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 The International Programme on Chemical Safety (IPCS) is a joint venture of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization. The main objective of the IPCS is to carry out and disseminate evaluations of the effects of chemicals on human health and the quality of the environment. Supporting activities include the development of epidemiological, experimental laboratory, and risk-assessment methods that could produce internationally comparable results, and the development of manpower in the field of toxicology. Other activities carried out by the IPCS include the development of know-how for coping with chemical accidents, coordination of laboratory testing and epidemiological studies, and promotion of research on the mechanisms of the biological action of chemicals. WHO Library Cataloguing in Publication Data 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 The World Health Organization welcomes requests for permission to reproduce or translate its publications, in part or in full. Applications and enquiries should be addressed to the Office of Publications, World Health Organization, Geneva, Switzerland, which will be glad to provide the latest information on any changes made to the text, plans for new editions, and reprints and translations already available. (c) World Health Organization 1993 Publications of the World Health Organization enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Convention. All rights reserved. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. CONTENTS ENVIRONMENTAL HEALTH CRITERIA FOR 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 184.108.40.206 Bone marrow depression; aplastic anaemia 220.127.116.11 Immunological effects 18.104.22.168 Chromosomal effects 22.214.171.124 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 126.96.36.199 Haematotoxicity; bone marrow depression 188.8.131.52 Mechanism of action and metabolism 184.108.40.206 Immunotoxicity 9.3.2 Genotoxicity and carcinogenic effects 220.127.116.11 Mechanism of carcinogenicity 18.104.22.168 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 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 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. 22.214.171.124 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. 126.96.36.199 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. 188.8.131.52 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). 184.108.40.206 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. 220.127.116.11 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). 18.104.22.168 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. 22.214.171.124 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. 126.96.36.199 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. 188.8.131.52 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). References Aksoy M (1980) Different types of malignancies due to occupational exposure to benzene. A review of recent observations in Turkey. Environ Res, 23: 181-190. Aksoy M & Erdem S (1978) Follow-up study on the mortality and the development of leukaemia in 44 pancytopaenic patients with chronic exposure to benzene. Blood, 52: 285-292. Aksoy M, Dincol K, Akgun T, Erdem S, & Dincol G (1971) Haematological effects of chronic benzene poisoning in 217 workers. Br J Ind Med, 28: 296-302. Aksoy M, Dincol K, Akgun T, Erdem S, & Dincol G (1972) Details of blood changes in 32 patients with pancytopaenia associated with long-term exposure to benzene. Br J Ind Med, 29: 56-64. Aksoy M, Erdem S, Hepyüksel T, & Dincol G (1974a) Chronic exposure to benzene as a possible contributory etiologic factor in Hodgkin's Disease. Blood, 38: 93-298. Aksoy M, Erdem S, & Dincol G (1974b) Leukemia in shoe-workers exposed chronically to benzene. Blood, 44: 837-841. Allen PD (1987) Residential population exposure to ambient benzene in California. Sacramento, California, California Air Resources Board. Anderson D & Richardson CR (1981) Issues relevant to the assessment of chemically induced chromosome damage in vivo and their relationship to chemical mutagenesis. Mutat Res, 90: 261-272. Andrews LS & Snyder R (1986) Toxic effects of solvents and vapors. In: Casarett LJ & Doull J ed. Toxicology: The basic science of poisons, 3rd ed. New York, McMillan Publishing Co., chapter 20, p 641. Antoine SR, Delon IR, & O'Dell-Smith RM (1986) Environmentally significant volatile organic pollutants in human blood. Bull Environ Contam Toxicol, 36(3): 364-371. Appuhn E & Goldeck H (1957) [Early and late disorders of haematopoiesis caused by benzene and its homologues.] Arch Gewerbepathol Gewerbehyg, 15: 399-428 (in German). Ashby J, de Serres FJ, Draper M, Ishidate M Jr, Margolin BH, Matter BE, & Shelby MD ed. (1985) Evaluation of short-term tests for carcinogens. Report of the International Programme on Chemical Safety's collaborative study on in vitro assays. Amsterdam, Oxford, New York, Elsevier Science Publishers (Progress in Mutation Research, Volume 5). ATSDR (1989) Toxicological profile for benzene. Atlanta, Georgia, Agency for Toxic Substances and Disease Registry, 173 pp (ATSDR/TP-88/03; PB/89/209464/AS). ATSDR (1991) Toxicological profile for benzene. Atlanta, Georgia, Agency for Toxic Substances and Disease Registry, 193 pp. Au WW, Ramanujam VMS, Ward JB Jr, & Legator MS (1991) Chromosome aberrations in lymphocytes of mice after sub-acute low-level inhalation exposure to benzene. Mutat Res, 260: 219-224. Baarson K, Snyder CA, & Green J (1982) The hematotoxic effects of inhaled benzene on peripheral blood, bone marrow, and spleen cells are increased by ingested ethanol. Toxicol Appl Pharmacol, 64: 393-404. Baarson K, Snyder CA, & Albert RE (1984) Repeated exposures of C57B1 mice to 10 ppm inhaled benzene markedly depressed erythropoietic colony formation. Toxicol Lett, 20: 337-342. Bailer AJ & Hoel DG (1989) Metabolite-based internal doses used in a risk assessment of benzene. Environ Health Perspect, 82: 177-184. Bailey JC & Schmidl B (1989) A survey of hydrocarbons emitted in vehicle exhaust gases, over a range of driving speeds and conditions from a representative sample of the 1986-87 UK vehicle fleet. Stevenage, Herts, Warren Spring Laboratory (DTI) (Report No. LR 673). Bandow H, Washida N, & Akimoto H (1985) Ring-cleavage reactions of aromatic hydrocarbons studies by FT-IR spectroscopy. I. Photooxidation of toluene and benzene in the NOx-air system. Bull Chem Soc Jpn, 58: 2531-2540. Barale R, Giorgelli F, Migliore L, Ciranno R, Casini D, Zucconi D, & Loprieno N (1985) Benzene induces micronuclei in circulating erythrocytes of chronically treated mice. Mutat Res, 144: 193-196. Barrett RH (1985) Assays for unscheduled DNA synthesis in HeLa S3 cells. In: Ashby J, de Serres FJ, Draper M, Ishidate M Jr, Margolin BH, Matter BE, & Shelby MD ed. Short-term tests for carcinogens: Report of the International Programme on Chemical Safety's collaborative study on in vitro assays. Amsterdam, Oxford, New York, Elsevier Science Publishers, pp 347-352 (Progress in Mutation Research, Volume 5). Battersby NS & Wilson V (1989) Survey of the anaerobic biodegradation potential of organic chemicals in digesting sludge. Appl Environ Microbiol, 52(2): 433-439. Baxter HG, Blakemore R, Moore JP, & Coker DT (1980) The measurement of airborne benzene vapour. Ann Occup Hyg, 23: 117-132. Bayer CW, Black MS, & Galloway LM (1988) Sampling and analysis techniques for trace volatile organic emissions from consumer products. J Chromatogr Sci, 26: 168-173. Bechtold WE, Sabourin PJ, & Henderson RF (1988) A reverse isotope dilution method for determining benzene and metabolites in tissues. J Anal Toxicol, 12(7/8): 176-179. Bechtold WE, Lucier G, Birnbaum LS, Yin SN, Li GL, & Henderson RF (1991) Muconic acid determinations in urine as a biological exposure index for workers occupationally exposed to benzene. Am Ind Hyg Assoc J, 52(11): 473-478. Bechtold WE, Sun JD, Birnbaum LS, Yin SN, Li GL, Kasicki S, Lucier G, & Henderson RF (1992) S-Phenylcysteine formation in haemoglobin as a biological exposure index to benzene. Arch Toxicol, 66: 