1989). In the kidneys of rats,
    increased sodium concentrations were accompanied by equally
    decreased potassium concentrations (Davis  et al., 1980). All these
    changes occurred at similar or higher intraperitoneal doses than
    those at which renal necrosis was observed.

         Distinct renal functional changes in adult rats have been
    observed at intraperitoneal doses of 100-400 mg/kg body weight.
    These include a decrease in urine-concentrating ability (polyuria)
    (Lock & Ishmael, 1979; Berndt & Mehendale, 1979; Davis  et al.,
    1980; Stonard  et al., 1987), a reduced glomerular filtration rate
    (Davis  et al., 1980) and a reduction of  in vivo renal clearance
    of inulin, urea,  p-aminohippuric acid (PAH), tetraethyl-ammonium
    bromide (TEA) (Lock & Ishmael, 1979) and imipramine (Davis  et al.,
    1980). When organic ion transport was assessed  in vitro in renal
    cortical slices of rats and mice that had been exposed to an
    intraperitoneal dose of 100 mg/kg, 200 mg/kg body weight or more,
    the transport of anions (PAH) was found to be reduced, but the
    transport of the cation (TEA), aminoisobutyrate was not (or was only
    slightly reduced) in rats (Lock & Ishmael, 1979; Berndt & Mehendale,
    1979; Kluwe  et al., 1982; Hook  et al., 1982, 1983). In male
    adult mice, transport of PAH and TEA was reduced from
    intraperitoneal doses of 12.5 and 25.0 mg/kg body weight,
    respectively. The anion transport was reduced in adult females (Lock
     et al., 1984).

    8.8.3.2  Short- and long-term effects

         The short- and long-term effects of hexachlorobutadiene on the
    kidneys of experimental animals have already been discussed in
    sections 8.2 , 8.5 and 8.7. Based on the studies of Kociba  et al.
    (1971, 1977a,b), Schwetz  et al. (1977), Harleman & Seinen (1979),
    Stott  et al. (1981) and Yang  et al. (1989), the oral NOAEL for
    renal toxicity is 0.2 mg/kg body weight per day. Results of the
    studies are summarized in Table 15. Female rats and mice were found
    to be distinctly more susceptible than males upon oral exposure for

    13 weeks (Yang  et al., 1989; Yang, 1991); this was also observed
    in the single-exposure mortality studies (section 8.1).

    8.9  Factors modifying toxicity; toxicity of metabolites

    8.9.1  Factors modifying toxicity

    8.9.1.1  Surgery

         Complete protection from the nephrotoxic effects of
    hexachlorobutadiene was observed in rats that had been fitted with a
    biliary cannula before being given a single oral dose of 200 mg/kg
    body weight. Administration of bile, collected from rats dosed
    orally with the compound, to naive rats produced marked renal
    toxicity but no liver toxicity (Nash  et al., 1984).

    8.9.1.2  Inhibitors and inducers of mixed-function oxidases (MFO)

         In the majority of studies, the effects of MFO inhibitors
    (piperonyl butoxide, SKF 525A) and MFO inducers (Aroclor 1254,
    isosafrole, ß-naphthoflavone, phenobarbitone) on the nephro-toxicity
    induced by hexachlorobutadiene in rats and mice were absent or
    negligible (Lock & Ishmael, 1981; Hook  et al., 1982; Lock  et al.,
    1984; Davis, 1984). Furthermore, phenobarbitone pretreatment for 7
    days at 0.05% in drinking-water enhanced the renal toxicity induced
    by intraperitoneal doses of hexachloro-butadiene in weanling rats
    (Hook  et al., 1983).

    8.9.1.3  Inhibitors of gamma-glutamyltranspeptidase (EC 2.3.2.2)

         Male rats pretreated with Acivicin (L-(alphaS, 5S)-
    alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid), an
    inhibitor of gamma-glutamyltranspeptidase (down to 3% of control
    activity in this study), and subsequently exposed intraperitoneally
    to hexachlorobutadiene, did not show a decrease in nephrotoxicity
    compared to rats treated with hexachlorobutadiene alone. It was
    concluded that gamma-glutamyltranspeptidase inhibition did not limit
    the formation of nephrotoxic metabolites (Davis, 1988).

         Male Swiss OF1 mice, pretreated with Acivicin and
    subsequently exposed to a single oral dose of hexachlorobutadiene
    (80 mg/kg body weight), showed a decrease in nephrotoxicity compared
    to mice treated with hexachlorobutadiene alone, as measured by
    alkaline phosphatase staining (De Ceaurriz & Ban, 1990). The Task
    Group noted that only one marker for nephrotoxicity was employed in
    this study.


        Table 15.  No-observed-adverse-effect level (NOAEL) calculated from short-term and long-term
               studies of exposure to hexachlorobutadiene by oral administration

                                                                                                                                              

    Species (Strain)         Age         Sex       Number of    Duration of      Dose (mg/kg           NOAEL (mg/kg   References
                                                    animals        study         body weight           body weight
                                                   per group      per day)        per day)

                                                                                                                                              

    Rat (Wistar-derived)     weanling    male          6        2 weeks      7.3, 18.2, 44.7               < 7.3    Harleman & Seinen
                                         female        6                                                            (1979)

    Rat (Sprague-Dawley)     adult       male          5        3 weeks      0.2, 20                        0.2     Stott et al. (1981)

    Rat (Sprague-Dawley)     adult       female        4        30 days      1, 3, 10, 30, 65, 100           3      Kociba et al. (1971)

    Rat (Sprague-Dawley)     adult       male        10-12      3 months     0.2, 2.0, 20                   0.2     Schwetz et al.
                                         female      20-24                                                          (1977)

    Rat (Sprague-Dawley)     adult       male        39-40      24 months    0.2, 2.0, 20                   0.2     Kociba et al.
                                         female      39-40                                                          (1977a,b)

    Mouse (B6C3F1)           adult       male          5        2 weeks      4.3, 14.3, 43, 143, 430       < 4.3    Yang et al.
                                         female        5                                                            (1989); Yang (1991)

    Mouse (B6C3F1)           adult       male         10        13 weeks     0.1, 0.4, 1.5, 4.9, 18.8       1.5     Yang et al.
                                         female       10                     0.2, 0.5, 1.8, 4.5, 19.2      < 0.2    (1989); Yang (1991)
                                                                                                                                              
    

    8.9.1.4  Inhibitors of cysteine conjugate ß-lyase

         Male Swiss OF1 mice, pretreated with the two ß-lyase inhibitors
    amino-oxyacetic acid (AOAA) and DL-propargylglycine (PPG) and
    subsequently exposed to a single oral dose of hexachlorobutadiene
    (80 mg/kg body weight), showed a decrease in nephrotoxicity compared
    to mice treated with hexachlorobutadiene alone, as measured by
    alkaline phosphatase staining (De Ceaurriz & Ban, 1990). The Task
    Group again noted that only one marker for nephrotoxicity was
    employed in this study.

    8.9.1.5  Inhibitors of organic anion transport

         Pre-treatment of male rats with probenecid
    [(4-(dipropyl-amino)sulfonyl)] benzoic acid (105 µmol/kg body
    weight), an inhibitor of organic anion transport, did not alter the
    increase in plasma urea or decrease in renal clearance of
     p-aminohippuric acid induced by hexachlorobutadiene (Hook  et al.,
    1982). However, in female rats, a higher dose (500 µmol/kg body
    weight) of probenecid totally protected against the renal toxicity,
    both functional and morphological, produced by ACPB (Lock & Ishmael,
    1985). In addition this dose of probenecid protected female rats
    against the toxic effects produced by CPB and GPBN as well as the
    parent chemical (Lock & Ishmael, 1985). Male mice pre-treated with
    probenecid were also protected against the nephrotoxicity produced
    by hexachlorobutadiene (Ban & De Ceaurriz, 1988). The Task Group
    noted that this latter study used only one marker for
    nephrotoxicity.

    8.9.1.6  Non-protein sulfhydryl scavengers

         Depletion of hepatic and renal non-protein sulfhydryl content
    (glutathione) by diethylmaleate in rats appears to potentiate the
    nephrotoxicity of hexachlorobutadiene as measured by a number of
    functional markers such as plasma urea (Hook  et al., 1982; Baggett
    & Berndt, 1984, Davis  et al., 1986). However, the Task Group noted
    that no information was available on the metabolism of
    hexachlorobutadiene to help interpret these studies.

    8.9.2  Toxicity of metabolites

         This section discusses the renal toxicity of some metabolites
    of hexachlorobutadiene, the formation of which was discussed in
    section 6.2. These metabolites are 1-(glutathion- S-yl)-1,2,3,4,4-
    pentachloro-1,3-butadiene (GPB), 1-(cystein- S-yl)-1,2,3,4,4-
    pentachloro-1,3-butadiene (CPB), and 1-( N-acetylcystein- S-yl)-
    1,2,3,4,4-pentachloro-1,3-butadiene (ACPB). Their mutagenic activity
    was described along with that of hexachlorobutadiene in section 8.6.

    8.9.2.1  In vitro studies

         GPB decreased the viability of isolated renal epithelial cells
    of male rats, as measured by leakage of lactate dehydrogenase (EC
    1.1.1.27), with a very steep dose-response curve and a lag period of
    30 min. No cytotoxicity was observed when the GPB metabolism was
    blocked by anthglutin, an inhibitor of gamma-glutamyl-transpeptidase
    (EC 2.3.2.2) or amino-oxyacetic acid (AOAA), an inhibitor of renal
    cysteine conjugate ß-lyase (EC 4.4.1.13). The cytotoxicity of GPB
    was related to an impairment of mitochondrial function, as shown by
    loss of mitochondrial Ca2+ and ATP and inhibition of respiration
    and thiol depletion (Jones  et al., 1986b). Likewise, GPB produced
    a concentration-dependent nephro-toxicity in the isolated perfused
    rat kidney, as indicated by the appearance in the urine of alkaline
    phosphatase, gamma-glutamyl-transpeptidase and glucose (Jones  et
     al., 1986a). These changes were prevented by Acivicin and by AOAA
    (Schrenk  et al., 1988a).

         In the study of Schrenk  et al. (1988a), CPB also caused a
    marked nephrotoxicity in the isolated perfused kidney, which could
    be prevented by AOAA. In isolated rabbit renal tubules, CPB was
    observed to decrease the accumulation of  p-amino-hippuric acid and
    tetraethylammonium (Jaffe  et al., 1983), to affect mitochondrial
    function as shown by effects on cell respiration, and to decrease
    the glutathione content and, after a lag period of 60 min, cell
    viability (Schnellmann  et al., 1987). The effects on respiration
    resulted initially from the uncoupling of oxidative phosphorylation,
    followed later by inhibition of state 3 respiration (Schnellmann  et
     al., 1987). Impaired mitochondrial function was observed in
    CPB-exposed isolated rat renal cortical mitochondria as an inability
    to retain Ca2+, collapse of the membrane potential, impaired state
    3 respiration with succinate as substrate, and nonenzymatic
    depletion of thiol content. The latter effect was blocked by AOAA.
    From these results it was concluded that the reactive intermediate
    formed from CPB interacts with the inner mitochondrial membrane
    (Wallin  et al., 1987). CPB also inhibited rat kidney mitochondrial
    DNA, RNA and protein synthesis, and AOAA blocked this effect.
    Moreover, CPB converted supercoiled DNA to relaxed circular DNA and
    shorter linear fragments (Banki & Anders, 1989). Chen  et al.
    (1990) observed a decreased viability of isolated human renal
    proximal tubular cells upon exposure to CPB, which was again blocked
    by AOAA. Using radiolabelled ACPB and rat renal cortical slices, it
    was established that ACPB is transported by the same renal
    mechanisms involved in the movement of many organic anions into
    tubular fluid. This carrier-mediated transport is reduced by
    specific inhibitors like probenecid and sulfinpyrazone, a
    competitive and metabolic inhibitor like 2,4-dinitrophenol, and the
    transport substrate  p-aminohippuric acid (Lock  et al., 1986).
    This was confirmed by recent studies on the mechanism of uptake of
    GPB and CPB in the isolated perfused rat kidney (Schrenk  et al.,
    1988b). Probenecid has also been reported to protect renal proximal

    tubular cells against ACPB-induced cytotoxicity, as determined by
    monitoring proline incorporation into renal proteins (Bach  et al.,
    1986).

    8.9.2.2  In vivo studies

         A single oral dose of 138 mg/kg body weight (0.27 mmol) of GPB
    or a single equimolar oral dose of 100 mg ACPB/kg body weight in
    polyethylene glycol to male rats caused marked nephrotoxicity
    similar in both biochemical and histopathological aspects to that
    observed with an oral dose of 200 mg/kg body weight (0.97 mmol) of
    hexachlorobutadiene (Nash  et al., 1984). When rats received
    intraperitoneally GPB, CPB or ACPB in polyethylene glycol at single
    doses between 6.25 and 100 mg/kg body weight, increases in plasma
    urea level and renal proximal tubular necrosis were observed at dose
    levels of > 6.25 mg/kg body weight in females and 10 or 12.5 mg/kg
    body weight in males. The conjugates exhibited a similar pattern of
    nephrotoxicity at equimolar doses and were more nephrotoxic than the
    parent compound (Lock & Ishmael, 1985; Ishmael & Lock, 1986). All
    compounds tested were more toxic to female rats than males (Ishmael
    & Lock, 1986). Probenecid pretreatment protected the rats against
    the nephrotoxicity of these metabolites. Probenecid was shown to
    block the active tubular secretion of ACPB and to reduce the extent
    of covalent binding to renal protein (Lock & Ishmael, 1985). In
    mice, GPB and ACPB were also shown to be more toxic than the parent
    compound: renal necrosis was found following single intraperitoneal
    doses of 5.0 mg hexachlorobutadiene/kg body weight, 3.1 mg GPB/kg
    body weight and 3.0 mg ACPB/kg body weight in corn oil, which were
    the lowest doses tested (Lock  et al., 1984). A single
    intraperitoneal dose of 10 mg CPB/kg body weight in DMSO and water
    caused dose-related damage in the pars recta of renal proximal
    tubules in male mice (Jaffe  et al., 1983).

         The nephrotoxicity of some structural analogues of the
    above-mentioned conjugates, e.g.  S-(1,2-dichlorovinyl)-L-cysteine,
    has been investigated extensively and has revealed a remarkable
    similarity (Anders  et al., 1987; Lock, 1988).

    8.10  Mechanisms of toxicity - mode of action

    8.10.1  Mechanisms of toxicity

         The following evidence supports the hypothesis that the
    nephrotoxicity, mutagenicity and carcinogenicity of
    hexachloro-butadiene is dependent on the biosynthesis of the toxic
    sulfur conjugate GPB. This conjugate is mainly synthetized in the
    liver and further metabolized in the bile, gut, and kidneys to the
    CPB. Cysteine conjugate ß-lyase-dependent activation of CPB to a
    reactive thioketene in the proximal tubular cells finally results in
    covalent binding to cellular macromolecules.

    1.   The nephrotoxicity of hexachlorobutadiene in rats was prevented
         by the implantation of a biliary cannula; administration of
         bile from hexachlorobutadiene-treated rats to naive rats
         resulted in nephrotoxicity identical to the nephrotoxicity
         caused by hexachlorobutadiene (see section 8.9.1.1).

    2.   Inhibitors of renal organic anion transport protected rats
         against the nephrotoxicity of hexachlorobutadiene and its
         sulfur conjugates. Inhibition of the organic anion transport
         also protected isolated kidney cells against the nephrotoxicity
         of hexachlorobutadiene-derived sulfur-conjugates (see sections
         8.9.1.3 and 8.9.2.1).

    3.   Anthglutin, Acivicin and aminooxyacetic acid, specific
         inhibitors of gamma-glutamyltranspeptidase and cysteine
         conjugate ß-lyase protected against the cytotoxicity of
         hexachloro-butadiene-derived sulfur-conjugates in freshly
         isolated rat renal proximal tubular cells (see section
         8.9.2.1).

    4.   Synthetic sulfur-conjugates of hexachlorobutadiene show a
         higher nephrotoxicity than the parent compounds in rats and
         mice and produce renal damage identical to the renal damage
         induced by hexachlorobutadiene, based on clinical chemistry and
         histopathological examination (see section 8.9.2.2).

