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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY



    ENVIRONMENTAL HEALTH CRITERIA 150





    BENZENE







    This report contains the collective views of an international group of
    experts and does not necessarily represent the decisions or the stated
    policy of the United Nations Environment Programme, the International
    Labour Organisation, or the World Health Organization.

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

    First draft prepared by Dr E.E. McConnell,
    Raleigh, North Carolina, USA

    World Health Orgnization
    Geneva, 1993


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    WHO Library Cataloguing in Publication Data

    Benzene.

        (Environmental health criteria ; 150)

        1.Benzene - adverse effects  2.Benzene - toxicity
        3.Environmental exposure       I.Series

        ISBN 92 4 157150 0        (NLM Classification: QV 633)
        ISSN 0250-863X

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    CONTENTS

    ENVIRONMENTAL HEALTH CRITERIA FOR BENZENE

    1.   SUMMARY AND CONCLUSIONS

         1.1   Identity, physical and chemical
               properties, analytical methods
         1.2   Sources of human exposure
         1.3   Environmental transport, distribution
               and transformation
         1.4   Environmental levels and human exposure
         1.5   Kinetics and metabolism
         1.6   Effects on laboratory mammals and
                in vitro test systems
               1.6.1   Systemic toxicity
               1.6.2   Genotoxicity and carcinogenicity
               1.6.3   Reproductive toxicity, embryotoxicity
                       and teratogenicity
               1.6.4   Immunotoxicity
         1.7   Effects on humans
         1.8   Conclusions

    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
               2.4.1   Environmental samples
               2.4.2  Biological materials

    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

    4.   ENVIRONMENTAL TRANSPORT, DISTRIBUTION
         AND TRANSFORMATION

         4.1   Transport and distribution between media
         4.2   Environmental degradation
               4.2.1   Abiotic degradation
               4.2.2   Biodegradation
               4.2.3   Bioconcentration

    5.   ENVIRONMENTAL LEVELS AND HUMAN
         EXPOSURE

         5.1   Environmental levels
               5.1.1   Air
               5.1.2   Water
               5.1.3   Soil and sediments
               5.1.4   Food
         5.2   General population exposure
         5.3   Occupational exposure during manufacture,
               formulation or use

    6.   KINETICS AND METABOLISM IN LABORATORY
         ANIMALS AND HUMANS

         6.1   Absorption
               6.1.1   Air
               6.1.2   Oral
               6.1.3   Dermal
         6.2   Distribution
               6.2.1   Inhalation exposure
               6.2.2   Oral and dermal exposures
         6.3   Metabolic transformation
         6.4   Elimination and excretion
               6.4.1   Inhalation exposure
               6.4.2   Oral exposure
               6.4.3   Dermal exposure
         6.5   Retention and turnover
         6.6   Reaction with body components
         6.7   Modelling of pharmacokinetic data for benzene

    7.   EFFECTS ON LABORATORY MAMMALS AND
          IN VITRO TEST SYSTEMS

         7.1   Single exposure
         7.2   Short-term and long-term exposures
         7.3   Skin and eye irritation
         7.4   Reproductive toxicity, embryotoxicity
               and teratogenicity
         7.5   Mutagenicity and related end-points
               7.5.1    In vitro studies
               7.5.2    In vivo studies
         7.6   Carcinogenicity
               7.6.1   Inhalation studies
               7.6.2   Oral and subcutaneous studies
         7.7   Special studies
               7.7.1   Immunotoxicity
               7.7.2   Neurotoxicity
         7.8   Factors modifying toxicity
         7.9   Mechanism of toxicity

    8.   EFFECTS ON HUMANS

         8.1   General population and occupational exposure
               8.1.1   Acute toxicity
               8.1.2   Effects of short- and long-term exposures
                       8.1.2.1   Bone marrow depression; aplastic
                                 anaemia
                       8.1.2.2   Immunological effects
                       8.1.2.3   Chromosomal effects
                       8.1.2.4   Carcinogenic effects

    9.   EVALUATION OF HUMAN HEALTH RISKS

         9.1   General population
         9.2   Occupational exposure
         9.3   Toxic effects
               9.3.1   Short-term and long-term exposures;
                       organ toxicity
                       9.3.1.1   Haematotoxicity; bone marrow
                                 depression
                       9.3.1.2   Mechanism of action and
                                 metabolism
                       9.3.1.3   Immunotoxicity
               9.3.2   Genotoxicity and carcinogenic effects
                       9.3.2.1   Mechanism of carcinogenicity
                       9.3.2.2   Human carcinogenesis
         9.4   Other toxicological end-points
         9.5   Conclusions

    10.  RECOMMENDATIONS FOR PROTECTION OF HUMAN HEALTH

    11.  FURTHER RESEARCH

    12.  PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES

    REFERENCES

    RESUME ET CONCLUSIONS

    RESUMEN Y CONCLUSIONES
    

    WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR BENZENE

     Members

    Dr D. Anderson, BIBRA (British Industrial Biological Research
       Association), Toxicology International, Carshalton, Surrey, United
       Kingdom  (Vice-Chairman)

    Dr H.A. Greim, Institute of Toxicology, Association for Radiation and
       Environmental Research, Neuherberg, Germany  (Chairman)

    Dr R.F. Henderson, Inhalation Toxicology Research Institute, Lovelace
       Biomedical and Environmental Research Institute, Albuquerque, New
       Mexico

    Dr R. Hertel, Fraunhofer Institute for Toxicology, Hanover, Germany
       (now at the Bundesgesundheitsamt, Berlin) Professor A.-A.M. Kamal,
       Ain Shams University, Abbassia, Cairo, Egypt

    Dr S. Parodi, Istituto Nazionale per la Ricerca sul Cancro, Genoa,
       Italy

    Dr R.A. Rinsky, Division of Surveillance, Hazard Evaluations and Field
       Studies, National Institute of Occupational Safety and Health,
       Cincinnati, Ohio, USA

    Dr R. Snyder, Department of Pharmacology and Toxicology, Rutgers
       University, Piscataway, New Jersey, USA

    Dr G.M.H. Swaen, Department of Occupational Medicine, University of
       Limburg, Maastricht, The Netherlands

    Dr S.-N. Yin, Chinese Academy of Preventive Medicine, Institute of
       Occupational Medicine, Beijing, China

     Observers

    Dr M. Bird, Exxon Biomedical Sciences, East Millstone, New Jersey, USA

    Dr J. Gamble, Exxon Biomedical Sciences, East Millstone, New Jersey,
       USA

    Dr J. Kielhorn, Fraunhofer Institute for Toxicology, Hanover, Germany

    Dr K. Levsen, Fraunhofer Institute for Toxicology, Hanover, Germany

    Dr G. Raabe, Mobil Research, Princeton, New Jersey, USA

     Secretariat

    Dr G.C. Becking, International Programme on Chemical Safety,
       Interregional Research Unit, World Health Organization, Research
       Triangle Park, North Carolina, USA  (Secretary)

    Dr M. Kogevinas, International Agency for Research on Cancer, Lyon,
       France

    Dr E.E. McConnell, Raleigh, North Carolina, USA  (Rapporteur)

    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 BENZENE

       A WHO Task Group on Environmental Health Criteria for Benzene met
    at the Fraunhofer Institute of Toxicology and Aerosol Research,
    Hanover, Germany, from 2 to 6 December 1991, the meeting being
    sponsored by the German Ministry of the Environment.  Dr R.F. Hertel
    welcomed the participants on behalf of the host institute.  Dr G.C.
    Becking, IPCS, welcomed the participants on behalf of Dr M. Mercier,
    Director of the IPCS, and the three IPCS Cooperating organizations
    (UNEP/ILO/WHO).  The Group reviewed and revised the draft document and
    made an evaluation of the risks for human health from exposure to 
    benzene.

       The first draft was prepared by Dr E.E. McConnell, Raleigh, North
    Carolina, USA.  Extensive scientific comments on the first  draft were
    received from governments, research institutions, and  industry; in
    particular: Exxon Biomedical Sciences; CONCAWE;  Mobil Research;
    Health and Welfare Canada; IARC; RIVM, The  Netherlands; Fraunhofer
    Institute and Ministry of Health,  Germany; National Institute of
    Environmental Health Sciences,  National Institute of Occupational
    Safety and Health, and Agency  for Toxic Substances and Disease
    Registry, USA; Department of  Health, United Kingdom; and National
    Chemical Inspectorate  (KEMI), Sweden.  These comments were
    incorporated into the  second draft by the Secretariat.

       Dr H. Greim, Chairman of the Task Group, Dr C. Pohlenz-Michel and
    Dr H. Sterzl-Eckert of GSF-Institute of Toxicology  deserve special
    thanks for the time taken after the Task Group to  ensure the
    scientific accuracy of the final draft monograph.

       Dr G.C. Becking (IPCS Central Unit, Interregional Research  Unit)
    and Dr P.G. Jenkins (IPCS Central Unit, Geneva) were responsible for
    the overall scientific content and technical editing, respectively, of
    this monograph.  The efforts of all who helped in the preparation and
    finalization of this publication are gratefully acknowledged.

    ABBREVIATIONS

    ALMS        Atomic line molecular spectrometry

    CHO         Chinese hamster ovary

    FID         flame ionization detection

    GC          gas chromatography

    MS          mass spectrometry

    SCE         sister chromatid exchange

    SMR         standardized mortality ratio

    S-PMA       S-phenyl-mercapturic acid

    TWA         time-weighted average

    1.  SUMMARY AND CONCLUSIONS

    1.1  Identity, physical and chemical properties, analytical methods

         Benzene is a stable colourless liquid at room temperature and
    normal atmospheric pressure.  It has a characteristic aromatic odour,
    a relatively low boiling point (80.1 °C) and a high vapour pressure,
    which causes it to evaporate rapidly at room temperature, and is
    highly flammable.  It is slightly soluble in water but miscible with
    most other organic solvents.

         Analytical methods are available for the detection of benzene in
    various media (air, water, organs/tissues).  The choice between gas
    chromatography (GC) with flame ionization or photoionization detection
    and mass spectrometry (MS) depends upon the sensitivity required and
    levels of benzene expected.  Detection of benzene in the workplace
    usually involves collection on charcoal and GC/MS analysis after
    desorption.  Where sensitivity in the mg/m3 range is sufficient,
    portable direct-reading instruments and passive dosimeters are
    available.  If greater sensitivity is required, methods to detect
    benzene at levels as low as 0.01 µg/m3 (air) or 1 ng/kg (soil or
    water) have been reported.

    1.2  Sources of human exposure

         Benzene is a naturally occurring chemical found in crude
    petroleum at levels up to 4 g/litre.  It is also produced in extremely
    large quantities (14.8 million tonnes) worldwide.  Emissions arise
    during the processing of petroleum products, in the coking of coal,
    during the production of toluene, xylene and other aromatic compounds,
    and from its use in consumer products, as a chemical intermediate and
    as a component of gasoline (petrol).

    1.3  Environmental transport, distribution and transformation

         Benzene in air exists predominantly in the vapour phase, with
    residence times varying between a few hours and a few days, depending
    on environment and climate, and on the concentration of hydroxyl
    radicals, as well as nitrogen and sulfur dioxides.  It can be removed
    from air by rain, leading to contamination of surface and ground
    water, in which it is soluble at about 1000 mg/litre.

         Due primarily to volatilization, the residence time of benzene in
    water is a few hours, with little or no adsorption to sediments.

         Benzene in soil can be transported to air via volatilization and
    to surface waters by run off.  If benzene is buried or is released
    well below the surface, it will be transported into ground water.

         Under aerobic conditions, benzene in water or soil is rapidly
    (within hours) degraded by bacteria to lactate and pyruvate through

    phenol and catechol intermediates.  However, under anaerobic
    conditions (for example, in ground water) bacterial degradation is
    measured in weeks and months rather than hours.  In the absence of
    bacterial degradation benzene can be persistent.  It has not been
    shown to bioconcentrate or bioaccumulate in aquatic or terrestrial
    organisms.

    1.4  Environmental levels and human exposure

         The presence of benzene in gasoline (petrol), and as a widely
    used industrial solvent can result in significant and widespread
    emissions to the environment.  Outdoor environmental levels range from
    0.2 µg/m3 in remote rural areas to 349 µg/m3 in industrial centres
    with a high density of automobile traffic.  During refuelling of
    automobiles, levels up to 10 mg/m3 have been measured.

         Benzene has been detected at levels as high as 500 µg/m3 in
    indoor residential air.  Cigarette smoke contributes significant
    amounts of benzene to the levels reported in indoor air, with smokers
    inhaling approximately 1800 µg benzene/day compared to 50 µg/day by
    non-smokers.

         In many countries, occupational exposures seldom exceed a
    time-weighted average of 15 mg/m3.  However, the actual levels
    reported depend upon the industry studied and in some industrially
    developing countries exposures can be considerably higher.

         Water and food-borne benzene contributes only a small percentage
    of the total daily intake in non-smoking adults (between about 3 and
    24 µg/kg body weight per day).

    1.5  Kinetics and metabolism

         Benzene is well absorbed in humans and experimental animals after
    oral and inhalation exposures, but in humans dermal absorption is
    poor.  Approximately 50% absorption occurs in humans during continuous
    exposures to 163-326 mg/m3 for several hours.  After a 4-h exposure
    to 170-202 mg/m3, retention in the human body was approximately 30%,
    with 16% of the retained dose having been excreted as unchanged
    benzene in expired air.  Women may retain a greater percentage of
    inhaled benzene than men.  Benzene tends to accumulate in tissues
    containing high amounts of lipids, and it crosses the placenta.

         Benzene metabolism occurs mainly in the liver, is mediated
    primarily through the cytochrome P-450 IIE1 enzyme system and involves
    the formation of a series of unstable reactive metabolites. In rodents
    the formation of two putative toxic metabolites, benzoquinone and
    muconaldehyde, appears to be saturable.  This may have important
    implications for dose-response relationships, because a higher
    proportion of the benzene will be converted to toxic metabolites at
    low doses than at high doses.  The metabolic products are excreted

    primarily in the urine.  Appreciable levels of the known metabolites
    phenol, catechol and hydroquinone are found in bone marrow.  Phenol is
    the predominant urinary metabolite in humans and is mainly found as an
    ethereal sulfate conjugate until levels approach 480 mg/litre, at
    which time glucuronides are detected.  Recent studies suggest that
    benzene toxicity is the result of the interactive effects of several
    benzene metabolites formed in both the liver and the bone marrow.

         Inhaled benzene had been found to bind to rat liver DNA to the
    extent of 2.38 µmoles/mole DNA phosphate.  Seven deoxyguanosine
    adducts and one deoxyadenine adduct have been detected in rabbit bone
    marrow mitochondrial DNA.

    1.6  Effects on laboratory mammals and in vitro test systems

    1.6.1  Systemic toxicity

         Benzene appears to be of low acute toxicity in various animal
    species, with LD50 values after oral exposure ranging between 3000
    and 8100 mg/kg body weight in the rat.  Reported LC50 values range
    from 15 000 mg/m3 (8 h) in mice to 44 000 mg/m3 (4 h) in rats.

         Benzene is a moderate eye irritant and is irritating to rabbit
    skin after multiple applications of the undiluted chemical.  No
    information is available on the skin-sensitizing potential of benzene.

         Exposure of mice to benzene by inhalation results in a
    significant lowering of blood parameters such as haematocrit,
    haemoglobin level, and erythrocyte, leucocyte and platelet counts. 
    Long-term exposure at high doses results in bone marrow aplasia. 
    Similar, but less severe, findings were noted in rats.

    1.6.2  Genotoxicity and carcinogenicity

         Benzene has given negative results in mutagenicity assays
     in vitro.

         In  in vivo studies, benzene or its metabolites cause both
    structural and numerical chromosome aberrations in humans and
    laboratory animals.  In addition, benzene administration results in
    the production of sister chromatid exchanges and polychromatic
    erythrocytes with micronuclei.  Benzene can reach germ cells, after
    intraperitoneal dosing, as shown by the production of abnormalities in
    sperm head morphology.

         Benzene has been reported to cause the production of several
    types of neoplasms in both rats and mice after either oral dosing or
    inhalation exposures.  These include various types of epithelial
    neoplasms, e.g., Zymbal gland, liver, mammary tissue and nasal cavity
    neoplasms, and a few lymphomas and leukaemias. 

         In those inhalation studies where a positive carcinogenic
    response was reported, exposure levels were between 100 and 960
    mg/m3 for 5-7 h/day, 5 days/week.  Oral benzene doses of between 25
    and 500 mg/kg body weight in mice and rats resulted in the production
    of neoplasms.  The length of exposure was usually 1-2 years.

    1.6.3  Reproductive toxicity, embryotoxicity and teratogenicity

         Benzene crosses the placental barrier freely.  There are no data
    showing that it is teratogenic after numerous experiments in
    experimental animals even at maternally toxic doses.  However, it has
    been shown to be fetotoxic following inhalation exposure in mice (1600
    µg/m3, 7 h/day, gestation days 6-15) and in rabbits. 

    1.6.4  Immunotoxicity

         Benzene depresses the proliferative ability of B- and T-cell
    lymphocytes.  Host resistance to infection in several laboratory
    species has been reduced by exposure to benzene.

    1.7  Effects on humans

         It is known that benzene produces a number of adverse health
    effects.  The most frequently reported health effect of benzene is
    bone marrow depression leading to aplastic anaemia.  At high levels of
    exposure a high incidence of these diseases is probable. 

         Benzene is a well-established human carcinogen.  Epidemio-logical
    studies of benzene-exposed workers have demonstrated a causal
    relationship between benzene exposure and the production of
    myelogenous leukaemia.  A relationship between benzene exposure and
    the production of lymphoma and multiple myeloma remains to be
    clarified.

         The Task Group was of the opinion that the epidemiological
    evidence is not capable of distinguishing between a) a small increase
    in mortality from leukaemia in workers exposed to low levels of
    benzene, and b) a non-risk situation. 

    1.8  Conclusions

         It was concluded that a time-weighted average of 3.2 mg/m3
    (1 ppm) over a 40-year working career has not been statistically
    associated with any increase in deaths from leukaemia.  Because this
    is a human carcinogen, however, exposures should be limited to the
    lowest level technically feasible.  Increases in exposure level to
    over 32 mg/m3 (10 ppm) should be avoided.  Benzene and
    benzene-containing products such as petrol should never be used for
    cleaning purposes.

         Traditionally, bone marrow depression, i.e. anaemia leucopenia or
    thrombocytopenia, in the workplace has been recognized as the first
    stage of benzene toxicity and appears to follow a dose-response
    relationship.  In other words, the higher the dose, the greater the
    likelihood of observing decreases in circulating blood cells.

         Exposure to high benzene levels (160-320 mg/m3) for one year
    would most likely produce bone marrow toxicity in a large percentage
    of the workers and aplastic anaemia in some cases, but little effect
    would be expected at lower doses.  Exposure to both high and low doses
    would be expected to produce benzene toxicity after 10 years of
    continuous exposure.  Thus, a high level of both bone marrow
    depression and aplastic anaemia would be seen at the higher doses and
    some damage would also be seen at lower doses.  The observation of any
    of these effects, regardless of the level of exposure, should indicate
    the need for improved control over benzene exposures.

         There is no evidence of benzene being teratogenic at doses lower
    than those that produce maternal toxicity, but fetal toxicity has been
    demonstrated.

         Neurotoxicity and immunotoxicity of benzene has not been well
    studied in experimental animals or humans.

    2.  IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS

    2.1  Identity

    Chemical structure:

    CHEMICAL STRUCTURE

    Chemical formula:     C6H6

    CAS number:           71-43-2

    RTECS number:         CY1400000

    Common name:          Benzene

    IUPAC name:           Benzene

    Common synonyms:      Annulene, benzine, benzol, benzole, benzol coal
                          naphtha, cyclohexatriene, mineral naphtha,
                          motor benzol, phenyl hydride, pyrobenzol,
                          pyrobenzole

    Purity:               Nitration grade >99%.  Benzol 90 contains
                          80-85% benzene, 13-15% toluene and 2-3% xylene.
                          Commercial grades are free of H2S and SO2
                          and have a maximum of 0.15% non-aromatics
                          compounds.

    2.2  Physical and chemical properties

         Benzene is a naturally occurring colourless liquid at room
    temperature (20 °C) and ambient pressure (760 mmHg), and has a
    characteristic aromatic odour.  The principal physical and chemical
    properties of benzene are shown in Table 1.

    2.3  Conversion factors

         1 ppm = 3.2 mg/m3 at 20 °C at normal atmospheric pressure
         1 mg/m3 = 0.31 ppm

    2.4  Analytical methods

         This section does not provide an exhaustive list of the
    analytical methods available for detecting and quantifying benzene in
    various media.  However, those methods that are well established and
    have been used in studies of human exposure and in experiments on the
    biological effects of benzene will be described briefly.

        Table 1.  Some physical and chemical properties of benzenea
                                                                        

    Physical form (20 °C)                        clear colourless liquid

    Relative molecular mass                      78.11

    Flash point                                  -11.1 °C

    Flammable limits                             1.3-7.1%

    Melting/freezing point                       5.5 °C

    Boiling point                                80.1 °C at 760 mmHg

    Density                                      0.878

    Relative vapour density
      (air = 1)                                  2.7

    Vapour pressure (26 °C)                      13.3 kPa

    Solubilities:
      water                                      1800 mg/litre at 25 °C
      non-aqueous solvents                       miscible with most

    Odour threshold                              4.8-15.0 mg/m3

    Taste threshold (water)                      0.5-4.5 mg/litre

    Log  n-octanol/water partition
      coefficient                                1.56-2.15

    Sorption coefficient (log Koc -
     distribution coefficient between
     benzene adsorbed to soil organic
     carbon and benzene in solution)             1.8-1.9
                                                                        

    a  Data from:  GDCh (1988),  RIVM (1988) and ATSDR (1989)
    
         The analytical methods used for the determination of benzene
    depend upon the media sampled and the level of sensitivity required. 
    In all cases proper sampling and sample storage are essential
    prerequisites, particularly as microgram and nanogram quantities are
    often found in environmental samples.

         Some of the commonly used methods for the detection of benzene in
    various media are summarized in Table 2.

    2.4.1  Environmental samples

         Methods are available for the determination of benzene in air,
    water sediments, soil, foods, cigarette smoke, and petroleum and
    petroleum products.  Most involve separation by gas chromatography
    (GC) with detection by flame ionization (FID) or photoionization (PID)
    or by mass spectrometry (MS). 

         The measurement of benzene in air (ambient and workplace) usually
    involves a preconcentration step in which the sample is passed through
    a solid absorbent (Baxter et al., 1980; Pellizzari, 1982; Roberts et
    al., 1984; Clark et al., 1984b; Reineke & Bächmann, 1985; Harkov et
    al., 1985; Gruenke et al., 1986; OSHA, 1987; Bayer et al., 1988;
    Brown, 1988a,b).  Commonly used adsorbents are TenaxR resin, silica
    gel, and activated carbon.  Preconcentration of benzene can also be
    accomplished by direct on-column cryogenic trapping (Reineke &
    Bächmann, 1985; Holdren et al., 1985; Fung & Wright, 1986), or benzene
    can be analysed directly (Clark et al., 1984a; Hadeishi et al., 1985;
    Bayer et al., 1988).  As noted in Table 2, the limit of detection of
    the GC/FID or GC/PID techniques is in the low ppb (µg/m3) to low ppt
    (ng/m3) range whereas the GC/MS method has a limit of detection in
    the low ppb (µg/m3) range (Gruenke et al., 1986).  Although GC/FID
    and GC/PID provide greater sensitivity than GC/MS, the latter is
    generally considered more reliable for the measurement of benzene in
    samples containing multiple components with similar GC elution
    characteristics.  Atomic line molecular spectrometry (ALMS) has been
    developed to monitor benzene and other organic compounds in ambient
    air samples (Hadeishi et al., 1985). The detection limit is 800
    µg/m3 (250 ppb).

         Benzene in the workplace can be measured by portable
    direct-reading instruments, real-time continuous monitoring systems
    and passive dosimeters (OSHA, 1987) having sensitivities in the ppm
    (mg/m3) range.  In the USA, the more sensitive procedure of
    preconcentration on charcoal followed by GC/MS analysis is generally
    preferred (OSHA, 1987).

         Benzene in aqueous media is usually isolated by the
    purge-and-trap method (Brass et al., 1977; Hammers & Bosman, 1986)
    followed by GC/MS, GC/FID or GC/PID analysis (Harland et al., 1985;
    Blanchard & Hardy, 1986; Michael et al., 1988).  An inert gas such as
    nitrogen is used to purge the sample, the benzene is trapped on an
    absorbent such as TenaxR or activated charcoal, and this is followed
    by thermal desorption.  The sensitivity of these methods is in the low
    to sub µg/litre range with good recoveries and precision for most
    methods.


    
    Table 2.  Analytical methods for the determination of benzene
                                                                                                                                              

    Sample                Preparation                                   Analytical methoda     Detection limitb    Reference
                                                                                                                                              

    Air                   silica gel trap                               indicator tube         4.9 mg/m3           Koljkowsky (1981)

    Air                   charcoal trap, CS2 desorption                 GC/FID                 3.2 µg/m3           Baxter et al. (1980)

    Air (ambient)         Tenax GC sorbent, thermal desorption          capillary GC/MS        NR                  Pellizzari (1982)
                                                                        computer analysis

    Air                   Tenax GC trap, thermal desorption,            C/FID/MS               0.01 µg/m3          Roberts et al. (1984)
                          cryogenic focusing

    Air (ambient)         direct injection                              GC/PID                 0.82 µg/m3          Clark et al. (1984a)

    Air                   direct analysis                               UV Spect.              800 µg/m3           Hadeishi et al. (1985)

    Air                   Tenax or cryogenic trap, thermal desorption   GC/FID                 NR                  Holdren et al. (1985)

    Air near landfills/   Tenax GC trap, thermal decomposition          GC/FID/ECD/MS          0.03 µg/m3          Harkov et al. (1985)
     waste sites

    Air                   silica gel trap, thermal desorption           GC/MS                  0.32 µg/m3          Gruenke et al. (1986)

    Air (ambient)         cryogenic trap, thermal desorption            GC/PID                 16 ng/m3            Reineke &
                                                                        GC/FID                 77 ng/m3            Bachmann (1985)
    Air (ambient)         charcoal trap (badge or tube, desorb with     GC/FID                 0.96 µg/m3          Fung & Wright (1986)
                          CS2

    Air                   solid sorbent trap, thermal desorption        GC/MS                  NR                  Bayer et al. (1988)

    Air (occupational)    activated charcoal sorbent, CS2 desorption    GC/FID                 0.64 mg/m3 (in      Brown (1988a)
                                                                                               12 litres)
                                                                                                                                              

    Table 2 (contd).
                                                                                                                                              

    Sample                Preparation                                   Analytical methoda     Detection limitb    Reference
                                                                                                                                              

    Air (occupational)    porous polymeric sorbent, thermal desorption  GC/FID                 0.83 µg/m3          Brown (1988b)

    Water (drinking)      purge and trap                                GC/MS                  0.2 µg/litre        Brass et al. (1977)

    Water (surface or     helium purge, Tenax GC trap, thermal          GC/MS                  0.1 µg/litre        Fentiman et al. (1979)
     effluents)           desorption

    Water                 purge with inert gas, Tenax trap, thermal     GC/MS                  NR                  Harland et al. (1985)
                          desorption

    Water                 N2 purge, Tenax GC trap, thermal desorption   GC/FID                 1 ng/litre          Hammers & Bosman (1986)

    Water                 filter through silicone polycarbonate         GC/FID                 7.2 µg/litre        Blanchard & Hardy (1986)
                          membrane into inert gas stream

    Water                 purge with inert gas, Tenax trap, thermal     HRGC/MS                0.1 µg/litre        Michael et al. (1988)
                          desorption to on-column cryogenic trap

    Soil                  N2 purge, Tenax GC trap                       GC/FID                 0.1 µg/kg           Fentiman et al. (1979)

    Soil                  N2 purge, Tenax trap, thermal desorption      GC/FID                 1 ng/kg             Hammers & Bosman (1986)

    Sediment              N2 purge, Tenax trap, thermal desorption      GC/MS                  0.01 µg/kg          Ferrario et al. (1985)

    Mainstream            filter smoke and direct to GC/MS; for         HRGC/MS                NR                  Brunnemann et al. (1989)
     cigarette smoke      passive smoke collect air in cryogenic
                          methanol-filled impingers

    Jet fuel fumes        sample on charcoal, methylene chloride,       HPLC/UV                0.29 mg/m3          Dibben et al. (1989)
                          ethyl acetate desorption; column elution
                          with acetonitrile
                                                                                                                                              

    Table 2 (contd).
                                                                                                                                              

    Sample                Preparation                                   Analytical methoda     Detection limitb    Reference
                                                                                                                                              

    Blood                 N2 purge, Tenax GC-silica gel trap            GC/MS                  0.5 µg/litre        Antoine et al. (1986)

    Blood                 extract with toluene, centrifuge; analyse     GC/FID                 100 µg/litre        Jirka & Bourne (1982)
                          toluene layer

    Blood                 add heparinized sample to isotonic saline     HRGC/PID               0.4 µg/litre        Pekari et al. (1989)
                          in headspace via equilibrate with heat

    Breath                collect on Tenax GC, thermal desorption       HRGC/MS                9.8 ng/m3           Pellizzari et al. (1988)

    Breath                collect on Tenax GC, thermal desorption into  GC/MS                  5.2 µg/m3           Wallace et al. (1985)
                          on-column cryogenic trap

    Urine                 extraction                                    GC/MS                  2 µmol/litre        Stommel et al. (1989)
                                                                                               (as S-PMA)

    Urine (phenol         enzyme and acid digestion; ethyl ether        GC/FID                 1 mg/litre          Buchet (1988)
     and conjugates)      extraction

    Urine (muconic        sample mixed with methanol, centrifuge,       HPLC/UV                0.1 mg/litre        Inoue et al. (1989)
     acids)               analyse supernatant, elute with methanol -
                          acetic acid

    Tissues               add butyl hydroxytoluene to buffered homo-    RID-HPLC/UV            20 pg/g             Bechtold et al. (1988)
                          genate, centrifuge, analyse supernatant
                                                                                                                                              

    a    GC = gas chromatography; FID = flame ionization detection; PID = photoionization detection; MS = mass spectrometry;
         HRGC = high resolution (capillary) gas chromatography; RID = reverse isotope dilution; HPLC = high performance liquid chromatography;
         UV = ultraviolet detection
    b    NR = not reported
    

         Benzene in soil, sediment and food samples is usually determined
    by purge-and-trap methods (Harland et al., 1985; Ferrario et al.,
    1985; Hammers & Bosman, 1986), with headspace analysis (Kiang & Grob,
    1986) and liquid extraction (Kozioski, 1985) techniques being used
    less frequently.  Detection limits as low as 1 ng/kg have been
    reported after GC/FID or GC/MS analysis, but recoveries and precision
    are frequently low.