303-309. Beliles RP & Totman LC (1989) Pharmacokinetically based risk assessment of workplace exposure to benzene. Regul Toxicol Pharmacol, 9: 186-195. Bennett GF (1989) Impact of toxic chemicals on local wastewater treatment plant and the environment. Environ Geol Water Sci, 13(3): 201-212. Bentley P, Sschassmann H, Sims P, & Oesch F (1976) Epoxides derived from various polycyclic hydrocarbons as substrates of homogeneous and microsome-bound epoxide hydratase. A general assay and kinetic properties. Eur J Biochem, 69: 97-103. Blanchard RD & Hardy JK (1986) Continuous monitoring device for the collection of 23 volatile organic priority pollutants. Anal Chem, 58: 1529-1532. Blank IH & McAuliffe DJ (1985) Penetration of benzene through human skin. J Invest Dermatol, 85: 522-526. Bois FY, Woodruff TJ, & Spear RC (1991a) Comparison of three physiologically based pharmacokinetic models of benzene disposition. Toxicol Appl Pharmacol, 110: 79-88. Bois FY, Smith MT, & Spear RC (1991b) Mechanisms of benzene carcinogenesis: application of a physiological model of benzene pharmacokinetics and metabolism. Toxicol Lett, 56: 283-298. Bond AE, Thompson VL, & Ortman GC (1986) Self service station vehicle refueling exposure study. In: Proceedings of the 1986 EPA/APCA Symposium on Measurement of Toxic Air Pollutants. Pittsburgh, Pennsylvania, Air Pollution Control Association, pp 458-466. Bond GG, McLaren EA, Baldwin CL, & Cook RR (1986) An update of mortality among chemical workers exposed to benzene. Br J Ind Med, 43: 685-691. Bradley MO (1985) Measurement of DNA single-strand breaks by alkaline elution in rat hepatocytes. In: Ashby J, de Serres FJ, Draper M, Ishidate M Jr, Margolin BH, Matter BE, & Shelby MD ed. Evaluation of short-term tests for carcinogens: Report of the International Programme on Chemical Safety's collaborative study on in vitro assays. Amsterdam, Oxford, New York, Elsevier Science Publishers, pp 353-357 (Progress in Mutation Research, Volume 5). Brodfuehrer JI, Chapman DE, Wilke TJ, & Powis G (1990) Comparative studies of the in vitro metabolism and covalent binding of 14C-benzene by liver slices and microsomal fraction of mouse, rat and human. Drug Metab Dispos, 18: 20-27. Brown, RH (1988a) Determination of benzene, toluene, and xylene in industrial air by charcoal tube, solvent desorption and gas chromatography. In: Fishbein L & O'Neill JK ed. Environmental carcinogens method of analysis and exposure measurement - Volume 10: Benzene and alkylated benzenes. New York, London, Oxford University Press, pp 225-233. Brown RH (1988b) Determination of benzene, toluene, and xylene in industrial air by porous polymer adsorption tube, thermal desorption and gas chromatography. In: Fishbein L & O'Neill JK ed. Environmental carcinogens method of analysis and exposure measurement - Volume 10: Benzene and alkylated benzenes. New York, London, Oxford University Press, pp 235-242. Bruckmann P, Beier R, & Krautscheid S (1983) [Measurements of emissions of hydrocarbons in areas of high exposure in the Rhine-Ruhr district.] Staub-Reinhalt-Luft, 43: 404 (in German). Bruckmann P, Kersten W, Funcke W, Balfanz E, König J, Theisen J, Ball M, & Päpke O (1988) The occurrence of chlorinated and other organic trace compounds in urban air. Chemosphere, 17(12): 2363-2380. Brunnemann KD, Kagan MR, Cox JE, & Hoffmann D (1989) Determination of benzene, toluene and 1,3-butadiene in cigarette smoke by GC-MSD. Exp Pathol, 37: 108-113. Brunnemann KD, Kagan MR, Cox JE, & Hoffmann D (1990) Analysis of 1,3-butadiene and other selected gas-phase components in cigarette mainstream and sidestream smoke by gas chromatography - mass selective detection. Carcinogenesis, 11(10): 1863-1868. Bryce-Smith D & Gilbert A (1976) The organic photochemistry of benzene. I. Tetrahedron, 32: 1309-1326. Buchet JP (1988) Determination of phenol and its glucurono- and sulfoconjugates in urine by gas chromatography. In: Fishbein L & O'Neill JK ed. Environmental carcinogens methods of analysis and exposure measurement - Volume 10: Benzene and alkylated benzenes. New York, London, Oxford University Press, pp 281-286. Burmaster DE (1982) The new pollution. Groundwater contamination. Environment, 24: 33-36. Busby WF Jr, Wang J-S, Stevens EK, Padykula RE, Aleksejczyk RA, & Berchtold GA (1990) Lung tumourigenicity of benzene oxide, benzene dihydrodiols and diolepoxides in the BLV: Ha newborn mouse assay. Carcinogenesis, 11: 1473-1478. Carpenter C, Shaffer C, Weil C, & Smyth H (1944) Studies on the inhalation of 1,3-butadiene; with comparison of its narcotic effect with benzol, toluol, and styrene and a note on the elimination of styrene by the human. J Ind Hyg Toxicol, 26: 69-78. CEC (1993) Occupational exposure limits - Criteria document for benzene. Luxembourg, Commission of the European Communities, Directorate-General Employment, Industrial Relations and Social Affairs (EUR 14491 EN) (Health and Safety Series). Chang SS & Peterson RJ (1977) Symposium: The basis of quality in muscle foods. Recent developments in the flavor of meat. J Food Sci, 42: 298-306. Chepiga TA, Yang CS, & Snyder R (1991) Benzene metabolism in two purified, reconstituted rat hepatic mixed function oxidase systems. In: Witmer CM, Snyder R, Jollow DJ, Kalf GF, Kocsis JJ, & Sipes IG ed. Biological reactive intermediates: IV. Molecular and cellular effects and their impact on human health. New York, London, Plenum Press, pp 261-265. Choy WN, MacGregor JT, & Shelby MD (1985) Induction of micronuclei by benzene in B6C3F1 mice: Retrospective analysis of peripheral blood smears from the NTP carcinogenesis bioassay. Mutat Res, 143: 55-59. Ciranni R, Barale R, Marrazzini A, & Loprieno N (1988) Benzene and the genotoxicity of its metabolites. I. Transplacental activity in mouse fetuses and in their dams. Mutat Res, 208: 61-67. Clark AI, McIntyre AE, Lester JN, & Perry R (1984a) Ambient air measurements of aromatic and halogenated hydrocarbons at urban, rural and motorway locations. Sci Total Environ, 39: 265-279. Clark AI, McIntyre AE, & Lester JN (1984b) A comparison of photoionization detection gas chromatography with a Tenax GC sampling tube procedure for the measurement of aromatic hydrocarbons in ambient air. J Environ Anal Chem, 17: 315-326. Coate WB, Hoberman AM, & Durloo RS (1984) Inhalation teratology study of benzene in rats. Adv Mod Environ Toxicol, 6: 187-198. Colenutt BA & Thornburn S (1980) Gas-chromatographic analysis of trace hydrocarbon pollutants in water samples. Int J Environ Stud, 15: 25-32. Collins JJ, Conner P, Friedlander BR, Easterday PA, Nair RS, Rashmi S, & Braun J (1991) A study of the hematological effects of chronic low-level exposure to benzene. J Occup Med, 33(5): 619-626. CONCAWE (1986) Review of European oil industry benzene exposure data. The Hague, European Oil Companies Organization for Environmental and Health Protection (Report No. 3/86). Cornish HH & Ryan RC (1965) Metabolism of benzene in non-fasted, fasted, and arylhydroxylase inhibited rats. Toxicol Appl Pharmacol, 7: 767-771. Cronkite EP (1986) Benzene hematotoxicity and leukemogenesis. Blood Cells, 12: 129-137. Cronkite EP, Bullis J, Inoue T, & Drew RT (1984) Benzene inhalation produces leukemia in mice. Toxicol Appl Pharmacol, 75: 358. Cronkite EP, Drew RT, Inoue T, & Bullis JE (1985) Benzene hematotoxicity and leukemogenesis. Am J Ind Med, 7: 447-456. Cronkite EP, Drew RT, Inoue T, Hirabayashi Y, & Bullis JE (1989) Hematotoxicity and carcinogenicity of inhaled benzene. Environ Health Perspect, 82: 97-108. Crump K & Allen B (1984) Quantitative estimates of risk of leukemia from occupational exposure to benzene. Washington, DC, US Department of Labour (OSHA Docket H-059b, Exhibit 152, Annex B). Dann T (1987) Ambient benzene levels in urban areas of Canada. Report of the Technology Development and Technical Services Branch, Conservation and Protection. Ottawa, Environment Canada. Dannecker W, Schröder B, & Stechmann H (1990) Organic and inorganic substances in highway tunnel exhaust air. Sci Total Environ, 93: 293-300. Da Silva C, Fan X, & Castagna M (1989) Benzene-mediated protein kinase c activation. Environ Health Perspect, 82: 91-95. Dean BJ (1978) Genetic toxicology of benzene, toluene, xylenes and phenols. Mutat Res, 47: 75-97. Dean BJ (1985a) Recent findings on the genetic toxicology of benzene, toluene, xylenes and phenols. Mutat Res, 154: 153-181. Dean BJ (1985b) Summary report on the performance of cytogenetic assays in cultured mammalian cells. In: Ashby J, de Serres FJ, Draper M, Ishidate M Jr, Margolin BH, Matter BE, & Shelby MD ed. Evaluation of short-term tests for carcinogens. Report of the International Programme on Chemical Safety's collaborative study on in vitro assays. Amsterdam, Oxford, New York, Elsevier Science Publishers, pp 69-83 (Progress in Mutation Research, Volume 5). Decouflé P, Blattner WA, & Blair A (1983) Mortality among chemical workers exposed to benzene and other agents. Environ. Res., 30: 16-25. De Flora S, Zanacchi P, Camoirano A, Bennicelli C, & Badolati GS (1984) Genotoxic activity and potency of 135 compounds in the Ames reversion test and in a bacterial DNA-repair test. Mutat Res, 133: 161-198. Deichmann WB, MacDonald WE, & Bernal E (1963) The hemopoietic tissue toxicity of benzene vapors. Toxicol Appl Pharmacol, 5: 201-224. Dibben MJ, Thomas TC, & Anderson MP (1989) Is benzene a problem in fuel operations: A new analytical method to overcome potential interferences in the present methods. Liq Chromatogr-Gas Chromatogr Int, 2: 54, 56, 58. Dorfman LM, Taub IA, & Buhler RE (1962) Pulse radiolysis studies. I. Transient spectra and reaction-rate constants in irradiated aqueous solutions of benzene. J Chem Phys, 36: 3051-3061. Douglas GR, Blakely DH, Liu-Lee VW, Bell RDL, & Bayley JM (1985) Alkaline sucrose sedimentation, sister-chromatid exchange and micronucleus assays in CHO cells. In: Ashby J, de Serres FJ, Draper M, Ishidate M Jr, Margolin BH, Matter BE, & Shelby MD ed. Evaluation of short-term tests for carcinogens: Report of the International Programme on Chemical Safety's collaborative study on in vitro assays. Amsterdam, Oxford, New York, Elsevier Science Publishers, pp 359-366 (Progress in Mutation Research, Volume 5). Dowty BJ, Laseter JL, & Storer J (1976) The transplacental migration and accumulation in blood of volatile organic constituents. Pediatr Res, 10: 696-701. Drew RT & Fouts JR (1974) The lack of effects of pretreatment with phenobarbital and chlorpromazine on the acute toxicity of benzene in rats. Toxicol Appl Pharmacol, 27: 183-193. Eastmond DA, Smith MT, & Irons RD (1987) An interaction of benzene metabolites reproduces the myelotoxicity observed with benzene exposure. Toxicol Appl Pharmacol, 91: 85-95. Eisenreich SJ, Looney BB, & Thornton JD (1981) Airborne organic contaminants in the Great Lakes ecosystem. Environ Sci Technol, 15: 30-38. Environment Agency, Japan (1989) Chemicals in the environment. Report on environmental survey and wildlife monitoring of chemicals in F.Y. 1986 and 1987. Tokyo, Environment Agency, Department of Environmental Health, Office of Health Studies. Erexson GL, Wilmer JL, & Kligerman AD (1985) Sister chromatid exchange in human lymphocytes exposed to benzene and its metabolites in vitro. Cancer Res, 45: 2471-2477. Erexson GL, Wilmer JL, Steinhagen WH, & Kligerman AD (1986) Induction of cytogenetic damage in rodents after short-term inhalation of benzene. Environ Mutagen, 8: 29-40. Erf LA & Rhoads CP (1939) The hematological effects of benzene (benzol) poisoning. J Ind Hyg Toxicol, 20(8): 421. Ewing BB, Chain ESK, Cook JC, Evans CA, Hopke PK, & Perkins EG (1977) Monitoring to detect previously unrecognized pollutants in surface waters. Washington, DC, US Environmental Protection Agency (EPA-560/6/77/015A). Fel'dt EG (1985) [Evaluation of the mutagenic hazards of benzene and some of its derivatives.] Gig i Sanit, 7: 21-23 (in Russian with English summary). Fentiman AF, Neher MB, Kinzer GW, Sticksel PR, & Coutant RW (1979) Environmental monitoring benzene. Washington, DC, US Environmental Protection Agency, Office of Toxic Substances (EPA-560/679/006) (Prepared by Battelle Columbus for US EPA). Ferrario JB, Lawler GC, Delon IR, & Laseter JL (1985) Volatile organic pollutants in biota and sediments of Lake Pontchartrain. Bull Environ Contam Toxicol, 34(2): 246-255. Fishbein L (1984) An overview of environmental and toxicological aspects of aromatic hydrocarbons. I. Benzene. Sci Total Environ, 40: 189-218. Forni A, Pacifico E, & Limonta A (1971a) Chromosome studies in workers exposed to benzene or toluene or both. Arch Environ Health, 22: 373-378. Forni AM, Cappellini A, Pacifico E, & Vigliani EC (1971b) Chromosome changes and their evolution in subjects with past exposure to benzene. Arch Environ Health, 23: 385-391. Franz TJ (1984) Percutaneous absorption of benzene. In: Applied toxicology of hydrocarbons. Princeton, New Jersey, Princeton Scientific Publishers, Inc., pp 61-70 (Advances in Modern Environmental Toxicology, Volume VI). Fung KK & Wright BJ (1986) Monitoring of benzene in ambient air with organic vapor badges. J Air Pollut Control Assoc, 36: 819-821. Furnas DW & Hine CH (1958) Neurotoxicity of some selected hydrocarbons. Arch Ind Health, 18: 9-15. Gad-El-Karim MM, Harper BL, & Legator MS (1984) Modifications in the myeloclastogenic effect of benzene in mice with toluene, phenobarbital, 3-methylcholanthrene, Aroclor 1254 and SKF-525A. Mutat Res, 135: 225-243. Galton DAG (1986) The myelodysplastic syndromes. Scand J Haematol, 36: 11-20. Garner RC (1985) Summary report on the performance of gene mutation assays in mammalian cells in culture. In: Ashby J, de Serres FJ, Draper M, Ishidate M Jr, Margolin BH, Matter BE, & Shelby MD ed. Evaluation of short-term tests for carcinogens. Report of the International Programme on Chemical Safety's collaborative study on in vitro assays. Amsterdam, Oxford, New York, Elsevier Science Publishers, pp 85-94 (Progress in Mutation Research, Volume 5). GDCh (Society of German Chemists) (1988) [Advisory Committee on Existing Chemicals of Environmental Relevance. Benzene.] Weinheim, VCH Verlagsgesellschaft, 82 pp (BUA Report 24) (in German with English summary). Geist CR, Kelly LD, Schoenheit CM, & Praed JE (1983) Learning impairments following postnatal exposures to benzene. Percept Motor Skills, 57: 1083-1086. Gerarde HW (1960) Toxicology and biochemistry of aromatic hydrocarbons. Amsterdam, Oxford, New York, Elsevier Science Publishers. Gerner-Smidt P & Friedrich U (1978) The mutagenic effect of benzene, toluene and xylene studied by the SCE technique. Mutat Res, 58: 313-316. Geyer H, Politzki G, & Freitag D (1984) Prediction of ecotoxicological behaviour of chemicals: relationship between n-octanol/water partition coefficient and bioaccumulation of organic chemicals by the alga Chlorella. Chemosphere, 13: 269-284. Ghantous H & Danielsson BRG (1986) Placental transfer and distribution of toluene, xylene, and benzene, and their metabolites during gestation in mice. Biol Res Pregnancy, 7: 98-105. Gilman AG, Goodman LA, & Gilman A (1985) The Goodman and Gilman's pharmacological basis of therapeutics, 7th ed. New York, MacMillan Publishing Co. Gilmour SK, Kalf GF, & Snyder R (1986) Comparison of the metabolism of benzene and its metabolite phenol in rat liver microsomes. In: Kocsis JJ, Jollow DJ, Witmer CM, Nelson J, & Snyder R ed. Biological reactive intermediates. III - Mechanisms of action in animal models and human disease. New York, Plenum Publishing Co., pp 223-235. Glatt H, Padykula R, Berchtold GA, Ludewig G, Platt KL, Klein J, & Oesch F (1989) Multiple activation pathways of benzene leading to products with varying genotoxic characteristics. Environ Health Perspect, 82: 81-89. Gofmekler VA (1968) [The embryotropic action of benzene and formaldehyde in experimental administration by inhalation.] Gig i Sanit, 33: 12-16 (in Russian with English summary). Goldstein BD (1988) Benzene toxicity: occupational medicine. State Art Rev, 3: 541-554. Goldstein BD, Snyder CA, Laskin S, Brombert I, Albert RE, & Nelson N (1982) Myelogenous leukemia in rodents inhaling benzene. Toxicol Lett, 13: 169-173. Goldwater LJ (1941) Disturbances in the blood following exposure to benzol. J Lab Clin Med, 