    5.   Hexachlorobutadiene and its sulfur-conjugates are genotoxic in
         bacteria; bioactivation by glutathione conjugation is required
         for hexachlorobutadiene genotoxicity. The ultimate mutagen is
         formed by cysteine conjugate ß-lyase-dependent cleavage of CPB
         (see section 8.6).

    6.   Hexachlorobutadiene induces renal tumours in rats only at doses
         that produce marked nephrotoxicity (see sections 8.7 and
         8.8.2.2).

    8.10.2  Mode of action

         The  in vitro studies of Jones  et al. (1986b), Wallin  et
     al. (1987) and Schnellmann  et al. (1987) on the cytotoxicity of
    sulfur-conjugates to renal tubular cells (section 8.9.1.2) point to
    renal cortical mitochondria as the major target for
    sulfur-conjugates of hexachlorobutadiene, analogous to that
    established for close structural analogues (Dekant  et al., 1990b).
    The hypothesis proposes an interaction of the reactive metabolite
    with the inner mitochondrial membrane, which ultimately causes
    respiratory insufficiency.

    9.  EFFECTS ON HUMANS

    9.1  General population exposure

         Hexachlorobutadiene has been found in postmortem examinations,
    but not in living persons. No pathogenic effects have been recorded
    (see section 5.2).

    9.2  Occupational exposure

         Two reports on certain disorders among agricultural workers in
    vineyards where hexachlorobutadiene has been used as a fumigant
    (Krasniuk  et al., 1969; Burkatskaya  et al., 1982) cannot be
    evaluated, since such workers are known to be occupationally exposed
    to additional substances.

         In two cytogenetic studies of occupationally exposed workers
    from the same plant engaged in the production of
    hexachloro-butadiene, an increase in the frequency of chromosomal
    aberrations in peripheral blood lymphocytes was observed (German,
    1986). The workers were exposed to hexachloro-butadiene
    concentrations that ranged from 1.6 to 16.9 mg/m3. The Task Group
    noted that exposure concentrations were determined by the factory
    and that the frequency of chromosome aberrations was not associated
    with the period of employment.

    9.3  In vitro metabolism studies

         The following studies have been reported:

    a)   Purified human liver microsomal glutathione- S-transferase 
         and human liver cytosol metabolize hexachlorobutadiene to form
         GPB (McLellan  et al., 1989; Oesch & Wolf, 1989).

    b)   The enzyme cysteine conjugate ß-lyase has been isolated and 
         purified from human kidney cytosol (Lash  et al., 1990). The 
         activity of the human cytosolic enzymes with a structurally 
         related compound (1,2,2-trichlorovinyl-L-cysteine) is about 
         10-fold lower than that of rat renal cytosol (Green  et al.,
         1990).

    c)   Studies in isolated human proximal tubular cells have shown 
         that CPB causes a ß-lyase-dependant cytotoxicity (Chen  et al.,
         1990).

         These limited studies suggest that humans have the ability to
    metabolize hexachlorobutadiene to toxic metabolites.

    9.4  Extrapolation of NOAEL from animals to humans

         Conversion of equivalent doses across species can utilize
    allometric relationships that relate physiological and anatomical
    variables across species. Physiological and metabolic rates have
    been shown to relate closely to body weight to the power 0.75
    (Boxenbaum, 1982). The equivalent NOAEL in humans (mg/kg body weight
    per day) can be determined from the following equation:

                              Wa
                     dh = da (--)0.25
                              Wh

    where

         d = dose rate (mg/kg body weight per day) in humans (dh) or
             animals (da)
         wa = weight (kg) of animals (mice 30 g; rats 400 g)
         wh = weight of humans (70 kg)

    The NOAEL for humans, based on the NOAEL in mice, is:

                                               
         dh= (0.2 mg/kg body weight per day) (0.03)0.25
                                               70

           = 0.03 mg/kg body weight per day

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

    10.1  Evaluation of human health risks

    10.1.1  Hazard identification

         The following evaluation is based on toxicity studies in
    experimental animals; however, there are some human  in vitro data
    which indicate that hexachlorobutadiene metabolism can occur by a
    similar route to that shown in experimental animals.

         Hexachlorobutadiene is slightly to moderately toxic based on
    acute oral experiments with adult rats, and moderately to highly
    toxic based on acute oral experiments with weanling rats (based on
    the WHO pesticide toxicity classification). The specific toxicity of
    the compound to the kidneys is higher for females than males.
    Following acute dermal exposure of rabbits, the compound was found
    to be weakly toxic.

         Regardless of species studied and the route of exposure (ip,
    oral, inhalation, dermal) in both short- and long-term studies, the
    target organ for toxicity is the kidney. Bioactivation to produce a
    reactive sulfur metabolite occurs following conjugation with
    glutathione. The monoglutathione conjugate of hexachloro-butadiene
    is processed to the cysteine- S-conjugate, which is then a
    substrate for renal cysteine conjugate ß-lyase. Hexachloro-butadiene
    produces a dose-dependent necrosis of the renal proximal tubules,
    which is followed by regenerative and/or proliferative changes. On
    the basis of both short- and long-term studies in rats and mice
    orally exposed to hexachlorobutadiene, the no-observed-adverse-
    effect level (NOAEL) is 0.2 mg/kg body weight per day. In one
    short-term inhalation study (12 days, 6 h/day), the NOAEL was
    53 mg/m3.

         The vapour of hexachlorobutadiene was found to be irritating to
    the eyes and nose of rats in one short-term inhalation study. The
    undiluted compound appeared corrosive in an experiment with rabbits.
    Based on these limited data, the vapour should be regarded as
    irritating to human mucous membranes and the liquid should be
    regarded as corrosive.

         In a well-conducted Magnusson-Kligman test hexachloro-butadiene
    was a sensitizing agent both with and without adjuvant. Therefore,
    the compound should be regarded as a sensitizing agent for humans.

         In reproductive studies, reduced birth weight and neonatal
    weight gain in rats were observed, but these effects may be
    attributed to maternal toxicity. Developmental toxicity to rat
    fetuses was observed in two teratogenicity tests, but again only at
    levels that were also toxic to the dams. This developmental toxicity
    included reduced birth weight, a 1- to 2-day delay in heart

    development, and dilated ureters, but no gross abnormalities were
    observed.

          In vitro studies have shown that hexachlorobutadiene and, to
    a much greater extent, its sulfur metabolites induce mutations in
     Salmonella typhimurium. In one study of exposure to
    hexachloro-butadiene by inhalation or oral administration, an
    increased frequency of chromosomal aberrations was observed in mouse
    bone marrow cells. There is limited evidence for the genotoxicity of
    hexachlorobutadiene in animals, and insufficient evidence in humans.

         The long-term oral administration of hexachlorobutadiene to
    rats induced an increased frequency of renal tubular neoplasms, but
    only at doses that caused marked nephrotoxicity; at the lowest dose,
    no adverse effects were observed.

         The Task Group concluded that there is limited evidence for the
    carcinogenicity of hexachlorobutadiene in animals (one study in one
    rodent strain) and insufficient evidence in humans.

    10.1.2  Exposure

         Hexachlorobutadiene is mainly a waste product. As such, it can
    be encountered in different environmental compartments, but
    predominantly in sediment and biota (see also 10.2.1). Exposure of
    the general public therefore mainly occurs indirectly via
    drinking-water and food of high lipid content. Assuming a maximum
    concentration of 2.5 µg/litre in contaminated drinking-water and
    10 µg/kg wet weight in contaminated fatty food items (meat, fish,
    milk) and daily intakes of 2 litres drinking-water, 0.3 kg meat,
    0.2 kg fish and 0.5 kg milk, a maximum total daily intake of
    0.2 µg/kg body weight can be calculated for a 70-kg person.

    10.1.3  Hazard evaluation

         The NOAEL for mice or rats exposed to hexachlorobutadiene is
    0.2 mg/kg body weight per day (see Table 15), from which a NOAEL of
    0.03-0.05 mg/kg body weight day has been derived for humans (see
    section 9.4).