         Methods have been reported for the analysis of benzene in other
    environmental media such as cigarette smoke (Brunnemann et al., 1989,
    1990) and in petroleum products such as petrol (gasoline) (Poole et
    al., 1988; Dibben et al., 1989).

    2.4.2  Biological materials

         Benzene levels in exhaled breath, blood, and body tissues have
    been analysed by GC/FID, GC/PID or GC/MS, and benzene metabolites in
    urine have been measured using GC/FID and high-performance liquid
    chromatography (HPLC) with ultraviolet detection.

         Prior to analysis, breath samples are usually collected on a
    solid sorbent such as activated charcoal, silica gel or TenaxR GC
    and thermally desorbed (Wallace et al., 1985; Pellizzari et al.,
    1988).  Headspace analysis has also been used to analyse levels of
    benzene in exhaled breath (Gruenke et al., 1986).  Greater selectivity
    is achieved if capillary columns are used for high-resolution gas
    chromatography (HRGC) (Pellizzari et al., 1988).

         Three methods have been used to extract benzene from blood, i.e.
    purge-and-trap (Antoine et al., 1986), headspace analysis (Gruenke et
    al., 1986; Pekari et al., 1989) and solvent extraction (Jirka &
    Bourne, 1982).  Sensitivity for the first two procedures is in the sub
    to low µg/litre range, whereas solvent extraction is less sensitive
    (low to mid µg/litre).

         Total phenolic metabolites of benzene have been determined in
    urine following hydrolysis, extraction with ethyl ether and GC/FID
    analysis (Buchet, 1988).  The technique of HPLC/UV has been used to
    determine the trans, trans-muconic acid metabolites of benzene in
    urine (Inoue et al., 1989).  A more sensititive GC/MS method to
    monitor muconic acid in the urine of exposed workers has been
    developed by Bechtold et al. (1991).  Biological monitoring methods
    using urine measure concentrations of phenolic conjugates, the major
    metabolites of benzene (Buchet, 1988).  Such methods, however, lack
    adequate specificity and sensitivity for low levels of benzene
    exposure.  A method based on the determination of the minor metabolite
    S-phenyl-mercapturic acid (S-PMA) appears to overcome these
    deficiencies (Stommel et al., 1989).  Benzene and its organic-soluble
    metabolites have been determined quantitatively in rodent tissues
    using GC/MS and reverse isotope dilution (RID) combined with
    semipreparative HPLC/UV (Bechtold et al., 1988).  A method using

    ion-pairing HPLC was used to analyse water-soluble metabolites of
    benzene in liver and in urine (Sabourin et al., 1988).

         Schrenk & Bock (1990) have developed an HPLC method for the
    determination of metabolites secreted by isolated hepatocytes. 
    Brodfuehrer et al. (1990) have reported on the determination of
    benzene metabolites in liver slices of rat, mouse and man.

    3.  SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

         Benzene is released to the environment from both natural and
    man-made sources, the latter accounting for the major part of the
    emissions.

    3.1  Natural occurrence

         Benzene is a naturally occurring organic compound.  It is a
    component of petroleum (1-4%) (IARC, 1989) and can be found in sea
    water (0.8 µg/litre) in the vicinity of natural deposits of petroleum
    and natural gas (Reynolds & Harrison, 1982).

    3.2  Anthropogenic sources

         Major anthropogenic sources of benzene include automobile
    exhaust, automobile-refuelling operations and industrial emissions. 
    Automobile exhaust probably accounts for the largest anthropogenic
    source in the general environment.  Cigarette smoke, off-gassing from
    building material and structural fires all lead to increased
    atmospheric benzene levels.  People are exposed to benzene mainly
    through the inhalation of contaminated air, particularly in areas of
    heavy automobile traffic and around gasoline (petrol) stations and
    other facilities for storage and distribution of petrol, and through
    tobacco smoke from both active and passive smoking (ATSDR, 1991). 
    Other sources of exposure have been reported to include industrial
    emissions and consumer products (Wallace et al., 1987).  However,
    certain individuals may be exposed to potentially high concentrations
    of benzene in drinking-water as a result of seepage from underground
    petroleum storage tanks, landfills, waste streams, or natural gas
    deposits (ATSDR, 1991).  Individuals employed in industries that
    produce or use benzene or benzene-containing products are probably
    exposed to much higher levels than the general population.  Industrial
    discharge, landfill leachate, and disposal of benzene-containing waste
    are also anthropogenic sources.

    3.2.1  Production levels and processes

         Benzene ranks sixteenth in production volume for chemicals
    produced in the USA, with an estimated production of 4.39 x 105
    tonnes (1.6 x 109 gallons) in the USA in 1991 (ATSDR, 1991) and 1480
    x 103 tonnes in western Europe in 1986 (GDCh, 1988) (Table 3).  In
    the USA over 90% of the benzene produced is derived from petroleum
    sources (ATSDR, 1991), i.e. refinery streams (catalytic reformates),
    pyrolysis of gasoline, and toluene hydrodealkylation.  In western
    Europe 55% of the benzene production is from gasoline pyrolysis, 10%
    from coking of coal, and the remaining production is divided
    approximately equally between catalytic reformate and the
    hydrodealkylation of toluene (GDCh, 1988).

    
    Table 3.  World production of benzene in thousands of tonnes
              for 1981a
                                                                        
                                            Capacity       Production
                                                                        

    North & South America (total)              9350            6150

    Asia (total)                               3550            2460

    Western Europe (total)                     6950            3800

    Eastern Europe (total)                     5840            2340

    Japan                                      2880            2060

    USA                                        8030            5190

    USSR                                       3250            1700

    Other countries                             100              50

    World                                    25 800          14 800
                                                                        

    a  From: RIVM (1988)
         Benzene in petrol is not included.
    
         Given the high production volume, widespread use, and physical
    and chemical properties of benzene, there is a high potential for
    large amounts to be released to the environment.  However, accurate
    data on the amounts released are difficult to obtain.  The data in
    Table 4 are given to show the relative amounts of benzene released to
    the air from various industrial sources in several countries.  It is
    evident that the largest amounts released are from the use of
    gasoline.  In California (USA), the 1984 benzene emission inventory
    totalled 17 500 tonnes (Allen, 1987), with motor vehicle exhaust
    accounting for 71% of this amount.  Total emissions of benzene from
    industrial sources within the USA have been reported to be 33 000 to
    34 000 tonnes (US EPA, 1989).  Recent emission data related to
    automobile use in the USA are difficult to obtain, but in 1980 such
    emissions were between 40 000 and 80 000 tonnes (IARC, 1982).  In
    Germany approximately 80% of the air emissions reported are due to the
    use of motor vehicles, whereas coke ovens account for 3.9% of such
    emissions.  Other sources are gasoline storage and transport (6.2%)
    and industrial furnace emissions (4.0%).

        Table 4.  Major emissions of benzene into the atmosphere in tonnes per yeara

                                                                                  
                               Road traffic    Refineries   Remaining    Total
                                                            sources
                                                                                  

    Belgium/Luxembourg              4950            60          750         5760

    Canada                        25 895           654         7601       34 150

    Denmark                         2600            10          390         3000

    France                        30 000           200         4000       34 200

    Germany (FRG)                 62 000           200       11 000       73 200

    Greece                          4700            30          700         5430

    Ireland                         1650             0          200         1850

    Italy                         29 000           190         4200       33 390

    Netherlands                     7300            80          980         8360

    United Kingdom                29 000           150         4200       33 350

    European Community
     (total)                     171 200           920       26 420      198 540
                                                                                  

    a  From: RIVM (1988).  Calculated using crude oil consumption figures from 1982.
    
    3.2.2  Uses

         Benzene has a large number of industrial, commercial and
    scientific uses.  Approximately, 10% of the total use of benzene is in
    gasoline (RIVM, 1988), where levels average < 1% by weight in the USA
    (US EPA, 1985) and 2.5-3.0% v/v in western Europe (GDCh, 1988).

         Along with other aromatic compounds, benzene is important in the
    production of organic chemicals, particularly styrene (Table 5).  The
    major uses of benzene as a chemical intermediate are summarized in
    Table 5.  There are no data indicating a major deviation from this
    pattern of use, which was reported in 1981.

    
    Table 5.  Industrial uses of benzene in 1981.  Benzene in petrol
              has not been incorporateda
                                                                              
    Production of:                USA       Japan      Western    Netherlands
                                                       Europe
                                                                              
    Ethylbenzene/styrene         51.1       50.4         48.6         73

    Cumene/phenol                20.6       12.1         19.3         16

    Cyclohexane                  13.8       25.6         13.4         11

    Alkylates                     3.0        3.7          5.2          -

    Maleic acid anhydride         2.8        2.5          3.3          -

    Nitrobenzene/aniline          5.3        -            6.7          -

    Chlorinated benzenes          2.6        5.7          2.0          -

    Other products                0.8        -            1.5          -
                                                                              

    a    From: RIVM (1988).  Data shown as a percentage of the total benzene
         consumed in each area.
    
         In the past, benzene was used widely as a solvent, but this use
    is declining in most developed countries; it represents < 2% of
    current use.  However, it is still used as a solvent in scientific
    laboratories, industrial paints, rubber cements, adhesives, paint
    removers, degreasing agents, production of artificial leather and of
    rubber goods, and in the shoe industry (Mara & Lee, 1978; Windholz et
    al., 1983; Gilman et al., 1985).  For many solvent uses, benzene has
    been replaced by other less toxic organic solvents.  However, in the
    past significant human exposure occurred when benzene was used as a
    paint stripper, a carburettor cleaner, in the production of denatured
    alcohol and rubber cements, and in arts and crafts supplies (Young et
    al., 1978).  It has also been reported that benzene vapours could be
    detected from such products as carpet glue, textured carpet, liquid
    detergent and furniture wax (Wallace et al., 1987).

    4.  ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION

    4.1  Transport and distribution between media

         Benzene is released into the environment from both natural and
    man-made sources, although the latter are the most significant.  The
    volatility and solubility are the most important properties which
    influence its environmental transport (see Table 1).  Benzene enters
    the atmosphere from direct emissions and volatilization from soil and
    water surfaces.

         The high volatility of benzene (vapour pressure of 13.3 Kpa at
    26 °C), its solubility in water (1800 mg/litre at 25 °C) and a Henry's
    law constant of 5.5 x 10-3 atm/m3 per mole at 20 °C suggest that
    benzene will partition to the atmosphere from surface water (Mackay &
    Leinonen, 1975).  These authors have calculated a t´ in water of 4.8
    h (1 metre deep at 25 °C).  Benzene in air is fairly soluble in water
    and is removed from the atmosphere by rain (Ogata & Miyake, 1978).
    However, once it has been deposited on soil or water, volatilization
    will return a portion back to the atmosphere.

         Benzene is not expected to adsorb to bottom sediments for several
    reasons: (1) the Koc (soil/organic carbon sorption coefficient)
    (Table 1) does not predict adsorption to particles; (2) the solubility
    of benzene in water, and (3) the volatility of benzene.

         Benzene released to soil can partition to the atmosphere through
    volatilization, to surface water through run-off, and to ground water
    if released well below the surface.  Evaporation from surface soil is
    expected to be rapid (Hine & Mookerjee, 1975).  With a Koc of 60-83,
    benzene is considered fairly mobile in soil (Kenaga, 1980; Karickhoff,
    1981).  Leaching of benzene into ground water from soil is influenced
    by several parameters including type of soil (sand versus clay),
    amount of rainfall, depth of ground water and extent of benzene
    degradation.

    4.2  Environmental degradation

    4.2.1  Abiotic degradation

         In air benzene exists predominantly in the vapour phase
    (Eisenreich et al., 1981).  Degradation of benzene in air occurs
    mainly by reactions with hydroxy, alkoxy and peroxy radicals, oxygen
    atoms and ozone, of which the reaction with hydroxy radicals is the
    most important.  The rate constant for the reaction has been measured
    often (Tully et al., 1981).  Assuming an average hydroxy radical
    concentration of 1.25 x 106 molecules/cm3 and a rate constant of
    1.3 x 10-12 cm3/molecule per second, a t´ of 5.3 days was
    calculated for benzene (RIVM, 1988).  In areas of high traffic density
    where there is a higher concentration of hydroxy radicals (1 x 108
    molecules/cm3) and increased levels of NOx, the 24-h average t´

    for benzene has been reported as 3-10 days (GDCh, 1988).  Under these
    conditions phototransformation products may include phenol,
    nitrobenzenes, nitrophenol and various ring-opened dicarbonyl
    compounds (Bandow et al., 1985).  Direct photolysis of benzene in the
    troposphere is unlikely since the UV-visible spectrum of benzene shows
    no appreciable absorbance at wavelengths longer than 260 nm
    (Bryce-Smith & Gilbert, 1976).  This hypothesis was supported by Korte
    & Klein (1982).  No degradation was seen after 6 days irradiation of
    benzene in the laboratory with light of wavelength longer than 290 nm.

    4.2.2  Biodegradation

         Benzene in surface and ground water is biodegradable by a variety
    of microorganisms under both aerobic and anaerobic conditions (RIVM,
    1988).  Under both conditions the mechanism of biodegradation seems to
    involve the formation of catechol via  cis-1,2-dihydroxy-
    1,2-dihydrolbenzene followed by ring cleavage (Högn & Jaenicke, 1972;
    Korte & Klein, 1982).

         Karlson & Frankenberger (1989) studied the aerobic biodegradation
    of benzene in ground water utilizing a mixed bacterial culture
    containing petroleum-degrading bacteria from ground water and soil
    bacteria capable of using gasoline as a sole carbon source.  Under
    closed agitated conditions without added nutrients, benzene levels
    dropped from 480 µg/litre to 218 µg/litre in 48 h.  However, when
    nitrogen was added the reaction was much more rapid, with benzene
    levels decreasing to 35 µg/litre in 20 h.  The biodegradation of
    benzene in ground water and river water appears to follow first-order
    rate kinetics, with t´ values of 28 and 16 days, respectively,
    having been reported for ground water and river water (Vaishnav &
    Babeu, 1987).

         Korte & Klein (1982) studied the fate of benzene on soil
    utilizing composting waste.  Of the benzene applied to the waste only
    2-2.5% remained  in situ whereas 35% volatilized.  These authors
    concluded that benzene does not usually remain on soil long enough for
    biodegradation to play an important role in its removal.  A model
    developed to predict the environmental fate of benzene following
    losses of gasoline from underground tanks indicated that approximately
    1% of the benzene would be degraded (Tucker et al., 1986).

         Benzene is not usually biodegradable under anaerobic conditions
    (GDCh, 1988).  However, Wilson et al. (1986) using samples of landfill
    leachate showed under methogenic conditions in an anaerobic glove-box
    that, although no significant benzene biodegradation occurred during
    the first 20 weeks of incubation, after 40 weeks benzene
    concentrations were reduced by 72%.  Using anaerobic digesting sludge,
    Battersby & Wilson (1989) examined the degradation of benzene under
    methanotrophic conditions.  Benzene, at a level of 50 mg carbon/litre,
    remained undegraded after 11 weeks of digestion.  Although it is
    slowly degraded under anaerobic conditions, benzene levels in sewage

    influents up to 6 mg/litre do not affect sewage treatment processes
    using activated sludge systems (Bennett, 1989).  Jackson & Brown
    (1970) reported no toxic effects of benzene on the anaerobic digestion
    of sewage sludges until levels of between 50 and 200 mg/litre had been
    reached.

    4.2.3  Bioconcentration

         Benzene is not expected to bioconcentrate to any great extent in
    aquatic or terrestrial organisms given the reported values for log
    Pow (octanol/water) of 2.13 and for bioconcentration factor (BCF) of
    24 (Miller & Wasik, 1985).  The BCF for freshwater algae was reported
    to be 30 (Geyer et al., 1984), for water fleas ( Daphnia sp.) it was
    153-225, depending on the concentration of benzene in their food, and
    for goldfish it was 4.3 (Ogata et al., 1984).

    5.  ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

    5.1  Environmental levels

    5.1.1  Air

         Examples of benzene concentrations in urban and rural areas are
    given in Table 6.  Daily median benzene air concentrations in the USA
    have been reported as: remote areas, 0.51 µg/m3 (0.16 ppb); rural
    areas, 1.50 µg/m3 (0.47 ppb); and urban/suburban areas, 5.76 µg/m3
    (1.8 ppb) (Shah & Singh, 1988).

         The concentration appears to depend largely on the density of
    automobile traffic and local weather conditions (Wallace, 1989a). 
    Although the median level in USA urban areas is 5.76 µg/m3 (1.8 ppb)
    (Shah & Singh, 1988), levels as high as 112 µg/m3 (35 ppb) have been
    observed (US EPA, 1987).  Maximum levels of 510 µg/m3 (Wallace et
    al., 1985) and 210.6 µg/m3 (Singh et al., 1982) have been reported
    in two cities in the USA.  In addition to the concentrations of
    benzene shown in Table 6, the following levels of benzene have been
    reported in the urban air of European cities:  London, 10-12 µg/m3
    background and 28-31 µg/m3 kerbside (Bailey & Schmidl, 1989);
    Hamburg, Elb Tunnel, 80.5-95.3 µg/m3 (Dannecker et al., 1990) and a
    residential site 9.3 µg/m3 (Bruckmann et al., 1988); and Stockholm,
    average values of 147.7 µg/m3 on a busy street in the city centre
    and 7.7 µg/m3 on a quiet street in the city centre (Jonsson et al.,
    1985).  Country wide averages in Germany have been reported to be 1-10
    µg/m3 (0.31-3.1 ppb) (GDCh, 1988) and in three urban areas of Canada
    they were 2.9-19.6 µg/m3 (0.9-6.0 ppb) (Government of Canada, in
    press).  Benzene levels, along with other pollutants, may increase
    during periods of still air.

         Concentrations of benzene in the atmosphere of cities where
    chemical factories use or produce benzene are more variable.  In the
    USA, benzene concentrations have been shown to vary between 0.4 and 16
    µg/m3 (Pellizzari, 1982).  Levels of 3.2 mg/m3 (1 ppm) have been
    measured in the breathing zone during the refuelling of automobiles
    (Bond et al., 1986a).

         In Frankfurt, Germany, the highest benzene levels have been
    measured in the vicinity of coke ovens (maximum, 166.2 µg/m3;
    average, 57.2 µg/m3), near industrial refineries (maximum, 102
    µg/m3; average, 13.4 µg/m3), and in congested traffic areas
    (maximum, 171.8 µg/m3; average, 16.9 µg/m3) (GDCh, 1988).

         It has been reported that people living near petrochemical plants
    in New Jersey, USA, have no greater exposure to benzene than the
    general population throughout the area (Wallace et al., 1985).  Of
    particular interest in this study was the observation that in Bayonne,
    New Jersey, benzene levels (arithmetic means) in indoor air (29.7
    µg/m3) were greater than those reported for outside air (8.6
    µg/m3) (Table 6).


    
    Table 6.  Examples of the concentrations of benzene measured in air
                                                                                       

                                          Concentration (µg/m3)
                                                                
    Location (year)                       Mean           Maximum     Reference
                                                                                  

    Montreal, PQ, Canada (1984-1986)      18.6             81.8      Dann (1987)

    Toronto, ONT, Canada (1984-1986)       9.1             37.8      Dann (1987)

    Houston, TX, USA (1980)               18.8            122.9      Singh et al. (1982)

    Elizabeth & Bayonne, NJ, USA           8.6             91        Wallace et al.
      (outdoor air) (1981)                                           (1985)

    Elizabeth & Bayonne, NJ, USA          29.7            510        Wallace et al.
      (indoor air) (1981)                                            (1985)

    Pittsburgh, PA, USA (1981)            16.3            210.6      Singh et al. (1982)

    Oslo, Norway (1980)                   40              114        Wathne (1983)

    Rhine area, Germany (1983)          4.6-22.4            -        Bruckmann et al.
                                                                     (1983)

    Black Forest, Germany (1983)           2.0              -        Bruckmann et al.
                                                                     (1983)

    London, England (1983)                23               85        Clark et al. (1984a)

    England (1983) (45 km from London)     6               16        Clark et al. (1984a)

    Bilthoven, Netherlands (1982-1983)     2.8             10.4      RIVM (1988)
                                                                                       
    

         The principal source of benzene detected in indoor air appears to
    be cigarette smoke, making active smoking and exposure to passive
    smoke important sources of exposure to benzene for the general
    population.  The mainstream cigarette smoke from one cigarette
    contains between 6 and 73 µg of benzene (Brunnemann et al., 1989). 
    Benzene has been found at higher levels in the homes of smokers
    (16 µg/m3) than those of nonsmokers (9.2 µg/m3) during the autumn
    and winter, whereas levels in the summer were comparable in both
    domiciles (4.8 and 4.4 µg/m3, respectively) (Wallace & Pellizzari,
    1986).  Levels of benzene in a smoke-filled bar in the USA were found
    to be 26 to 36 µg/m3 (Brunnemann et al., 1989).

         Preliminary studies have indicated the release into indoor air of
    low levels of benzene from consumer products such as adhesives,
    building materials and paints (Wallace et al., 1987).

    5.1.2  Water

         Rain water in the United Kingdom has been found to contain
    benzene levels as high as 87.2 µg/litre (Colenutt & Thorburn, 1980)
    (Table 7).

         Concentrations as high as 330 µg/litre have been found in
    contaminated well water on the east coast of the USA (Burmaster,
    1982).  Benzene levels in open ocean samples from the relatively
    unpolluted waters of the Gulf of Mexico were found to be 0.005-0.015
    µg/litre (Sauer, 1981) and in polluted waters levels were 0.005-0.04
    µg/litre (Sauer, 1981).

         Representative concentrations of benzene in various sources of
    water are given in Table 7.

         Benzene concentrations in fresh surface waters are generally less
    than 1 µg/litre. In the USA, early studies reported 1-7 µg/litre in
    polluted areas (Ewing et al., 1977) whereas McDonald et al. (1988)
    reported levels of between 0.004 and 0.91 µg/litre in river water
    taken downstream from a chemical plant.  Levels between 0.2 and 0.8
    µg/litre were reported in the River Rhine in 1976 (Merian & Zander
    (1982).  In Japan, a survey of 112 water samples revealed benzene in
    only 19 of the samples at levels varying from 0.03 to 2.1 µg/litre
    (Environment Agency, Japan, 1989).

         The limited data available indicate that benzene concentrations
    in drinking-water are also in the µg/litre range.  Otson (1987)
    reported that levels in 10 drinking-water supplies in Canada did not
    exceed 1 µg/litre.  At a detection limit of 0.1 µg/litre, benzene was
    found in 13, 3 and 2 out of 14 samples of treated water in the summer,
    winter and spring, respectively.  Previously, Otson et al.  (1982) had
    reported detectable (> 1 µg/litre) levels of benzene in 50 to 60% of
    samples taken, the mean concentrations varying between 1 and 3
    µg/litre.  In the USA, water from contaminated wells contained 30 to

    330 µg benzene/litre.  In the same area, most samples of
    drinking-water taken from surface sources had non-detectable
    concentrations of benzene, and a maximum level of 4.4 µg/litre was
    detected (Burmaster, 1982).

    5.1.3  Soil and sediments

         In general, soil contamination does not lead directly to
    significant levels of human exposure because of rapid volatilization
    to air.  Benzene in soil is usually the result of direct contamination
    by spillage or leakage.  It has been found at levels ranging from 
    < 2 to 191 µg/kg in soils in the vicinity of five industrial 
    facilities using or producing benzene in the USA (Fentiman et al., 
    1979).  Soil concentrations in the Netherlands are low, the measured 
    concentrations being less than those found in ground water, 
    i.e. < 0.005 to 0.03 µg/litre (RIVM, 1988).

         Benzene was detected in 37 out of 98 bottom sediments in Japan at
    levels ranging from 0.5 to 30 µg/kg (Environment Agency, Japan, 1989). 
    In Lake Pontchartrain, Louisiana, Ferrario et al. (1985) reported
    sediment levels of 8 to 21 µg/kg wet weight.  Between 1980 and 1982,
    benzene was detected in 9% of the sediment samples taken from 335
    observation sites in the USA, the median level being < 5 µg/kg
    (Staples et al., 1985).


    
    Table 7.  Levels of benzene in water

                                                                                                                                         

    Source              Location                Concentration (µg/litre)   Comments                             References
                                                                                                                                              

    Rainwater           United Kingdom                    87.2             appear high for unknown reason(s)    Colenutt & Thorburn (1980)

                        Germany (Berlin)                 0.1-0.5                                                Lahmann et al. (1977)

    Surface water       USA (Brazos River,              0.004-0.9          downriver chemical plant outfall     McDonald et al. (1988)
                        TX)

                        USA (13 sampling                  1-13             both upstream and downstream near    Fentiman et al. (1979)
                        locations)                                         industrial outfall

                        USA (Potomac River)                < 2             detection limit, 2 µg/litre          Hall et al. (1987)

                        Switzerland (Lake                 0.03                                                  Grob & Grob (1974)
                        Zurich)

                        United Kingdom            > 7.2 (98.4 maximum)     average of 61 of 154 samples above   SAC (1989)
                        (80 water bodies             for all samples       0.1 µg/litre detection limit
                        across UK)

                        Netherlands                       < 0.1            sampling in 1979                     RIVM (1988)
                        (Rhine River)

                        Germany                          < 0.1-1           occasionally up to 200 µg/litre      Reynolds & Harrison (1982)
                                                                                                                                              

    Table 7 (contd).

                                                                                                                                         
                      
    Source              Location                Concentration (µg/litre)   Comments                             References
                                                                                                                                         

    Sea water           Gulf of Mexico               0.005 to 0.015        unpolluted waters; sampling          Sauer (1981)
                                                                           during 1977

                        USA (Brazos River               0.004-0.2          flows into Gulf of Mexico            McDonald et al. (1988)
                        estuary, TX)

                        Atlantic Ocean                 0.06 x 10-3         open sea                             OECD (1986)

                        Baltic Sea                   0.1-4.6 x 10-3        open sea                             OECD (1986)

    Drinking-water      USA                            0.1 to 0.3                                               US EPA (1980)

                        Canada (Ontario)              < 0.1 to 0.2         10 treatment plants surveyed         Otson (1987)

                        Germany                          < 0.1-1           occasionally up to 10 µg/litre       Reynolds & Harrison (1982)

    Ground water        USA (Nebraska)                1.6 (median)         63 private wells, 3.2% of samples    Goodenhauf & Atkinson (1986)
                                                      1.8 (maximum)        contained benzene

                        Germany                         0.02-0.05                                               Korte & Klein (1982)

                        USA (New York, New               30-300            contaminated well water              Burmaster (1982)
                        Jersey, Connecticut)

                        Netherlands                    0.005-0.03          unpolluted areas                     RIVM (1988)
                                                                                                                                              
    

    5.1.4  Food

         Data on the occurrence of benzene in food are limited.  However,
    early studies reported low levels of benzene in a variety of foods. 
    Some of the higher levels have been reported in Jamaican rum (120
    µg/litre), irradiated beef, (19 µg/kg), heat-treated canned beef
    (2 µg/kg) and eggs (500-1900 µg/kg) (IARC, 1982).  Other foods where
    it has been found but not quantified include haddock fillet, dry red
    beans, blue cheese, cheddar cheese, cayenne pepper, pineapple, roasted
    filberts, cooked potato peels, cooked chicken, hothouse tomatoes,
    strawberries, blackcurrants, roasted peanuts, soybean milk and codfish
    (Chang & Peterson, 1977).  Benzene was detected at levels of 220 and
    260 µg/kg wet weight in one sample of clams and oysters from Lake
    Pontchartrain in Louisiana, USA (Ferrario et al., 1985).  These
    findings were not repeated when a second sample was analysed.