26: 957. Gollmer L, Graf H, & Ulrich V (1984) Characterization of the benzene monooxygenase system in rabbit bone marrow. Biochem Pharmacol, 33: 3597-3602. Goodenkaug O & Atkinson JC (1986) Occurrence of volatile organic chemicals in Nebraska ground water. Ground Water, 24(2): 231-233. Gorsky LD & Coon MJ (1985) Evaluation of the role of free hydroxyl radicals in the cytochrome P-450 catalyzed oxidation of benzene in microsomes and reconstituted enzyme systems from rabbit liver. J Biol Chem, 258: 7311. Gossett RW, Brown DA, & Young DR (1983) Predicting the bioaccumulation of organic compounds in marine organisms using octanol/water partition coefficients. Mar Pollut Bull, 14: 387-392. Government of Canada (in press) Canadian environmental protection act - Priority assessment report on benzene. Ottawa, Environment Canada/Health and Welfare Canada. Green JD, Leong BKJ, Lobue J, Goldstein BD, & Albert RE (1978) Inhaled benzene fetotoxicity in rats. Toxicol Appl Pharmacol, 46: 9-18. Green JD, Snyder CA, Lobue J, Goldstein BD, & Albert RE (1981a) Acute and chronic dose/response effect of benzene inhalation on the peripheral blood, bone marrow, and spleen cells of CD-1 male mice. Toxicol Appl Pharmacol, 59: 204-214. Green JD, Snyder CA, Lobue J, Goldstein BD, & Albert RE (1981b) Acute and chronic dose/response effects of inhaled benzene on multipotential hematopoietic stem (CFU-S) and granulocyte/macrophase progenitor (GM-CFU-C) cells in CD-1 mice. Toxicol Appl Pharmacol, 58: 492-503. Grob K & Grob G (1974) Organic substances in potable water and its precursor. Part II. Applications in the area of Zurich. J Chromatogr, 90: 303-313. Gruenke LD, Craig JC, Wester RC, & Maibach HI (1986) Quantitative analysis of benzene by selected ion monitoring/gas chromatography/mass spectrometry. J Anal Toxicol, 10(11/12): 225-232. Hadeishi T, Pollard M, McLaughlin R, & Koga M (1985) Development of an optical monitor for toxic organic compounds in air. Research Triangle Park, North Carolina, US Environmental Protection Agency (EPA/600/4-85-043). Hall LW Jr, Hall WS, Bushong SJ, & Herman RL (1987) In situ striped bass (Morone saxatilis) contaminant and water quality studies in the Potomac River. Aquat Toxicol, 10: 73-99. Hammers WE & Bosman HFPM (1986) Quantitative evaluation of a simple dynamic headspace analysis technique for non-polar pollutants in aqueous samples at the ng/kg level. J Chromatogr, 360: 425-432. Hancock DG, Moffitt AE Jr, & Hay EB (1984) Hematological findings among workers exposed to benzene at a coke oven by-product recovery facility. Arch Environ Health, 39: 414-418. Hanke J, Dutkiewicz T, & Piotrowski J (1961) [The absorption of benzene through the skin in men.] Med Pracy, 12: 413-426 (in Polish). Harkov R, Gianti SJ, Bozzelli JW, & Laregina JE (1985) Monitoring volatile organic compounds at hazardous and sanitary landfills in New Jersey. J Environ Sci Health, A20(5): 491-501. Harland BJ, Whitby FJ, & Comber MHI (1985) Measurement of volatile aromatic hydrocarbons in an industrial estuary. Int J Environ Anal Chem, 20: 295-311. Hattemer-Frey HA, Travis CC, & Land ML (1990) Environmental partitioning and human exposure. Environ Res, 53: 221-232. Henderson RF, Sabourin PJ, Bechtold WE, Griffith WC, Medinsky MA, Birnbaum LS, & Lucier GW (1989) The effect of dose, dose rate, route of administration, and species on tissue and blood levels of benzene metabolites. Environ Health Perspect, 82: 9-17. Hine J & Mookerjee PK (1975) The intrinsic hydrophilic character of organic compounds. Correlations in terms of structural contributions. J Org Chem, 40: 292-298. Hinson JA, Freeman JP, Potter DW, Mitchum RK, & Evans FE (1985) Mechanism of microsomal metabolism of benzene to phenol. Mol Pharmacol, 27: 574-577. Hite M, Pecharo M, Smith I, & Thornton S (1980) The effect of benzene in the micronucleus test. Mutat Res, 77: 149-155. Högn T & Jaenicke L (1972) Benzene metabolism in Moraxella species. Eur J Biochem, 30: 369-375. Holdren MW, Smith DL, & Smith RN (1985) Comparison of ambient air sampling techniques for volatile organic compounds. Washington, DC, US Environmental Protection Agency (EPA/600/S4-85-067; PB86-120953/GAR). Hornung RW, Ward E, Morris JA, & Rinsky RA (1989) Letter to the editor. Haematologic effects of benzene: a thirty-five year longitudinal study of rubber workers. Toxicol Ind Health, 5(6): 1153-1155. HSE (1982) Toxicity review No. 4: Benzene. London, Health and Safety Executive, Her Majesty's Stationery Office, 36 pp. Hsieh GC, Sharma RP, & Parker RDR (1988a) Subclinical effects of groundwater contaminants. I. Alteration of humoral and cellular immunity by benzene in CD-1 mice. Arch Environ Contam Toxicol, 17: 151-158. Hsieh GC, Parker RDR, & Sharma RP (1988b) Subclinical effects of groundwater contaminants. II. Alteration of regional brain monoamine neurotransmitters by benzene in CD-1 mice. Arch Environ Contam Toxicol, 17: 799-805. Hudak A & Ungvary G (1978) Embryotoxic effects of benzene and its methyl derivatives: toluene and xylene. Toxicology, 11: 55-63. Huff JE, Haseman JK, Demarini DM, Eustis S, Maronpot RR, Peters AC, Persing RL, Chrisp CE, & Jacobs AC (1989) Multiple-site carcinogenicity of benzene in Fischer 344 rats and B6C3F1 mice. Environ Health Perspect, 82: 125-163. Hurley JF, Cherrie JW, & MacLaren W (1991) Exposure to benzene and mortality from leukaemia: results from coke oven and other coal product workers. Br J Ind Med, 48: 502-504. IARC (1982) Benzene. In: Some industrial chemicals and dyestuffs. Lyon, International Agency for Research on Cancer, pp 93-148 (Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Volume 29). IARC (1987a) Benzene. In: Genetic and related effects: an updating of selected IARC Monographs from Volumes 1 to 42. Lyon, International Agency for Research on Cancer, pp 91-95 (IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Supplement 6). IARC (1987b) Formaldehyde. In: Overall evaluations of carcinogenicity: An updating of IARC Monographs Volumes 1 to 42. Lyon, International Agency for Research on Cancer, pp 211-216 (IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Supplement 7). IARC (1989) Occupational exposures in petroleum refining; crude oil and major petroleum fuels. Lyon, International Agency for Research on Cancer, 322 pp (IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume 45). Ikeda M & Ohtsuji H (1971) Phenobarbital-induced protection against toxicity of toluene and benzene in the rat. Toxicol Appl Pharmacol, 20: 30-43. Infante PF, Wagoner JK, Rinsky RA, & Young RJ (1977) Leukaemia in benzene workers. Lancet, 2: 76-78. Inoue O, Seiji K, & Nakatsuka H (1989) Urinary t,t-muconic acid as an indicator of exposure to benzene. Br J Ind Med, 46: 122-127. Irons RD (1985) Quinones as toxic metabolites of benzene. J Toxicol Environ Health, 16: 673-678. Irons RD, Dent JG, Baker TS, & Rickert DE (1980) Benzene is metabolized and covalently bound in bone marrow in situ. Chem-Biol Interact, 30: 241-245. Irons RD, Neptun DA, & Pfeifer RW (1981) Inhibition of lymphocyte transformation and microtubule assembly by quinone metabolites of benzene: Evidence for a common mechanism. J Reticuloendothel Soc, 30: 359-372. IRPTC (1987) IRPTC Legal file 1986 on benzene - Volume 1. Geneva, International Register of Potentially Toxic Chemicals, United Nations Environment Programme. Jackson S & Brown VM (1970) Effect of toxic wastes on treatment processes and watercourses. Water Pollut Control, 69(3): 292-313. Jerina DM & Daly JW (1974) Arene oxides: a new aspect of drug metabolism. Science, 185: 573-582. Jerina D, Daly J, Witkop B, Zaltzman-Norenberg P, & Udenfriend S (1968) Role of the arene oxide-oxepin system in the metabolism of aromatic substrates. I. In vitro conversion of benzene oxide to a premercapturic acid and a dihydrodiol. Arch Biochem Biophys, 128: 176-183. Jirka AM & Bourne S (1982) Gas-chromatographic analysis for benzene in blood. Clin Chem, 28: 1492-1494. Johansson I & Ingelman-Sundberg M (1988) Benzene metabolism by ethanol-, acetone-, and benzene-inducible cytochrome P-450(IIE1) in rat and rabbit liver microsomes. Cancer Res, 48: 5387-5390. Johnston