         The Task Group considered the margin of safety of 150 between
    the estimated NOAEL in humans and the maximum total daily intake
    (see section 10.1.2) to be sufficient to protect the general
    population against the adverse effects of hexachlorobutadiene.

    10.2  Evaluation of effects on the environment

    10.2.1  Hazard identification

         Hexachlorobutadiene is a chemically stable compound. Complete
    aerobic biodegradation has been observed following adaptation of the
    inoculum. Partial biodegradation was found to occur in a pilot
    sewage treatment plant. Based on these observations and the chemical
    structure, it can be concluded that hexachlorobutadiene is not
    readily biodegradable, but can be considered to be inherently
    biodegradable. Experimental photolysis of hexachlorobutadiene in the
    presence of a surface was rapid, but in the absence of a surface the
    compound is believed to be persistent. Degradation in the atmosphere
    is assumed to occur by a rather slow reaction with hydroxyl
    radicals. A half-life of up to 2.3 years has been calculated.

         Once hexachlorobutadiene is released into the environment,
    intercompartmental transport will occur chiefly by volatilization
    from water and soil, adsorption to particulate matter in water and
    air, and subsequent sedimentation or deposition. In view of a strong
    adsorption potential to organic matter, the compound accumulates in
    sediment and will not migrate rapidly in soils. Both field and
    laboratory exposure data support these conclusions.

         Field and laboratory data also support the high
    bioaccumu-lation potential in aquatic and benthic organisms which
    can be expected on the basis of the lipophilic nature of the
    compound. However, no evidence has been obtained for
    biomagnification.

         Hexachlorobutadiene is moderately to highly toxic to aquatic
    organisms; crustaceans and fish are the most sensitive species. The
    lowest E(L)C50 for freshwater organisms is 0.09 mg/litre
    (goldfish). The lowest chronic NOEC is 3 µg/litre (goldfish).
    Applying the preliminary effect assessment extrapolation procedure,
    as adopted in the OECD Workshop on Aquatic Effect Assessment (OECD,
    1990), an Environmental Concern Level of 0.1 µg/litre can be
    established.

         The toxicity data on terrestrial organisms are insufficient to
    establish any toxicity threshold.

    10.2.2  Exposure

         Current environmental levels in surface waters are generally
    below 0.2 µg/litre, rising to 1.3 µg/litre in highly polluted
    rivers. Levels in the upper sediment can be as high as 120 µg/kg in
    heavily polluted rivers or estuaries. In older sediment layers much
    higher concentrations can be measured. The concentrations in
    freshwater biota measured since 1980 generally do not exceed

    100 µg/kg fresh weight, but in a polluted area can reach 120 mg/kg
    in the lipid of fish.

    10.2.3  Hazard evaluation

         It can be concluded that away from point sources the maximum
    predicted environmental concentration (PEC) is twice the
    extrapolated Environmental Concern Level of 0.1 µg/litre. Aquatic
    organisms therefore may be at risk in polluted surface waters.

         In view of the rather high concentrations of the compound
    measured in some sediments, adverse effects on benthic organisms
    cannot be excluded.

         Considering the toxicity of the substance to mammals (the NOAEL
    for rats or mice is 0.2 mg/kg body weight per day) and its high
    bioaccumulating potential, the consumption of benthic or aquatic
    organisms in polluted surface water by other species may give cause
    for concern. For example, an otter weighing 10 kg and consuming 1 kg
    fish per day in waters containing 0.2 µg hexachlorobutadiene/litre
    could ingest 1200 µg/day (assuming a bioconcentration factor for
    fish of 6000, leading to a concentration of 1200 µg/kg wet weight)
    or 120 µg/kg body weight per day, which is above the calculated
    NOAEL value for the otter (calculated as in section 9.4).

    11.  FURTHER RESEARCH

         Hexachlorobutadiene is primarily a waste product and hence an
    environmental contaminant having only limited use as a fumigant in
    some parts of the world. The Task Group identified the following
    areas for which additional information is needed:

    a)   the degradation of hexachlorobutadiene in the environment
         focusing on photodegradation and biodegradation;

    b)   the terrestrial toxicity of hexachlorobutadiene including tests
         on benthic organisms;

    c)   the genotoxic activity of hexachlorobutadiene  in vivo. A
         further test for micronucleus or chromosome aberration
         induction in mouse bone marrow cells would strengthen the 
         available data;

    d)   the metabolism of hexachlorobutadiene and its glutathione-
         derived conjugates by human liver and renal enzymes and
         inter-individual variability.

    12.  PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES

         The carcinogenic risk of hexachlorobutadiene was evaluated by
    the International Agency for Research on Cancer in 1979 (IARC,
    1979).

         The summary of data reported and the evaluation of the IARC
    monograph on hexachlorobutadiene is reproduced here.

     Experimental data

         Hexachlorobutadiene was tested in one experiment in rats by
    oral administration: it produced benign and malignant tumours in the
    kidneys of animals of both sexes. It was tested inadequately in one
    experiment in mice by intraperitoneal injection.

     Human data

         No case reports of epidemiological studies were available to
    the Working Group.

         The occurrence of hexachlorobutadiene as a by-product in the
    production of various chlorinated hydrocarbons for over 50 years and
    its use in some areas as a pesticide indicate that widespread human
    exposure in both the occupational and general environment occurs.
    This is confirmed by reports of its occurrence in the environment.

     Evaluation

         There is limited evidence that hexachlorobutadiene is
    carcinogenic in rats.

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    RESUME

    1.  Identité, propriétés physiques et chimiques, méthodes d'analyse

         L'hexachlorobutadiène est un liquide ininflammable,
    incombustible, limpide et huileux à la température et la pression
    ordinaires. Il est peu soluble dans l'eau mais miscible à l'éther et
    à l'éthanol.

         On peut le mettre en évidence et le doser par chromatographie
    en phase gazeuse. Les limites de détection sont de 0,03 µg/m3 dans
    l'air, 0,001 µg/litre dans l'eau, de 0,7 µg/kg de matière humide
    dans le sol ou les sédiments et de 0,02 µg/litre dans le sang. Dans
    les tissus, cette limite est de 0,47 µg/kg de tissus frais.

    2.  Sources d'exposition humaine et environnementale

         L'hexachlorobutadiène n'existe pas à l'état naturel. C'est
    essentiellement un sous-produit de la fabrication des hydro-carbures
    chlorés que l'on retrouve dans les fractions lourdes. La production
    annuelle mondiale (dans les fractions lourdes) a été estimée à
    10 000 tonnes en 1982. L'hexachlorobutadiène peut être utilisé pour
    la récupération des gaz contenant du chlore dans les ateliers de
    fabrication du chlore et comme liquide de lavage pour éliminer du
    courant gazeux certains composés organiques volatils. On l'utilise
    également dans les gyroscopes, comme fluide calo-porteur, dans les
    transformateurs, comme liquide isolant ou liquide hydraulique ainsi
    que comme solvant des élastomères, comme intermédiaire et comme
    fumigant.

    3.  Transport, distribution et transformation dans l'environnement

         Les principales voies de pénétration dans l'environnement sont
    les émissions résultant des déchets et les utilisations qui
    entraînent la dispersion du produit. Le transport
    inter-compartimental s'effectue principalement par volatilisation,
    adsorption sur les matières particulaires puis dépôt ou
    sédimentation. L'hexachloro-butadiène ne migre pas facilement dans
    le sol et s'accumule dans les sédiments. Dans l'eau, on le considère
    comme persistant, sauf turbulences importantes. Il n'y a pas
    d'hydrolyse. Le produit semble être facilement biodégradable par
    voie aérobie, encore que le phénomène n'ait pas été étudié à fond.
    L'hexachlorobutadiène présent sur les surfaces subit une photolyse.
    Outre le dépôt, on estime que la réaction de l'hexachlorobutadiène
    avec les radicaux hydroxyles constitue un mode de piégeage important
    de ce composé dans la troposphère, la demi-vie atmosphérique
    estimative de l'hexachlorobutadiène pouvant aller jusqu'à 2,3 ans.
    Le produit a un potentiel élevé de bioaccumulation, comme l'ont
    confirmé les observations en laboratoire et sur le terrain. Ainsi,
    on a trouvé des facteurs de bioconcentration à l'état stationnaire
    (obtenus expérimentalement par rapport au poids de tissus frais)

    respectivement égaux en moyenne à 5800 et 17 000 chez la truite
    arc-en-ciel. On n'a pas observé d'amplification biologique au
    laboratoire ou sur le terrain.