         Benzene was detected in 37 out of 114 samples of fish in Japan
    within the range of 3-88 µg/kg (Environment Agency, Japan, 1989). 
    Gossett et al. (1983) reported that livers of marine fish caught in
    polluted waters near Los Angeles, USA contained levels of benzene in
    the range 15-52 µg/kg.

    5.2  General population exposure

         Benzene is ubiquitous in the environment.  Most of the general
    population is exposed to benzene through a variety of sources.  The
    most important source of exposure for the general population is
    through breathing air contaminated from man-made sources (including
    cigarette smoking), with inhalation exposures accounting for more than
    99% of the general population exposure (Hattemer-Frey et al., 1990). 
    Inhalation exposures occurring during the refuelling of automobiles
    with gasoline can also be important.  It has been estimated that a
    person is exposed to levels of benzene of about 3.2 mg/m3 while
    refuelling a vehicle with regular grade gasoline (Bond et al., 1986a),
    which adds about 10 µg of benzene to the average daily intake.  Other
    sources of inhalation exposure include air near hazardous waste sites
    or industrial facilities, and emissions from consumer products,
    including off-gassing from particle board (ATSDR, 1991).  Based on
    extensive studies in the USA, it appears that facilities manufacturing
    chemicals, drinking-water, food and beverages, and petroleum refining
    operations play only a minimal role in the total exposure of the
    general population to benzene (Wallace, 1989b).

         Attempts have been made to quantify the level of benzene exposure
    in the general population (Wallace, 1989a,b; Government of Canada, in
    press).  These studies make various assumptions as to the relative
    importance and amounts of benzene from various sources, many supported
    only in unpublished reports.  However, they all agree that personal
    sources (use of products emitting benzene, driving or riding in
    automobiles), automobile exhaust and smoking (active and passive) are
    major sources of benzene to the general population.  By far the

    greatest source of benzene exposure arises from active smoking (about
    1800 µg from about 30 cigarettes/day) (Wallace, 1989b).

         In both the USA and Canada, daily intakes from food and water are
    minimal (up to about 1.4 µg/day).  Intake from ambient and indoor air
    is extremely variable depending upon whether one resides in an
    industrial or large urban centre or a more rural environment, but it
    has been calculated to be about 90 µg/day for a 70-kg adult in Canada
    and between 180 and 1300 µg for adults in the USA.  Other sources are
    passive smoking (50 µg/day) and automobile-related activities (50
    µg/day).  For an average non-smoking 70-kg Canadian exposed to passive
    smoke and various consumer products, the total daily intake of benzene
    has been calculated to be approximately 230 µg, with an active smoker
    taking in an additional 1800 µg daily (Government of Canada, in
    press).  Within the USA, daily intakes for non-smokers have been
    calculated to range between 430 and 1530 µg/day (Wallace 1989a,b). 
    The higher levels and wider range of exposures in the USA probably
    reflect higher levels of benzene detected in the ambient air of large
    cities and the variations from city to city.

    5.3  Occupational exposure during manufacture, formulation or use

         Occupational exposure occurs mainly during the production,
    handling and use of benzene and its derivatives.  Surveys of
    occupational exposure have been reported by Fishbein (1984), UBA
    (1982) and Weaver et al. (1983).

         Table 8 presents the number of workers in several industrial
    sectors exposed to various time-weighted average (TWA) benzene
    concentrations.  These data are from the USA only and are presented to
    show the workers at highest risk within an industrialized country. 
    Without data to the contrary, it should be assumed that the data in
    Table 8 are, in general, representative of other industrialized
    countries.  The table does not include workers in firms not covered by
    the US OSHA regulations, those under other US jurisdictions, those
    using chemicals containing low levels of benzene, and tank maintenance
    firms.  However, these data do show that in seven major industries in
    the USA employing 237 812 potentially exposed workers, approximately
    95% of the workers were exposed to air levels below 16 mg/m3, i.e.
    less than 50% of the 32 mg/m3 TWA.  Similarly, most workers in
    Sweden are exposed to values less than 16 mg/m3, with occasional
    short-term exposures to 32 mg/m3 being reported among workers in
    refineries and bulk petrol terminals (Nordlinder & Ramnäs, 1987).

         CONCAWE (1986) reported on benzene exposure data measured over
    recent years in European countries during the manufacture and
    distribution of gasoline.  These data represent 8-h TWA exposure
    levels in various sectors of the oil industry.  The report concluded
    that such exposures are normally below 3.2 mg/m3 (1 ppm) for
    refinery unit operators, road tanker drivers and service station
    attendants.  Under some conditions, 8-h TWA exposures may exceed 3.2



        Table 8.  Percentage of employees in the USA potentially exposed to benzenea

                                                                                                                          

                                 Percentage of observations in each exposure category according to
                                        range of 8-h TWA benzene concentrations (mg/m3)
                                                                                                     

    Industry sector                     0.3-0.32    0.33-1.6    1.61-3.2    3.3-16.0    16.1-32   32+     Total number of
                                                                                                          employees
                                                                                                     
                                                                                                                          

    Petrochemical plantsb                             74.6                    23.0        2.4     0.0            4300

    Petroleum refineriesc,d               64.6        26.1         4.6         3.8        0.5     0.4          47 547

    Coke and coal chemicalse               0.0        39.3        27.6        27.5        4.4     1.3             947

    Tyre manufacturersc                   53.4        37.5         6.3         2.8        0.0     0.0          65 000

    Bulk terminalsc                       57.8        32.8         5.3         3.7        0.3     0.1          27 095

    Bulk plantsc                          57.8        32.8         5.3         3.7        0.3     0.1          45 323

    Transportation via tank
      truckc                              68.4        23.1         5.3         2.9        0.1     0.2          47 600
                                                                                                                      
    Total                                                                                                     237 812
                                                                                                                      
                                                                                                                          

    a  Adapted from: OSHA (1987)
    b  Percentages represent the proportion of workers whose average exposures are in each category.
    c  Percentages represent the proportion of sampling results in each exposure category.
    d  Data do not reflect respirator use and sampling biases.
    e  Excludes workers employed at the coke ovens.
    

    mg/m3 for operators and supervisors in road tanker filling, in rail
    car and marine loading, and in drum filling, but only rarely do these
    exceed 32 mg/m3 (10 ppm).  Additional information on occupational
    exposure levels in these industries is provided in IARC (1989).

         Yin et al. (1987) reported benzene concentrations in Chinese
    facilities producing paint and manufacturing shoes.  While the
    majority of the exposures were less than 40 mg/m3, concentrations in
    excess of 1000 mg/m3 were found in over 500 workplaces.  In
    addition, area samples were taken in 50 255 workplaces where benzene
    or benzene mixtures were used (Yin et al., 1987).  The geometric mean
    concentration of benzene in these workplaces was 18.1 mg/m3, and
    64.6% of the workplaces had concentrations of less than 40 mg/m3.

    6.  KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS

    6.1  Absorption

         The primary route of benzene exposure and subsequent toxicity is
    via inhalation.  Dermal and oral exposures are of minimal importance
    in terms of total daily intake of the general population.

    6.1.1  Air

         Studies in rats and mice suggest that the uptake of benzene from
    the lungs is nonlinearly related to the exposure concentration, i.e.
    the lower the concentration the greater the absorption above
    approximately 320 mg/m3 (100 ppm) (Sabourin et al., 1987).  The
    percentage of inhaled benzene that was retained decreased from 33% to
    15% when exposure in rats for 6 h was increased from 32 to 3200
    mg/m3) (10-1000 ppm); the values for mice decreased from 50% to 10%
    absorption.

         Several studies of benzene exposure via inhalation in humans
    suggest a lung absorption factor of about 50% for continuous exposure
    to 160-320 mg/m3 (50-100 ppm) for several hours (Nomiyama &
    Nomiyama, 1974a,b; Snyder et al., 1981).  Results from men and women
    exposed to benzene concentrations of 170-200 mg/m3 (52-62 ppm) for
    4 h showed that retention decreased with the duration of exposure and
    reached a constant level after 2 h (Nomiyama & Nomiyama, 1974a,b). 
    Retention (difference between uptake and elimination) was estimated to
    be 30% of the inhaled dose (Nomiyama & Nomiyama, 1974a,b).  Absorption
    was greatest during the first 5 min and reached a constant level
    between 15 min and 3 h of continuous exposure.

    6.1.2  Oral

         Animal studies support the view that absorption after oral
    exposure occurs readily and rapidly.  Over 90% of the total
    radioactivity of orally administered doses of 14C-benzene to rabbits
    (340-500 mg/kg body weight) was absorbed and eliminated in the air and
    urine (Parke & Williams, 1953).  Similar studies in mice and rats
    indicate that > 97% of oral doses (0.5 to 150 mg/kg body weight) was
    absorbed in these species (Sabourin et al., 1987).

         Definitive studies in humans on the rate of absorption of benzene
    after ingestion are not available.  However, cases of accidental or
    intentional ingestion suggest that it is absorbed readily.  Estimated
    oral doses from 9 to 30 g have proved fatal (Sandmeyer, 1981).

    6.1.3  Dermal

         Dermal absorption of benzene has been shown to occur in rhesus
    monkeys, minipigs, and hairless mice (Franz, 1984; Susten et al.,
    1985).  Absorption was less than 1% following one application of

    liquid benzene.  However, the rate of absorption was high, with the
    highest urinary excretion of the absorbed dose being observed in the
    first 8 h (Franz, 1984).  Maibach & Anjo (1981) measured greater skin
    penetration after multiple applications of benzene or after
    applications to abraded skin.

         It has been shown that benzene is absorbed through the skin of
    humans.  One study found that on average 0.023% of the benzene applied
    to skin was absorbed; the remainder quickly volatilized (Franz, 1984). 
    Hanke et al. (1961) reported an hourly absorption of 0.4 mg/cm2 when
    the forearm was bathed in liquid benzene.

         It has been estimated that an adult working in ambient air
    containing benzene at a concentration of 32 mg/m3 (10 ppm) would
    absorb 7.5 µl/h via inhalation and 1.5 µl/h via whole body (2 m2)
    dermal exposure (Blank & McAuliffe, 1985).  The authors also estimated
    that 100 cm2 of smooth and bare human skin in contact with gasoline
    containing 5% benzene would absorb 7.0 µl/h.

    6.2  Distribution

    6.2.1  Inhalation exposure

         In experimental animals, absorbed benzene is distributed
    throughout several compartments, with the parent compound being
    preferentially stored in fat and fatty tissues.

         Steady state benzene concentrations in rats exposed via
    inhalation to 1600 mg/m3 (500 ppm) for 6 h were: blood, 11.5 mg/kg;
    bone marrow, 37.7 mg/kg; and fat, 164.0 mg/kg (Rickert et al., 1979). 
    Benzene was also found in the kidney, lung, liver, brain and spleen. 
    Levels of the benzene metabolites phenol, catechol and hydroquinone
    were higher in bone marrow than blood, with phenol being eliminated
    more rapidly after exposure than catechol or hydroquinone.  Ghantous
    & Danielsson (1986) exposed pregnant mice to a benzene concentration
    of 6400 mg/m3 (2000 ppm) for 10 min and found benzene and its
    metabolites in lipid-rich tissues such as brain and fat, as well as in
    perfused tissues such as liver and kidney.  Benzene was also found in
    the placenta and fetuses immediately following exposure.

         Studies on humans exposed to 170-202 mg/m3 (52-62 ppm) for 4 h
    showed that 46.9% of the dose was taken up by the subjects; 30.2% was
    retained and 16.8% was excreted as unchanged benzene in expired air
    (Nomiyama & Nomiyama, 1974a,b).  As far as retention is concerned,
    there is apparently no difference between men and women.  Most data on
    distribution of benzene in humans come from case studies.  As in
    animals, benzene is distributed in several organs, with lipid-rich
    tissues containing the highest levels.  For example, one autopsy study
    of a youth showed 20 mg/litre in blood; 390 mg/kg in brain; 16 mg/kg
    in liver; and 22 mg/kg in abdominal fat (Winek & Collom, 1971). 
    Benzene can cross the human placenta and has been found in cord blood

    at amounts equal to or greater than those in the mother (Dowty et al.,
    1976).

    6.2.2  Oral and dermal exposures

         Low et al. (1989) studied the tissue distribution of
    radioactivity arising from the administration of 14C-labelled
    benzene (0.15, 1.5, 15, 150 or 500 mg/kg body weight) by oral gavage
    to Sprague-Dawley rats.  At the lowest two dose levels,
    radioactivity/kg body weight was highest in the liver and kidney 1 h
    after dosing; intermediate levels were found in the blood, and the
    lowest levels in the Zymbal gland, nasal cavity and mammary gland. 
    When doses of 15 mg/kg or more were administered, there were larger
    increases in the levels found in mammary glands and bone marrow than
    in other tissues.  In these studies, it is difficult to differentiate
    between benzene distribution and the distribution of metabolites.

         After 48 h following dermal application to male rats of
    14C-benzene (0.004 mg/cm2) the highest percentage of administered
    radioactivity was found in the kidney (0.026%), followed by the liver
    (0.013%) and treated skin (0.11%) (Skowronski et al., 1988).

         No reports are available regarding the distribution of benzene in
    humans after oral or dermal exposures.

    6.3  Metabolic transformation

         The metabolism of benzene in animals and humans appears to be
    qualitatively similar (Snyder, 1987; Snyder et al., 1987).  There is
    no indication that the route of administration has any marked effect
    on the metabolites formed.

         Benzene metabolism occurs primarily in the liver through the
    cytochrome P-450 IIE1 system (Johansson & Ingelman-Sundberg, 1988;
    Koop et al., 1989; Nakajima et al., 1990; Chepiga et al., 1991) and,
    to a lesser extent, in such target tissues as the bone marrow (Kalf,
    1987).  The first step in benzene metabolism is oxidative, yielding
    ring-hydroxylated compounds (Fig. 1).  There is also a cytochrome
    P-450 in bone marrow capable of metabolizing benzene (Gollmer et al.,
    1984).  The hydroxylated compounds (phenol, catechol, hydroquinone and
    1,2,4-trihydroxy-benzene) are excreted in the urine as ethereal
    sulfates and glucuronides (Fig. 2).  Conjugation with glutathione and
    urinary mercapturic acid is considered as an additional detoxification
    pathway (Fig. 1).  The opening of the benzene ring, presumably at the
    epoxide or the dihydrodiol stage, is thought to yield
    trans,trans-muconaldehyde (Latriano et al., 1986) which is further
    oxidized via a semialdehyde to trans,trans-muconic acid (Kirley et
    al., 1989) (Fig. 1 and Fig. 3).

         The immediate result of the oxidative metabolism (Fig. 1) is the
    formation of a system in equilibrium between benzene oxide and its
    oxepin.  Although the oxepin is a postulated structure, the strongest
    evidence for the formation of the epoxide is the demonstration that
    the addition of the enzyme epoxide hydrolase to microsomes used to
    metabolize benzene results in the accumulation of benzene dihydrodiol
    (Tunek et al., 1978).  No other intermediate would yield the
    dihydrodiol.  Further evidence that the epoxide is an intermediate was
    presented by Hinson et al. (1985), who proposed that the NIH shift
    should occur if the epoxide was an intermediate.  Using deuterated
    benzene, he detected the postulated labelled products and concluded
    that the epoxide was formed and that cyclohexadienone is a key
    intermediate.

         On the other hand, Johansson & Ingelman-Sundberg (1988) have
    argued that the first step in benzene metabolism is catalysed by a
    hydroxy radical generated by cytochrome P-450 LM2 from rabbit liver. 
    Hydroxy radical attack on the benzene ring was first postulated as a
    feasible chemical mechanism by Dorfman et al. (1962) on the basis of
    pulse radiolysis studies, and was applied to benzene hydroxylation in
    biological systems by Simic et al. (1989) and Karam & Simic (1989). 
    Gorsky & Coon (1985) were unable to repeat the work of Johansson &
    Ingelman-Sundberg (1988) but argued that the essential distinction
    between the experiments was that the Swedish group used an extremely
    low substrate concentration, far below the Km of the enzyme, and
    under these circumstances cytochrome P-450 is uncoupled and is known
    to generate hydrogen peroxide.  At concentrations of benzene in the
    usual substrate range employed, the enzyme is fully coupled, peroxide
    is not generated, and the mechanism proceeds via the epoxide
    intermediate.

         The formation of phenol occurs by the spontaneous, non-enzymatic
    rearrangement of the epoxide.  Hydroquinone and catechol can then be
    formed by hydroxylation of phenol (Sawahata & Neal, 1983; Gilmour et
    al., 1986).  Catechol can also be formed by a sequential series of
    reactions beginning with the hydration of benzene oxide to yield
    benzene dihydrodiol, followed by the oxidation of the dihydrodiol by
    a dehydrogenase (Jerina & Daly, 1974; Bentley et al., 1976; Vogel et
    al., 1980).  The latter reaction cannot be observed in microsomal
    preparations since the dehydrogenase is a cytoplasmic enzyme.  Phenol,
    hydroquinone, catechol, and its further hydroxylation product,
    1,2,4-trihydroxy-benzene, can be conjugated with ethereal sulfate or
    glucuronic acid (Parke & Williams, 1953).  In a series of studies on
    benzene metabolism and toxicity, performed by Low et al. (1991) it was
    found that whereas phenylsulfate, a major conjugated metabolite of
    benzene, was found in many tissues after the administration of
    14C-benzene, none was found in the Zymbal gland, a significant
    target tissue.  These authors postulated that phenylsulfate was taken
    up by a transport system into the gland, and hydrolysed to yield the
    free phenol, which was then further metabolized to form reactive
    intermediates responsible for the carcinogenic activity of benzene in

    FIGURE 1

    FIGURE 2

    FIGURE 3

    the Zymbal gland.  This is the first suggestion that conjugation
    products, normally thought of as only a mechanism for urinary
    excretion, could also be considered to act as a transporting mechanism
    for bringing metabolites from the liver to target tissues.

         Parke & Williams (1953) reported that phenyl mercapturic acid was
    a urinary end product of benzene metabolism.  This observation was
    supported by the report of Jerina et al. (1968) who incubated
    glutathione with rat liver cytoplasm and benzene oxide and found that
    the principal metabolite was S-phenylglutathione.  Norpoth (1988) has
    developed a method for the determination of phenylmercapturic acid in
    human urine as a measure of exposure to benzene based on these
    observations.  However, Lunte & Kissinger (1983) showed that
     p-benzoquinone, an oxidation product of hydroquinone, forms
    glutathione conjugates non-enzymatically.  Lau et al. (1989) reported
    that 1,2,3 or 4 glutathione molecules could conjugate with
     p-benzoquinone.  Nerland & Pierce (1990) showed the occurrence of
     N-acetyl- S-(2,5-dihydroxyphenyl)l-cysteine as a urinary metabolite
    of benzene in rats.  Stommel et al. (1989) found that the metabolite
    phenylmercapturic acid increased proportionally in rats and humans as
    the inhaled dose rose to 1600 mg/m3 (500 ppm).  Thus, the array of
    mercapturic acid metabolites of benzene has expanded and the full
    extent of metabolites of this structure may not yet be fully
    appreciated.

         In summary, the postulated metabolic pathways for benzene are
    shown in Figures 1, 2 and 3.  The formations of mercapturic acids,
    ethereal sulfates and glucuronides are generally considered
    detoxification pathways leading to the excretion of benzene
    metabolites via the kidney (Henderson et al., 1989).  All other
    pathways lead to potentially toxic metabolites.  This hypothesis is
    discussed in more detail in section 7.9.

         In both rats and mice the formation of toxic metabolites via the
    epoxide pathway appears to be a saturable process, which suggests that
    both metabolism and toxicity would be non-linear.  In other words, the
    proportion of toxic metabolites formed would decrease once the
    saturation level is reached, whereas detoxification pathways appear to
    be low-affinity high-capacity reactions (Henderson et al., 1989;
    Medinsky et al., 1989a).  It has been shown that mice metabolize
    benzene faster and converted more of the benzene to toxic metabolites
    than rats (Henderson et al., 1989).  Because of this it has been
    suggested that metabolism in mice favours toxification pathways (e.g.,
    formation of benzoquinone and muconaldehyde), while in rats metabolism
    is primarily detoxification (phenyl conjugates and phenylmercapturic
    acids) (Medinsky et al., 1989a).  The percentage of benzene or its
    metabolites remaining in the body decreased in rats (from 33% to 15%)
    and mice (from 50% to 10%) as exposure increased from 32 to 3200
    mg/m3 (10 to 1000 ppm) (Sabourin et al., 1987).

         Model simulations for total benzene metabolized and for profiles
    of benzene metabolites formed after the administration of varying
    doses of benzene to rats and mice (Medinsky et al., 1989b,c) have
    suggested that the production of hydroquinone and muconic acid
    metabolites predominates at lower exposure concentrations, whereas at
    high exposure levels the detoxification pathways account for a larger
    fraction of benzene metabolized.  In addition, these model simulations
    have confirmed that mice metabolize more benzene on a µmole/kg body
    weight basis than rats after inhalation exposures, whereas rats
    metabolize more benzene than mice at oral doses greater than 50 mg/kg
    body weight.  After either oral or inhalation exposures mice
    preferentially form more of the putative toxic metabolites
    hydroquinone and muconic acid (Medinsky et al., 1989b).  It has also
    been reported by Witz et al. (1990b) that DBA/ZN mice (a strain
    sensitive to the haematotoxicity of benzene), excrete greater amounts
    of trans, trans-muconic acid than the less sensitive C57BL/6 strain
    after equivalent exposures to benzene. 

    6.4  Elimination and excretion

    6.4.1  Inhalation exposure

         In animals, expired air is the main route of elimination of
    unmetabolized benzene, while urine is the major route of excretion of
    benzene metabolites (with very little faecal excretion).  Rickert et
    al. (1979) found a biphasic pattern of excretion of unmetabolized
    benzene in rats after a 6-h exposure to 1600 mg/m3 (500 ppm), with
    half-times of 0.7 h for the rapid phase and 13.1 h for the slow phase. 
    The major route of excretion after inhalation exposures of rats and
    mice to 32-3200 mg/m3 (10-1000 ppm) appeared to be dependent upon
    the concentration inhaled (Sabourin et al., 1987).  Under these
    conditions mice received 150-200% of the dose given to rats on a per
    kg body weight basis.  The faecal excretion was < 3.5% in rats and 
    < 9% in mice.  At doses up to 416 mg/m3 (130 ppm), less than 6% of 
    the radioactivity was eliminated in expired air, whereas at the highest
    concentrations 48% of the dose was eliminated as unchanged chemical in
    rats and 14% in mice.  The total urinary excretion of metabolites at
    these high concentrations was 5-37% higher in mice than in rats.

         Findings in humans after inhalation exposure to benzene are
    similar to those in experimental animals; unmetabolized chemical is
    eliminated in expired air whereas metabolites of benzene are excreted
    in urine, primarily as the sulfate and glucuronide conjugates of
    phenol.  Nomiyama & Nomiyama (1974a,b) found similar expiratory
    patterns in men and women exposed for 4 h to benzene at concentration
    between 166 and 198 mg/m3 (52-62 ppm).  The proportion of the
    absorbed benzene that was excreted via the lungs was approximately 17%
    (Nomiyama & Nomiyama, 1974a,b).

    6.4.2.  Oral exposure

         Parke & Williams (1953) administered radiolabelled benzene
    (approximately 340 mg/kg body weight) by oral gavage to rabbits and
    reported that 43% of the label was recovered as unmetabolized benzene
    in expired air.  Urinary excretion accounted for 33% of the dose,
    mainly in the form of conjugated phenol (23.5%).  Other phenols
    excreted were hydroquinone (4.8%), catechol (2.2%), and hydroxyquinol
    (1,2,4-trihydroxybenzene) (0.3%).  Muconic acid accounted for 1.3% and
    L-phenylmercapturic acid for 0.5%, and 5-10% of the radiolabel
    remained in the tissues or was excreted in the faeces.  The excretion
    of benzene and its metabolites in rats and mice at various oral doses
    (0.5-300 mg/kg body weight) was studied by Sabourin et al. (1987).  In
    both species the excretion of urinary metabolites up to a dose of 15
    mg/kg accounted for 80% of the administered dose.  Above that level
    there was an increase in the elimination of 14C in expired air. 
    Equal amounts of unmetabolized benzene were eliminated in both species
    up to dose levels of 50 mg/kg.  At dose levels of between 15 and 50
    mg/kg body weight, metabolism appears to become saturated in rodents. 
    In rats, 50% of a 150-mg/kg dose of 14C-benzene was eliminated in
    expired air, while in the mouse 69% of this dose was exhaled (Sabourin
    et al., 1987).

         No studies were found regarding the excretion of benzene in
    humans after oral exposures.

    6.4.3  Dermal exposure

         After the dermal application of 14C-benzene (0.0026 to 0.0036
    mg/cm2) to monkeys and minipigs, Franz (1984) collected urine
    samples every 5 h for 2-4 days.  The rate of excretion was highest
    over the first 10 h, the total excretion of radioactivity being higher
    in the monkey (0.03 to 0.14% of the applied dose, with an average of
    0.06%) than in the minipigs (0.03-0.05%, with an average of 0.04%). 
    Using a glass cap to minimize volatilization from the skin, Skowronski
    et al. (1988) treated male rats dermally with 14C-benzene (0.004
    mg/cm2).  After 48 h, 86.2% of the initial dose was excreted in the
    urine and 12.8% was eliminated in expired air.  Phenol was the major
    urinary metabolite detected in the 0-12 h sample (37.7% of dose), and
    smaller quantities of hydroquinone, catechol and benzenetriol were
    also detected.

         In a study of four male human subjects, Franz (1984) applied
    14C-benzene dermally (0.0024 mg/cm2).  A mean of 0.023% (range
    0.006-0.054%) of the applied radiolabel was recovered in the urine
    over a 36-h period.  More than 80% of the excretion occurred within 
    8 h of application.

    6.5  Retention and turnover

         Steady state levels of benzene were found within 4 h in blood, 
    6 h in fat, and 2 h in bone marrow when male rats were exposed to a
    benzene concentration of 1600 mg/m3 (500 ppm) by inhalation for 6 h. 
    After exposure ceased, about 70% of the benzene was eliminated
    unchanged in the expired air and about 30% was excreted in urine as
    water-soluble metabolites within 15 h.  The half-life (t´) for
    elimination from these tissues was 0.4-0.8 h, except in the case of
    adipose tissue where elimination occurred with a t´ of 1.6 h.  The
    elimination of unchanged benzene in expired air was biphasic, the t´
    being 0.7 h for the first phase and 13.1 h for the slower phase.  Free
    phenol, catechol and hydroquinone were detected in blood and bone
    marrow after exposure ceased.  The phenol level declined rapidly over
    a 9-h observation period, whereas catechol and hydroquinone levels in
    both tissues remained constant over this period (Rickert et al.,
    1979).

         After intraperitoneal injection, oral gavage, or inhalation
    exposures of labelled benzene in rats and mice, over 95% of the
    administered radioactivity was excreted within 40 h (Sabourin et al.,
    1987; Henderson et al., 1989).  Approximately 90% of the metabolites
    was excreted in the urine.  According to the authors, these studies
    indicate that benzene is rapidly metabolized and excreted in the urine
    within 40 h of dosing by any route of administration.

    6.6  Reaction with body components

         3H-benzene metabolites have been shown to bind irreversibly to
    proteins in both mouse liver and bone marrow (Snyder et al., 1978a). 
    Benzene metabolites have also been shown to bind  in vivo to mouse
    protein in blood (Sun et al., 1990), liver, bone marrow and spleen
    (Longacre et al., 1981a,b).  Covalent binding increased both with dose
    and frequency of dosing.  Covalent binding of benzene metabolites to
    protein appears to be mediated by microsomal enzymes (Tunek et al.,
    1978) and has been suggested to be the result of binding by
    hydroquinone and catechol (Wallin et al., 1985).  The finding of high
    levels of phenylcysteine adducts in the haemoglobin of benzene-exposed
    rats suggests that benzene oxide also reacts with proteins to form
    adducts (Bechtold et al., 1992).  Benzene metabolism and covalent
    binding to proteins have been demonstrated  in situ in bone marrow
    (Irons et al., 1980b).

         Lutz (1979) has attempted to quantify the extent to which
    chemicals covalently bind to DNA using the concept of the covalent
    binding index (CBI).  The implication is that the higher the CBI, the
    more likely a chemical will be a carcinogen.  The calculated value for
    benzene, based on binding to liver nuclear DNA, was 1.7.  (To put this
    value in perspective, CBI values for some common carcinogens were:
    aflatoxin B1, > 1000; 2-acetyl-aminifluorene, 100 to several hundred;
    polycyclic aromatic hydrocarbons, 10 to 30.)  No values were given for

    benzene in bone marrow, but Snyder et al. (1978a) compared covalent
    binding of benzene residues per g dry weight of liver and bone marrow
    and found that, depending upon the dose of benzene, covalent residues
    bound to liver ranged from 500 to 800 nmoles/g, whereas binding to
    bone marrow ranged from 18 to 96 nmoles/g.  Thus, there appeared to be
    less covalent binding in the target organ, i.e. bone marrow, than in
    the metabolizing organ, i.e. liver.