RV, Pinkerton MN, & Mensik DC (1979) Haematological and myelogenous effects of inhaled benzene in the pig and rat. J Toxicol Environ Health, 5: 1025-1035. Jonek J, Olkowski Z, & Zieleznik B (1965) Histochemical studies on the spinal cord of mice poisoned with benzene. Acta Histochem, 20: 286-296. Jonsson A, Persson KA, & Grigoriadis V (1985) Measurements of some low molecular-weight oxygenated, aromatic and chlorinated hydrocarbons in ambient air and in vehicle emissions. Environ Int, 11: 383-392. Jowa L, Witz G, Snyder R, Winkle S, & Kalf GF (1990) Synthesis and characterization of deoxyguanosine-benzoquinone adduct. J Appl Toxicol, 10(1): 47-54. Kalf GF (1987) Recent advances in the metabolism and toxicity of benzene. CRC Crit Rev Toxicol, 18: 141-159. Kalf GF, Schlosser MJ, Renz JF, & Priozzi SJ (1989) Prevention of benzene-induced myelotoxicity by nonsteroidal anti-inflammatory drugs. Environ Health Perspect, 82: 57-64. Karam LR & Simic MG (1989) Mechanisms of free radical chemistry and biochemistry of benzene. Environ Health Perspect, 82: 37-41. Karickhoff SW (1981) Semi-empirical estimation of sorption of hydrophobic pollutants on natural sediments and soils. Chemosphere, 10(8): 833-846. Karlson U & Frankenberger WT Jr (1989) Microbial degradation of benzene and toluene in groundwater. Bull Environ Contam Toxicol, 43: 505-510. Keller KA & Snyder CA (1986) Mice exposed in utero to low concentrations of benzene exhibit enduring changes in their colony forming hematopoietic cells. Toxicology, 42: 171. Kenaga EE (1980) Predicted bioconcentration factors and soil sorption coefficients of pesticides and other chemicals. Ecotoxicol Environ Saf, 4: 26-38. Kiang PH & Grob RL (1986) A headspace technique for the determination of volatile compounds in soil. J Environ Sci Health, A21: 71-100. Kimmel CA & Wilson JG (1973) Skeletal deviations in rats: malformations or variations? Teratology, 8: 309-316. Kimura ET, Ebert DM, & Dodge PW (1971) Acute toxicity and limits of solvent residue for sixteen organic solvents. Toxicol Appl Pharmacol, 19: 699-704. Kipen HM, Cody RP, Crump KS, Allen BC, & Goldstein BD (1988) Hematologic effects of benzene: a thirty-five year longitudinal study of rubber workers. Toxicol Ind Health, 4(4): 411. Kipen HM, Cody RP, & Goldstein BD (1989) Use of longitudinal analysis of peripheral blood counts to validate historical reconstructions of benzene exposure. Environ Health Perspect, 82: 199-206. Kirley TA, Goldstein BD, Maniara WM, & Witz G (1989) Metabolism of trans,trans-muconaldehyde, a microsomal hematotoxic metabolite of benzene by purified yeast aldehyde dehydrogenase and a mouse liver soluble fraction. Toxicol Appl Pharmacol, 100: 360-367. Kissling M & Speck B (1972) Further studies on experimental benzene induced aplastic anemia. Blut, 25: 97-103. Kocsis JJ, Harkaway S, Santoyo MC, & Snyder R (1968) Dimethyl sulfoxide: interactions with aromatic hydrocarbons. Science, 160: 427-428. Koljkowsky P (1981) Indicator-tube method for the determination of benzene in air. Analyst, 94: 918-920. Korte F & Klein W (1982) Degradation of benzene in the environment. Ecotoxicol Environ Saf, 6(4): 311-327. Kozioski RP (1985) Determination of benzene and toluene in soils and plant material by azeotropic distillation. Bull Environ Contam Toxicol, 34: 10-16. Kuna RA & Kapp RW (1981) Embyrotoxic/teratogenic potential of benzene vapor in rats. Toxicol Appl Pharmacol, 57: 1-7. Lahmann E, Seifert B, & Ullrich D (1977) The pollution of ambient air and rain water by organic components of motor vehicle exhaust-gases. In: Kasuga S ed. Proceedings of the 4th International Clean Air Congress. Tokyo, Japanese Union of Air Pollution and Prevention Association, pp 595-597. Lange A, Smolik R, Zatonski W, & Szymanska J (1973a) Leukocyte agglutinins in workers exposed to benzene, toluene and xylene. Int Arch Arbeitsmed, 31: 45-50. Lange A, Smolik R, Zatonski W, & Szymanska J (1973b) Serum immunoglobulin levels in workers exposed to benzene, toluene and xylene. Int Arch Arbeitsmed, 31: 37-44. Latriano L, Goldstein BD, & Witz G (1986) Formation of muconaldehyde, an open-ring metabolite of benzene, in mouse liver microsomes: an additional pathway for toxic metabolites. Proc Natl Acad Sci (USA), 83: 8356-8360. Leong B (1977) Experimental benzene intoxication. In: Laskin S & Goldstein B ed. Benzene toxicity: A critical revaluation. J Toxicol Environ Health, 3(2): 45-61. Li G-L, Yin N, Watanabe T, Nakatsuka H, Kasahara M, Abe H, & Ikeda M (1986) Benzene-specific increase in leukocyte alkaline phosphatase activity in rats exposed to vapors of various organic solvents. J Toxicol Environ Health, 19: 581. Longacre S, Kocsis J, & Snyder R (1981a) Influence of strain differences in mice on the metabolism and toxicity of benzene. Toxicol Appl Pharmacol, 60: 398-409. Longacre S, Kocsis J, Witmer CM, Lee EW, Sammett D, & Snyder R (1981b) Toxicological and biochemical effects of repeated administration of benzene in mice. J Toxicol Environ Health, 7: 223-237. Low LK, Meeks JR, Norris KJ, Mehlman MA, & Macherer CR (1989) Pharmacokinetics and metabolism of benzene in Zymbal gland and other key target tissues after oral administration in rats. Environ Health Perspect, 82: 215-222. Low LK, Reddy MV, Blackburn GR, & Mackerer CR (1991) Benzene mechanistic research. Final report of collaborative study by Mobil, Amoco Ashland, Standard Oil and Sun Company. Princeton, New Jersey, Mobil Oil Corporation, 76 pp. Lunte SM & Kissinger PT (1983) Detections and identification of sulfhydryl conjugates of p-benzoquinone in microsomal incubations of benzene and phenol. Chem-Biol Interact, 47: 195-212. Lutz WK (1979) In vivo covalent binding of organic chemicals to DNA as a quantitative indicator in the process of chemical carcinogenesis. Mutat Res, 65: 289-356. Lutz WK & Schlatter C (1977) Mechanisms of the carcinogenic action of benzene: irreversible binding to rat liver DNA. Chem-Biol Interact, 18(2): 241-246. McDonald TJ, Kennicutt MC II, & Brooks JM (1988) Volatile organic compounds at a coastal Gulf of Mexico site. Chemosphere, 17(1): 123-136. MacKay D & Leinonen PJ (1975) Rate of evaporation of low-solubility contaminants from water bodies to atmosphere. Environ Sci Technol, 9: 1178-1180. Maibach HI & Anjo DM (1981) Percutaneous penetration of benzene and benzene contained in solvents in the rubber industry. Arch Environ Health, 36: 256-260. Maltoni C, Ciliberti A, & Carretti D (1982a) Experimental contributions in identifying brain potential carcinogens in the petrochemical industry. Ann NY Acad Sci, 381: 216-249. Maltoni C, Conti B, & Scarnato C (1982b) Squamous cell carcinomas of the oral cavity in Sprague-Dawley rats following exposure to benzene by ingestion. Med. Lav, 4: 441-445. Maltoni C, Cotti G, Valgimigli L, & Mandrioli A (1982c) Hepatocarcinomas in Sprague-Dawley rats following exposure to benzene by inhalation. Med Lav, 4: 446-450. Maltoni C, Conti B, & Cotti G (1983) Benzene: a multipotential carcinogen. Results of long-term bioassays performed at the Bologna Institute of Oncology. Am J Ind Med, 4: 589-630. Maltoni C, Conti B, Cotti G, & Belpoggi F (1985) Experimental studies on benzene carcinogenicity at the Bologna Institute of Oncology: current results and ongoing research. Am J Ind Med, 7: 415-446. Maltoni C, Ciliberti A, Cotti G, Conti B, & Belpoggi F (1989) Benzene, an experimental multipotential carcinogen: results of the long-term bioassays performed at the Bologna Institute of Oncology. Environ Health Perspect, 82: 109-124. Mara SJ & Lee SS (1978) Assessment of human exposures to atmospheric benzene. Research Triangle Park, North Carolina, US Environmental Protection Agency (EPA/450/378-031). Marcus WL (1990) Chemical of current interest: benzene. Cancer risk from benzene. Adv Mod Environ Toxicol, 17: 127-188. Medinsky MA, Sabourin PJ, Henderson RF, Lucier GW, & Birnbaum LS (1989a) Differences in the pathways for metabolism of benzene in rats and mice simulated by a physiological model. Environ Health Perspect, 82: 43-49. Medinsky MA, Sabourin PJ, Lucier G, Birnbaum LS, & Henderson RF (1989b) A toxicological model