    4.  Niveaux dans l'environnement et exposition humaine

         Le dosage de l'hexachlorobutadiène dans l'air des villes a
    donné dans tous les cas des valeurs inférieures à 0,5 µg/m3. Dans
    les régions écartées, les concentrations sont inférieures à
    1 pg/m3. Dans les lacs et les cours d'eau d'Europe, on a
    enregistré des concentrations pouvant aller jusqu'à 2 µg/litre mais
    les valeurs moyennes sont généralement inférieures à 100 ng/litre.
    Dans la région des grands lacs au Canada, on a obtenu des valeurs
    beaucoup plus faibles (autour de 1 ng/litre). En revanche la teneur
    des sédiments du fond peut, dans cette zone, atteindre 120 µg/kg de
    poids sec. Les couches sédimentaires plus anciennes, remontant aux
    environs de 1960, présentaient des teneurs plus élevées (jusqu'à
    550 µg/kg de matière humide). On a montré que la concentration dans
    les sédiments augmentait avec la granulométrie des particules.

         A en juger par la concentration de l'hexachlorobutadiène dans
    les organismes aquatiques, les oiseaux et les mammifères, le composé
    s'accumule mais ne subit pas d'amplification biologique. Dans les
    eaux polluées, on a relevé des concentrations dépassant 1000 µg/kg
    de tissus frais chez plusieurs espèces et même 120 mg/kg (par
    rapport aux lipides) chez une espèce. Les concentrations actuelles
    restent généralement inférieures à 1000 µg/kg de poids frais à
    distance des points de décharge industrielle.

         On a décelé la présence du composé dans l'urine, le sang et les
    tissus humains. Dans certaines denrées alimentaires ayant une
    fraction lipidique importante, on en a relevé jusqu'à 40 µg/kg et
    dans un cas, plus de 1000 µg/kg.

         D'après une étude, le niveau d'exposition pourrait atteindre
    1,6 à 12,2 mg/m3 et les concentrations urinaires, 20 mg/litre.

    5.  Cinétique et métabolisme

         Après administration par voie orale, l'hexachlorobutadiène est
    rapidement absorbé chez l'animal de laboratoire mais le taux de
    résorption après inhalation ou exposition par voie cutanée n'a pas
    été étudié. Chez le rat et la souris, le composé se répartit
    principalement dans le foie, les reins et les tissus adipeux. Il est
    rapidement excrété. On a mis en évidence une fixation aux protéines
    et aux acides nucléiques dans le foie et les reins.

         La biotransformation du composé chez l'animal de laboratoire se
    révèle être un processus saturable. Elle s'effectue principalement
    par l'intermédiaire du glutathion, l'hexachlorobutadiène étant
    d'abord transformé en conjugué du  S-glutathion. La métabolisation

    de ce conjugué se poursuit ensuite, en particulier au niveau de la
    membrane constituant la bordure en brosse des cellules des tubules
    rénaux, pour donner un métabolite sulfuré réactif qui est
    probablement responsable des effets néphrotoxiques, génotoxiques et
    cancérogènes observés.

    6.  Effets sur les êtres vivants dans leur milieu naturel

         L'hexachlorobutadiène est modérément à très toxique pour les
    organismes aquatiques. Certaines espèces de poissons et de crustacés
    se sont révélées être les plus sensibles, les valeurs de la CL50 à
    96 h. allant de 0,032 à 1,2 et de 0,09 à environ 1,7 mg/litre,
    respectivement pour les crustacés et les poissons. Chez les
    poissons, le rein est organe-cible important.

         On a établi la valeur de la dose sans effets observables à
    0,003 mg/litre, à partir des résultats d'un certain nombre
    d'épreuves à long terme sur certaines espèces d'algues et de
    poissons; cela permet de considérer ce composé comme très toxique
    pour les organismes aquatiques. Parmi les points d'aboutissement
    biologiques étudiés figuraient la toxicité générale, la
    neurotoxicité, la biochimie, l'hématologie, l'anatomopathologie et
    la reproduction. Lors d'une étude de 28 jours portant sur les
    premiers stades de la vie de  Pimephales promelas, une espèce de
    vairon, on a observé que la reproduction n'était pas affectée à des
    concentrations allant jusqu'à 0,017 mg/litre, alors qu'à 0,013 et
    0,017 mg/litre il y avait accroissement de la mortalité et réduction
    du poids du corps. La dose sans effets observables était de
    0,0065 mg/litre.

         On n'a décrit qu'une seule épreuve fiable portant sur des
    organismes terrestres. Lors d'une épreuve de 90 jours sur des
    cailles japonaises qui recevaient une alimentation contenant ce
    composé à des concentrations allant de 0,3 à 30 mg/kg de nourriture,
    on a constaté que la survie des oisillons n'était réduite qu'à
    partir de 10 mg/kg de nourriture.

    7.  Effets sur les animaux de laboratoire et les systèmes
        d'épreuves in vitro

    7.1  Toxicité générale

         L'hexachlorobutadiène est légèrement à modérément toxique pour
    le rat adulte, modérément toxique pour le raton juste sevré et
    extrêmement toxique pour les rattes juste sevrées après
    administration d'une seule dose par voie buccale. Les principaux
    organes-cibles sont le rein et dans une bien moindre mesure, le
    foie.

         D'après les données obtenues sur l'animal d'expérience, les
    vapeurs d'hexachlorobutadiène sont irritantes pour les muqueuses et

    le liquide est corrosif. On peut considérer ce composé comme un
    agent sensibilisateur.

         Chez le rat, la souris et le lapin, l'hexachlorobutadiène
    provoque une nécrose, liée à la dose, des tubules proximaux du rein.
    Les rats mâles adultes sont moins sensibles à la néphrotoxicité que
    les femelles ou les jeunes mâles. Les souriceaux sont plus sensibles
    que les souris adultes sans qu'on puisse observer de différences
    entre les deux sexes. Chez la ratte adulte, la dose intrapéritonéale
    unique la plus faible à laquelle on ait observé une nécrose rénale
    était de 25 mg/kg de poids corporel; elle était de 6,3 mg/kg de
    poids corporel chez les souris adultes mâles et femelles. A des
    doses égales ou supérieures à celles qui entraînaient une nécrose,
    on a observé des modifications biochimiques et une nette
    amélioration de la fonction rénale.

         Lors de six épreuves à court terme où le composé a été
    administré par la voie orale, deux études de reproduction et une
    étude d'alimentation à long terme portant sur des rats, c'est
    également le rein qui s'est révélé être l'organe-cible. Parmi les
    effets liés à la dose, on notait une diminution du poids relatif des
    reins et une dégénérescence de l'épithélium des tubules. La dose
    sans effets nocifs observables au niveau des reins, tirée d'une
    étude de deux ans sur le rat, était de 0,2 mg/kg de poids corporel
    et par jour. Une étude de 13 semaines sur des souris a montré que
    cette dose était de 0,2 mg/kg de poids corporel et par jour pour cet
    animal. Chez les deux espèces, les femelles adultes étaient plus
    sensibles que les mâles adultes.

         Lors d'une étude d'inhalation à court terme (six heures par
    jour pendant 12 jours) on a observé des effets analogues au niveau
    des reins avec une concentration nominale de vapeur
    d'hexa-chlorobutadiène égale à 267 mg/m3; cette concentration a
    également entraîné des difficultés respiratoires, ainsi qu'une
    dégénérescence des corticosurrénales.

    7.2  Reproduction, embryotoxicité et teratogétogénicité

         Deux études d'alimentation portant sur la reproduction ont été
    effectuée sur des rats à des doses quotidiennes allant jusqu'à 20 et
    75 mg/kg de poids corporel respectivement; elles ont fait ressortir
    une réduction du poids de naissance et du gain de poids néonatal aux
    doses toxiques pour la mère. La dose quotidienne de 75 mg/kg de
    poids corporel, qui était hautement toxique, s'est révélée
    suffisante pour empêcher la conception et la nidation intra-utérine.
    On n'a pas observé d'anomalies du squelette.