         Inhaled benzene has been found to bind to rat liver DNA to the
    extent of 2.38 µmoles/mole DNA phosphate (Lutz & Schlatter, 1977). 
    Studies of the covalent binding of benzene metabolites to DNA have
    resulted in the postulation of several structures for DNA adducts
    derived from benzene.  Bone marrow mitochondria from rabbits were
    incubated sequentially with 3H-deoxyguanosine triphosphate and
    14C-benzene to evaluate DNA adducts formed from benzene metabolites
    (Snyder et al., 1987b).  These authors identified at least seven
    deoxyguanosine adducts and one deoxyadenine adduct.  Covalent
    N-7-phenyl-guanine adducts have been isolated from rat urine after
    intraperitoneal dosing with 330-400 mg benzene/kg body weight (Norpoth
    et al., 1988).  Thus, Jowa et al. (1990) postulated the formation of
    an adduct between  p-benzoquinone and deoxyguanosine which had the
    structure (3'OH)benztheno(1,N2)deoxyguanosine. The structure of an
    adduct formed between  p-benzoquinone and deoxyadenosine-3'-phos
    phate was suggested to be 3'-hydroxy-1,N6-benztheno-2'-deoxy-
    adenosine-3'-phosphate (Pongracz & Bodell, 1991).  Reddy et al.
    (1990), however, reported that they were unable to detect DNA adducts
    derived from benzene in the rat  in vivo, despite having observed
    them in Zymbal gland cells  in vitro, using the 32P-post labelling
    technique.

    6.7  Modelling of pharmacokinetic data for benzene

         In order to obtain better insight into the interspecies
    variations in the uptake, metabolic fate and excretion of benzene and
    its metabolites, both compartmentally (Bailer & Hoel, 1989; Beliles &
    Totman, 1989) and physiologically based (Medinsky et al., 1989b,c;
    Paxman & Rappaport, 1990; Travis et al., 1990; Bois et al., 1991a,b),
    pharmacokinetic models have been developed.  These models have been
    used as an aid to risk assessment by facilitating extrapolation
    between species where various exposure regimens had been utilized. 
    Also, such models are useful for identifying gaps in knowledge that
    have been highlighted by poor fits of the experimental data to the
    models developed.

    7.  EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS

         Benzene has been shown to produce a number of biological
    responses in experimental animals.  The acute effects of benzene at
    high doses reflect its activity as a general anaesthetic and can lead
    to central nervous system (CNS) depression, loss of consciousness and
    coincidental sensitization of the myocardium to catecholamines. 
    Chronic exposure can result in bone marrow depression expressed as
    leucopenia, anaemia and/or thrombocytopenia, leading to pancytopenia
    and aplastic anaemia.  The immunotoxic effects of benzene are probably
    related to bone marrow depression.  In animal cancer bioassays it is,
    primarily, epithelial tumours that have been reported, whereas in
    humans the carcinogenic response is leukaemia.  A third type of
    biological impact is the production of clastogenic responses such as
    chromosome aberrations, sister chromatid exchange and micronuclei. 
    Benzene has also been suggested to produce fetotoxic effects.

    7.1  Single exposure

         Consistent with many other aromatic hydrocarbons (Patty, 1981),
    benzene appears to be of low acute toxicity when administered to
    various animal species by various routes of administration (Table 9). 
    Other reported oral LD50 values for reagent grade benzene in male
    rats vary from as low as 930 mg/kg to as high as 5600 mg/kg body
    weight (Wolf et al., 1956; Cornish & Ryan, 1965; Kimura et al., 1971;
    Withey & Hall, 1975).  The LD50 after intraperitoneal injection in
    female rats was reported to be 2940 mg/kg (Drew & Fouts, 1974) and in
    mice it was 300 mg/kg body weight (Kocsis et al., 1968).  Young rats
    are more sensitive (in terms of LD50) than older ones (Table 9).

         The LC50 in female rats was estimated to be 43 770 mg/m3 (13
    700 ppm) after a single 4-h exposure (Drew & Fouts, 1974).

         Benzene has a narcotic effect after oral administration in rats
    (Withey & Hall, 1975) and after inhalation in mice (Uyeki et al.,
    1977).  The threshold narcotic effect after inhalation has been
    estimated to be approximately 13 000 mg/m3 (Leong, 1977). 
    Inhalation of air saturated with benzene resulted in ventricular
    tachycardia and occasionally ventricular fibrillation and death in
    rats, cats, rabbits and primates (Nahum & Hoff, 1934).  Respiratory
    failure was also observed during narcosis. Pathological findings after
    sudden death are congestion of various organs, particularly the lungs
    and liver (Jonek et al., 1965).

         No information on the acute toxicity/lethality in animals after
    dermal exposure has been reported.


        Table 9.  Toxicity of benzene in animals after acute exposure

                                                                                                                                    

    Route                         Species             Parameter      Value                       Reference
                                                                                                                                    

    Oral                          rat (14 days old)   LD50           3000 mg/kg body weight      Kimura et al. (1971)

    Oral                          rat (young adult)   LD50           3300 mg/kg body weight      Kimura et al. (1971)

    Oral                          rat (old adult)     LD50           4900 mg/kg body weight      HSE (1982); Kimura et al. (1971)

    Oral                          rat                 LD50           8100 mg/kg body weight      Cornish & Ryan (1965)

    Inhalation (4 h)              rat                 LC50           44 660 mg/m3 body weight    Drew & Fouts (1974)

    Inhalation (7 h)              rat                 LC50           32 600 mg/m3 body weight    HSE (1982)

    Inhalation (2 h)              mouse               lethal dose    61 125 mg/m3 body weight    Jonek et al. (1965)

    Intraperitoneal injection     rat                 LD50           2940 mg/kg body weight      Drew & Fouts (1974)

    Intraperitoneal injection     mouse               LD50           300 mg/kg body weight       Kocsis et al. (1968)
                                                                                                                                    
    

    7.2  Short-term and long-term exposures

         The studies discussed in this section, some of which are
    summarized in Table 10, have a duration of less than one year. 
    Lifetime (> 1 year) studies are discussed in section 7.6 and
    summarized in Tables 15 to 17.

         In short-term inhalation studies, three out of eight male rats
    died within 24 h after exposure for five periods of 25-35 min to a
    benzene concentration of 128 000 mg/m3 (40 000 ppm) and two out of
    ten died after exposure for 12.5-30 min daily to 32 000 mg/m3
    (10 000 ppm) for 1-12 days (Furnas & Hine, 1958).

         Male and female rats and mice exposed to benzene vapour at
    concentrations of 3.2, 32, 96 or 960 mg/m3 (1, 10, 30 or 300 ppm)
    for 6 h/day, 5 days/week for 13 weeks, and sacrificed at various time
    points during the study, showed no haematological effects up to 96
    mg/m3 (30 ppm) (Ward et al., 1985).  However, at 960 mg/m3 (300
    ppm) mice exhibited significant decreases in haematocrit, haemoglobin,
    erythrocyte count, leucocyte count, platelet count and the percentage
    of lymphocytes.  There was an increase in erythrocyte volume and mean
    corpuscular haemoglobin.  These changes were first observed on days 14
    (males) or 28 (females).  Most of the haematological effects were also
    detected in rats but were of lesser severity.  Compound-related
    histopathological findings included myeloid hypoplasia, depletion of
    the periarteriolar lymphoid sheaths in the spleen, lymphoid depletion
    in the mesenteric lymph nodes, and increased extramedullary
    haematopoiesis in the spleen.  These lesions persisted throughout the
    study and increased in severity with time.  The only histopathological
    lesion observed in rats was slightly reduced cellularity in the bone
    marrow of the femur.

         A dose-related increase in leucocyte alkaline phosphatase levels
    and a decrease in leucocyte levels was observed in female rats exposed
    via inhalation to 320, 960, 3200 or 9600 mg/m3 (100, 300, 1000 or
    3000 ppm) for 7 or 14 days, but not in those exposed to 64 or 160
    mg/m3 (20 or 50 ppm) (Li et al., 1986).  In a study on male mice,
    with doses of 3.5, 32, 330, 980, 1930, 4080, 7730 and 15 600 mg/m3
    (1.1, 9.9, 103, 306, 603, 1276, 2416 and 4862 ppm), granulocytopenia,
    lymphocytopenia and reduced bone marrow and splenic cellularity were
    observed after exposure to > 330 mg/m3 for 5 h/day for 5 days but
    not at lower levels (Green et al., 1981a).  These authors found
    splenic lesions at levels as low as 32 mg/m3 when exposure was
    extended to 10 weeks.


        Table 10.  Toxicity of benzene in animals after short-term and long-term inhalation exposuresa

                                                                                                                                         

    Species        Dose (mg/m3)    Exposure period                     Effects                                        References
                                                                                                                                         

    Rat                 48         7 h/day; 5 days/week;     no adverse effects, blood benzene level            Deichmann et al. (1963)
                                   28 weeks                  at term 90 µg/litre

    Rat                 100        7 h/day; 5 days/week;     no adverse effects, blood benzene level            Deichmann et al. (1963)
                                   7 weeks                   at term 290 µg/litre

    Rat                 150        7 h/day; 5 days/week;     slight leucopenia, blood benzene level             Deichmann et al. (1963)
                                   32 weeks                  at term 420 µg/litre

    Rat                3200        18 h/day; 7 days/week;    reversible haemotological effects,                 Nau et al. (1966)
                                   15 weeks                  reversible leucopenia within 6 months

    Rat               3.2-960      6 h/day; 5 days/week;     slight changes in haematological cell counts       Ward et al. (1985)
                                   13 weeks                  and lower cellularity in bone marrow of femur

    Rat (female)      64-9600      1-2 weeks                 dose-related increase in leucocyte alkaline        Li et al. (1986)
                                                             phosphatase and decreased leucocyte counts
                                                             at exposures of 960 mg/m3 or more

    Mouse          3.5 to 15 600   6 h/day; 1 week           doses > 330 mg/m3 resulted in granulocytopenia,    Green et al. (1981a,b)
                                                             lymphocytopenia, decreased splenic cellularity
                                                             and decreased stem cell production

    Mouse (male)       30.7        6 h/day; 5 days/week;     some increase in spleen weight and some            Green et al. (1981a)
                                   10 weeks                  increased cellularity
                                                                                                                                              

    Table 10 (contd).

                                                                                                                                         

    Route                         Species             Parameter      Value                       Reference
                                                                                                                                         

    Mouse (male)        960        6 h/day; 5 days/week;     increased mortality, marked lymphocytopenia,       Green et al. (1981a)
                                   26 weeks                  reduced red blood cell count, reduced spleen
                                                             weight, many morphological abnormalities in
                                                             circulating red blood cells and neutrophils

    Mouse (male)        32         6 h/day; 5 days/week;     depression in the numbers of splenic nucleated     Baarson et al. (1984)
                                   25.5 weeks                cells and of circulating red cells and lymphocytes

    Mouse             3.2-960      6 h/day; 5 days/week;     only at 960 mg/m3 decreased blood cell counts,     Ward et al. (1985)
                                   13 weeks                  haematocrit, myeloid hypoplasia, and numbers of
                                                             splenic and lymph node lesions

    Mouse             32-1280      6 h/day; 5 days/week;     reduced bone marrow cellularity and number of      Cronkite et al. (1985)
                                                             pluripotent stem cells at 320 mg/m3 or more

    Guinea-pig          280        7 h/day; 5 days/week;     slight leucopenia, increased weight of kidneys     Wolf et al. (1956)
                                   4.5 weeks

    Pig               64, 320      6 h/day; 5 days/week;     leucopenia at 320 and 1600 mg/m3, reversible       Johnston et al. (1979)
                     and 1600      3 weeks                   9-16 weeks after exposure
                                                                                                                                              

    a    To avoid duplication, toxicity from life-time exposures (over 1 year) is discussed in section 7.6
         (carcinogenicity) and Tables 15 to 17.
    

         Cronkite et al. (1985) exposed male and female mice by inhalation
    to benzene at concentrations of 32, 80, 320, 960 or 1280 mg/m3 (10,
    25, 100, 300 or 400 ppm) for 2 weeks (6 h/day, 5 days per week).  At
    320 mg/m3 or more, reduced bone marrow cellularity and a decreased
    number of pluripotent stem cells in bone marrow were reported.  Under
    similar conditions of exposure to 960 mg/m3 for 16 weeks, these
    authors reported a lower level of stem cells in bone marrow, which
    returned to 92% of control values after 25 weeks post-exposure.
    Complete reversibility within 2 weeks was reported after 2 or 4 weeks
    of exposure to 960 mg/m3.

         Long-term (> 6 months) exposure studies at benzene levels above
    approximately 160 mg/m3 (50 ppm) have shown effects on circulating
    leucocytes (especially leucopenia).  For example, rats exposed via
    inhalation to 150 mg/m3 (47 ppm) (7 h/day, 5 days per week for 8
    months) showed slight leucopenia, while those exposed to 100 mg/m3
    (31 ppm) for 4 months and those exposed to 48 mg/m3 (15 ppm) failed
    to show such changes (Deichmann et al., 1963).  The lowest reported
    exposure in animals that resulted in haematological effects was in
    mice (Baarson et al., 1984).  These authors reported that male mice
    exposed via inhalation to 32 mg/m3 (10 ppm) (6 h/day, 5 days/week)
    for 25.5 weeks showed a decrease in the number of circulating
    erythrocytes and lymphocytes, a decrease in the number of nucleated
    cells in the spleen and a depression of the  in vitro colony-forming
    ability of erythroid precursor cells (CFU-E).

         Uyeki et al. (1977) demonstrated a depression of stem cell
    activity in mice using the spleen colony-forming technique (CFU-S)
    after exposure to a benzene concentration of 15 000 mg/m3 (4680 ppm)
    for 3 days (8 h/day).

         Haematotoxicity was also noted after oral exposure in rats and
    mice.  The animals were dosed by gavage with benzene in corn oil for
    120 days at 25, 50, 100, 200, 400 or 600 mg/kg body weight (5
    days/week).  Five animals in the control, 200- and 600-mg/kg groups
    were sacrificed at 60 days (Huff et al., 1989).  A dose-related
    leucopenia was observed in both male and female rats and lymphoid
    depletion in the B-cells of the spleen was observed in both the 200-
    and 600-mg/kg groups at 60 days.  In mice no compound-related
    histopathological effects were observed, but a dose-related leucopenia
    was observed in both males and females.

         No information was found on the haematotoxicity of benzene after
    dermal exposure has been reported.

         Additional studies on the effects of long-term exposure to
    benzene in experimental animals are described in section 7.6
    (carcinogenicity) and in Tables 15 to 17.

    7.3  Skin and eye irritation

         Benzene is considered a moderate eye irritant (as shown in the
    rabbit eye test).  Two drops of benzene caused moderate conjunctival
    irritation and very slight, transient corneal injury (Wolf et al.,
    1956).

         Undiluted benzene was irritating to the skin (ear) of rabbits
    after 10-20 applications (Wolf et al., 1956).  Erythema, oedema,
    exfoliation, blistering and moderate necrosis were observed after 20
    applications.

         There is no information available on the skin-sensitizing
    potential of benzene.  However, no such effect is expected based on
    the experience with other aromatic hydrocarbons (GDCh, 1988).

    7.4  Reproductive toxicity, embryotoxicity and teratogenicity

         Benzene does not appear to be a potent reproductive toxin in
    experimental animals.  Guinea-pigs and rabbits exposed to benzene by
    inhalation (7 h/day, 5 days/week) for up to 6 months showed variable
    results; guinea-pigs showed a slight increase in testicular weight at
    280 mg/m3 (88 ppm) and rabbits showed slight degeneration of the
    seminiferous germinal epithelium at 256 mg/m3 (80 ppm) (Wolf et al.,
    1956).  Mice, but not rats, exposed to benzene vapour at a
    concentration of 960 mg/m3 (300 ppm) (6 h/day, 5 days/week) for 13
    weeks showed bilateral cysts in the ovaries and degeneration and
    atrophy of the testes (Ward et al., 1985).  At concentrations of 3.2,
    32 or 96 mg/m3 (1, 10 or 30 ppm) these changes were also seen in
    mice, but they were of doubtful biological significance (Ward et al.,
    1985).  There was a complete absence of litters in female rats exposed
    to 670 mg/m3 (210 ppm) for 10-15 days before mating and for 3 weeks
    after mating (Gofmekler, 1968).  It is not known if this represents a
    problem in mating and fertility, or one of maternal or fetal toxicity. 
    Lower exposures (1 to 64 mg/m3; 0.30 to 20 ppm) produced no such
    effects.

         Numerous studies on experimental animals have failed to detect
    teratogenic effects, even at doses of benzene clearly toxic to the
    dam.  A few of these studies are summarized in Table 11.  However,
    benzene has been reported to be fetotoxic in mice, as shown by a
    decrease in fetal weight and skeletal variants (missing sternebrae and
    extra ribs) in the offspring of dams exposed to 1600 mg/m3 (500 ppm)
    for 7 h on gestation days 6-15 (Murray et al., 1979).  Similar effects
    were seen in rabbits exposed on gestation days 6-18 to the same
    levels.  However, these effects are not usually considered to be
    significant compound-related malformations (Kimmel & Wilson, 1973). 
    In mice, exposure to 500 or 1000 mg/m3 on gestation days 6-15
    resulted in a decrease in fetal weight and an increase in dead or
    resorbed fetuses, but no statistically significant increase in
    malformations (Ungvary & Tatrai, 1985).


    
    Table 11.  Teratogenic effects of benzene in the mouse and rabbita

                                                                                                                                              

    Animals        Route        Exposure level      Maternal weight   Fetal body    Resorptions/   Skeletal     Malformations   References
                                                         gain           weight      fetal death/   variants
                                                                                    abortions
                                                                                                                                              

    New Zealand    inhalation   1600 mg/m3                (-)             (-)          (-)        slightly **       (-)         Murray et al.
     rabbit                                                                                                                     (1979)

    New Zealand    inhalation   500 mg/m3                 (-)             (-)          (-)            (-)           (-)         Ungvary & Tatrai
     rabbit                     1000 mg/m3                 *               *           **b            **            **          (1985)

     CF-1 mouse    inhalation   1600 mg/m3                (-)              *           (-)            **            (-)         Murray et al.
                                                                                                                                (1979)

    CFLP mouse     inhalation   500 mg/m3                 (-)              *          **c,d           **            (-)         Ungvary & Tatrai
                                1000 mg/m3                (-)              *          **c,d           **            (-)         (1985)

    CD-1 mouse     gavage       0.3 ml/kg body weight     (-)              *           (-)                          (-)         Nawrot &
                                0.5 ml/kg body weight     (-)              *          **c,d                         (-)         Staples (1979)
                                1.0 ml/kg body weight     (-)              *          **c,d           (-)           (-)
                                                                                                                                              

    a  (-) indicates no significant difference from controls; * indicates decrease compared with controls;
       ** indicates increase compared with controls.
    b  Fetal death
    c  Abortions
    d  Resorptions

    Table 12.  Teratology studies in rats after inhalation of benzenea

                                                                                                                                              

    Exposure level   Exposure period   Maternal weight    Resorptions   Fetal weight   Skeletal variants   Malformations     Reference
       (mg/m3)           (h/day)            gain
                                                                                                                                              

           32               6               (-)              (-)            (-)              (-)               (-)       Coate et al. (1984)
           32               7               (-)              (-)            (-)              (-)               (-)       Kuna & Kapp (1981)
           32               6               (-)              (-)            (-)              (-)               (-)       Coate et al. (1984)
          128               6               (-)              (-)            (-)              (-)               (-)       Coate et al. (1984)
          150              24                *               (-)             *               (-)               (-)       Tatrai et al. (1980a)
          160               7                *               (-)             *               **                (-)       Kuna & Kapp (1981)
          320               6               (-)              (-)            (-)              (-)               (-)       Green et al. (1978)
          320               6               (-)              (-)             *               (-)               (-)       Coate et al. (1984)
          400              24                *               (-)             *               **                (-)       Tatrai et al. (1980b)
          450              24                *               **              *               **                (-)       Tatrai et al. (1980a)
          960               6               (-)              (-)            (-)              **                (-)       Green et al. (1978)
         1000              24                *               (-)             *               **                (-)       Hudak & Ungvary (1978)
         1500              24                *               **              *               **                (-)       Tatrai et al. (1980a)
         1600               7                *               (-)             *               **                (-)       Kuna & Kapp (1981)
         3000              24                *               **              *               **                (-)       Tatrai et al. (1980a)
         7000               6                *               (-)             *               **                (-)       Green et al. (1978)
                                                                                                                                              

    a  (-) indicates no significant difference compared with controls; * indicates decrease compared with controls;
         ** indicates increase compared with controls
    

         Several studies in rats show similar results to those in mice and
    rabbits (Table 12).  Maternal and fetal weight were decreased at
    levels > 160 mg/m3 (> 50 ppm) as were the number of skeletal
    variants observed.  No malformations were noted in any of the studies
    even at doses as high as 7100 mg/m3 (2200 ppm).

         It is noteworthy that haematopoietic changes were observed in the
    fetuses and offspring of mice exposed to 16, 32 or 64 mg/m3 (5, 10
    or 20 ppm) for 6 h/day on gestation days 6-15 (Keller & Snyder, 1986). 
    The changes included a decrease in the number of erythroid
    colony-forming cells (at all dose levels) and granulocytic
    colony-forming cells at the two highest levels.  When the offspring
    were re-exposed to benzene as adults the decrease in these progenitor
    cells was greater than in adult mice exposed to benzene at the same
    levels for the first time.

    7.5  Mutagenicity and related end-points

         Benzene has been widely studied regarding the production of gene
    mutations in  in vitro tests, chromosomal effects both  in vitro and
     in vivo, and effects on DNA (binding, synthesis and damage).  An
    overview of the testing up to 1985 for the mutagenicity of benzene is
    shown in Fig. 4 (IARC, 1987a).  Detailed reviews have also been
    published (Dean, 1978, 1985a; Huff et al., 1989; ATSDR, 1991),
    therefore only some of the many studies are shown in Tables 13
    ( in vitro) and 14 ( in vivo).

    7.5.1  In vitro studies

         As shown in Table 13 and Fig. 4, benzene has consistently given
    negative results in assays for point mutations in bacteria using
    standard test conditions.  In such studies, six tester strains have
    been used with benzene concentrations ranging from 0.1 to 528 µg per
    plate both with and without metabolic activation (HSE, 1982).  Other
    studies using doses as high as 880 mg/plate failed to cause mutations
    in  Salmonella typhimurium (Dean, 1978).  In some 10  in vitro gene
    mutation tests carried out in various human, mouse and Chinese hamster
    cells, as part of an international collaborative study, mixed results
    were obtained with benzene (Ashby et al., 1985; Venitt, 1985).  When
     S. typhimurium (strain TA1535) was incubated with benzene in a
    desicator to enhance exposure, a doubling of revertants was noted at
    10 ppm only in the presence of a post-mitochondrial activating system
    (Glatt et al., 1989).


    
    Figure 4.  Tabular summary of  in vitro and  in vivo tests on benzene for mutagenicity and related end-pointsa

                                                                                                                                               

                Non-mammalian systems                                                                   Mammalian systems
                                                                                                                                             

                                                                                         In vitro                                   In vivo
                                                                                                                                             
    Prokaryotes       Lower        Plants      Insects                  Animal cells                  Human cells         Animals         Humans
                   eukaryotes
                                                                                                                                               

     D      G      R    G    A       G       R    G    C       D    G    S    M    C    A    T      D    G    S   C      S    M    C      S    C

                                                                                                                                               

    +1      -      +    +    +1      +       +1   ?    -1      +    +    -    -1   +    +    +      -1   +1   ?   +1     +    +    +      -1   +
                                                                                                                                               

    a  Adapted from: IARC (1987a)
         A = aneuploidy; C = chromosomal aberrations; D = DNA damage; G = gene mutation; M = micronuclei; R = mitotic recombination and gene
         conversion; S = sister chromatid exchange; T = cell transformation; + = considered to be positive for the specific end-point and
         level of biological complexity; +1 = considered to be positive, but only one valid study was available to the Working Group;
         - = considered to be negative; -1 = considered to be negative, but only one valid study was available to the Working Group; ? =
         considered to be equivocal or inconclusive (e.g., there were contradictory results from different laboratories; there were confounding
         exposures; the results were equivocal)
    
        Table 13.  Some  in vitro genotoxicity studies of benzene

                                                                                            

    End-point             Test system                 Resultsa        References
                                                                                            

    Gene mutations

    Ames test          Salmonella typhimurium            -/-        De Flora et al.
                                                        -/+        (1984); Venitt (1985);
                                                                   Glatt et al. (1989)

    Azaguanine         Salmonella typhimurium            -/         Seixas et al.
    resistance                                                     (1982)

    TK test           mouse L5178Y cells                -/-        Oberly et al. (1984)

    TK, ouabain,      total of 15 studies using       mixedb       Garner (1985)
    HGPRT loci        various human, mouse and
                      Chinese hamster cells

    Chromosome abnormalities

    Chromosome        human lymphocytes                mixed       Gerner-Smidt &
    aberrations                                                    Friedrich (1978);
                                                                   Morimoto (1976)

                      total of 8 studies using         mixed       Dean (1985b)
                      Chinese hamster or human
                      cells

    Sister            Chinese hamster ovary and         -/-        Dean (1985b)
    chromatid         V79 cells and rat RL4
    exchange          cells
                                                                                            

    Table 13 (contd).

                                                                                            

    End-point             Test system                 Resultsa        References
                                                                                            

    Sister            human lymphocytes                mixed       Morimoto (1983);
    chromatid                                                      Morimoto et al.
    exchange                                                       (1983); Erexson
                                                                   et al. (1985)

    Micronuclei       Chinese hamster ovary             -/-        Douglas et al.
                      cells                                        (1985)

    Other effects

    DNA breaks        Rat hepatocytes                  ND/-        Bradley (1985)

                      Chinese hamster V79 cells         -/-        Swenberg et al.
                                                                   (1976)

                      Chinese hamster ovary             -/-        Douglas et al.
                      cells                                        (1985)

                      Mouse L5178Y cells                -/         Pellack-Walker &
                                                                   Blumer (1986)

    Unscheduled       rat hepatocytes                  ND/-        Probst & Hill
    DNA synthesis                                                  (1985)

                      HeLa cells                        -/-        Barrett (1985)

    DNA synthesis     Hela cells                        -/-        Painter & Howard
    inhibition                                                     (1982)
                                                                                            

    a  Without/with an exogenous metabolic activation system; ND = no data
    b  The IPCS CSSTT working group disagreed over data analysis and therefore
         called the results inconclusive
    

         Whereas the results of  in vitro studies for mutations by
    benzene have been largely negative, there is some evidence that
    treatment of human and animal cells  in vitro with benzene can lead
    to chromosomal abnormalities.  However, as shown in Table 13, mixed
    results have been obtained.

         The ring-opened metabolite of benzene, trans,trans-muconal-dehyde
    (MUC) has been tested for mutagenic and clastogenic activity in CHO
    cells and  Salmonella typhimurium bacteria, and for its effects on
    DNA synthesis in primary rat liver hepatocytes (Witz et al., 1990a). 
    Only minimal mutational activity in bacteria was reported (in only
     S. typhimurium strain TA97 of the five strains tested).  However, at
    a concentration of 0.4 to 0.8 µg/ml media, MUC resulted in a
    dose-related increase in micronuclei in CHO cells.  No effect on
    unscheduled DNA synthesis or in the HG PRT assay in CHO cells was
    reported.  Using  S. typhimurium (point mutations) and V79 cells
    (sister chromatid exchange, acquisition of thioguanine or ouabain
    resistance, and induction of micronuclei), 13 other potential benzene
    metabolites were examined for genotoxicity.  Each metabolite showed a
    specific spectrum of activity, the highest genotoxic activity in most
    systems being exhibited by quinone, hydroquinone, anti-diol epoxide
    and catechol (Glatt et al., 1989).

         The negative mutagenic data found in studies where benzene was
    added to standard systems  in vitro may well have been caused by the
    technique used in these studies.  Benzene is metabolically activated
    to reactive metabolites by cytochrome P-450 IIE1, a natural
    constituent of liver microsomes.  In many of these studies
    insufficient activation of benzene may have occurred.  Post & Snyder
    (1983) demonstrated that enzyme induction with benzene increased
    benzene metabolism by increasing the activity of an enzyme having a
    low Km and a high turnover rate for benzene, which is now thought to
    be cytochrome P-450 IIE1.  These authors also found that phenobarbital
    induction reduced benzene metabolism until very high substrate
    concentrations were reached.  Chepiga et al. (1991), using purified
    reconstituted cytochrome P-450, have shown that, whereas the Km
    value for benzene as a substrate for cytochrome P-450 IIE1 is quite
    low, much higher concentrations are required for benzene to be
    metabolized by cytochrome P-450 IIB1, which also has a low affinity
    for benzene.  Thus, the lack of positive results in mutagenesis tests
    involving benzene may have been due to the low activity of
    benzene-activating enzymes in these preparations.