for simulation of benzene metabolism. Exp Pathol, 37: 150-154. Medinsky MA, Sabourin PJ, Henderson RF, Lucier GW, & Birnbaum LS (1989c) A physiological model for simulation of benzene metabolism by rats and mice. Toxicol Appl Pharmacol, 99: 193-206. Merian E & Zander M (1982) Volatile aromatics. In: Hutzinger O ed. The handbook of environmental chemistry, vol 3, Part B: Anthropogenic compounds. Berlin, Heidelberg, New York, Springer-Verlag, pp 117-161. Michael LC, Pellizzari ED, & Wiseman RW (1988) Development and evaluation of a procedure for determining volatile organics in water. Environ Sci Technol, 22: 565-570. Miller MM & Wasik SP (1985) Relationships between octanol-water partition coefficient and aqueous solubility. Environ Sci Technol, 19(6): 522-529. Morimoto K (1976) Analysis of combined effects of benzene with radiation on chromosomes in cultured human leukocytes. Jpn J Ind Health, 18: 23-24. Morimoto K (1983) Induction of sister chromatid exchanges and cell division delays in human lymphocytes by microsomal activation of benzene. Cancer Res, 43: 1330-1334. Morimoto K, Wolff S, & Koizumi A (1983) Induction of sister-chromatid exchanges in human lymphocytes by microsomal activation of benzene metabolites. Mutat Res, 119: 355-360. Moszczynski P (1981) Organic solvents and T-lymphocytes. Lancet, 1: 438. Murray FJ, John JA, Rampy LW, Kuna RA, & Schweta BA (1979) Embryotoxicity of inhaled benzene in mice and rabbits. Am Ind Hyg Assoc J, 40: 993-998. Nahum LH & Hoff HE (1934) The mechanism of sudden death in experimental benzol poisoning. J Pharmacol Exp Ther, 50: 336. Nakajima T, Okuyama S, & Yonekura I (1985) Effects of ethanol and phenobarbital administration on the metabolism and toxicity of benzene. Chem-Biol Interact, 55: 23-38. Nakajima T, Elovaara E, Park SS, Gelbain HV, Hietanen E, & Vainio H (1990) Monoclonal antibody-directed characterization of benzene, ethoxy-resorufin and pentoxy-resorufin metabolism in rat liver microsomes. Biochem Pharmacol, 40: 1255-1261. Nau CA, Neal J, & Thornton M (1966) C9-C12 fractions obtained from petroleum distillates. Arch Environ Health, 12: 382-393. Nawrot PS & Staples RE (1979) Embryo-fetal toxicity and teratogenicity of benzene and toluene in the mouse. Teratology, 19: 41A. Nerland DE & Pierce WM (1990) Identification of N-acetyl- S-(2,5-dihydroxyphenyl)-L-cysteine as a urinary metabolite of benzene, phenol and hydroquinone. Drug Metab Dispos, 18: 958-961. Nomiyama K & Nomiyama H (1974a) Respiratory retention, uptake and excretion of organic solvents in man. Benzene, toluene, n-hexane, trichloroethylene, acetone, ethyl acetate and ethyl alcohol. Int Arch Arbeitsmed, 32: 75-83. Nomiyama K & Nomiyama H (1974b) Respiratory elimination of organic solvents in man. Benzene, toluene, n-hexane, trichloroethylene, acetone, ethyl acetate and ethyl alcohol. Int Arch Arbeitsmed, 32: 85-91. Nordlinder R & Ramnäs O (1987) Exposure to benzene at different work places in Sweden. Ann Occup Hyg, 31: 345-355. Norpoth K, Stücker W, Krewer E, & Müller G (1988) Biomonitoring of benzene exposure by trace analyses of phenylguanine. Arch Occup Environ Health, 60: 163-168. NTP (1986) Toxicology and carcinogenesis studies of benzene (CAS No. 71-43-2) in F344/N rats and B6C3F1 mice (gavage studies). Research Triangle Park, North Carolina, US Department of Health and Human Services, National Toxicology Program (NTP TR 289; NIH Publication 86-2545). Oberly TJ, Bewsey BJ, & Probst GS (1984) An evaluation of the L5178Y TK+/- mouse lymphoma forward mutation assay using 42 chemicals. Mutat Res, 125: 291-306. OECD (1986) Control of toxic substances in the atmosphere: Benzene. Paris, Organisation for Economic Cooperation and Development (Environment Monographs No. 5). Ogata M & Miyake Y (1978) Disappearance of aromatic hydrocarbons and organic sulphur compounds from fish flesh reared in crude oil suspension. Water Res, 12: 1041-1044. Ogata M, Fujisawa K, Ogino Y, & Mano E (1984) Partition coefficients as a measure of bioconcentration potential of crude oil compounds in fish and shellfish. Bull Environ Contam Toxicol, 33: 561-567. OSHA (Occupational Safety and Health Administration) (1987) Occupational exposure to benzene. Fed Reg, 52(176): 34460-34578. Otson R (1987) Purgeable organics in Great Lakes raw and treated water. Int J Environ Anal Chem, 31: 41-53. Otson R, Williams DT, & Bothwell PD (1982) Volatile organic compounds in water at thirty Canadian potable water treatment facilities. J Assoc Off Anal Chem, 54: 1370-1374. Ott MG, Townsend JC, Fishbeck WA, & Langner RA (1978) Mortality among workers occupationally exposed to benzene. Arch Environ Health, 33: 3-10. Paci E, Buiatti E, Costantini AS, Miligi L, Pucci N, Scarpelli A, Petriolo G, Simonata L, Winkelmann R, & Kaldor JM (1989) Aplastic anemia, leukemia and other cancer mortality in a cohort of shoe workers exposed to benzene. Scand J Work Environ Health, 15: 313-318. Painter RB & Howard R (1982) The HeLa-DNA synthesis inhibition test as a rapid screen for mutagenic carcinogens. Mutat Res, 92: 427-437. Parke DV & Williams RT (1953) Studies in detoxication. 49. The metabolism of benzene containing [14C1]benzene. Biochem J, 54: 231-238. Patty F (1981) Industrial hygiene and toxicology, 3rd revis ed. New York, Interscience Publishers. Paustembach DJ, Price PS, Ollison W, Blank C, Jernigan JD, Bass RD, & Peterson HD (1992) Reevaluation of benzene exposure for the pliofilm (rubberworker). J Toxicol Environ Health, 36: 177-231. Paxman D & Rappaport SM (1990) Analysis of OSHA's short-term-exposure limit for benzene. Regul Toxicol Pharmacol, 11: 275-287. Pekari K, Riellola M-L, & Aitio A (1989) Simultaneous determination of benzene and toluene in blood using head-space gas chromatography. J Chromatogr, 491: 309-320. Pellack-Walker P & Blumer JL (1986) DNA damage in L5178YS cells following exposure to benzene metabolites. Mol Pharmacol, 30: 42-47. Pellizzari ED (1982) Analysis for organic vapor emissions near industrial and chemical waste disposal sites. Environ Sci Technol, 16(11): 781-785. Pellizzari ED, Zweidinger RA, & Sheldon LS (1988) Determination of benzene, toluene and xylene in breath samples by gas chromatography/mass spectrometry. In: Fishbein L & O'Neill JK ed. Environmental carcinogens method of analysis and exposure measurement - Volume 10: Benzene and alkylated benzenes. New York, London, Oxford University Press, pp 267-279. Philip P & Krogh-Jensen M (1970) Benzene induced chromosome abnormalities in rat bone marrow cells. Acta Pathol Microbiol Scand, A78: 489-490. Pollini G, Biscaldi GP, & Robustelli della Cuna G (1969) [Chromosome changes in lymphocytes five years after benzene haemopathy.] Med Lav, 60: 743-758 (in Italian). Pongracz K & Bodell WJ (1991) Detection of 3@-hydroxy-1, N6-benzetheno-2'-deoxyadenosine 3'-phosphate by 32P postlabelling of DNA reacted with p-benzoquinone. Chem Res Toxicol, 4: 199-202. Poole SK, Furton KG, & Poole CF (1988) Determination of benzene and toluene in gasoline by gas chromatography using a liquid organic salt column. J Chromatogr Sci, 26: 67-73. Post GB & Snyder R (1983) Effects of enzyme induction on microsomal benzene metabolism. J Toxicol Environ Health, 11: 811. Post GB, Snyder R, & Kalf GF (1985) Inhibition of RNA synthesis and interleukin-2 production in lymphocytes in vitro by benzene and its metabolites, hydroquinone and p-benzoquinone. Toxicol Lett, 29: 161-167. Probst GS & Hill LE (1985) Tests for the induction of DNA-repair synthesis in primary cultures of adult rat hepatocytes. In: Ashby J, de Serres FJ, Draper M, Ishidata M Jr, Margolin BH, Matter BE, & Shelby MD ed. Evaluation of short-term tests for carcinogens: Report of the International Programme on Chemical Safety's collaborative study on in vitro assays. Amsterdam, Oxford, New York, Elsevier Science Publishers, pp 381-386 (Progress in Mutation Research, Volume 5). Reddy MV, Bleicher WT, Blackburn GR, & Mackerer CR (1990) DNA adduction by phenol hydroquinone, or benzoquinone in vitro but not in vivo: nuclease P1-enhanced 32P-postlabelling of adducts as labeled nucleoside biphosphates, dinucleotides and nucleoside