         Lors de deux études de tératogénicité, des rats ont été exposés
    soit à des vapeurs d'hexachlorobutadiène à des concentrations allant
    de 21 à 160 mg/m3, six heures par jour du sixième au vingtième
    jour de la gestation, soit par voie intrapéritonéale à une dose
    quotidienne de 10 mg/kg de poids corporel (du premier au quinzième

    jour de la gestation). Des effets nocifs ont été notés sur le
    développement des foetus, qui consistaient en une réduction du poids
    de naissance, un retard dans le développement cardiaque, une
    dilatation des uretères, mais pas de malformations macroscopiques.
    Le retard de développement a été observé à des doses qui étaient
    également toxiques pour les mères.

    7.3  Génotoxicité et cancérogénicité

         L'hexachlorobutadiène provoque des mutations géniques dans
    l'épreuve d'Ames sur salmonelle dans des conditions particulières
    qui favorisent la formation de produits de conjugaison avec le
    glutathion. Lors d'une étude  in vivo on a observé des aberrations
    chromosomiques qui n'ont en revanche pas été constatées lors de deux
    autres études  in vitro. Une étude  in vitro portant sur des
    cellules ovariennes de hamster chinois a révélé une augmentation de
    la fréquence des échanges entre chromatides soeurs. On a fait état
    de la très forte mutagénicité des métabolites sulfurés de
    l'hexachlorobutadiène. D'autres études  in vitro ont montré que ce
    composé provoquait une synthèse non programmée de l'ADN dans des
    cultures de fibroblastes embryonnaires de hamsters de Syrie, effets
    qui n'étaient pas observés dans des cultures d'hépatocytes de rats.
    Le composé provoque également une synthèse non programmée de l'ADN
     in vivo mais n'induit pas de mutations létales récessives liées au
    sexe chez  Drosophila melanogaster.

         Lors de la seule étude à long terme (deux ans) qui ait été
    effectuée, des rats ont reçu une alimentation contenant de
    l'hexachlorobutadiène à des doses quotidiennes respectives de 0,2, 2
    ou 20 mg/kg de poids corporel et seule la dose la plus élevée a
    provoqué un accroissement de l'incidence des tumeurs malignes au
    niveau des tubules rénaux.

    7.4  Mécanismes de la toxicité

         La néphrotoxicité, la mutagénicité et la cancérogénicité de
    l'hexachlorobutadiène sont liées à la biosynthèse d'un conjugué
    sulfuré toxique, le 1-glutathion- S-yl-1,2,3,4,4-pentachloro-
    butadiène. Ce conjugué est principalement synthétisé dans le foie et
    métabolisé ensuite dans la bile, l'intestin et les reins en
    1-cystéine- S-yl-1,2,3,4,4-pentachlorobutadiène (CPB). L'activation
    du CPB en thiocétène réactif (qui dépend de la cystéine-conjuguée-
    béta lyase) au niveau des cellules des tubules proximaux, aboutit en
    définitive à la formation de liaisons covalentes avec les
    macromolécules cellulaires.

    8.  Effets sur l'homme

         On n'a pas décrit d'effets pathogènes sur la population dans
    son ensemble.

         On possède deux rapports faisant état de troubles chez des
    ouvriers agricoles qui utilisaient de l'hexachlorobutadiène comme
    fumigant mais il est vrai qu'ils avaient également été exposés à
    d'autres substances. On a également observé un accroissement de la
    fréquence des aberrations chromosomiques dans les lymphocytes du
    sang périphériques de travailleurs employés à la production
    d'hexachlorobutadiène et qui avaient été exposés à des
    concentrations de 1,6 à 12,2 mg/m3.

    9.  Evaluation des risques pour la santé humaine et des
        effets sur l'environnement

    9.1  Evaluation des risques pour la santé humaine

         Comme très peu d'études ont été effectuées sur l'homme,
    l'évaluation repose essentiellement sur les animaux de laboratoire.
    Toutefois les données  in vitro limitées dont on dispose au sujet
    de l'homme incitent à penser que le métabolisme de
    l'hexachloro-butadiène est analogue chez l'homme et l'animal.

         On estime que les vapeurs d'hexachlorobutadiène sont irritantes
    pour les muqueuses et que le liquide est corrosif. Ce composé doit
    également être considéré comme un agent sensibilisateur.

         Les principaux organes-cibles de son action toxique sont les
    reins et dans une mesure bien moindre, le foie. Sur la base des
    études à court et à long terme effectuées sur des rats et des
    souris, la dose quotidienne sans effets nocifs observables est
    évaluée à 0,2 mg/kg de poids corporel. On l'a estimée à 53 mg/m3
    lors d'une étude d'inhalation à court terme chez le rat (12 jours,
    six heures par jour).

         L'action toxique sur le développement, de même que la réduction
    du poids de naissance et du gain de poids néonatal n'ont été
    observés qu'à des doses toxiques pour la mère.

         On a observé que l'hexachlorobutadiène produisait des mutations
    géniques, des aberrations chromosomiques, un accroissement des
    échanges entre chromatides soeurs et une synthèse non programmée de
    l'ADN, encore que certaines études aient donné des résultats
    négatifs. On ne possède que des indices limités en faveur d'une
    génotoxicité de l'hexachlorobutadiène chez l'animal, indices qui
    sont insuffisants en ce qui concerne l'homme.

         On a constaté que l'administration d'hexachlorobutadiène par
    voie orale pendant une longue période à des rats accroissait la
    fréquence des tumeurs malignes au niveau des tubules rénaux, mais il
    s'agissait uniquement de doses élevées fortement néphrotoxiques. En
    ce qui concerne la cancérogénicité de cette substance, les indices
    sont limités chez l'animal et insuffisants chez l'homme.

         En se basant sur la dose quotidienne sans effets nocifs
    observables estimée à 0,2 mg/kg de poids corporel chez la souris ou
    le rat, on a fixé à 0,03-0,05 mg/kg de poids corporel la dose
    quotidienne sans effets nocifs observables chez l'homme. La marge de
    sécurité entre la dose estimative sans effets nocifs observables et
    la dose journalière maximale totale ingérée estimée en se basant sur
    une absorption du composé par l'intermédiaire d'une eau de boisson
    et de produits alimentaires contaminés à forte teneur en lipides,
    est égale à 150.

    9.2  Evaluation des effets sur l'environnement

         L'hexachlorobutadiène est modérément à fortement toxique pour
    les organismes aquatiques: les crustacés et les poissons sont les
    espèces les plus sensibles. On a fixé à 0,1 œg/litre la
    concentration écologiquement préoccupante. On estime que la
    concentration maximale prévisible dans l'environnement à distance
    des sources ponctuelles de pollution est égale à deux fois la dose
    écologique-ment préoccupante extrapolée et, par voie de conséquence,
    que les organismes aquatiques peuvent être menacés dans les eaux de
    surface polluées. On ne peut exclure des effets nocifs sur le
    benthos.

         Compte tenu de la toxicité de l'hexachlorobutadiène pour les
    mammifères, la consommation de benthos ou d'organismes aquatiques
    par d'autres espèces pourrait être préoccupante.

    RESUMEN

    1. Identidad, propiedades físicas y químicas, métodos de análisis

         El hexaclorobutadieno es un líquido no inflamable,
    incombus-tible, claro, oleoso e incoloro a temperatura y presión
    ordinarias. Es poco soluble en el agua, pero miscible con éter y
    etanol.

         La sustancia puede detectarse y determinarse cuantitativamente
    por métodos de cromatografía de gases. Los límites de detección son
    de 0,03 µg/m3 de aire, 0,001 µg/litro de agua, 0,7 µg/kg de peso
    húmedo en el suelo o en sedimentos y de 0,02 µg/litro de sangre. Se
    ha determinado un nivel de 0,47 µg/kg de peso húmedo de tejido.

    2.  Fuentes de exposición humana y ambiental

         No hay indicaciones de que el hexaclorobutadieno exista como
    producto natural. Es principalmente un subproducto de la fabricación
    de hidrocarburos clorados y se presenta en las fracciones pesadas
    (como residuo). La producción anual mundial del compuesto en las
    fracciones pesadas en 1982 se estimó en 10 000 toneladas.