    7.5.2  In vivo studies

         No data are available on the production by benzene of gene
    mutations  in vivo.  Benzene, or its metabolites, cause both
    structural and numerical chromosome aberrations in humans (see chapter
    8), laboratory animals and cells in culture (see section 7.6.1), as
    well as sister chromatid exchanges (SCE) and micronuclei in
    polychromatic erythrocytes.  Some  in vivo studies are summarized in
    Table 14.

         Chromosomal changes occur after exposure of experimental animals
    by the subcutaneous, oral, intraperitoneal or inhalation routes. 
    Philip & Krogh-Jensen (1970) administered 1750 mg benzene/kg body
    weight subcutaneously to rats and noted an increase in chromatid
    aberrations 12 and 24 h post-dosing but not after 36 h.  This suggests
    damage to S and/or G2 phase cells and a rapid elimination of the
    alterations.  In a study by Kissling & Speck (1972), the subcutaneous
    administration of 1750 mg benzene/kg body weight to rabbits 3 times
    weekly for 18 weeks led to tetraploidy in one animal as well as a high
    percentage (58%) of bone marrow cells having chromosomal aberrations. 
    Siou et al. (1981) reported an increase in chromosome aberrations in
    the bone marrow cells of mice treated orally with doses greater than
    56 mg benzene/kg body weight on 2 successive days before sacrifice.

         At high levels of benzene administered by inhalation (10 000
    mg/m3 for 4 h), a marked increase in SCEs was noted in mouse bone
    marrow cells (Tice et al., 1980).  In a later experiment a significant
    increase was reported in SCEs in mouse bone marrow cells when the
    animals were exposed by inhalation to 91 mg/m3 for 4 h (Tice et al.,
    1982).  Erexson et al. (1986) reported a significant increase in the
    levels of SCEs in peripheral lymphocytes after 6 h of exposure to 32
    mg/m3 in mice and 9.6 mg/m3 in rats.  At these levels, the
    frequency of micronuclei in bone marrow smears was also increased. 
    After 6 weeks of exposure of mice (22 h/day, 7 days/week) at benzene
    levels of between 0.128 and 3.2 mg/m3, increased chromosomal
    aberrations in lymphocytes from the spleen were reported by Au et al.
    (1991).  These changes reached a significance of P = 0.05 only in
    female mice (in males P = 0.15).

         The frequency of micronuclei was increased in the bone marrow of
    mice treated orally at doses ranging from 56 to 2200 mg/kg body weight
    (Hite et al., 1980; Siou et al., 1981).  A dose-related increase in
    micronuclei in circulating erythrocytes was seen at 120 days in mice
    treated by oral gavage with 25, 50, 100, 200, 400 or 600 mg benzene/kg
    body weight.  A significant increase was seen at all doses, with male
    mice being more sensitive (Choy et al., 1985).


    
    Table 14.  Some mammalian  in vivo genotoxicity studies on benzene

                                                                                                                                    

    Route of          Test system          Results    Exposure concentration                              Reference
    administration                                    and duration
                                                                                                                                    

    Chromosome aberrations

    Inhalation        mouse bone marrow       +       14 to 74 mg/m3, 7 days                              Zhurkov et al. (1983)
                                              -       10 000 mg/m3, 4 h                                   Tice et al. (1980)
                                              -       9600 mg/m3, 4 h                                     Tice et al. (1982)
                                              +       9600 mg/m3, 4 h, phenobarbital pretreatment         Tice et al. (1982)

                      rat bone marrow         +       3.2-3200 mg/m3, 6 h                                 Styles & Richardson (1984)

    Oral              mouse bone marrow       +       6 doses of between 9 and 2200 mg/kg, daily          Siou et al. (1981)
                                                      for 2 days
                                              +       5-80 mg/kg body weight, daily for 2 days            Zhurkov et al. (1983)

                      Chinese hamster         -       2 doses of 2200 and 8800 mg/kg, daily for 2 days    Siou et al. (1981)
                      bone marrow

    Intraperitoneal   rat bone marrow         +       1 dose of 878 mg/kg body weight; 24 h prior to      Anderson & Richardson
                                                      sacrifice                                           (1981)

    Micronuclei

    Inhalation        mouse lymphocytes       +       32, 320 and 3200 mg/m3, 6 h                         Erexson et al. (1986)
                      mouse lymphocytes       +       > 67 mg/m3, 4-10 days                               Toft et al. (1982)
                      rat lymphocytes         +       0.3 to 96 mg/m3, 6 h                                Erexson et al. (1986)
                                                                                                                                    

    Table 14 (contd).

                                                                                                                                    

    Route of          Test system          Results    Exposure concentration                              Reference
    administration                                    and duration
                                                                                                                                    

    Oral              mouse bone marrow       +       6 doses of between 9 and 2200 mg/kg, daily          Siou et al. (1981)
                                                      for 2 days
                      mouse bone marrow       +       440 mg/kg body weight, 2 doses, 24 h apart          Gad El-Karim et al. (1984)
                      mouse bone marrow       +       55 to 1760 mg/kg, daily for 2 days                  Hite et al. (1980)
                      mouse circulating       +       26 to 440 mg/kg body weight, 5 days                 Barale et al. (1985)
                      erythrocytes            +       25 to 600 mg/kg, 120 days                           Choy et al. (1985)

                      Chinese hamster         -       2 doses of 2200 and 8800 mg/kg, daily for 2 days    Siou et al. (1981)

    Sperm head abnormality

    Intraperitoneal   mouse (spermatogonia    +       88 to 880 mg/kg, daily for 5 days                   Topham (1980)
                      treated)
                                                                                                                                    
    

         There were statistically significant alterations in sperm head
    morphology after intraperitoneal doses of 88 to 880 mg/kg body weight
    were administered to mice for 5 days and the sperm were examined 5
    weeks later (Topham, 1980).

         Witz et al. (1990a) reported that administration of
    trans,trans-muconaldehyde, a microsomal metabolite of benzene, to
    B6C3F1 mice (< 0.1-6.0 mg/kg body weight intraperitoneally)
    resulted in the production of SCEs.  The lowest dose producing a
    significant increase was 3 mg/kg. No increase in the frequency of
    micronuclei was reported.

         There has been no clear demonstration of dominant lethal effects
    in animals following benzene exposure.  Fel'dt (1985) found no
    significant dominant lethal effect in mice following oral
    administration of up to 320 mg benzene/kg body weight.  No dominant
    lethal effect was reported in rats by Dean (1978) after the
    intraperitoneal injection of 440 mg benzene/kg.  However, Ciranni et
    al. (1988) demonstrated the induction of micronuclei in the bone
    marrow cells of pregnant mice and in fetal liver cells after a single
    exposure to benzene or its metabolites.

    7.6  Carcinogenicity

         In several studies benzene has been shown to be carcinogenic in
    experimental animals after exposure by inhalation and after oral
    (gavage) dosing.  These experiments are summarized in Tables 15-18. 
    As indicated in these Tables, several types of neoplasms have been
    reported to be associated with exposure to benzene.  Various types of
    lymphomas/leukaemias have been found, but the majority of neoplasms
    are of epithelial origin, i.e. Zymbal gland, liver, mammary gland and
    the oro-nasal cavity.  These results support the hypothesis that
    benzene exposure in experimental animals can produce cancer at
    multiple sites.  A review of such studies has been published by Huff
    et al. (1989).

    7.6.1  Inhalation studies

         The experimental design and major effects noted in several
    inhalation cancer bioassays on benzene are summarized in Table 15.


    
    Table 15.  Inhalation studies on the carcinogenicity of benzene in experimental animals

                                                                                                                                               

    Species           Number of animals      Exposure concentration     Effects                                                       Reference
                                             and duration
                                                                                                                                               

    Mouse AKR/J,      dosed, 60;             960 mg/m3, 6 h/day,        mean lifetime for dosed group, 11 weeks; mean lifetime          Snyder
    males (8 weeks    control, 60            5 days/week, lifetime      for control group, 39 weeks, death due to aplastic              et al.
    old)                                     (about 70 weeks)           anaemia; bone marrow hypoplasia; no evidence for any            (1978b)
                                                                        tumours at autopsy in test animals

    Mouse C57BL,      dosed, 40;             960 mg/m3, 6 h/day,        mean lifetime for dosed group, 41 weeks; mean lifetime          Snyder
    males (8 weeks    control, 40            5 days/week, lifetime      for control group, 75 weeks; anaemia, lymphocytopenia,          et al.
    old)                                     (about 70 weeks)           neutrophilic bone marrow hyperplasia; 6/40 lymphocytic          (1980)
                                                                        lymphoma with thymic involvement (P < 0.01), 1/40 plasma
                                                                        cytoma, 1/40 haematocytoblastic leukaemia, 2/40 control
                                                                        mice lymphocytic lymphoma without thymic involvement

    Mouse AKR/J,      dosed, 50;             320 mg/m3, 6 h/day,        mean life-span for dosed group, 39 weeks; for control,          Snyder
    males (8 weeks    control, 50            5 days/week, lifetime      47 weeks; anaemia, lymphocytopenia, neutrophilia, bone          et al.
    old)                                     (about 70 weeks)           marrow hyperplasia 10/49 dosed, 1/50 control                    (1980)

    Mouse Charles     number of              320 and 960 mg/m3,         two mice of high-dose group developed myelogenous               Snyder
    river CD-1,       animals                6 h/day, 5 days/week,      leukaemia                                                       et al.
    males             unknown                lifetime                                                                                   (1978b)

    Mouse CD-1,       dosed, 60;             intermittent exposure      greater mortality than with continuous exposure to 3480         Snyder
    C57BL/6, male     control, 60            at 960 mg/m3 for 1         mg/m3 for 10 weeks; elevated incidences of malignant            et al.
                      for each               week followed by 2         tumours in both strains; 35% incidence of Zymbal gland          (1988)
                      strain                 weeks non-exposure;        tumours in C57BL (control 0%), lung adenomas in CD-1
                                             6 h/day, 5 days/week       (26% versus 7% controls), and no significant increase
                                             for lifetime               in incidence of leukaemia or lymphomas
                                                                                                                                               

    Table 15 (contd).

                                                                                                                                               

    Species           Number of animals      Exposure concentration     Effects                                                       Reference
                                             and duration
                                                                                                                                               
    Mouse CD-1,       dosed, 80;             short-term exposure to     only CD-1 strain showed increased tumour incidence; 46%
    C57BL/6, male     control, 80            3840 mg/m3, 6 h/day,       incidence of lung adenomas (control 24%) in addition to
                      for each               5 days/week for 10         other malignant and benign tumours; no increase in
                      strain                 weeks; observation for     incidence of leukaemia or lymphoma; marked haematotoxicity
                                             lifetime after cessation   noted (anaemia and lymphocytopenia)
                                             of exposure

    Mouse C57BL/6,    dosed, 118; control,   960 mg/m3, 6 h/day, 5      48 weeks after treatment survival rates were: dosed (D)         Cronkite
    female (7-9       116 (groups reduced    days/week for 16 weeks;    80/90; control (C) 87/88; of the 10 exposed mice that           et al.
    weeks old)        to 90 and 88           observation period for     died, 6 had thymic lymphomas, 2 had unspecified lymphomas;      (1985)
                      respectively for       lifetime                   at end of study tumour incidence reported: leukaemia all
                      haemopoietic                                      types (D) 20/89, (C) 8/88; lymphomas (thymic) (D) 10/89,
                      stem cell assays                                  (C) 1/88; lymphomas (non-thymic) (D) 6/89, (C) 2/88

    Rat Sprague-      dosed, 45;             960 mg/m3, 6 h/day,        no evidence of leukaemic response or pre-leukaemic              Snyder
    Dawley, male      control, 25            5 days/week, for 99        effects nor for tumours incidence at any other site             et al.
                                             weeks                                                                                      (1978b)

    Rat Sprague-      dosed, 40;             320 mg/m3; 6 h/day,        incidence of total and malignant tumours not significantly      Snyder
    Dawley, male      control, 40            5 days/week, lifetime      elevated over controls; several rare tumours in dosed           et al.
    (6 weeks old)                            (about 123 weeks)          group, not in controls: 4 liver, 2 Zymbal gland and 1           (1984)
                                                                        chronic myelogenous leukaemia

    Rat Sprague-      dosed, 70 male,        640 mg/m3; 4 h/day,        at end of experiment (150 weeks) no information on survival;    Maltoni
    Dawley            59 female; control,    5 days/week for 7 weeks    at 118 weeks survival rates of dosed (D) and control animals    et al.
                      158 male, 149 female   then 7 h/day for 8         (C) comparable; tumour incidence at 150 weeks;  Zymbal            (1983,
                                             weeks; exposure  in           gland (D) 4/70 male, 1/59 female with (C) 2/158 male and        1985,
                                              utero  from day 12 of       0/149 female;  oral cavity (D) 2/70 male, 6/59 female with       1989)
                                             gestation through          (C) 0/158 male and 0/149 female; leukaemias (D) 4/70 male,
                                             lactation                  4/58 female with (C) 12/158 male and 1/148 female;
                                                                         hepatomas (D) 2/70 male, 5/59 female with (C) 1/158 male
                                                                        and 0/149 female
                                                                                                                                               

    Table 15 (contd).

                                                                                                                                               

    Species           Number of animals      Exposure concentration     Effects                                                       Reference
                                             and duration
                                                                                                                                               

    Rat Sprague-      Breeders: dosed        640 mg/m3, 4 h/day,        at termination (150 weeks) no information on survival;          Maltoni
    Dawley (breeders  (D), 54; control       5 days/week, 7 weeks;      at 118 weeks  breeder  (D) 5/54, (C) 11/60;  offspring  (D)         et al.
    13 weeks old)     (C), 60                7 h/day, 5 days/week,      male 11/75, (D) female 23/65 with (C) males 39/158 and          (1983,
                                             12 weeks; and then         females 47/149; (C)  breeders  had 2/60 with malignant            1985,
                                             960 mg/m3, 7 h/day,        mammary tumours and (C)  offspring  had 3/158 and female          1989)
                                             5 days/week, 85 weeks      8/149 malignant mammary tumours; male 1/158 and 0/149
                                                                        with leukaemias; tumour incidence in (D)  breeders was:
                      Offspring: dosed,      Breeders: from day         Zymbal gland carcinoma 3/54; oral cavity carcinoma 2/54;
                      75 male and 65         12 of gestation            nasal carcinoma 1/54; malignant mammary tumours 6/54;
                      female; control:       Offspring: in utero,       hepatomas 1/54 and leukaemias 0/54; in  offspring (D)
                      158 male and 149       through lactation          male 6/75 and female 8/65 Zymbal gland carcinoma; male
                      female                 and for 104 weeks          1/75 and female 10/65 oral cavity carcinoma; male 1/75
                                                                        and female 2/65 nasal cavity carcinoma; male 1/75 and
                                                                        female 1/65 skin carcinoma; male 0/75 and female 3/65
                                                                        forestomach carcinomas; male 0/75 and female 9/65
                                                                        malignant mammary tumours; male 2/75 and female 7/65
                                                                        hepatomas; and male 6/75 and female 0/65 leukaemias
                                                                                                                                               
    

         Snyder et al. (1980) reported the development of malignant
    lymphomas in mice after the exposure of male C57BL mice for about 70
    weeks to 960 mg benzene/m3.  Goldstein et al. (1982) exposed
    Sprague-Dawley (SD) rats and three strains of mice (AKR, C57BL and
    CD-1) to 320 and 960 mg/m3 for their lifetime and reported a small,
    but not statistically significant, increase in the incidence of
    granulocytic leukaemia in CD-1 mice (2 cases) and one case of chronic
    myelogenous leukaemia in SD rats (these are rare neoplasms in these
    strains).  Snyder et al. (1984) in a subsequent full report of this
    study, also noted increases in the incidence of liver tumours and
    Zymbal gland carcinomas.  The incidence of malignant lymphoma in male
    AKR mice exposed to 320 mg/m3 was not significantly greater than
    that in controls (Snyder et al., 1984).  At about the same time
    Maltoni et al. (1983) reported that Zymbal gland carcinomas were
    observed in SD rats exposed to benzene (960 mg/m3) for 86 weeks.  At
    the end of the observation period (150 weeks), female breeder rats and
    their offspring had not developed increased levels of leukaemia but
    had an increased incidence of other tumours such as oral and nasal
    cavity carcinomas, malignant mammary carcinomas and hepatomas (Maltoni
    et al., 1982c, 1983, 1989).

         Benzene-induced leukaemia in experimental animals has been
    reported (Cronkite et al., 1984, 1985, 1989; Cronkite, 1986).  In an
    attempt to mimic more closely patterns of human exposure to benzene,
    C57BL/6 and CBA/Ca mice were exposed to 960 mg/m3 (300 ppm) (6
    h/day, 5 days/week) for 16 weeks, followed by an observation period of
    82 weeks.  A highly significant increase in leukaemia was noted in
    C57BL/6 mice (Cronkite et al., 1984), and a biphasic response was
    reported regarding mortality and lymphoma appearance (Cronkite et al.,
    1985).  The first increase in lymphomas was noted at about 150 days
    post-exposure, and there was increased mortality between 330 and 390
    days.  A second increase in lymphomas as well as solid tumours
    occurred at 420 days post-exposure, the mortality again increasing at
    570 days post-exposure.  Cronkite et al. (1989) reported that benzene
    at a concentration of 960 mg/m3 for 76 weeks was leukaemogenic in
    both male and female CBA/Ca mice.

         Snyder et al. (1988) reported that benzene exposure patterns in
    CD-1 and C57BL mice, which were closely related to the occupational
    setting (intermittent for lifetime as well as short-term high doses
    for a portion of the normal life span), resulted in marked
    haematotoxicity as well as being tumorigenic.  Neither of the benzene
    exposure patterns induced elevated incidences of leukaemia/lymphoma in
    either strain.  Elevated incidences of malignant tumours (Zymbal gland
    and lung) were noted in both strains after intermittent (1 week
    exposure, 2 weeks non-exposure) exposure (960 mg/m3, 6 h/day, 5
    days/week) over the full lifetime, whereas an increase in lung tumour
    incidence was noted in only the CD-1 strain after 10 weeks of exposure
    to 3840 mg/m3 (6 h/day, 5 day/week) followed by a lifetime of
    non-exposure.

         Table 16 presents a summary of the lowest dose levels at which
    various authors reported a possible causal relationship between
    benzene exposure and the end-point studied.

    7.6.2  Oral and subcutaneous studies

         Several experiments using oral (gavage) administration of benzene
    to experimental animals are summarized in Table 17, and some of the
    major effects reported are given.  Oral exposure to benzene has
    resulted in the induction of neoplasms in 13 different tissue/organs,
    namely Zymbal gland, oral and nasal cavities, mammary gland, liver,
    forestomach, skin, harderian gland, preputial gland and ovary, and the
    haemopoietic and lympho-reticular systems (Maltoni et al., 1983, 1985,
    1989; NTP, 1986; Huff et al., 1989).  Table 17 indicates the
    similarity in protocols used, namely 25-500 mg benzene/kg body weight
    per day via gavage, 4 to 5 times weekly for 52-104 weeks and
    termination after 103-144 weeks.  The lowest dose of benzene that
    produced specific neoplasms varied from 25 mg/kg body weight for the
    adenomas of the lung, harderian gland and liver of mice to 500 mg/kg
    body weight for lymphoreticular neoplasms in rats.

    7.7  Special studies

    7.7.1  Immunotoxicity

         The proliferative ability of B- and T-cell lymphocytes was
    depressed in a short-term (6 h/day for 6 days) dose-response study
    (32, 96, 320 and 960 mg/m3) on benzene in mice (Rozen et al., 1984). 
    Liposaccharide-induced B-cell proliferation was depressed at levels as
    low as 32 mg/m3 (the range of many occupational exposures), and
    phytohaemagglutinin-induced T-cell response was depressed at 96
    mg/m3.  Peripheral lymphocyte counts were lower at all exposure
    levels, but erythrocyte counts were depressed only at 320 and 960
    mg/m3.  In a subsequent study it was shown that a benzene
    concentration of 960 mg/m3 (6 h/day, 5 days/week), administered for
    115 days to mice, reduced the number of both B-cells in the spleen and
    bone marrow and T-cells in the thymus and spleen and reduced their
    response to mitogens (Rozen & Snyder, 1985).  Other studies have shown
    that polyhydroxylated derivatives of benzene are potent inhibitors of
    T- and B-cell function  in vitro (Irons et al., 1982).


    
    Table 16.  Carcinogenic-related end-points observed in animals exposed to benzene by inhalationa

                                                                                                                                              

    End-points                            SD rat       C57BL/6J mouse       CD-1 mouse       CBA/Ca mouse         Reference
                                                                                                                                              

    Lymphocytic lymphoma                                960/488 days                                              Snyder et al. (1980)
    Myelogenous leukaemia (acute)                                            960/life                             Goldstein et al. (1982)
    Myelogenous leukaemia (chronic)      320/life                            960/life                             Goldstein et al. (1982)
    Zymbal gland carcinoma               320/life                                                                 Snyder et al. (1984)
                                                          960/lifeb          960/lifeb                            Snyder et al. (1988)
    Hepatoma                              640-960                                                                 Maltoni et al. (1982a)
                                                                                                960/16            Cronkite (1986)
    Lung adenoma                                                            3840/lifec                            Snyder et al. (1988)
    Nasal carcinoma                     640-960/104                                                               Maltoni et al. (1983)
    Thymic lymphoma                                        960/16                                                 Cronkite et al. (1984)
    Lymphoma (unspecified)                                 960/16                                                 Cronkite et al. (1984)
    Liver tumour                         320/life                                                                 Snyder et al. (1984)
    Granulocytic leukaemia               320/life                                                                 Snyder et al. (1984)
    Leukaemia                                                                                   960/16            Cronkite (1986)
                                                                                                                                              

    a  Doses are expressed in mg/m3 given 4-7 h/day, 5 days/week over a number of weeks or lifetime (e.g., 960 mg/m3/16
       indicates that 960 mg/m3 was given for 16 weeks); exposures shown are the lowest for which the author claims
       possible causal relationship.
    b  960 mg/m3 dose intermittent, i.e. 1 week followed by 2 weeks non-exposure.
    c  Exposed to 3840 mg/m3 for 10 weeks followed by lifetime non-exposure.

    Table 17.  Long-term toxicity/carcinogenicity of benzene in experimental animals after oral administrationa

                                                                                                                                              

    Species/groups     Number of animals     Exposure concentration   Effects                                                       Reference
                                                  and duration
                                                                                                                                              

    Rat Sprague-       high dose: 30 m,      50 or 250 mg/kg,         only tumours reported in (C) were 4/30 f with malignant       Maltoni et
    Dawley, males &    35 f; low dose:       4-5 times/week           mammary tumours and 1/30 with leukaemia; in dosed animals     al. (1982b,
    females (13        30 m, 30 f;           for 52 weeks;            Zymbal gland carcinoma in 2/30 f in low-dose group and        1983, 1985,
    weeks old)         controls: 30 m,       death at 144             8/35 f in high-dose group; oral cavity carcinomas in 2/35 f   1989)
                       30 f                  weeks                    in high-dose group; malignant mammary tumours in 4/30 f in
                                                                      low-dose group and 7/35 f in high-dose group; hepatomas in
                                                                      1/35 m in high-dose group and leukaemias in 2/30 f of
                                                                      low-dose group and 4/35 m and 1/35 f in high-dose group

    Rat Sprague-       dose: 40 m, 40 f;     500 mg/kg, 4-5           only tumours reported in (C) were 1/50 m with Zymbal gland    Maltoni et
    Dawley, males &    control: 50 m, 50 f   days/week for            carcinomas; 1/50 skin carcinomas; 7/50 f malignant mammary    al. (1982b,
    females (6 weeks                         104 weeks;               tumours; 3/50 m hepatomas; 3/50 m and 1/50 f with             1983, 1985,
    old)                                     observed until           leukaemias; in (D) following tumours reported: Zymbal
                                             natural death            gland carcinomas 18/40 m and 16/40 f; oral cavity
                                                                      carcinomas 21/40 m and 20/40 f; nasal cavity carcinomas
                                                                      3/40 m and 1/40 f; skin carcinomas 9/40 m; forestomach
                                                                      acanthomas and dysplasias 10/40 m and 7/40 f;  in situ
                                                                      carcinomas (forestomach) 6/40 f; invasive carcinomas
                                                                      (forestomach) 1/40 m; hepatomas 3/40 m and 1/40 f; 
                                                                      angiosarcomas 2/40 m and 3/40 f; leukaemias 1/40 m and
                                                                      3/40 f
                                                                                                                                              

    Table 17 (contd).

                                                                                                                                              

    Species/groups     Number of animals     Exposure concentration   Effects                                                       Reference
                                                  and duration
                                                                                                                                              

    Rat F344/N,        dose groups: 60 m,    all groups dosed         number of survivors in control and dosed groups,              NTP (1986);
    males & females    60 f; control:        5 days/week for          respectively; males: 32/50, 29/50, 24/50, 16/50; females:     Huff et al.
    (7-8 weeks old)    60 m, 60 f            103 weeks; males:        46/50, 38/50, 33/50, 25/50                                    (1989)
                                             50, 100 or 200
                                             mg/kg per day            in control and dosed groups, respectively: Zymbal gland
                                             females: 25, 50 or       carcinomas, males: 2/50, 6/50, 10/50, 17/50; females:
                                             100 mg/kg per day        0/50, 5/50, 5/50, 14/50

                                                                      oral cavity squamous cell papilloma: m 1/50, 60/50, 11/50
                                                                      and 13/50; f 1/50, 4/50, 8/50, 5/50

                                                                      oral cavity squamous cell carcinoma: m 0/50, 3/50, 5/50,
                                                                      70/50; f 0/50, 1/50, 4/50, 5/50

                                                                      skin squamous cell papilloma: m 0/50, 2/50, 1/50, 5/50

                                                                      skin squamous cell carcinoma: m 0/50, 5/50, 3/50, 8/50
                                                                                                                                              

    Table 17 (contd).

                                                                                                                                              

    Species/groups     Number of animals     Exposure concentration   Effects                                                       Reference
                                                  and duration
                                                                                                                                              

    Mouse B6C3F1,      dose: 60 m, 60 f;     males and females:       numbers of survivors in control and dosed groups,             NTP (1986)
    male & females     control: 60 m, 60 f   25, 50 or 100 mg/kg      respectively: males: 28/50, 22/50, 18/50, 7/50; females:
    (6-8 weeks old)                          per day, 5 days/week     30/50, 25/50, 24/50, 16/50
                                             for 103 weeks
                                                                      control and dosed groups, respectively: Zymbal gland
                                                                      carcinomas, males: 0/49, 1/48, 4/50, 21/49; females: 0/49,
                                                                      0/45, 1/50, 3/49

                                                                      mammary carcinomas and carcinosarcomas occurred at higher
                                                                      incidences in the mid- and/or high-dose groups; increased
                                                                      tumour incidences in dosed mice were also noted elsewhere
                                                                      including the haemopoietic system, adrenals, ovary, liver,
                                                                      lung and preputial gland
                                                                                                                                              

    a  m = male animals, f = female animals, (C) = control groups, and (D) = dosed groups
    

         The primary antibody response to fluid tetanus toxoid was reduced
    by 74-89% in mice that were exposed to 1280 mg benzene/m3 (6 h/day)
    for 5, 7 or 22 days of exposure (Stoner et al., 1981).  No effect was
    seen at 160 mg/m3.  These investigators concluded that the threshold
    level for repression of primary antibody response was between 160 and
    640 mg/m3.

         Host resistance to infection by  Listeria monocytogenes in mice
    was reduced after exposure to benzene (Rosenthal & Snyder, 1985).  The
    infection rate, as determined by bacterial counts in the spleen, was
    increased by 730% on day 4 post-infection after exposure to 960
    mg/m3 for 5 days, but not at lower benzene exposures.  In contrast,
    increased bacterial counts were seen at all doses of benzene greater
    than 32 mg/m3 when benzene exposure was continued after exposure to
     L. monocytogenes.

         Both the humoral and cellular immune responses in CD-1 mice were
    altered by oral administration of benzene at 8, 40 or 180 mg/kg body
    weight daily for 4 weeks.  A dose-response reduction in peripheral
    blood lymphocytes was reported whereas there was no effect on the
    levels of neutrophils and other white blood cells (Hsieh et al.,
    1988a).  A dose-related biphasic splenic lymphocyte proliferative
    response to B- and T-cell mitogens was also reported.  At the 8 mg/kg
    dose the response was enhanced, while at the 40 and 180 mg/kg per day
    doses a depression was observed. A similar biphasic response was
    reported for cell-mediated immunity.