monophosphates. Carcinogenesis, 11: 1349-1357. Reineke FJ & Bächmann K (1985) Gas chromatographic determination of C2-C8 hydrocarbons and halocarbons in ambient air by simultaneous use of three detectors. J Chromatogr, 323: 323-329. Renova NW (1962) [Concerning auto-immunity shifts in chronic occupational benzol poisoning.] Gig Tr Prof Zabol, 7: 38-42 (in Russian). Reynolds LF & Harrison DW (1982) Study of discharges of: benzene, chloroform and carbon tetrachloride into the aquatic environment and the best technical means for the reduction of water pollution from such discharges. Final Report. Brussels, Commission of the European Communities (BL/A/2198). Rickert DE, Baker TS, Bus JS, Barrow CS, & Irons RD (1979) Benzene disposition in the rat after exposure by inhalation. Toxicol Appl Pharmacol, 49: 417-423. Rinsky RA, Alexander B, Smith MD, Hornung R, Filloon TG, Young RJ, Okun AH, & Landrigan PJ (1987) Benzene and leukemia: an epidemiological risk assessment. New Engl J Med, 316: 1044-1050. RIVM (1988) Integrated criteria document on benzene. Bilthoven, The Netherlands, National Institute for Public Health and Environmental Protection, 138 pp (Report No. 758476003). Roberts JM, Rehsenfeld FC, & Liu SC (1984) Measurements of aromatic hydrocarbon ratios and nitrogen oxide concentrations in the rural troposphere: observation of air mass photochemical aging and nitrogen oxides removal. Atmos Environ, 18(11): 2421-2432. Roghani M, Da Silva C, Guvelli D, & Castagna M (1987) Benzene and toluene activate protein kinase C. Carcinogenesis, 8: 1105-1107. Rosenthal GJ & Snyder CA (1985) Modulation of the immune response to Listeria monocytogenes by benzene inhalation. Toxicol Appl Pharmacol, 80: 502. Rozen MG & Snyder CA (1985) Protracted exposure of C57B1/6 mice to 300 ppm benzene depresses B- and T-lymphocyte numbers and mitogen responses. Evidence for thymic and bone marrow proliferation in response to the exposures. Toxicology, 37(1-2): 13. Rozen MG, Snyder CA, & Albert RE (1984) Depressions in B- and T-lymphocyte mitogen-induced blastogenesis in mice exposed to low concentrations of benzene. Toxicol Lett, 20: 343. Rushton L & Alderson M (1981a) An epidemiological survey of eight oil refineries in Britain. Br J Ind Med, 38: 225-234. Rushton L & Alderson M (1981b) Case control study to investigate the association between exposure to benzene and deaths from leukemia in oil refinery workers. Br J Cancer, 43: 77-84. Sabourin PJ, Chen BT, Lucier G, Birnbaum LS, Fisher E, & Henderson RF (1987) Effect of dose on the absorption and excretion of [14C] benzene administered orally or by inhalation in rats and mice. Toxicol Appl Pharmacol, 87: 325-336. Sabourin PJ, Bechtold WE, & Henderson RF (1988) A high pressure liquid chromatographic method for the separation and quantitation of water-soluble radiolabeled benzene metabolites. Anal Biochem, 170: 316-327. Sabourin PJ, Sun JD, MacGregor JT, Wehr CM, Birnbaum LS, Lucier G, & Henderson RF (1990) Effect of repeated benzene inhalation exposures on benzene metabolism, binding to hemoglobin, and induction of micronuclei. Toxicol Appl Pharmacol, 103: 452-462. SAC (1989) Analysis of potentially dangerous substances in UK water (Prepared under contract to DOE). London, SAC Scientific Ltd (DOE Reference No. PECD 7/7/306). Sandmeyer EE (1981) Aromatic hydrocarbons. In: Clayton GD & Clayton FE ed. Patty's Industrial hygiene and toxicology, 3rd revis ed. New York, Interscience Publishers, vol 2, pp 3253-3283. Sasiadek M, Jagielski J, & Smolik R (1989) Localization of breakpoints in the karyotype of workers professionally exposed to benzene. Mutat Res, 224: 235-240. Sauer TC Jr (1981) Volatile organic compounds in open ocean and coastal surface waters. Org Geochem, 3: 91-101. Sawahata T & Neal RA (1983) Biotransformation of phenol to hydroquinone and catechol by rat liver microsomes. Mol Pharmacol, 23: 453-460. Schrenk D & Bock KW (1990) Metabolism of benzene in rat hepatocytes. Influence of inducers on phenol glucuronidation. Drug Metab Dispos, 18(5): 720-725. Seixas G, Andon BM, Hollingshead PG, & Thilly WG (1982) The aza-arenes as mutagens for Salmonella typhimurium. Mutat Res, 102: 201-212. Sellyei M & Kelemen E (1971) Chromosome study in a case of granulocytic leukemia with "pelgerisation" seven years after benzene pancytopenia. Eur J Cancer, 7: 83-85. Shah JJ & Singh HB (1988) Distribution of volatile organic chemicals in outdoor and indoor air. A national VOCs data base. Environ Sci Technol, 22(12): 1381-1388. Simic MG, Bergtold DS, & Karam LR (1989) Generation of oxy radicals in biosystems. Mutat Res, 214: 3-12. Singh HB, Salas L, & Stiles RE (1982) Distribution of selected gaseous organic mutagens and suspected carcinogens in ambient air. Environ Sci Technol, 16: 872. Siou G, Conan L, & El Haitem M (1981) Evaluation of the clastogenic action of benzene by oral administration with 2 cytogenetic techniques in mouse and Chinese hamster. Mutat Res, 90: 273-278. Skowronski GA, Turkall RM, & Abdel-Rahman MS (1988) Soil adsorption alters bioavailability of benzene in dermally exposed male rats. Am Ind Hyg Assoc J, 49(10): 506-511. Smart RC & Zannoni VG (1985) Effect of ascorbate on covalent binding of benzene and metabolites to isolated tissue preparations. Toxicol Appl Pharmacol, 77: 334-343. Snyder CA (1987) Benzene in Ethel Browning's toxicity and metabolism of industrial solvents. Volume 1: Hydrocarbons, 2nd ed. Amsterdam, Oxford, New York, Elsevier Science Publishers, pp 3-37. Snyder R, Lee EW, & Kocsis JJ (1978a) Binding of labelled metabolites to mouse liver and bone marrow. Res Commun Chem Pathol Pharmacol, 20: 191-194. Snyder CA, Goldstein BD, Sellakumar A, Wolman S, Bromberg I, Erlichman MN, & Laskin S (1978b) Hematotoxicity of inhaled benzene to Sprague Dawley rats and AKR mice at 300 ppm. J Toxicol Environ Health, 4: 605-618. Snyder CA, Goldstein BD, Sellakumar A, Bromberg I, Laskin S, & Albert RE (1980) The inhalation toxicology of benzene: incidence of hematopoietic neoplasms and hematotoxicity in AKR/J and C57BL/6J mice. Toxicol Appl Pharmacol, 54: 323-331. Snyder R, Longacre SL, Witmer CM, & Kocsis JJ (1981) Biochemical toxicology of benzene. In: Hodgson E, Bend JR, & Philpot RM ed. Reviews in biochemical toxicology 3. Amsterdam, Oxford, New York, Elsevier/North Holland, pp 123-153. Snyder CA, Goldstein BD, Sellakumar AR, & Albert RE (1984) Evidence for hematotoxicity and tumorigenesis in rats exposed to 100 ppm benzene. Am J Ind Med, 5: 429-434. Snyder R, Jowa L, Witz G, Jakf G, & Rushmore T (1987) Formation of reactive metabolites from benzene. Arch Toxicol, 60(1-3): 61-64. Snyder CA, Sellakumar AR, James DJ, & Albert RE (1988) The carcinogenicity of discontinuous inhaled benzene exposures in CD-1 and C57B1/6 mice. Arch Toxicol, 62: 331-335. Snyder R, Dimitriadis E, Guy R, Hu P, Cooper K, Bauer H, Witz G, & Goldstein BD (1989) Studies on the mechanism of benzene toxicity. Environ Health Perspect, 82: 31-35. Staples CA, Werner AF, & Hoogheem TJ (1985) Assessment of priority pollutant concentrations in the United States using STORET database. Environ Toxicol Chem, 4: 131-142. Stommel P, Müller G, Stücker W, Verkoyen C, Schöbel S, & Norpoth K (1989) Determination of S-phenylmercapturic acid in the urine - an improvement in the biological monitoring of benzene exposure. Carcinogenesis, 10: 279-282. Stoner RD, Drew RT, & Bernstein DM (1981) Benzene inhalation effect upon tetanus antitoxin responses and leukemogenesis in mice. In: Mahlum DD, Gray RH, & Felix WD ed. Coal conversion and the environment. Oak Ridge, Tennessee, US Department of Energy, Technical Information Center, pp 445-461. Styles JA & Richardson CR (1984) Cytogenetic effects of benzene: dosimetric studies on rats exposed to benzene vapor. Mutat Res, 135: 203-209. Sun JD, Medinsky MA, & Birnbaum LS (1990) Benzene hemoglobin adducts in mice and rats: Characterization of formation and physiological modeling. Fundam Appl Toxicol, 15: 468-475. Susten A, Dames B, & Burg J (1985) Percutaneous penetration of benzene in hairless mice: An estimate of dermal absorption during tire-building operations. Am J Ind Med, 7: 