         El hexaclorobutadieno puede utilizarse para recuperar gas que
    contiene cloro en plantas productoras de cloro y como líquido de
    lavado para eliminar ciertos compuestos orgánicos volátiles de las
    corrientes de gases. También se ha utilizado como fluido en
    giróscopos, como transmisor de calor, transformador, fluido aislante
    y fluido hidráulico, disolvente para elastómeros y como
    intermediario y sustancia para fumigar.

    3.  Transporte, distribución y transformación en el medio  ambiente

         Las principales vías de ingreso en el medio ambiente son las
    emisiones de residuos y el uso dispersivo. El paso de un entorno a
    otro ocurre principalmente por volatilización, adsorción a
    corpúsculos de materia y subsiguiente deposición o sedimentación. El
    hexaclorabutadieno no migra rápidamente en el suelo y se acumula en
    el sedimento. Se considera persistente en el agua a menos que haya
    mucha turbulencia. No produce hidrólisis. La sustancia parece ser
    fácilmente biodegradable aeróbicamente, aunque su biodegradabilidad
    no se ha investigado a fondo. El hexaclorobutadieno se fotoliza en
    las superficies. Se supone que, además de la deposición, la reacción
    con radicales hidroxilo es un importante sumidero de
    hexaclorobutadieno en la troposfera y su semivida atmosférica
    estimada es de hasta 2,3 años. La sustancia tiene un elevado
    potencial de bioacumulación, que se ha comprobado mediante
    observaciones en laboratorio y sobre el terreno. En la trucha arco
    iris se han determinado experimentalmente factores de
    bioconcentración en estado estacionario de 5800 y 17 000 como
    promedio, sobre la base del peso húmedo. No se ha observado
    biomagnificación en laboratorio ni sobre el terreno.

    4.  Niveles ambientales y exposición humana

         Se ha determinado la presencia de hexaclorubutadieno en el aire
    urbano; en todos los casos, los niveles eran inferiores a
    0,5 µg/m3. Las concentraciones en lugares aislados son inferiores
    a 7 pg/m3. En las aguas de lagos y ríos de Europa se han
    registrado concentraciones de hasta 2 µg/litro, pero los niveles
    medios son generalmente inferiores a 100 ng/litro. En la región de
    los Grandes Lagos del Canadá se han detectado niveles muy inferiores
    (de aproximadamente 1 ng/litro). Allí los niveles en el sedimento
    del fondo pueden ser de 120 µg/kg de peso en seco. En capas más
    antiguas de sedimento, de 1960 aproximadamente, se encontraron
    concentraciones más elevadas (de hasta 550 µg/kg de peso húmedo). Se
    ha demostrado que la concentración en el sedimento aumenta con el
    tamaño de la partícula de sedimento.

         Las concentraciones de hexaclorobutadieno en organismos
    acuáticos, aves y mamíferos indican bioacumulación pero no
    biomagnificación. En las aguas contaminadas se han detectado niveles
    de más de 1000 µg/kg de peso húmedo en varias especies y de
    120 mg/kg (base grasa) en una especie. Lejos de los efluentes
    industriales, los niveles actuales se mantienen en general por
    debajo de 100 µg/kg de peso húmedo.

         Se ha detectado la presencia del compuesto en la orina, en la
    sangre y en tejidos humanos. En ciertos alimentos que contienen una
    elevada fracción lipídica se han encontrado hasta unos 40 µg/kg y,
    en un caso, más de 1000 µg/kg.

         Un estudio señala exposiciones ocupacionales de
    1,6-12,2 mg/m3 y en la orina niveles de hasta 20 mg/litro.

    5.  Cinética y metabolismo

         Se ha observado que los animales de laboratorio absorben
    rápidamente el hexaclorobutadieno después de la administración oral,
    pero no se ha investigado la velocidad de absorción después de la
    inhalación o de la exposición dérmica. En ratas y ratones, el
    compuesto se distribuye principalmente al hígado, a los riñones y al
    tejido adiposo. Se excreta rápidamente. Se ha demostrado que se fija
    a las proteínas y ácidos nucleicos del hígado y de los riñones.

         La biotransformación del compuesto en animales de
    experimentación parece ser un proceso saturable. Se produce
    principalmente a través de una vía mediada por el glutatión, en la
    cual el hexaclorobutadieno se convierte inicialmente en conjugados
    de  S-glutatión. Estos conjugados pueden seguir metabolizándose,
    especialmente en el ribete en cepillo de las membranas de las
    células de los tubos renales, produciendo un metabolito sulfuroso
    reactivo que probablemente explique la nefrotoxicidad, genotoxicidad
    y carcinogenicidad observadas.

    6.  Efectos en organismos presentes en el medio ambiente

         El hexaclorobutadieno es de moderadamente a muy tóxico para los
    organismos acuáticos. Los más sensibles que se hayan observado han
    sido especies de peces y crustáceos; los valores de la CL50 en
    96 horas oscilan entre 0,032 y 1,2 mg/litro en crustáceos y entre
    0,09 y 1,7 mg/litro en peces. Se ha demostrado que el riñón es un
    órgano muy afectado en los peces.

         Sobre la base de varias pruebas a largo plazo con especies de
    algas y de peces, se ha establecido un nivel sin efectos observados
    de 0,003 mg/litro; así pues, el compuesto se clasifica como muy
    tóxico para las especies acuáticas. Los valores extremos
    investigados comprenden parámetros de toxicidad general,
    neurotoxicidad, bioquímicos, hematológicos, patológicos y
    relacionados con la reproducción. En una prueba de 28 días de
    duración en la que se examinaron las primeras fases de la vida de
    carpas se observó que la reproducción no se veía afectada con
    concentraciones de hasta 0,017 mg/litro, mientras que con
    concentraciones de 0,013 y 0,017 mg/litro se observaron un aumento
    de la mortalidad y una disminución del peso corporal. El nivel sin
    efectos observados era de 0,0065 mg por litro.

         Se ha descrito una sola prueba fiable con organismos
    terrestres. En una prueba de 90 días con codornices japonesas
    alimentadas con una dieta que contenía el compuesto en
    concentraciones de 0,3 a 30 mg/kg de dieta se observó que la
    supervivencia de los polluelos disminuía a partir de 10 mg/kg de
    dieta.

    7.  Efectos en animales de experimentación y en sistemas de
        prueba in vitro

    7.1  Toxicidad general

         Después de la ingestión de una dosis oral única, el
    hexaclorobutadieno es de levemente a moderadamente tóxico para las
    ratas adultas, moderadamente tóxico para las ratas macho destetadas
    y muy tóxico para las ratas hembras destetadas. Los principales
    órganos afectados son el riñón y, en grado mucho menor, el hígado.

         Los datos obtenidos con animales indican que el vapor de
    hexaclorobutadieno es irritante para las membranas mucosas y el
    líquido es corrosivo. La sustancia debe considerarse como un agente
    sensibilizador.

         En los riñones de ratas, ratones y conejos, el
    hexacloro-butadieno causa en los tubos proximales del riñón una
    necrosis que depende de la dosis. Las ratas macho adultas son menos
    vulnerables a la toxicidad renal que las hembras adultas y que los
    machos jóvenes. Los ratones jóvenes son más vulnerables que los
    adultos y no se observaron diferencias entre un sexo y otro. En las

    ratas hembra adultas la dosis intraperitoneal única más baja con la
    cual se observó necrosis renal fue de 25 mg/kg de peso corporal y en
    ratones adultos, machos y hembras, fue de 6,3 mg/kg de peso
    corporal. Se observaron cambios bioquímicos y alteraciones
    funcionales marcados en los riñones con dosis iguales o mayores que
    las asociadas con necrosis.

         Asimismo, en seis pruebas orales de corto plazo, dos estudios
    sobre reproducción y un estudio de largo plazo sobre la dieta
    realizados con ratas, el riñón fue el principal órgano afectado. Los
    efectos relacionados con la dosis comprenden una reducción del peso
    relativo del riñón y una degeneración del epitelio de los tubos. El
    nivel sin efectos nocivos observados de toxicidad renal en ratas en
    un estudio de dos años fue de 0,2 mg/kg de peso corporal por día. En
    un estudio de 13 semanas efectuado en ratones se obtuvo un nivel sin
    efectos nocivos observados de 0,2 mg/kg de peso corporal por día.
    Las hembras adultas de ambas especies eran más vulnerables que los
    machos adultos.