    7.7.2  Neurotoxicity

         The neurotoxicity of benzene in experimental animals has not been
    well studied.  Benzene caused light narcosis after 3 min of exposure
    to a level of about 144 000 mg/m3 (45 000 ppm) in rabbits (Carpenter
    et al., 1944).  At this dose, tremors were noted after 5 min, loss of
    pupillary reflex to strong light after 6 min, involuntary blinking
    after 15 min, and death after 36 min.  Learning defects have been
    reported in rats exposed three times intraperitoneally to 550 mg
    benzene/kg body weight on days 9, 11 and 13 postpartum (Geist et al.,
    1983).

         Male adult CD-1 mice received ( ad libitum for 4 weeks)
    drinking-water containing 31, 166 and 790 mg benzene/litre (estimated
    daily doses of 8, 40 and 180 mg/kg body weight).  No treatment-related
    behavioural changes were observed in the test animals.  However, oral
    ingestion of benzene was found to alter the levels of norepinephrine,
    serotonin, dopamine and catecholamine in several brain regions (Hsieh
    et al., 1988b).

    7.8  Factors modifying toxicity

         The toxicity of benzene can be modified by several factors,
    including species or strain of animal exposed, dose received, and
    patterns of exposure.

         As shown in Figures 1-3 (section 6.3) benzene metabolism is
    complex and involves several detoxification pathways, as well as two
    pathways which form the putative metabolites muconaldehyde and
    benzoquinone.  Henderson et al. (1989) and Sabourin et al. (1987)
    demonstrated species differences for the metabolism of benzene, i.e.
    mice exhibited a higher rate of metabolism with the production of more
    putative toxic metabolites.  These authors also reported that
    increasing the dose (by oral or inhalation routes) in both rats and
    mice resulted in a higher proportion of benzene being metabolized by
    detoxification pathways.

         The myelotoxicity of benzene in mice is much more pronounced
    following a discontinuous dosing regimen than following continuous
    exposure (Tice et al., 1989).  These effects of exposure route and
    regimen suggest that the toxicity of benzene is dependent on cell
    cycle kinetics in the bone marrow.

         Evidence indicates that benzene must be metabolized prior to
    producing adverse effects on the haemopoietic system or leading to
    carcinogenic and clastogenic effects (Snyder et al., 1981; Irons,
    1985).  Therefore, agents or other factors that alter the metabolism
    of benzene can also modify its toxicity.

         Ethanol and benzene induce formation of cytochrome P-450 IIE1 in
    rabbit and rats (Johansson & Ingelman-Sundberg, 1988).  The toxicity
    of benzene is enhanced by ethanol, increasing the severity of anaemia
    induced by benzene, lymphocytopenia and reduction in bone marrow
    cellularity (Baarson et al., 1982).

         Phenobarbital that induces specific isoenzymes of P-450 increases
    the rate of benzene metabolism  in vivo in rats resulting in
    increased resistance against the leucopenic action of benzene (Ikeda
    & Ohtsuji, 1971; Nakajima et al., 1985).

         Sabourin et al. (1990) found no evidence of the induction of
    benzene metabolism by repeated exposure of rodents to benzene.

    7.9  Mechanism of toxicity

         It has become increasingly clear that the impact of benzene on
    the bone marrow is conferred by a combination of metabolites, rather
    than by a single metabolite.  Thus, Eastmond et al. (1987) showed that
    there was an interaction between phenol and hydroquinone when the two
    were co-administered, which resulted in a degree of myelotoxicity
    greater than additive.  In mice, Snyder et al. (1989) reported that

    whereas phenol alone did not decrease erythrocyte production,
    hydroquinone,  p-benzoquinone, and muconaldehyde were effective
    inhibitors of red cell production.  These authors also showed that
    phenol interacted with hydroquinone to produce greater-than-expected
    depression of erythrocyte synthesis.  A similar interaction was
    observed between phenol and catechol.  The most striking interaction
    was observed when doses of hydroquinone and muconaldehyde were
    selected which were ineffective in inhibiting erythropoiesis when
    given alone, but when given together produced cessation of red cell
    production.  Thus, bone marrow depression appears to be the result of
    the combined effects of these metabolites.  A further contributing
    factor, however, is the finding by Roghani et al. (1987) and Da Silva
    et al. (1989) that benzene stimulates the activity of membrane protein
    kinase c, an important regulatory enzyme.  The effect of its
    perturbation may combine with the biological effects of the various
    metabolites to yield the disease we term aplastic anaemia.

         It is clear that aplastic anaemia requires that benzene be
    metabolized to toxic metabolites.  It also appears that metabolism is
    required for the production of clastogenic responses.  While it seems
    likely that metabolism is important for the induction of tumours,
    there is very little data on this point.  One report, however,
    suggests a role for benzene metabolites in one type of carcinogenic
    response.  Busby et al. (1990) explored the ability of several known
    benzene metabolites, as well as postulated benzene metabolites, to
    induce lung tumours in newborn mice.  They examined the effectiveness
    of benzene oxide and enantiomers and racemates of benzene dihydrodiols
    and diol epoxides given orally using a prescribed regimen.  Lung
    tumour incidence and multiplicity were increased after treatment with
    benzene oxide, racemates of dihydrodiol and by diol epoxide-2. 
    Benzene and diol epoxide-1 were inactive in this system.

         Although it is well known that benzene produces bone marrow
    damage resulting from the production of benzene metabolites in liver,
    it is also well known that these metabolites do not produce
    hepatotoxicity.  From the mechanistic point of view, it appears that
    the liver is protected against damage from quinone metabolites of
    benzene by the enzymes DT-diaphorase (Smart & Zannoni, 1985) and
    carbonyl reductase (Wermuth et al., 1986).  These enzymes prevent the
    metabolic activation of phenolic metabolites to their otherwise toxic
    quinones.  The bone marrow is relatively deficient in these enzymes,
    but partial protection of the marrow has been afforded through the
    administration of high doses of ascorbic acid (Smart & Zannoni, 1985).

         Metabolic activation of benzene metabolites, once they reach the
    bone marrow, may lead to eventual toxicity.  For example, in bone
    marrow stromal macrophages, phenol (but not benzene) can be
    metabolized and in the process inhibit RNA synthesis in macrophages,
    thus possibly inhibiting the production of the haemopoietic factor
    (Post et al., 1985).  It has also been suggested (Kalf et al., 1989)
    that the cyclooxygenase component of prostaglandin synthetase plays a

    significant role in the metabolism of benzene and/or its metabolites
    in bone marrow.  Administration of indomethacin or other
    cyclooxygenase inhibitors protected against benzene-induced bone
    marrow depression and micronucleus formation.

         A general mechanism for benzene-induced bone marrow depression
    might be that benzene metabolites arising in the liver travel to the
    bone marrow where further metabolic activation occurs.  The newly
    generated metabolites, perhaps acting in concert with unmetabolized
    benzene in cell membranes, act upon target cells such as stem cells,
    progenitor cells and stromal cells in the marrow to produce bone
    marrow depression.  Chromosomal damage may ensue, which is reflected
    in clastogenesis observed in circulating lymphocytes or bone marrow
    cells.  The point in this series of events that leads to a
    leukaemogenic response requires further examination once an adequate
    model for the disease in animals has been established.

    8.  EFFECTS ON HUMANS

         Acute inhalation and oral exposures of humans to high
    concentrations of benzene have resulted in central nervous system
    depression and death.  The most noted effects resulting from
    longer-term exposure to lower levels of benzene are haematotoxicity,
    immunotoxicity and neoplasia.

    8.1  General population and occupational exposure

         The human health effects after exposure to benzene are
    qualitatively the same for the general population and those exposed in
    the workplace.  To avoid duplication, the effects on both groups
    (general population and workers) will be discussed together, with
    emphasis on exposure levels and duration of exposure.  The
    quantitative response will be determined from such levels of total
    daily intake.

    8.1.1  Acute toxicity

         Exposures in the general population that result in acute toxic
    effects are usually related to accidents and misuse or abuse of
    benzene.  Many deaths and serious health effects have resulted from
    benzene exposures after deliberate the "sniffing" of glue and other
    products which contain benzene as a solvent (Winek & Collom, 1971). 
    Blood levels in people who have died as a result of "sniffing" glue
    have ranged from 0.94 to 65 mg/litre (Winek et al., 1967; Winek &
    Collom, 1971).  Autopsy observations in these individuals included
    pulmonary haemorrhage and inflammation, renal congestion and cerebral
    oedema.

         It has been estimated that exposure to benzene concentrations of
    about 64 000 mg/m3 (20 000 ppm) for 5-10 min can result in
    fatalities, 24 000 mg/m3 (7500 ppm) for 30 min is dangerous to life,
    4800 mg/m3 (1500 ppm) for 60 min causes serious symptoms, 1600
    mg/m3 (500 ppm) for 60 min leads to symptoms of illness, and 160-480
    mg/m3 (50-150 ppm) for 5 h causes headache, lassitude, and weakness,
    while 80 mg/m3 (25 ppm) for 8 h is without clinical effect (Gerarde,
    1960).  The clinical signs of acute toxicity from benzene include CNS
    depression, cardiac arrhythmia, and eventually asphyxiation and
    respiratory failure if exposures are at the lethal level (Andrews &
    Snyder, 1986).  Mild CNS symptoms are rapidly reversible following
    cessation of exposure and there is no evidence that they result in
    neurological brain damage (Marcus, 1990).

         The single acute oral lethal dose in humans has been estimated to
    be 10 ml of benzene (8.8 g) (Thienes & Haley, 1972).  Clinical signs
    of toxicity after acute oral exposure include staggering gait,
    vomiting, shallow and rapid pulse, somnolence, loss of consciousness,
    delirium, pneumonitis, profound CNS depression, and collapse
    (Sandmeyer, 1981).  High but sublethal oral doses may produce one or

    more of the following symptoms: dizziness, visual disturbances,
    euphoria, excitation, pallor, flushing, breathlessness and
    constriction of the chest, headache, fatigue, sleepiness, and fear of
    impending death (Sandmeyer, 1981).  In addition to the autopsy
    findings noted above, ingestion of benzene has been reported to cause
    gastrointestinal ulceration (Appuhn & Goldeck, 1957).

         No studies on the acute toxicity of benzene after dermal exposure
    are available.

    8.1.2  Effects of short- and long-term exposures

         The most significant health effects from short- or long-term
    exposure to benzene are haematotoxicity, immunotoxicity, neurotoxicity
    and carcinogenicity.  Three types of bone marrow effects have been
    reported in response to benzene exposure; these are bone marrow
    depression leading to aplastic anaemia, chromosomal changes and
    carcinogenicity.

    8.1.2.1  Bone marrow depression; aplastic anaemia

         Several types of blood dyscrasias, including pancytopenia,
    aplastic anaemia, thrombocytopenia, granulocytopenia, lymphocytopenia
    and leukaemia, have been noted after exposure to benzene.  These
    changes are a continuum and not a discrete disease entity.  Which
    effect is noted will depend on the dose, length of exposure and the
    stage of stem cell development affected (Galton, 1986).  As in
    experimental animals, the primary target organ of benzene that results
    in haematological changes is the bone marrow.  It has been suggested
    that the cells at highest risk are the rapidly proliferating stem
    cells (Marcus, 1990).

         A study of 32 patients that were chronically exposed by
    inhalation to benzene levels of 480-2100 mg/m3 (150-650 ppm) for 4
    months to 15 years revealed pancytopenia with hypoplastic,
    hyperplastic or normoblastic bone marrow.  Eight of the 32 individuals
    showed thrombocytopenia which resulted in haemorrhage and infection
    (Aksoy et al., 1972).  Haematotoxicity after prolonged exposure has
    also been reported in rotogravure workers exposed for 6-60 months at
    concentrations of 36-3485 mg/m3 (11-1069 ppm) (Goldwater, 1941) and
    77-3400 mg/m3 (24-1060 ppm) (Erf & Rhoads, 1939), shoe factory
    workers exposed to 96-670 mg/m3 (30-210 ppm) for 3 months to 17
    years (Aksoy et al., 1971), and rubber factory workers exposed to up
    to 1600 mg/m3 (500 ppm) (Wilson, 1942).  Kipen et al. (1988)
    reported on rubber workers who were exposed to benzene during the
    1940s.  An inverse relationship was found between the mean yearly
    white blood cell count and the year that the count was made,
    suggesting that exposures to benzene were very high in the early
    1940s.  As estimated by Crump & Allen (1984), benzene exposures
    decreased from 438 mg/m3 (137 ppm) in 1940 to 102 mg/m3 (32 ppm)
    in 1948.  In a follow-up letter, Hornung et al. (1989) pointed out

    that a similar rise in white blood cells counts was seen in
    pre-employment physical examinations occurring over the same time
    period at the same facility, and that that the trend could not be
    attributed soley to benzene exposure.  In a study of 1008 male
    shoemakers in Florence, excess mortality from aplastic anaemia was
    observed (SMR = 1566, 95% CI 547-3264), based on 6 deaths.  All cases
    of aplastic anaemia occurred among workers first employed before 1964
    when the level of exposure to benzene was assumed to be highest (Paci
    et al., 1989).

         At levels less than 32 mg/m3 (10 ppm) no haematologic effects
    have been observed (Collins et al., 1991).  These authors found no
    haematological effects in 200 benzene-exposed workers (10 year TWA of
    0.03-4.5 mg/m3, 0.01-1.4 ppm) or in 268 control workers in the same
    plant.  In an earlier study of 70 workers in a coke oven by-product
    recovery facility, Hancock et al. (1984) measured the levels of red
    blood cells, white blood cells and haemoglobin in three groups exposed
    to different concentrations of benzene (average, 34 mg/m3, 10.5 ppm;
    range, 3.2-534 mg/m3, 1-167 ppm) and one non-exposed control group. 
    No significant differences between groups were noted in these
    haematological parameters.

         No data are available regarding haematotoxicity after short-term
    or chronic oral or dermal exposure.

    8.1.2.2  Immunological effects

         As noted in animal studies (section 7.7.1), the immunological
    manifestations of benzene toxicity are related to effects on the bone
    marrow, resulting in changes to both humoral and cellular acquired
    immunity.  Workers (76) exposed to benzene (10-22 mg/m3, 3-7 ppm),
    as well as to toluene and xylenes, for periods of 1-21 years were
    examined for the presence of leucocyte agglutinins and levels of
    circulating immunoglobulins.  In 10 out of 35 workers where blood was
    taken during working hours, the adverse effect of agglutinins reacting
    with autoleucocytes was noted (Lange et al., 1973a).  In addition, it
    was found that the sera from the 35 workers had increased levels of
    IgM and decreased levels of IgG and IgA immunoglobulins (Lange et al.,
    1973b).  The simultaneous exposure of these workers to solvents other
    than benzene makes it difficult to interpret these results. 
    Autoimmunity, as shown by the pressure of antibodies against
    leucocytes, platelets, and erythrocytes in the sera of exposed
    workers, has been reported (Renova, 1962).  Workers have been reported
    to have an increased susceptibility to allergies (Aksoy et al., 1971)
    when exposed to benzene concentrations as low as 96 mg/m3 (30 ppm).

         A loss of leucocytes was observed in several studies of workers
    reported to be exposed to benzene levels of 96-2080 mg/m3 (30-650
    ppm) (Aksoy et al., 1971, 1974a; Aksoy, 1987).  Signs of preleukaemia,
    including loss of leucocytes and other blood elements and enlarged
    spleens, were reported in one study (Aksoy et al., 1974a).  Kipen et

    al. (1989) and Yin et al. (1987) also reported decrease in circulating
    lymphocytes and other blood elements at benzene exposures ranging from
    48 to 240 mg/m3 (15-75 ppm).  The number of T-cell lymphocytes were
    found to have been reduced in workers exposed chronically to benzene,
    toluene and xylene (Moszczynski, 1981).

         In a study of workers exposed to low average concentrations of
    benzene (< 32 mg/m3), there was no difference in cell cycle
    kinetics of phytohaemagglutinin-stimulated lymphocytes in 66 male
    workers of a refinery population when compared with 33 control workers
    in the same refinery (Yardley-Jones et al., 1988).

         No studies are available regarding the immunotoxicity of benzene
    in humans after oral or dermal exposures.

    8.1.2.3  Chromosomal effects

         Both structural and numerical chromosomal aberrations have been
    observed fairly consistently in the lymphocytes and bone marrow cells
    of individuals occupationally exposed to benzene.  It is now generally
    accepted that benzene is a human clastogen (IARC, 1987a; Huff et al.,
    1989).  Increases in the number of both unstable and stable
    chromosomal aberrations were observed in men, even 2 years after
    cessation of workplace exposure (Tough & Court Brown, 1965).  Up to
    70% aneuploid lymphocytes were found in five women with benzene
    haemopathy (Pollini et al., 1969), the effects still being
    demonstrable 5 years post-exposure.  Similar effects were observed in
    the lymphocytes of workers in a rotogravure plant that had been
    exposed to very high levels of benzene 400-1700 mg/m3 (125-532 ppm)
    for 1-22 years (Forni et al., 1971a,b).

         Recent studies by Yardley-Jones et al. (1988, 1990) revealed much
    lower responses in the lymphocytes of workers exposed to low
    concentrations of benzene (average < 32 mg/m3).  In a study of 66
    refinery workers and 33 controls, no alteration in cell cycle kinetics
    was noted nor was there any increase in the level of SCEs
    (Yardley-Jones et al., 1988).  The lymphocytes from 48 of the workers
    and 29 of the controls were analysed for chromosomal aberrations. 
    According to Yardley-Jones et al. (1990), the increase in aberrations
    (particularly chromatid deletions and gaps) was of borderline
    significance in parametric statistical tests, but was significant
    using Fisher's exact test.  No lifestyle factors had any consistent
    effect on the incidence of chromosomal aberrations.

         In an attempt to determine whether benzene and its metabolites
    damage certain human chromosomes preferentially, Sasiadek et al.
    (1989) examined the karyotypes of 33 workers exposed to less than 99
    mg/m3 (31 ppm).  At these levels no clinical or haematological
    symptoms were noted in 31 workers, but pancytopenia was observed in
    two workers.  Nonrandom breaks and gaps were observed in the exposed
    group; chromosomes two, four and nine were more prone to breaks and

    chromosomes one and two more prone to gaps.  The results of this study
    are of limited value in view of the small number of controls and the
    fact that all participants smoked.

         Other studies that corroborate the clastogenicity of benzene in
    humans have been reviewed by IARC (1982), Dean (1985a) and Kalf
    (1987).

    8.1.2.4  Carcinogenic effects

         The fact that benzene is a human leukaemogen has been well
    established by epidemiological and case studies (IARC 1982, 1987b),
    most of which have dealt with industrial exposures.  The
    epidemiological studies reported have been selected because they
    contain sufficient quantitative data on exposure and effects to permit
    a discussion of the dose-response relationship.  Some case reports are
    summarized in Table 18, and prospective epidemiological studies are
    summarized in Table 19.  Of the two major classes of leukaemia
    (granulocytic and lymphocytic), the most consistent evidence for a
    causal relationship in humans has been found between benzene exposure
    and myeloid leukaemia (Goldstein, 1988).

         One case study followed the course of 44 patients with
    benzene-induced pancytopenia and found that 6 of them later developed
    leukaemia (Aksoy & Erdem, 1978).

         The first bridge between case reports and a formal
    epidemiological investigation was conducted in the early 1970s (Aksoy
    et al., 1974b).  These investigators reported on a series of cases
    from an estimated population of 28 500 Turkish shoe workers exposed
    since the 1950s to solvents and adhesives containing high levels of
    benzene.  Aplastic anaemia was first observed in 1961, and 26 patients
    with acute leukaemia were observed by 1967.  Peak exposure levels of
    benzene were reported to be 96-670 mg/m3 (30-210 ppm), with rare
    excursions to 2100 mg/m3 (650 ppm), for periods of 1 to 14 years
    (mean 9.7 years).  From this group of workers, a leukaemia incidence
    rate (number of cases per 100 000 per year) of 13 was estimated
    compared with a rate of 6 for the general population (Aksoy et al.,
    1974b).  It was unclear to the Task Group, from the description of the
    authors, which methods were used to ascertain cases and from which
    exposed population these cases were derived.

         A study showing an excess risk of leukaemia in a cohort of 748
    male workers producing "rubber hydrochloride" in three plants in two
    locations within the USA during 1940-1949 (a product made from natural
    rubber suspended in benzene) was first reported by Infante et al.
    (1977).  A follow-up study of this cohort was reported by Rinsky et
    al. (1981) and this was subsequently updated (Rinsky et al., 1987). 
    In this most recent follow-up, the cohort definition was expanded to
    include workers employed between 1940 and 1965 who had a cumulative
    exposure to benzene of 3.2 mg/m3 per day (1 ppm/day) or more.  The


    
    Table 18. Case studies of workers occupationally exposed to benzene

                                                                                                                                              

             Group studied                                Exposure               Condition observed           Author's      References
                                                                                                            conclusiona
                                                                                                                                              

    44 pancytopenic patients exposed to benzene     480-2100 mg/m3 (150-650      leukaemia                       PC         Aksoy & Erdem
    in adhesives                                    ppm); 4 months to 15         myeloid metaplasia               *         (1978)
                                                    years

    42 leukaemia patients and 21 patients with      not given                    leukaemia                       DC         Aksoy (1980)
    other malignancies; 47 were shoe workers, the                                multiple myeloma                PC
    remainder in other occupations using benzene                                 myeloblastic leukaemia           *
    solvents                                                                     acute erythroleukaemia           *
                                                                                 preleukaemia                     *
                                                                                 malignant lymphoma              PC
                                                                                 paroxymal nocturnal
                                                                                 haematuria                       *
                                                                                 lung cancer (all heavy
                                                                                 smokers)                         *
                                                                                                                                              

    Table 18 (contd).

                                                                                                                                              

             Group studied                                Exposure               Condition observed           Author's      References
                                                                                                            conclusiona
                                                                                                                                              

    6 of 94 Hodgkin's patients who had been         480670 mg/m3 (150-210        Hodgekin's disease              PC         Aksoy et al.
    exposed to benzene adhesives                    ppm); 1-28 years                                                        (1974a)

    A 35-year-old man who had used benzene          200-1640 mg/m3; 18 months    subacute granulocytic           DC         Sellyei & Kelemen
    8 years earlier as a paint solvent                                           leukaemia                                  (1971)

    6 leukaemia patients in different occupations   levels unknown; 1-20 years   haemocytoblastic leukaemia      DC         Vigliani & Saita
    all using benzene solvents                                                                                              (1964)

    A 51-year-old chemical worker exposed to        3.2 mg/m3 (< 2 ppm);         acute myelogenous               PC         Ott et 81. (1978)
    benzene 15 years earlier                        18 months                    leukemia
                                                                                                                                              

    a  DC = direct correlation; PC = possible correlation; * = no conclusion made

    Table 19. Epidemiological studies of workers exposed to benzene

                                                                                                                                              

            Group studied                     Exposure            Condition observed          Number of       SMRa      95% CI       References
                                                                                           observed deaths
                                                                                                                                              

    Incidence of leukaemia in           96-670 mg/m3              aplastic anaemia,              26            200        NR         Aksoy et al.
    Turkish shoe workers 1950-1965      rarely 2100 mg/m3;        acute leukaemia                                                    (1974b)
    (28 500 shoe, slipper and           1-15 years (mean,
    handbag workers)                    9.7 years)

    Mortality study of rubber           within legal limits of    malignomas of lymphatic        14            260        NR         Infante et
    workers exposed to benzene          period, i.e. 320 mg/m3    and haemopoietic                                     P < 0.05      al. (1977)
    between 1940 and 1949               (100 ppm) down to         systems; myeloid and
                                        32 mg/m3 (10 ppm)         monocytic leukaemia             7            506        NR
                                        for up to 10 years                                                             P < 0,02

    Mortality studyb of pliofilm        from < 40 ppm-years       lymphatic and haemopoietic     15            227      127-376      Rinskyetal.
    workers exposed to benzene          to > 400 ppm-years        neoplasms                                                          (1987)
    between 1940 and 1965 with
    a period at risk from 1950                                    leukaemia, total                9            337      159-641
    to 1981                                                       < 40 ppm-years                  2            109      12-394
                                                                  40-200 ppm-years                2            322      36-1165
                                                                  205-400 ppm-years               2           1186     133-4289
                                                                  > 400 ppm-years                 3           6637    1334-19 393

                                                                  multiple myeloma total          4            398     110-1047
                                                                  < 40 ppm-years                  3            458      92-1339
                                                                  > 40 ppm-years                  1           5347     70-29 753

    Mortality studyc of 956             > 0.3-114 mg/m3           leukaemia (total)               4            194      52-488       Bond et al.
    workers employed at a chemical      (> 0.1-35.5 ppm)          acute myelogenous               4            444     P < 0.05      (198613)
    company between 1940 and            estimated TWA for         leukaemia
    1982                                up to 34 years
                                                                                                                                              

    Table 19 (contd).

                                                                                                                                              

            Group studied                     Exposure            Condition observed          Number of       SMRa      95% CI       References
                                                                                           observed deaths
                                                                                                                                              

    Retrospective mortality study       < 3.2 mg/m3               leukaemia                       0             0                    Tsai et al.
    of workers (454) employed at        (< 1 ppm) in 84%                                                                             (1983)
    a Texas refinery between 1952       samples (median,
    and 1981                            1.6 mg/m3; 0.5 ppm)
                                        in benzene-related
                                        areas

    Mortality study of chemical         < 15, 15-60 and > 60      lymphatic and haemopoletic,                                        Wong
    workers in 7 plants, > 6            ppm-years                 total:                                                             (1987)
    months on job at single plant                                 non-exposed                     3            39        7-101
    between 1946 and 1975                                         < 15 ppm-years                  5            91       30-213
    (3636 males)                                                  15-60 ppm-years                 5            147      48-343
                                                                  60 ppm-years                    5            179      57-409

                                                                  leukaemia, total:
                                                                  non-exposed                     0             -
                                                                  < 15 ppm-years                 97          12-349
                                                                  15-60 ppm-years                78           2-434
                                                                  60 ppm-years                  276          57-806

    Retrospective cohort of 259         no benzene levels         lymphatic and haemopoietic      4            377     109-1024      Decouflé et
    male chemical workers               reported; benzene         neoplasms (workers with                                            al. (1983)
    employed between 1947 and           used in large             > 1 year of employment)
    1960                                quantities
                                                                                                                                              

    Table 19 (contd).

                                                                                                                                              

            Group studied                     Exposure            Condition observed          Number of       SMRa      95% CI       References
                                                                                           observed deaths
                                                                                                                                              

    Retrospective cohort of shoe        exposed for up to 29      aplastic anaemia (males);       4           1566     P < 0.05      Paci et al.
    workers (1008 males, 1005           years; levels of          leukaemia (males)               6            400      146-870      (1989)
    women) employed between             benzene not reported
    1939 and December 1984 and
    still employed in plant in
    January 1950

    Retrospective cohort study          grab samples; means       acute and chronic           30 cases         574     P < 0.01      Yin et al.
    (28 460 workers in 233 factories);  between 10 and 1000       leukaemia                   5 deaths                               (1987)
    reference population 28 257         mg/m3
    in 83 machine production,
    and clothing factories              50-500 mg/m3 found in
                                        most plants

    Mortality study of coke plant       non-exposed, coke         leukaemia (non-exposed)        13            135        NR         Swaen et al.
    workers (5639) with > 6 months      ovens and by-product      leukaemia (coke-oven)           6            163        NR         (1991)
    work between 1945 and 1969          workers; levels of        leukaemia (by-product)          7             85        NR
                                        benzene not reported

    Retrospective cohort of 391         mean time-weighted        leukaemia                                                          Hurley et
    benzole workers in 2 cohorts        average exposure of       by-product 1                    1             98       2-557       al. (1991)
    (cohort 1, 84 workers;              coke by-product           by product 2                    1             76       2-429
    cohort 2, 307 workers)              workers in Britain
                                        in 1980s 4.2 mg/m3
                                        (1.3 ppm)
                                                                                                                                              

    a    SMR - Standard mortality ratio
    b    Follow-up of cohort described by Infante et al. (1977)
    c    Follow-up of cohort described by Ott et al. (1978)
         NR = not reported
    

    study included 1165 white males followed from 1950 to 1981.  For the
    analysis the cohort was divided into four cumulative exposure groups:
    < 128 mg/m3-years (< 40 ppm-years), 128-640 mg/m3-years (40-200
    ppm-years), 640-1280 mg/m3-years (200-400 ppm-years) and > 1280
    mg/m3-years (> 400 ppm-years).  A statistically significant excess
    risk was observed for all lymphatic and haemopoietic neoplasms (15
    observed deaths compared to 6.6 expected; SMR = 227, 95% CI, 127-376). 
    There were nine deaths from leukaemia compared to an expected 2.66
    (SMR = 337, 95% CI, 159-641), and 4 deaths from multiple myeloma (SMR
    = 398, 95% CI, 110-1047).  A strong positive trend in leukaemia
    mortality was obtained with increasing cumulative exposure.  Within
    the four cumulative exposure groups there were 2,2,2, and 3 deaths
    with SMRs of 109, 322, 1186 and 6637, respectively.  In order to
    investigate further the shape of the exposure-response curve, Rinsky
    et al. (1987) conducted a nested case-control study by matching each
    of nine deaths due to leukaemia with ten controls.  A conditional
    logistic regression analysis described a significant positive
    association between estimated level and average duration of benzene
    exposure and leukaemia that was projected downward to levels of zero
    accumulated exposure over a working lifetime.  From this model it was
    calculated that an exponential relationship existed between benzene
    exposure and the developmental leukaemia.