         En una prueba de inhalación de corto plazo (6 horas por día
    durante 12 días) se observaron efectos semejantes en los riñones con
    una concentración de vapor nominal de 267 mg/m3, con la cual
    también se observaron trastornos respiratorios y degeneración de la
    corteza suprarrenal.

    7.2  Reproducción, embriotoxicidad y teratogenicidad

         Dos estudios sobre dieta y reproducción en ratas con dosis de
    hasta 20 y 75 mg/kg de peso corporal por día, respectivamente,
    mostraron una reducción del peso al nacer y un aumento del peso
    neonatal cuando se administraban a la madre dosis tóxicas de 20 y
    7,5 mg/kg de peso corporal, respectivamente. La dosis altamente
    tóxica de 75 mg/kg de peso corporal por día fue suficiente para
    impedir la concepción y la implantación uterina. No se observaron
    anormalidades del esqueleto.

         En dos pruebas de teratogenicidad en las que se expuso a las
    ratas o bien a vapor de hexaclorobutadieno en concentraciones que
    oscilaban entre 21 y 160 mg/m3 durante 6 horas diarias (desde el
    6° hasta el 20° día del embarazo) o bien a la administración
    intraperitoneal de 10 mg/kg de peso corporal por día (desde el 1° al
    15° día de embarazo) se observaron en el desarrollo del feto efectos
    tóxicos tales como una reducción del peso al nacer, un retraso del
    desarrollo del corazón y uréteres dilatados pero sin grandes
    malformaciones. El retraso del desarrollo se observó en niveles que
    también eran tóxicos para las madres.

    7.3  Genotoxicidad y carcinogenicidad

         En la prueba de Ames Salmonella se ha observado que el
    hexaclorobutadieno induce mutaciones genéticas en condiciones
    especiales que favorecen la formación de productos de conjugación

    con el glutatión. En un estudio  in vivo se observó que había
    inducido aberraciones cromosómicas, pero no se observaron tales
    aberraciones en dos estudios  in vitro. En una prueba  in vitro se
    observó que la frecuencia de los intercambios entre cromátidas
    hermanas había aumentado en las células ováricas de hámsters de
    China. Se ha señalado el gran potencial mutagénico de los
    metabolitos sulfurosos del hexaclorobutadieno. En estudios  in
     vitro, el compuesto indujo síntesis imprevistas de ADN en cultivos
    de fibroblastos de embriones de hámsters de Siria, pero no en
    cultivos de hepatocitos. Indujo síntesis imprevistas de ADN en ratas
     in vivo, pero no indujo mutaciones letales recesivas ligadas al
    sexo en Drosophila melanogaster.

         En el único estudio de largo plazo (dos años), en el cual las
    ratas recibieron una dieta que contenía hexaclorobutadieno en dosis
    de 0,2, 2 ó 20 mg/kg de peso corporal por día, se observó una mayor
    incidencia de neoplasias de los tubos renales únicamente con la
    dosis más elevada.

    7.4  Mecanismos de toxicidad

         La nefrotoxicidad, mutagenicidad y carcinogenicidad del
    hexaclorobutadieno depende de la biosíntesis del conjugado sulfuroso
    tóxico 1-glutatión- S-yl-1,2,3,4,4-pentaclorobutadieno. Este
    conjugado se sintetiza principalmente en el hígado y se metaboliza
    luego en la bilis, el intestino y los riñones convirtiéndose en
    1-cisteína- S-yl-1,2,3,4,4-pentaclorobutadieno (CPB). La activación
    de CPB, que depende del conjugado de cisteína beta-lyasa, en una
    tiocetena reactiva en las células de los tubos proximales finalmente
    da lugar a un enlace covalente con macromoléculas celulares.

    8.  Efectos en el ser humano

         No se han descrito efectos patogénicos en la población en
    general.

         Se conocen dos casos de trastornos padecidos por trabajadores
    agrícolas que utilizaban el hexaclorobutadieno como fumigante, pero
    esas personas también habían estado expuestas a otras sustancias. En
    los linfocitos de la sangre periférica de operarios que trabajaban
    en la producción de hexaclorobutadieno y estaban expuestos, según se
    informa, a concentraciones de 1,6 a 12,2 mg/m3 se observó una
    frecuencia mayor de aberraciones cromosómicas.

    9.  Evaluación de los riesgos para la salud humana y de los
        efectos en el medio ambiente

    9.1  Evaluación de los riesgos para la salud humana

         Como se han hecho muy pocos estudios en el ser humano, la
    evaluación se basa principalmente en estudios efectuados en animales
    de laboratorio. Sin embargo, los limitados datos existentes sobre el

    ser humano, obtenidos  in vitro, sugieren que el metabolismo del
    hexaclorobutadieno en el ser humano es semejante al observado en
    animales.

         Se considera que el vapor de hexaclorobutadieno irrita las
    membranas mucosas del ser humano y que en estado líquido es
    corrosivo. El compuesto también debe considerarse como un agente
    sensibilizador.

         Los principales órganos afectados por la toxicidad son los
    riñones y, en mucho menor grado, el hígado. En base a estudios de
    corto y largo plazo de ingestión por vía oral por ratas y ratones,
    se determinó un nivel sin efectos adversos observados de 0,2 mg/kg
    de peso corporal por día. En un estudio de inhalación en el corto
    plazo en ratas (12 días, a razón de 6 horas por día), el nivel sin
    efectos adversos observados fue de 53 mg/m3.

         Se observaron una reducción del peso al nacer y un aumento de
    peso neonatal únicamente con dosis tóxicas para la madre; lo mismo
    puede decirse de los efectos tóxicos para el desarrollo.

         Se ha observado que el hexaclorobutadieno induce mutaciones
    genéticas, aberraciones cromosómicas, aumento de los intercambios
    entre cromátidas hermanas y síntesis imprevistas de ADN, aunque
    algunos estudios han dado resultados negativos. Con respecto a la
    genotoxicidad del hexaclorubutadieno, las observaciones realizadas
    en animales son limitadas y las efectuadas en el ser humano
    insuficientes.

         Tras la administración oral a largo plazo del
    hexacloro-butadieno a ratas, se ha observado una mayor frecuencia de
    neoplasias de los tubos renales, pero solamente con dosis elevadas
    causantes de nefrotoxicidad notable. Hay indicios limitados de
    carcinogenicidad en animales e indicios insuficientes en el ser
    humano.

         En base al nivel sin efectos adversos observados en ratones y
    ratas, que es de 0,2 mg/kg de peso corporal por día, se ha estimado
    un nivel sin efectos adversos observados en el ser humano, que es de
    0,03 a 0,05 mg/kg de peso corporal por día. Hay un margen de
    seguridad de 150 entre el nivel sin efectos adversos observados
    estimado y la ingesta diaria total máxima estimada, suponiendo que
    el compuesto se absorba a través del agua de bebida contaminada y de
    alimentos con elevado contenido de lípidos.

    9.2  Evaluación de los efectos en el medio ambiente

         El hexaclorobutadieno es de moderadamente a muy tóxico para los
    organismos acuáticos; los crustáceos y peces son los más
    vulnerables. Se ha establecido un nivel de riesgo para el medio
    ambiente de 0,1 µg/litro. Se estima que la concentración ambiental
    prevista máxima lejos de las fuentes equivale al nivel de riesgo

    ambiental extrapolado multiplicado por dos y, por consiguiente, los
    organismos acuáticos tal vez estén en peligro en las aguas de
    superficie contaminadas. No pueden excluirse efectos adversos en
    organismos bentónicos.

         En vista de la toxicidad del hexaclorobutadieno para los
    mamíferos, el consumo de organismos bentónicos o acuáticos por otras
    especies tal vez sea motivo de inquietud.


See Also:
        Hexachlorobutadiene (IARC Summary & Evaluation, Volume 20, 1979)
        Hexachlorobutadiene (IARC Summary & Evaluation, Volume 73, 1999)