         When actual exposure measurements did not exist, Rinsky et al.
    (1987) estimated exposures to benzene by averaging historical annual
    measured benzene levels from seven existing industrial hygiene survey
    sources.  The majority of measurements occurred after 1963, but some
    data existed as early as 1946.  Where no sampling data could be found,
    exposure levels were estimated by interpolation from existing
    information.  Alterative exposure estimates and subsequent reanalyses
    have been developed by Crump & Allen (1984) and Paustenbach et al.
    (1992).  The differences in exposure estimates between Crump & Allen
    (1984) and Rinsky et al. (1987) centre primarily on assumptions of
    benzene exposure prior to 1946 where no historical data exist. 
    Paustenbach et al. (1992) gathered additional information and
    considered other factors that modified the estimates of exposure over
    the entire period during which rubber hydrochloride plants operated
    (1936 to 1976) to develop a new set of exposures over time.  For the
    most part the exposures estimated by Paustenbach et al. (1992) are
    higher than those reported by Rinsky et al. (1987) and Crump & Allen
    (1984).

         In a retrospective study of 594 employees of a chemical company
    exposed to levels of benzene between 0.3 and 114 mg/m3 (0.1-35.5
    ppm) for up to 34 years, no statistically significant increase in
    total mortality was reported (Ott et al., 1978).  There were three
    cases of myelocytic leukaemia compared to an expected incidence of 0.8
    cases (significant at the P < 0.05 level).  Some workers in this

    cohort were also exposed to vinyl chloride, arsenicals and several
    other potentially carcinogenic chemicals.  A follow-up study of these
    workers expanded the cohort by 362 potentially exposed workers (Bond
    et al., 1986b).  Four deaths from myelogenous leukaemia were reported
    and the SMR for all leukaemias was 194 (95% CI, 52-488).  The
    difference between observed and expected values was statistically
    significant only when myelogenous leukaemia was considered (4
    observed, 0.9 expected; P = 0.01).

         Wong (1987) reported a significant dose-response relationship
    between cumulative exposure to benzene and mortality from leukaemia
    and all lymphopoietic cancers combined.  The mortality experience of
    3536 workers who had continuous exposure to benzene was compared to
    that of an internal comparison group of 3074 workers not exposed to
    benzene but who had worked at the same plant.  The 3536 exposed
    workers were categorized into cumulative exposure categories of < 48
    mg/m3-years (< 15 ppm-years), 48 to 192 mg/m3-years (15 to 60
    ppm-years), and > 192 mg/m3-years (> 60 ppm-years).  There was an
    increasing trend in the SMRs for lymphatic and haemopoietic cancers as
    exposure increased (SMR = 35, 91, 147 and 175 for the non-exposed and
    the 3 exposure categories, respectively (P = 0.02).  The respective
    SMRs for leukaemia were 0, 97, 78 and 275 (P = 0.01).  It should be
    noted that none of the six leukaemia deaths was from acute myeloid
    leukaemia.  In addition, the highest category of exposure started at
    only 192 mg/m3-years (60 ppm-years), the equivalent of 32 mg/m3
    (10 ppm) annually for only a six-year working career.  The
    exposure-response relationship between cumulative benzene exposure and
    non-Hodgkin's lymphoma was of marginal statistical significance.

         A retrospective cohort mortality study was conducted on 259 male
    employees at a chemical plant in the USA where benzene had been used
    in large quantities (Decouflé et al., 1983).  The study group included
    workers employed between 1947 and 1960, and workers were followed
    until 1977.  Among workers with more than one year of employment, a
    statistically significant excess risk was observed for neoplasms of
    the lymphatic and haemopoietic systems (SMR=377; 95% CI 109-1024, 4
    deaths). No SMR was given specifically for mortalities to leukaemia
    and multiple myeloma.  Three of these deaths were leukaemias and the
    fourth a multiple myeloma.

         Coke-oven workers and some workers at coke by-product plants are
    exposed to low levels of benzene.  A study in the Netherlands examined
    5639 workers in a coke plant and a comparison group of 5740 workers in
    a nitrogen-fixation plant (Swaen et al., 1991), employed for at least
    six months between 1945 and 1965.  The SMR for leukaemia in by-product
    benzene plant workers was 85 (7 deaths), in coke-oven workers 163 (6
    deaths), and in non-exposed workers 135 (13 deaths).  Among the 222
    workers in the benzene plant, no indication of an increased leukaemia
    risk was found, but the expected number was small.

         Hurley et al. (1991) have reported preliminary results on the
    mortality of 6520 male coke plant workers from 27 plants in the United
    Kingdom.  Personal air samples were taken from 84 benzole workers from
    14 plants in one cohort with levels of < 0.6-22 mg/m3 (< 0.19-7
    ppm) and 307 benzole workers from 13 plants in the second cohort with
    levels of < 0.6-48 mg/m3 (< 0.19-14.99 ppm).  Mean time-weighted
    average concentrations for benzole house workers in the United Kingdom
    in the 1980s was considered to be about 4.2 mg/m3 (1.3 ppm).  No
    increased risk of mortality from leukaemia was reported in either
    cohort (cohort 1 SMR = 98, 95% CI 2-557, 1 death; and cohort 2 SMR =
    76, 95% CI 2-429, 1 death).  These SMRs have been calculated utilizing
    data from all 1293 by-product workers.  Only a proportion of the
    by-product workers had worked in the benzene plants where the greatest
    exposure to benzene occurred.  The SMR for leukaemia among 2349
    coke-oven workers was 34 for cohort 1 (95% CI 0-186, 1 death) and 35
    for cohort 2 (95% CI 1-192, 1 death).

         In a retrospective cohort study from China encompassing 28 460
    workers exposed to benzene in 233 factories, 30 cases of leukaemia
    (23 acute, 7 chronic) were found, as compared to four cases in a
    reference cohort of 28 257 workers in 83 machine production, textile
    and cloth factories (Yin et al., 1987, 1989).  The mortality rate (per
    100 000 person-years) from leukaemia was 14 among the exposed and 2
    among the unexposed (SMR, 574; P < 0.01).  Mortality was especially
    high for workers engaged in organic synthesis, painting and rubber
    production.  The mortality from leukaemia for cases that had
    previously experienced benzene poisoning was 701/100 000 person-years. 
    Grab-samples of benzene in air were taken during the time of the
    survey in workplaces where cases of leukaemia were observed; the mean
    concentrations varied over a wide range (from 10 to 1000 mg/m3) but
    the range was 50-500 mg/m3 in most locations.  The mortality
    incidence from all malignant neoplasms was higher in the exposed group
    (123 per 100 000) than in controls (55 per 100 000).  In addition, a
    statistically significant excess risk for lung cancer (SMR = 231) was
    observed in male workers exposed to benzene (Yin et al., 1989). 
    However, little description was given of the methods used to eliminate
    the possible confounding effects of smoking.

         A statistically significant excess risk for aplastic anaemia
    (SMR = 1566, 4 deaths) and for leukaemia (SMR = 400, 95% CI, 146-870,
    6 deaths) was reported for 1008 male workers in a shoe factory in
    Florence, Italy.  Included were workers employed on or after January
    1950 and vital status of cohort members was ascertained through 1984. 
    No workplace monitoring was reported.  The period of maximum benzene
    exposure was considered to be 1953-1962, given the amount of benzene
    used per day (about 30 kg) (Paci et al., 1989), and all cohort members
    who developed aplastic anaemia and leukaemia were employed during this
    period.

         The results of a cohort study of 34 781 workers in eight oil
    refineries in the United Kingdom which examined workers employed for
    1 year between 1950-1975 gave no indication of increased mortality
    from leukaemia (Rushton & Alderson, 1981a).  Within this cohort a
    nested case-control study was conducted to investigate the association
    between exposure to benzene and leukaemia (Rushton & Alderson, 1981b). 
    The 36 leukaemia cases and their controls were allocated to "low",
    "medium", and "high" benzene exposure categories based on their job
    history.  The authors reported a significant (P = 0.05) association in
    the combined medium and highly exposed workers compared to those in
    the low exposure group (RR = 3.0, 95% CI, 1.2-7.2).

         A study (Tsai et al., 1983) of 454 petroleum refinery workers in
    the USA employed between 1952 and 1978 in the petrochemical units
    showed no deaths from leukaemia (0.4 expected).  However, the median
    exposure to benzene throughout the refinery was 0.45 mg/m3 (0.14
    ppm), and only 16% of 1394 personal samples, taken between 1973 and
    1982 (inclusive), contained more than 3.2 mg/m3 (1 ppm).  The median
    exposure intensity in "benzene-related units: petrochemical units" was
    1.7 mg/m3 (0.53 ppm).  No significant changes in blood indices
    (counts of white and of red blood cells, haemoglobin, haematocrit,
    platelets, clotting and bleeding times) were reported.

    9.  EVALUATION OF HUMAN HEALTH RISKS

    9.1  General population

         Benzene is ubiquitous in the environment, resulting in the
    exposure of most humans to trace levels (or more) of this chemical. 
    Exposure in the general population is primarily to air-borne benzene
    and derives from active and passive tobacco smoke, industrial
    activity, and use of the automobile (gasoline fumes  from refilling,
    etc., and exhaust emissions).  Estimates of the daily amounts of
    benzene consumed in drinking-water and food-stuffs vary considerably
    and are of the order of µg/day.  Depending upon the assumptions made
    with respect to levels of benzene from tobacco products and
    foodstuffs, estimates for the exposure of the general smoking
    population in industrialized countries range from  2000 to 3500 µg
    benzene/day.  Adult (70 kg) non-smokers are considered to be exposed
    to about 200 to 1700 µg benzene/day (about 3 to 25 µg/kg body weight
    per day).  It would be helpful to have more information on total human
    exposure, particularly in developing countries.

    9.2  Occupational exposure

         The major factors controlling industrial exposure to benzene are
    process technology, worker practices and the efficiency and
    sophistication of engineering controls.  When appropriate engineering
    controls are in place, available monitoring data indicate that
    exposures of workers involved in the production, handling and use of
    benzene and benzene-containing materials vary from non-detectable
    levels to approximately  15 mg/m3 (8-h TWA), in addition to the
    amounts estimated for the general population.  In developing countries
    the exposure can be several times higher.  Due to the nature of the
    processes involved, a small percentage of workers may be exposed to
    more than 320 mg benzene/shift.  In some developing countries, benzene
    exposure may be sufficiently high to cause acute toxicity.  Dermal
    exposure to benzene has generally not been included in these
    estimates.  Validated benzene-specific biological markers of exposure
    to low levels are not available.

    9.3  Toxic effects

         Acute lethal doses of benzene in experimental animals cause
    narcosis, ventricular tachycardia and respiratory failure.  The
    threshold for narcotic effects in rats is about 13 000 mg/m3. 
    Reported oral LD50 values in rats vary from 3000 to 8100 mg
    benzene/kg body weight and the 4-h LC50 in rats has been reported to
    range between 32 600 and 44 600.  However, although the clinical
    pathological observations in animals are relevant to humans, the
    latter would not be expected to be exposed to such high levels for
    such long periods of time.

         In humans, exposure to high concentrations of benzene (e.g., 
    65 200 mg/m3 for 5-10 min) can result in central nervous system
    depression, cardiac arrhythmia, respiratory failure and death, while
    exposure to levels of benzene between 163 and 489 mg/m3 for 5 h
    leads to headaches, lassitude and general weakness.  The single acute
    oral dose that has been reported to be lethal to humans is 8800 mg/kg
    body weight.

    9.3.1  Short-term and long-term exposures; organ toxicity

         The most significant adverse effects from short- or long-term
    exposure to benzene are haematotoxicity, i.e. bone marrow suppression,
    immunotoxicity, genotoxicity and carcinogenicity.

    9.3.1.1  Haematotoxicity; bone marrow depression

         The level, timing and pattern of exposure are extremely important
    factors in determining the incidence and severity of haematological
    and bone marrow changes.  A significant species difference (rats and
    mice) in effects has also been reported, probably a reflection of the
    more rapid metabolism of benzene by mice and the production of a
    higher proportion of putative toxic metabolites than in rats.  In rats
    and mice, decreases in haematological cell counts (leucopenia and
    haematocrit) and in bone marrow cellularity generally occur only after
    several weeks of exposure to levels of benzene between 320 and 978
    mg/m3, the mouse being more sensitive than rats.  The lowest
    reported exposure in experimental animals leading to haematological
    effects was 32 mg/m3 (6 h/day, 5 days/week for 178 days) in male
    mice.  After oral administration of benzene to rats and mice for 120
    days, leucopenia was observed in both male and female rats, and
    lymphoid depletion in the B-cells of the spleen was observed in both
    male and females at doses of > 200 mg/kg body weight.  In mice, no
    histopathological effects were observed, but a dose-related leucopenia
    was observed in both males and females given 25, 50, 100, 200, 400 or
    600 mg/kg body weight (5 days/week).

    9.3.1.2  Mechanism of action and metabolism

         In humans, a spectrum of blood dyscrasias, including
    pancytopenia, aplastic anaemia, thrombocytopenia, granulocytopenia,
    lymphocytopenia, myeloid leukaemia and acute leukaemia, can result
    from benzene exposure.  The dose, length of exposure and the stage of
    stem cell development affected will determine which effect is
    observed.  Pancytopenia was noted in 32 patients exposed to 489-2119
    mg benzene/m3 for periods of between 4 months and 15 years.  At
    levels of benzene between 102 and 438 mg/m3, haematotoxicity in
    rubber factory workers was reported, the correlation being better
    between benzene exposure and WBC counts than in the case of RBC
    counts.  However, at levels of benzene less than 32 mg/m3, there is
    only weak evidence for a leukaemogenic effect; no haematological

    effects were noted in 200 workers exposed to 10 year TWA benzene
    levels of 0.03-4.5 mg/m3.

         The hepatic metabolism of benzene is responsible for
    detoxification of benzene via the formation of etheral sulfate,
    glucuronides and glutathione conjugates.  It also leads to the
    production of metabolites, such as hydroquinone,  p-benzoquinone,
    muconaldehyde and perhaps others, which appear to be required for the
    production of benzene toxicity in bone marrow.  Current concepts of
    the mechanism of benzene toxicity suggest that it is the result of the
    combined effects of several metabolites, perhaps acting in concert
    with unmodified benzene, to adversely alter the functions of stem
    cells, progenitor cells and stromal cells in the bone marrow.  It has
    been postulated that the specific intracellular targets are proteins
    and nucleic acids.  The terminal result of these biochemical insults
    is the development of aplastic anaemia.  It is likely that benzene
    metabolites damage chromosomes by causing DNA or protein adduct
    formation or generating oxidative damage to DNA, which may contribute
    to the chromosomal changes associated with benzene exposure.  Animal
    models exist for studying the mechanism of benzene-induced aplastic
    anaemia and chromosome damage.  However, no acceptable model for
    benzene-induced leukaemia has been developed.

         Since bone marrow depression and the production of leukaemia both
    involve the bone marrow, it is important to ascertain the relationship
    between these two biological effects of benzene.  The specific
    question in need of an answer is:  Is it necessary for aplastic
    anaemia to develop before leukaemia can be exhibited?  In humans, the
    continuum of effects of benzene on bone marrow involves first the
    production of either anaemia, leucopenia or thrombocytopenia, and this
    is followed by pancytopenia.  Aplastic anaemia is the ultimate form of
    bone marrow depression.  Leukaemia has also been associated with
    benzene exposure.  In addition to studying the relationship between
    these two phenomena, it is important to study the role of metabolites
    in each process.

         Several authors have recently used pharmacokinetic models to
    extrapolate from animal experiments to expected human dosimetry.  Some
    models require the use of physiological parameters, partition
    coefficients and metabolic data.  Of these, the data on such metabolic
    parameters in humans are least well known.  With increasing
    information on appropriate human metabolic data, such models will be
    useful in extrapolating from animals to humans and from high- to
    low-dose exposure.

    9.3.1.3  Immunotoxicity

         Benzene-induced immunological effects are probably a reflection
    of bone marrow toxicity.  Immunological function was depressed in mice
    exposed to benzene levels between 32 and 96 mg/m3, 6 h/day, for 6
    days.  In mice, polyhydroxylated metabolites of benzene have been

    shown to depress B- and T-cell functions.  Although the relevance of
    the animal data to human immunological functions has not been
    established, human immunological alterations have been observed after
    exposure to benzene.

         In workers exposed to, but not seriously intoxicated by, benzene
    (10-22 mg/m3), the serum complement levels of IgA and IgG were
    decreased but the levels of IgM were slightly increased.  An increased
    susceptibility to allergies was found in workers exposed to benzene
    levels as low as 96 mg/m3.  A loss of leucocytes and other blood
    elements has been noted at benzene levels ranging between 48 and 240
    mg/m3.  A study of low exposures to benzene (TWA: 32 mg/m3, 10
    ppm) showed no differences in mitogen-induced blastogenesis in exposed
    workers and controls.

    9.3.2  Genotoxicity and carcinogenic effects

          In vitro tests indicate that benzene is not mutagenic. 
    However, benzene, or its metabolites have been shown to cause both
    structural and numerical chromosomal aberrations in experimental
    animals as well as sister chromatid exchanges (SCE) and micronuclei in
    polychromatic erythrocytes.

         Humans with benzene haemopathy have been found to have a high
    percentage of aneuploid lymphocytes.  Increases in the number of both
    stable and unstable chromosomal aberrations were observed in workers
    exposed to high levels (408-1734 mg/m3) of benzene for 1 to 22
    years.  Therefore, benzene should be considered a clastogen in both
    animals and humans.

         Recent studies of workers exposed to lower concentrations of
    benzene (TWA: < 3.2-32 mg/m3, < 1-10 ppm) revealed no alteration
    in cell-cycle kinetics and no increase in SCEs.  Only a marginally
    significant increase in chromosomal aberrations (chromatid deletions
    and gaps) was noted at these exposure levels.

    9.3.2.1  Mechanism of carcinogenicity

         It is broadly accepted today, as a result of several studies in
    the field of molecular biology, that chromosomal rearrangements
    generating gene translocations, gene deletions and gene amplifications
    are relevant steps in the carcinogenic process.

         There is at present no adequate animal model for benzene-induced
    leukaemia in humans.  However, benzene has been shown to be
    carcinogenic in experimental animals after inhalation or oral
    exposure.  While several types of neoplasms have been reported to be
    associated with benzene exposure in rats and mice, these are primarily
    of epithelial origin, i.e. zymbal gland, liver, mammary gland and
    nasal cavity.  Lymphomas/leukaemias have been observed with lesser
    frequency.

         One study showed an increased incidence of lymphoma in the CBA/CA
    mouse.  No statistically significant increase was seen for lymphoid
    tumour incidence in the Sprague-Dawley rat, Wistar rat or Swiss mouse. 
    In the B6C3F1 mouse the incidence was not dose-related. 
    Dose-related (possibly linear) increases in epithelial tumours were
    observed in mice and rats.  The results in animals indicate that
    benzene is an experimental multipotential carcinogen, although there
    is not the leukaemogenic response seen in humans.  The lowest dose
    resulting in increased incidence of tumours was demonstrated in
    B6C3F1 mice (25 mg/kg body weight, 5 days per week for 103 weeks). 
    No useful information is available for doses below this level.

         Attempts to understand the mechanism of benzene-induced
    carcinogenesis can be based on basic principles of chemical
    carcinogenesis.  Thus, one mechanism of cancer has been postulated to
    be the result of autosomal mutations due to the formation of DNA
    adducts, which result in alteration of cell function and control of
    replication.  Secondly, cancer appears to be induced by a combination
    of multiple genetic and epigenetic events.

         Metabolic data suggest that several reactive metabolites of
    benzene are formed and these can potentially form adducts both with
    DNA and protein.  Binding of benzene metabolites to the protein
    components of the spindle apparatus has been suggested to inhibit
    mitosis.  The data for the formation of DNA adducts by reactive
    metabolites of benzene suggests that, compared with other carcinogens,
    benzene does not form large amounts of DNA adducts.  Furthermore,
    although there is evidence for DNA-adduct formation in liver,
    DNA-adduct formation in bone marrow has been a subject of some
    controversy.  Assuming that adduct formation is an initiating event,
    there is the possibility of a protective event if DNA repair or immune
    surveillance intervenes.  A promotional event might lead to a
    carcinogenic response.  It is possible that benzene acts as a promoter
    of its own initiation.

         While it is clear that a greater percentage of benzene
    metabolites are converted to reactive metabolites at low doses than at
    high doses, the total amounts of reactive metabolites increases with
    increasing dose.  Hence, it is clear that the severity of benzene
    toxicity increases with increasing dose.  By analogy, it is expected
    that increasing doses of benzene should yield more leukaemia in
    humans.

         The negative results obtained with  in vitro mutagenicity tests
    could be related to an inadequate production of mutagenic metabolites
    in the system.  The failure to produce leukaemia in animals may be due
    to lack of adequate formation of leukaemogenic metabolites or the need
    to produce bone marrow damage prior to the induction of leukaemia.  If
    the bone marrow must undergo a stage of aplastic anaemia prior to the
    development of leukaemia, one would have to estimate higher exposure
    to benzene and to define a threshold.  Alternatively, at doses lower

    than those producing aplastic anaemia, sufficient damage may occur to
    induce tumour promotion.

         Both animal and human studies show that benzene exposure produces
    bone marrow and chromosomal damage.  However, the various stages in
    carcinogenesis cited above have not been observed with benzene either
    because they do not occur in these species or because of technical
    problems.  The results of both animal and human studies suggest that
    benzene is a weak carcinogen on a molar basis.

    9.3.2.2  Human carcinogenesis

         Benzene is a well-established human leukaemogen.  There have been
    numerous epidemiological studies on the effects of benzene, most of
    which have dealt with chronic industrial exposures.  Increased
    leukaemia risk was identified in studies of shoemakers, chemical
    workers and workers in oil refineries, and in a nationwide study of
    benzene-exposed workers in different industries.  The most consistent
    evidence for a causal association in humans has been found between
    benzene exposure and myeloid leukaemia.  An exposure-response
    relationship was identified in some studies, the response being
    influenced both by exposure levels and duration of exposure.  In the
    study where estimated past exposures were based on the most extensive
    exposure measurements, a three-fold increased leukaemia risk was
    identified in workers exposed to benzene levels of 128-640
    mg/m3-years (40-200 ppm-years) (ppm-years is the average
    concentration times the duration of exposure in years, e.g., 4 ppm for
    10 years is equivalent to 40 ppm-years) and a statistically
    significant 12-fold risk for workers exposed to benzene levels between
    of 640-1280 mg/m3-years (200-400 ppm-years).  The scientific
    assessment of alternative exposure estimates for the rubber
    hydrochloride cohort has not yet been fully explored.  Of the two
    alternative exposures estimates so far postulated for this study, they
    both suggest a lower estimate of risk than that described above. 
    Other leukaemia types, multiple myeloma and other lymphomas were also
    reported.

         A statistically significant excess risk for multiple myeloma was
    found in the rubber hydrochloride study.  A marginally significant
    exposure-response relationship for malignant lymphomas was reported in
    a study on chemical workers.  Increased risk for skin, stomach and
    lung cancers has been reported in some studies, but these findings
    have not been consistent and may possibly be attributed to concomitant
    exposures to other chemicals or statistical artifact.

         Studies examining leukaemia risk in coke-oven workers, assumed to
    have been exposed to fairly low levels of benzene, have not identified
    excess leukaemia risk.  In these studies no attempt was made to
    examine leukaemia subtypes.  These studies do not provide enough
    evidence to prove that there is no risk of leukaemia as a result of
    exposure to these low concentrations of benzene.

         The Task Group is of the opinion that the epidemiological
    evidence presented so far is not capable of distinguishing between 
    (a) a small increase in leukaemia mortality in workers exposed to low
    benzene levels, and (b) a no-risk situation.

    9.4  Other toxicological end-points

         Benzene does cross the placenta of experimental animals, since
    haemopoietic changes have been observed in the fetuses and offspring
    of mice exposed to 16, 33 or 65 mg benzene/m3, 6 h/day during days
    6-15 of gestation.  Following inhalation exposure to high doses (500
    to 1600 mg/m3) in rabbits and mice, an increase in fetal resorptions
    or fetal death was observed.  However, benzene does not appear to be
    teratogenic, although it is fetotoxic, in experimental animals, and no
    evidence is available to permit the conclusion that it causes adverse
    reproductive effects in humans.

         The neurotoxicity of benzene in animals and humans has not been
    well studied.  An early study showed subtle changes such as reduced
    food intake and decreased hind-limb grip strength following exposures
    to 3300 and 9900 mg benzene/m3, and learning defects were observed
    in rats dosed orally with 550 mg benzene/kg body weight.

    9.5  Conclusions

         To assist Member States in the development of standards for
    benzene exposure, the Task Group concludes that a TWA of 3.2 mg/m3
    (1 ppm) over a 40-year working career has not been statistically
    associated with any increase in deaths from leukaemia.  However, since
    benzene is a human carcinogen, exposures should be limited to the
    lowest possible technically feasible level.  Increases in exposure to
    over 32 mg/m3 (10 ppm) should be avoided.  Benzene and
    benzene-containing products such as gasoline should never be used for
    cleaning purposes.

         Traditionally, bone marrow depression, i.e. anaemia, leucopenia
    or thrombocytopenia, in the workplace has been recognized as the first
    stage of benzene toxicity and appears to follow a dose-response
    relationship, i.e. the higher the dose, the greater the likelihood of
    observing decreases in circulating blood cell counts.

         Table 20 shows some "rough" estimates of the percentages of
    workers that might exhibit either bone marrow depression or frank
    aplastic anaemia after exposure to benzene for either 1 year or 10
    years at concentrations of 3.2, 32, 160 or 320 mg/m3 (1, 10, 50 or
    100 ppm).  These estimations are an interpretation of the literature
    using the experience of the Task Group.  The speculative nature of
    this table precludes its use in regulatory standard setting.  Exposure
    at high doses (160-320 mg/m3; 50-100 ppm) for one year would most
    likely produce bone marrow toxicity in a large percentage of the
    workers, and in some cases aplastic anaemia, but little effect would

    be expected at the lower doses.  Exposure to both high and low doses
    would be expected to produce benzene toxicity after 10 years.  Thus,
    a high level of both bone marrow depression and aplastic anaemia would
    be seen at the higher doses and some damage would also be seen at the
    lower doses.  The observation of any of these effects, regardless of
    the dose or period of exposure, should indicate the need for improved
    control of benzene exposure.

         There is no evidence of benzene being teratogenic at doses lower
    than those that produce maternal toxicity, but fetal toxicity has been
    demonstrated.

         The neurotoxicity and immunotoxicity of benzene have not been
    well studied in either experimental animals or humans.

    
    Table 20.  Estimated percentage of worker populations that might
               develop bone marrow depression or aplastic anaemia after
               chronic exposure to benzene ( Before using table note
                cautionary footnotea)

                                                                        

    Duration              Exposure              Bone marrow     Aplastic
                                                depression      anaemia
                                                                        

    1 year          320 mg/m3 (100 ppm)           90             10
                     160 mg/m3 (50 ppm)           50              5
                      32 mg/m3 (10 ppm)            1             0b
                      3.2 mg/m3 (1 ppm)           0b             0b

    10 years        320 mg/m3 (100 ppm)           99             50
                     160 mg/m3 (50 ppm)           75             10
                      32 mg/m3 (10 ppm)            5             0b
                      3.2 mg/m3 (1 ppm)           < 1            0b
                                                                        

    a  This estimation is an interpretation of the literature and is
       based on the experience of the Task Group.  The speculative
       nature of this table precludes its use in regulatory standard
       setting.
    b  Occasional cases may be observed.
    
    10.  RECOMMENDATIONS FOR PROTECTION OF HUMAN HEALTH

    a)   Benzene and benzene-containing products, including gasoline
         (petrol), should never be used for cleaning purposes.

    b)   Systematic information on occupational and non-occupational  
         exposure should be collected using the total human exposure  
         approach where possible.

    c)   The health risk of low-level benzene exposure is not clearly
         established.  Exposure should, therefore, be avoided as much as
         possible.

    d)   The occurrence of benzene in environmental media such as air and
         water where there exists potential human exposure should be
         evaluated.

    e)   A search for less toxic solvents to replace benzene in industrial
         processes should be encouraged.

    11.  FURTHER RESEARCH

    a)   Epidemiological studies of the risks of haematological
         malignancies, blood changes (red and white blood cells) and
         genotoxic effects at low and high exposure concentrations should
         have high priority.

    b)   Information on the mechanisms by which benzene induces neoplasms
         is required.  In particular, there is a need for animal models of
         benzene-induced haemopoietic malignancies similar to those seen
         in humans and a better understanding of the role of reactive
         intermediate.

    c)   Further studies are needed to elucidate the potential link
         between bone marrow suppression and the eventual occurrence of
         leukaemia.

    d)   Biological markers of benzene exposure, especially urinary
         muconic acid and macromolecular adducts, should be validated.

    e)   Individual susceptibility factors in benzene-induced toxicities
         should be investigated.

    f)   Animals and human studies are required to assist in validating
         physiologically based pharmacokinetic models using all routes of
         exposure.

    g)   The multigenerational effects of benzene exposure should be
         investigated.

    h)   Studies on immunotoxicological effects following benzene exposure
         should be performed.

    12.  PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES

         The carcinogenicity of benzene has been evaluated by the
    International Agency for Research on Cancer (IARC, 1982, 1987b).  It
    was concluded that there was sufficient evidence for the
    carcinogenicity of benzene in both animals and humans.

         A guideline value of 10 µg/litre was recommended by WHO (WHO,
    1984) for benzene in drinking-water based on data for the production
    of leukaemia after inhalation exposures in humans and using a linear
    multistage extrapolation model and a life-time risk level of 1 in
    100 000.  This guideline remained unchanged during the revisions
    recently completed (WHO, 1993).

         A Task Group convened by the WHO Regional Office for Europe
    concluded that an air quality guideline value could not be set for
    benzene in view of its carcinogenic activity in humans (WHO, 1987). 
    Assuming no threshold and an average relative risk model, it was
    calculated that at an air concentration of 1 µg benzene/m3, the
    estimated lifetime risk of leukaemia would be 4 x 10-6.

         Regulatory standards for benzene established by national bodies
    in some countries are summarized in the Legal File of the
    International Register of Potentially Toxic Chemicals (IRPTC, 1987). 
    Recently the Commission of the European Communities has proposed an
    occupational exposure limit for benzene of 1.6 mg/m3 (0.5 ppm) (CEC,
    1993).

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    RESUME ET CONCLUSIONS

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

         Le benzène est un liquide incolore, stable à la température
    ambiante et sous la pression atmosphérique normale.  Il possède une
    odeur aromatique caractéristique et du fait de son bas point
    d'ébullition (80,1 °C) et de sa forte tension de vapeur, il s'évapore
    rapidement et il est très inflammable.  Il est légèrement soluble dans
    l'eau mais miscible à la plupart des solvants organiques.

         Il existe des méthodes qui permettent de rechercher la présence
    de benzène dans différents milieux (air, eau, organes/tissus).  On
    peut utiliser à cette fin la chromatographie en phase gazeuse avec, au
    choix, une détection par ionisation de flamme, par photoionisation ou
    par spectrométrie de masse, en fonction de la sensibilité nécessaire
    et des concentrations attendues.  La recherche du benzène sur le lieu
    de travail s'effectue généralement par adsorption sur charbon actif
    puis analyse par chromatographie en phase gazeuse couplée à la
    spectrométrie de masse après désorption.  Lorsqu'on peut se contenter
    d'une sensibilité de l'ordre du mg/m3, on peut utiliser des
    instruments à lecture directe et des dosimètres passifs.  Si l'on
    désire une meilleure sensibilité, il existe des méthodes qui
    permettent de déceler la présence de benzène à des concentrations ne
    dépassant pas 0,01 µg/m3 d'air ou 1 ng/kg de terre ou d'eau.

    2.  Sources d'exposition humaine

         Le benzène existe à l'état naturel dans le pétrole brut à des
    concentrations pouvant atteindre 4 g/litre.  Il est également produit
    partout dans le monde en quantités extrêmement importantes (14,8
    millions de tonnes).  Des émissions peuvent se produire lors du
    traitement des produits pétroliers, de la cokéfaction du charbon, de
    la production de toluène, de xylène et autres dérivés aromatiques
    ainsi que lorsqu'il est utilisé dans certains produits de
    consommation, comme intermédiaire ou comme constituant de l'essence.

    3.  Transport, distribution et transformation dans l'environnement

         Dans l'air, le benzène est présent essentiellement en phase
    gazeuse, et sa durée de séjour varie de quelques heures à quelques
    jours, en fonction de l'environnement et du climat et aussi de la
    concentration des radicaux hydroxyles, des oxydes d'azote et de
    soufre.  Lorsqu'il est éliminé de l'air par la pluie, il peut
    contaminer les eaux de surface et les eaux souterraines dans
    lesquelles il est soluble à raison d'environ 1000 mg/litre.

         En raison principalement de sa volatilisation dans l'atmosphère,
    le temps de séjour du benzène dans l'eau est limité à quelques heures
    et il est peu ou pas adsorbé par les sédiments.

         Le benzène présent dans le sol peut passer dans l'air par
    volatilisation et dans les eaux de surface par ruissellement.  En cas
    d'enfouissement ou de libération à profondeur importante, il passe
    dans les eaux souterraines.

         En aérobiose, le benzène présent dans l'eau ou dans le sol est
    rapidement dégradé (quelques heures) par les bactéries en lactate et
    en pyruvate avec formation intermédiaire de phénol et de catéchol. 
    Cependant, en anaérobiose (par exemple dans les eaux souterraines) la
    dégradation bactérienne prend des semaines, voire des mois et non plus
    des heures.  Il y a persistance du benzène lorsque la dégradation
    bactérienne ne se produit pas.  Il ne semble pas subir de
    bioconcentration ou de bioaccumulation dans les organismes aquatiques
    ou terrestres.

    4.  Concentrations dans l'environnement et exposition humaine

         La présence de benzène dans l'essence et sa large utilisation
    comme solvant industriel peuvent conduire à des émissions importantes
    un peu partout dans l'environnement.  A l'extérieur, les
    concentrations vont de 0,2 µg/m3 dans les régions rurales écartées
    à 349 µg/m3 dans les zones industrielles où la circulation
    automobile est dense.  Lors du remplissage du réservoir d'un véhicule
    à moteur, les concentrations peuvent atteindre 10 mg/m3.

         On a décelé la présence de benzène à des concentrations
    atteignant 500 µg/m3 dans l'air intérieur des pièces de séjour.  La
    fumée de cigarette contribue de façon importante à la présence de
    benzène dans l'air intérieur, les fumeurs inhalant environ 1800 µg de
    benzène par jour contre 50 µg pour les non fumeurs.

         Dans de nombreux pays, l'exposition professionnelle dépasse
    rarement 15 mg/m3 en moyenne pondérée par rapport au temps. 
    Toutefois, les concentrations effectives  rapportées dépendent du type
    d'industries étudiées et elles peuvent être beaucoup plus élevées dans
    les pays en voie de développement industriel.

         L'eau et les aliments ne contribuent que pour une faible part à
    l'apport journalier de benzène chez les adultes non fumeurs (entre 3
    et 24 µg/kg de poids corporel et par jour).

    5.  Cinétique et métabolisme

         Le benzène est bien absorbé chez l'homme et les animaux de
    laboratoire après exposition par voie orale ou respiratoire, mais chez
    l'homme, le benzène n'est que faiblement absorbé par voie percutanée. 
    L'absorption se produit chez l'homme à hauteur d'environ 50% lors
    d'expositions continues pendant plusieurs heures à des concentrations
    de 163 à 326 mg/m3.  On a constaté qu'après quatre heures
    d'exposition à 170-202 mg/m3, le benzène était retenu par
    l'organisme humain dans la proportion d'environ 30%, 16% de cette dose

    étant rejetés tels quels dans l'air expiré. Le benzène inhalé est
    davantage retenu par l'organisme féminin que par l'organisme masculin. 
    Le benzène à tendance à s'accumuler dans les tissus à forte teneur en
    lipides et il traverse la barrière placentaire.

         Le métabolisme du benzène s'effectue principalement dans le foie,
    essentiellement par l'intermédiaire du système enzymatique du
    cytochrome P-450 IIE1 et il comporte la formation d'une série de
    métabolites réactifs instables. Chez les rongeurs, les processus de
    formation de deux métabolites toxiques supposés, la benzoquinone et le
    muconaldéhyde, se révèlent être saturables.  Ce phénomène peut avoir
    des conséquences importantes en ce qui concerne les relations
    dose-réponse car la proportion de benzène transformée en métabolites
    toxiques sera plus forte à faibles doses qu'à doses élevées.  Les
    produits du métabolisme sont principalement excrétés dans les urines. 
    Les métabolites reconnus du benzène: phénol, catéchol et hydroquinone
    - se retrouvent en quantité appréciable dans la moelle osseuse.  Le
    phénol est le principal métabolite urinaire chez l'homme et on le
    retrouve dans l'urine, essentiellement sous forme de sulfoconjugué
    jusqu'à ce que les concentrations atteignent 480 mg/litre, après quoi
    on observe la formation de glucuronides.  D'après des études récentes,
    la toxicité du benzène serait due à l'interaction des différents
    métabolites de ce composé qui se forment tant dans le foie que dans la
    moelle osseuse.

         Une fois inhalé, le benzène se fixe à l'ADN du foie chez le rat
    à raison de 2,38 µmol/mol d'ester phosphorique.  On a décelé dix
    adduits de désoxyguanosine et un adduit de désoxyadénine dans l'ADN
    mitochondrial de la moelle osseuse du lapin.

    6.  Effets sur les mammifères de laboratoire et les systèmes
        d'épreuves in vitro

    6.1  Toxicité générale

         Le benzène ne présente qu'une faible toxicité aiguë chez diverses
    espèces animales, les valeurs de la DL50 après exposition orale
    allant de 3000 à 8100 mg/kg de poids corporel chez le rat, par
    exemple.  On a fait état de valeurs de la CL50 allant de 15 000
    mg/m3 (8 h) chez la souris à 44 000 mg/m3 (4 h) chez le rat.

         Le benzène est modérément irritant pour la muqueuse oculaire et
    provoque une irritation dermique chez le lapin après plusieurs
    applications de produit non dilué.  On ne dispose d'aucune donnée sur
    le pouvoir de sensibilisation cutanée du benzène.

         L'exposition de souris à du benzène par la voie respiratoire
    entraîne une baisse sensible de certains paramètres hématologiques tel
    que l'hématocrite, le taux d'hémoglobine ainsi que le nombre
    d'érythrocytes, de leucocytes et de plaquettes.  Une exposition de

    longue durée à de fortes doses provoque une aplasie médullaire. Chez
    le rat les effets sont analogues mais moins graves.

    6.2  Génotoxicité et cancérogénicité

         Les tests de mutagénicité  in vitro ont donné des résultats
    négatifs.

         En ce qui concerne les études  in vivo, on observe que le
    benzène et ses métabolites entraînent des aberrations dans la
    structure et le nombre des chromosomes chez l'homme et les animaux de
    laboratoire.  En outre, l'administration de benzène provoque des
    échanges entre chromatides soeurs et la formation d'érythrocytes
    polychromatiques avec micronoyaux.  Après administration par la voie
    intrapéritonéale, le benzène peut atteindre les cellules germinales
    comme le montrent les anomalies observées dans la morphologie de la
    tête des spermatozoïdes.

         On a fait état de la formation de plusieurs types de cancers dus
    au benzène chez le rat et la souris après administration par voie
    orale ou exposition par la voie respiratoire.  Il s'agit de divers
    types de tumeurs malignes épithéliales concernant par exemple la
    glande de Zymbal, le foie, le tissu mammaire et les fosses nasales,
    avec en outre quelques lymphomes et leucémies.

         Dans les études comportant une exposition par inhalation et au
    cours desquelles on a relevé effectivement une action cancérogène, les
    doses allaient de 100 à 960 mg/m3, cinq à sept heures par jour et
    cinq jours par semaine.  L'administration par voie orale de benzène à
    des doses allant de 25 à 500 mg/kg de poids corporel à des souris et
    à des rats, a entraîné la formation de néoplasmes.  La durée
    d'exposition était généralement de un à deux ans.

    6.3  Effets toxiques sur la reproduction; embryotoxicité et tératogénicité

         Le benzène traverse facilement la barrière placentaire.  De
    nombreuses expériences au cours desquelles des animaux de laboratoire
    ont été soumis à des doses atteignant même les valeurs toxiques pour
    la mère n'ont pas permis de recueillir de données indicatives d'un
    effet tératogène.  Toutefois, on a montré que le benzène était
    foetotoxique après exposition par la voie respiratoire, chez la souris
    (1600 µg/m3, sept heures par jour, du sixième au quinzième jours de
    la gestation) et le lapin.

    6.4  Immunotoxicité

         Le benzène réduit l'aptitude des lymphocytes B et T à proliférer. 
    Chez plusieurs espèces d'animaux de laboratoire exposés au benzène, on
    a noté une diminution de la résistance de l'hôte aux infections.

    7.  Effets sur l'homme

         On sait que le benzène produit un certain nombre d'effets nocifs
    pour la santé humaine.  Le plus fréquemment cité de ces effets est une
    dépression médullaire qui conduit à une anémie aplasique. 
    L'exposition à de fortes doses de benzène entraîne probablement une
    forte incidence de ces maladies.

         Il est bien établi que le benzène est cancérogène pour l'homme. 
    Des études épidémiologiques effectuées sur les travailleurs exposés au
    benzène ont montré qu'il existait une relation causale entre
    l'exposition à cette substance et l'apparition d'une leucémie
    myéloïde.  La relation qui a été observée entre l'exposition au
    benzène et l'apparition de lymphomes ou de myélomes multiples reste à
    élucider.

         Le Groupe de travail a estimé que les données épidémiologiques ne
    permettent pas de distinguer entre (a) une faible augmentation de la
    mortalité par leucémie chez les travailleurs exposés à de faibles
    doses de benzène et (b) une situation où le risque n'existe pas.

    8.  Conclusions

         Le Groupe a conclu qu'une exposition moyenne pondérée par rapport
    au temps de l'ordre de 3,2 mg/m3 (1 ppm)  au cours d'une carrière de
    40 ans, n'entraîne pas, statistiquement parlant, de surmortalité par
    leucémie.  Toutefois, comme le benzène est cancérogène pour l'homme,
    il convient de limiter l'exposition à la dose la plus faible
    compatible avec les exigences techniques.  Il convient d'éviter
    également tout accroissement de l'exposition au-delà de la valeur de
    32 mg/m3 (10 ppm).  Le benzène et les produits qui en contiennent,
    comme l'essence, ne doivent jamais être utilisés comme agents de
    nettoyage.

         On admet traditionnellement que la dépression médullaire,
    c'est-à-dire une anémie, une leucopénie ou une thrombocytopénie,
    observées sur les lieux de travail, constituent le premier stade d'une
    intoxication par le benzène et que ces affections sont liées à la
    dose.  En d'autres termes, plus la dose est élevée, plus la
    probabilité d'observer une réduction des éléments figurés du sang est
    élevée.

         L'exposition à de fortes doses de benzène (160 à 320 mg/m3)
    pendant un an entraînerait selon toute probabilité une toxicité
    médullaire chez une proportion importante des travailleurs, voire une
    anémie aplasique chez certains d'entre eux, mais les effets ne
    seraient guère marqués à plus faibles doses.  Une exposition à de
    faibles et fortes doses devrait entraîner une intoxication benzénique
    au bout de dix années d'exposition continue.  On peut donc dire qu'à
    fortes doses, on observerait un nombre élevé de cas de dépression
    médullaire et d'anémie aplasique avec également quelques lésions à

    faibles doses.  Au cas où l'on observerait l'un quelconque de ces
    effets quel que soit le niveau d'exposition, il faudrait prendre des
    mesures pour améliorer le contrôle de cette exposition.

         Rien d'indique que le benzène soit tératogène à des doses plus
    faibles que celles qui sont toxiques pour la mère, toutefois on a
    observé une toxicité foetale.

         La neurotoxicité et l'immunotoxicité du benzène n'ont pas été
    bien étudiées, ni  chez l'animal de laboratoire ni chez l'homme.

    RESUMEN Y CONCLUSIONES

    1.  Identidad, propiedades físicas y químicas y métodos analíticos

         El benceno es un líquido incoloro y estable a temperatura
    ambiente y presión atmosférica normal.  Posee un olor aromático
    característico, un punto de ebullición relativamente bajo (80,1 °C) y
    una elevada presión de vapor, lo que hace que se evapore rápidamente
    a temperatura ambiente, y es altamente inflamable.  Es ligeramente
    soluble en agua, pero también es miscible en la mayoría de los otros
    disolventes orgánicos.

         Se dispone de métodos analíticos para detectar benceno en
    diversos medios (aire, agua, órganos/tejidos).  La elección entre
    cromatografía de gases (CG), con detección mediante ionización de
    llama o fotoionización, o espectrometría de masas (EM) depende de la
    sensibilidad requerida y de los niveles de benceno previstos.  La
    detección de benceno en el lugar de trabajo se realiza normalmente
    mediante captación con carbón vegetal, desorción, y análisis por CG o
    EM.  Si es suficiente una sensibilidad del orden de mg/m3, pueden
    emplearse instrumentos portátiles de lectura directa y dosímetros
    pasivos.  Para los casos en que hace falta una mayor sensibilidad, se
    han notificado métodos válidos para detectar benceno a niveles de sólo
    0,01 µg/m3 (aire) o 1 ng/kg (suelo o agua).

    2.  Fuentes de exposición humana

         El benceno es un producto químico natural, que se halla en el
    petróleo crudo a niveles de hasta 4 g/litro.  Además, es producido en
    muy grandes cantidades (14,8 millones de toneladas) en todo el mundo. 
    Se producen emisiones de benceno durante el procesamiento de los
    productos petroleros, durante la producción de coque a partir de
    carbón, durante la producción de tolueno, xileno y otros compuestos
    aromáticos, y como consecuencia de su empleo en productos de consumo,
    como compuesto intermedio y como componente de la gasolina.

    3.  Transporte, distribución y transformación en el medio ambiente

         El benceno presente en el aire se halla predominantemente en la
    fase de vapor, y su tiempo de persistencia varía entre unas horas y
    unos días, según el entorno y el clima, y en función de la
    concentración de radicales hidroxilo, así como de dióxidos de azufre
    y de nitrógeno.  El benceno presente en el aire es eliminado por la
    lluvia, con la consiguiente contaminación de las aguas superficiales
    y subterráneas, en las que es soluble a razón de aproximadamente 1000
    mg/litro.

         Debido fundamentalmente a la volatilización, el tiempo de
    persistencia del benceno en el agua es de unas cuantas horas, y su
    adsorción por los sedimentos es escasa o nula.

         El benceno presente en el suelo puede pasar al aire por
    volatilización, y a las aguas superficiales por la escorrentía.  Si es
    enterrado o liberado muy por debajo de la superficie, será
    transportado hasta las aguas subterráneas.

         En condiciones aerobias el benceno presente en el agua o el suelo
    es rápidamente (en cuestión de horas) degradado por las bacterias a
    lactato y piruvato, previa transformación en los productos intermedios
    fenol y catecol.  En cambio, en condiciones anaerobias (por ejemplo en
    las aguas subterráneas) la degradación bacteriana requiere semanas o
    meses en lugar de horas.  Si no hay degradación bacteriana el benceno
    puede persistir.  No hay pruebas de una bioconcentración o
    bioacumulación de benceno en organismos acuáticos o terrestres.

    4.  Niveles ambientales y exposición humana

         La presencia de benceno en la gasolina y su amplio uso como
    disolvente industrial puede dar lugar a emisiones importantes y
    generalizadas al medio ambiente.  Sus niveles ambientales al aire
    libre oscilan entre los 0,2 µg/m3 hallados en zonas rurales aisladas
    y los 349 µg/m3 detectados en centros industriales con alta densidad
    de tráfico de automóviles.  Durante las operaciones de
    reabastecimiento de combustible de los automóviles se han llegado a
    registrar niveles de hasta 10 mg/m3.

         En el aire del interior de las viviendas se han detectado niveles
    de benceno de hasta 500 µg/m3.  El humo del tabaco contribuye con
    importantes cantidades de benceno a los niveles registrados en el aire
    de los espacios interiores, cifrándose las cantidades inhaladas por
    los fumadores en aproximadamente 1800 µg de benceno al día, frente a
    50 µg/día los no fumadores.

         En numerosos países la exposición ocupacional rara vez supera una
    media ponderada respecto al tiempo de 15 mg/m3.  Sin embargo, los
    niveles reales notificados dependen de la industria estudiada, y en
    algunos países en fase de desarrollo industrial las exposiciones
    pueden ser considerablemente superiores.

         El benceno transmitido por el agua y los alimentos representa
    sólo un pequeño porcentaje de la ingesta diaria total de los adultos
    no fumadores (entre unos 3 y 24 µg/kg de peso corporal al día).

    5.  Cinética y metabolismo

         El benceno es fácilmente absorbido por el hombre y los animales
    experimentales que entran en contacto con el producto por exposición
    oral o inhalación, pero en la especie humana la absorción cutánea es
    escasa.  Con una exposición continua a niveles de 163-326 mg/m3
    durante varias horas, la absorción en el hombre es de aproximadamente
    un 50%.  Tras una exposición de 4 horas a niveles de 170-202 mg/m3,
    la retención en el organismo humano fue de aproximadamente un 30%,

    habiéndose excretado un 16% de la dosis retenida en forma de benceno
    inalterado a través del aire expirado.  Las mujeres suelen retener un
    mayor porcentaje del benceno inhalado que los hombres.  El benceno
    tiende a acumularse en los tejidos que contienen gran cantidad de
    lípidos y atraviesa la placenta.

         El metabolismo del benceno se produce principalmente en el
    hígado, depende básicamente del sistema enzimático del citocromo P-450
    IIE1 y conlleva la formación de una serie de metabolitos reactivos
    inestables.  En los roedores la formación de dos presuntos metabolitos
    tóxicos, la benzoquinona y el muconaldehído, parece ser saturable, lo
    que puede tener gran importancia desde el punto de vista de la
    relación dosis-respuesta, pues significa que a dosis bajas la
    proporción de benceno transformada en metabolitos tóxicos será mayor
    que a dosis altas.  Los productos metabólicos son excretados
    principalmente por la orina.  En la médula ósea se observan niveles
    importantes de los conocidos metabolitos fenol, catecol e
    hidroquinona.  En el hombre el metabolito urinario predominante es el
    fenol, que aparece sobre todo conjugado con sulfato como éter a
    niveles inferiores a 480 mg/litro, concentración a la cual se empiezan
    a detectar glucurónidos.  Estudios recientes llevan a pensar que la
    toxicidad del benceno se debe a la interacción de varios metabolitos
    bencénicos formados tanto en el hígado como en la médula ósea.

         Se ha observado que el benceno inhalado se une al ADN hepático de
    la rata a razón de 2,38 µmoles por mol de fosfato de ADN.  En el ADN
    mitocondrial de la médula ósea de conejo se han detectado siete
    aductos de desoxiguanosina y un aducto de desoxiadenina.

    6.  Efectos en los mamíferos de laboratorio y en las pruebas in vitro

    6.1  Toxicidad sistémica

         El benceno tiene al parecer una toxicidad aguda baja en diversas
    especies animales, oscilando las DL50 por exposición oral en la rata
    entre 3000 y 8100 mg/kg de peso corporal.  Las CL50 notificadas
    oscilan entre los 15 000 mg/m3 (8 h) del ratón y los 44 000 mg/m3
    (4 h) de la rata.

         El benceno tiene un efecto irritante moderado sobre los ojos, y
    aplicado reiteradamente y sin diluir también es irritante para la piel
    del conejo.  No se dispone de información sobre el potencial de
    sensibilización cutánea del benceno.

         Los ratones expuestos a benceno por inhalación presentan una
    disminución importante del valor de parámetros hemáticos tales como el
    hematocrito, la hemoglobina y el número de eritrocitos, leucocitos y
    plaquetas.  La exposición prolongada a altas dosis provoca aplasia de
    la médula ósea.  También en la rata se han observado efectos
    similares, aunque menos graves.

    6.2  Genotoxicidad y carcinogenicidad

         Las pruebas de mutagenicidad del benceno  in vitro han arrojado
    resultados negativos.

         En los estudios  in vivo el benceno o sus metabolitos causan
    aberraciones cromosómicas tanto estructurales como numéricas en el
    hombre y en los animales de laboratorio.  La administración de
    benceno, además, da lugar a intercambios entre cromatidios hermanos y
    a la producción de eritrocitos policromáticos con micronúcleos. 
    Administrado interperitonealmente el benceno puede alcanzar las
    células germinales, como demuestra la aparición de alteraciones
    morfológicas de la cabeza de los espermatozoides.

         Se ha notificado que la administración oral o la inhalación de
    benceno provocan tanto en la rata como en el ratón varios tipos de
    neoplasma, entre ellos diversos tipos de neoplasma epitelial, por
    ejemplo de la glándula de Zymbal, hígado, tejido mamario y cavidades
    nasales, y algunos linfomas y leucemias.

         En los estudios en que se observó una respuesta carcinógena
    positiva a la inhalación, los niveles de exposición oscilaban entre
    100 y 960 mg/m3 durante 5 a 7 h/día, cinco días por semana.  En el
    ratón y la rata, la administración oral de benceno a dosis de 25-500
    mg/kg de peso corporal provocó la aparición de neoplasmas; la duración
    de la exposición fue por lo general de 1 a 2 años.

    6.3  Toxicidad en la reproducción, embriotoxicidad y teratogenicidad

         El benceno atraviesa libremente la barrera placentaria.  Tras
    numerosos experimentos realizados con animales a dosis incluso tóxicas
    para la madre, no se ha obtenido ningún dato que demuestre que tenga
    efectos teratógenos.  No obstante, se ha demostrado que su inhalación
    tiene efectos fetotóxicos en el ratón (1600 µg/m3, 7 h/día, días 6
    a 15 de gestación) y en el conejo.

    6.4  Inmunotoxicidad

         El benceno deprime la capacidad de proliferación de los
    linfocitos B y T.  Se ha observado una menor resistencia a las
    infecciones en varias especies de laboratorio expuestas al benceno.

    7.  Efectos en el ser humano

         Es sabido que el benceno tiene varios efectos perjudiciales para
    la salud, entre los que destaca por su frecuencia la depresión de la
    médula ósea, que conduce a la anemia aplásica.  Unos niveles altos de
    exposición hacen probable una elevada incidencia de esas enfermedades.

         Está demostrado que el benceno tiene un efecto carcinógeno en el
    ser humano.  Los estudios epidemiológicos realizados sobre

    trabajadores expuestos al benceno han demostrado la existencia de una
    relación causal entre la exposición al benceno y la incidencia de
    leucemia mielógena.  Resta por aclarar si existe también una relación
    entre la exposición al benceno y la aparición de linfoma y mieloma
    múltiple.

         El Grupo de Estudio era de la opinión de que los datos
    epidemiológicos no permiten distinguir entre a) un ligero aumento de
    la mortalidad por leucemia entre los trabajadores expuestos a niveles
    bajos de benceno, y b) una situación sin riesgo.

    8.  Conclusiones

         Se llegó a la conclusión de que una media ponderada respecto al
    tiempo de 3,2 mg/m3 (1 ppm) a lo largo de 40 años de trabajo no
    determina un aumento estadísticamente significativo del número de
    defunciones por leucemia.  Debido a sus efectos carcinógenos sobre el
    hombre, sin embargo, las exposiciones se deberán limitar al nivel
    técnicamente más bajo posible.  Deberán evitarse las exposiciones
    superiores a 32 mg/m3 (10 ppm).  El benceno y los productos que lo
    contienen, como la gasolina, no se deberían emplear nunca en
    operaciones de limpieza.

         Tradicionalmente se ha considerado que la aparición de depresión
    de médula ósea - esto es, de anemia, leucopenia o trombocitopenia - en
    el lugar de trabajo representa la primera fase de toxicidad del
    benceno.  Esa manifestación obedece al parecer a una relación
    dosis-respuesta; en otras palabras, cuanto mayor la dosis, mayor
    también la probabilidad de observar una disminución del número de
    células sanguíneas circulantes.

         La exposición a altos niveles de benceno (160-320 mg/m3)
    durante un año tendría con toda probabilidad efectos tóxicos sobre la
    médula ósea en un elevado porcentaje de los trabajadores, y provocaría
    anemia aplásica en algunos casos, pero dosis menores apenas tendrían
    efectos.  En cambio, cabe prever que la exposición continua durante
    diez años a dosis altas o bajas tendría efectos tóxicos.  Así, con las
    dosis elevadas se observaría una alta incidencia tanto de depresión de
    la médula ósea como de anemia aplásica, y con las dosis más bajas se
    observarían también algunas lesiones.  La observación de cualquiera de
    esos efectos, con independencia del nivel de exposición, será
    reveladora de la necesidad de mejorar la vigilancia de la exposición
    al benceno.

         No hay indicios de que el benceno tenga efectos teratógenos a
    dosis inferiores a las que resultan tóxicas para la madre, pero sí se
    ha demostrado que tiene efectos tóxicos para el feto.

         La neurotoxicidad y la inmunotoxicidad del benceno no han sido
    suficientemente estudiadas ni en animales experimentales ni en el ser
    humano.


    See Also:
       Toxicological Abbreviations
       Benzene (ICSC)
       BENZENE (JECFA Evaluation)
       Benzene (PIM 063)
       Benzene  (IARC Summary & Evaluation, Supplement7, 1987)
       Benzene (IARC Summary & Evaluation, Volume 7, 1974)
       Benzene (IARC Summary & Evaluation, Volume 29, 1982)