323-335. Swaen GMH, Slangen JJM, Volovics A, Hayes RB, Scheffers T, & Sturmans F (1991) Mortality of coke plant workers in the Netherlands. Br J Ind Med, 48: 130-135. Swenberg JA, Petzold GL, & Harbach PR (1976) In vitro DNA damage/alkaline elution assay for predicting carcinogenic potential. Biochem Biophys Res Commun, 72: 732-738. Tatrai E, Ungvary GY, Hudak A, Rodics K, Loerincz M, & Barcza GY (1980a) Concentration of dependence of the embryotoxic effects of benzene inhalation in CFY rats. J Hyg Epidemiol Microbiol Immunol, 24: 363-371. Tatrai E, Rodics K, & Ungvary GY (1980b) Embryotoxic effects of simultaneously applied exposure of benzene and toluene. Folia Morphol (Praha), 28: 286-289. Thienes H & Haley TJ (1972) Clinical toxicology, 5th ed. Philadelphia, Pennsylvania, Lea & Febiger, pp 124-127. Tice RR, Costa DL, & Drew RT (1980) Cytogenetic effects of inhaled benzene in murine bone marrow: Induction of sister chromatid exchanges, chromosomal aberrations and cellular proliferation inhibition in DBA/2 mice. Proc Natl Acad Sci (USA), 77: 2148-2152. Tice RR, Vogt T, & Costa D (1982) Cytogenetic effects of inhaled benzene in murine bone marrow. In: Genotoxic effects of airborne agents. New York, London, Plenum Press, pp 257-275. Tice RR, Luke CA, & Drew RT (1989) Effect of exposure route, regimen, and duration on benzene-induced genotoxic and cytotoxic bone marrow damage in mice. Environ Health Perspect, 82: 65-74. Toft K, Olofsson T, Tunek A, & Berlin M (1982) Toxic effects on mouse bone marrow caused by inhalation of benzene. Arch Toxicol, 51: 295-302. Topham JC (1980) Do induced sperm-head abnormalities in mice specifically identify mammalian mutagens rather than carcinogens? Mutat Res, 74: 379-387. Tough IM & Court Brown WM (1965) Chromosome aberrations and exposure to ambient benzene. Lancet, 1: 684. Travis CC, Quillen JL, & Arms AD (1990) Pharmacokinetics of benzene. Toxicol Appl Pharmacol, 102: 400-420. Tsai SP, Wen CP, Weiss NS, Wong O, McClellan WA, & Gibson RL (1983) Retrospective mortality and medical surveillance studies of workers in benzene areas of refineries. J Occup Med, 25: 685-692. Tucker WA, Huang C, & Bral JM (1986) Validation of transport model. In: Benzene in Florida groundwater: an assessment of the significance to human health. Tallahassee, Florida, Florida Petroleum Council, American Petroleum Institute, pp 93-108. Tully FP, Ravishankara AR, Thopmson RL, Nicovich JM, Shah RC, Kreutter NM, & Wine PH (1981) Kinetics of the reactions of hydroxyl radicals with benzene and toluene. J Phys Chem, 85: 2262. Tunek A, Platt KL, Bentley P, & Oesch F (1978) Microsomal metabolism of benzene to species irreversibly binding to microsomal protein and effects of modification of this metabolism. Mol Pharmacol, 14: 920-929. UBA (1982) Air quality criteria for benzene. Berlin, Federal Office for the Environment (Report 6/82). Ungvary G & Tatrai E (1985) On the embryotoxic effects of benzene and its alkyl derivatives in mice, rats and rabbits. Arch Toxicol, 8: 425-430. US EPA (1985) Drinking water criteria document on benzene. Washington, DC, US Environmental Protection Agency, Office of Drinking Water (PB86-118122). US EPA (1987) June-September, 6-9 AM, ambient air benzene concentrations in 39 U.S. cities, 1984-1986. Research Triangle Park, North Carolina, US Environmental Protection Agency, Atmospheric Sciences Research Laboratory (EPA/600/D-87/160). US EPA (1989) National emissions standards for hazardous air pollutants: Benzene emissions from maleic anhydride plants, ethylbenzene styrene plants, benzene storage vessels, benzene equipment leaks, and coke by-product recovery plants. Fed Reg, 54: 38044-38139. Uyeki EM, Ashkar AE, Shoeman DW, & Bisel TU (1977) Acute toxicity of benzene inhalation to hemopoietic precursor cells. Toxicol Appl Pharmacol, 40: 49-57. Vaishnav DD & Babeu L (1987) Comparison of occurrence and rates of chemical biodegradation in natural waters. Bull Environ Contam Toxicol, 39: 237-244. Venitt S (1985) Summary report on the performance of the bacterial mutation assays. In: Ashby J, de Serres FJ, Draper M, Ishidate M Jr, Margolin BH, Matter BE, & Shelby MD ed. Evaluation of short-term tests for carcinogens. Report of the International Programme on Chemical Safety's collaborative study on in vitro assays. Amsterdam, Oxford, New York, Elsevier Science Publishers, pp 11-23 (Progress in Mutation Research, Volume 5). Vigliani EC & Saita G (1964) Benzene and leukemia. New Engl J Med, 271(17): 872-876. Vogel K, Bentley P, Platt KL, & Oesch F (1980) Rat liver cytoplasmic dihydrodiol dehydrogenase. J Biol Chem, 255: 9621-9625. Wallace LA (1989a) The exposure of the general population to benzene. Cell Biol Toxicol, 5(3): 197. Wallace LA (1989b) Major sources of benzene exposure. Environ Health Perspect, 82: 165-169. Wallace LA & Pellizzari ED (1986) Personal air exposures and breath concentrations of benzene and other volatile hydrocarbons for smokers and nonsmokers. Toxicol Lett, 35: 113-116. Wallace LA, Pellizzari E, & Hartwell TD (1985) Personal exposures, indoor-outdoor relationships, and breath levels of toxic air pollutants measured for 355 persons in New Jersey. Atmos Environ, 19: 1651-1661. Wallace LA, Pellizzari E, Leader B, Zelon H, & Sheldon L (1987) Emissions of volatile organic compounds from building materials and consumer products. Atmos Environ, 21(2): 385-393. Wallin H, Melin P, Schelin C, & Jergil B (1985) Evidence that covalent binding of metabolically activated phenol to microsomal proteins is caused by oxidised products of hydroquinone and catechol. Chem-Biol Interact, 55: 335-346. Ward CO, Kuna RA, Snyder NK, Alsaker RD, Coate WB, & Craig PH (1985) Subchronic inhalation toxicity of benzene in rats and mice. Am J Ind Med, 7: 457. Wathne BM (1983) Measurements of benzene, toluene and xylenes in urban air. Atmos Environ, 17: 1713. Weaver NK, Gibson RL, & Smith CW (1983) Occupational exposure to benzene in the petroleum and petrochemical industries. Adv Mod Environ Toxicol, 4: 63-75. Wermuth B, Platts K, Seidel A, & Oesch F (1986) Carbonyl reductase provides the enzymatic basis of quinone reduction in man. Biochem Pharmacol, 35: 1277. WHO (1984) Guidelines for drinking-water quality. Volume 1: Recommendations. Geneva, World Health Organization, 130 pp. WHO (1987) Benzene. In: Air quality guidelines for Europe. Copenhagen, World Health Organization, Regional Office for Europe, pp 45-58 (WHO Regional Publications, European Series No. 23). WHO (1993) Guidelines for drinking-water quality. Volume 1: Recommendations. Geneva, World Health Organization. Wilson RH (1942) Benzene poisoning in industry. J Lab Clin Med, 27: 1517-1521. Wilson BH, Smith GB, & Rees JF (1986) Biotransformation of selected alkylbenzenes and halogenated hydrocarbons in methanogenic aquifer material: A microcosm study. Environ Sci Technol, 20: 997-1002. Windholz M, Budavari S, Blumetti RF, & Otterbein ES (1983) The Merck Index: An encyclopedia of chemicals, drugs, and biologicals, 10th ed. Rahway, New Jersey, Merck and Co., Inc., p 151. Winek CL, Collom WD, & Wecht CH (1967) Fatal benzene exposure by glue sniffing. Lancet, March 25: 683 Winek CL & Collom WD (1971) Benzene and toluene fatalities. J Occup Med, 13: 259-261. Withey RJ & Hall JW (1975) The joint action of perchloroethylene with benzene or toluene in rats. Toxicology, 4: 5-15. Witz G, Gad SC, Tice RR, Oshiro Y, Piper CE, & Goldstein BD (1990a) Genetic toxicity of the benzene metabolite trans,trans-muconaldehyde in mammalian and bacterial cells. Mutat Res, 240: 295-306. Witz G, Maniara W, Mylavarapu V, & Goldstein BD (1990b) Comparative metabolism of benzene and trans,trans-muconaldehyde to trans,trans-muconic acid in DBA/2N and C57Bl/6 mice. Biochem Pharmacol, 40: 1275-1280. Wolf MA, Rowe VK, McCollister DD, Hollingsworth RL, & Oyen F (1956) Toxicological studies of certain alkylated benzenes and benzene. Arch Ind Health, 14: 387-398. Wong O (1987) An industry wide mortality study of chemical workers occupationally exposed to benzene: II. Dose response analyses. Br J Ind Med, 44: 382-395. Yardley-Jones A, Anderson D, Jenkinson PC, Lovell DP, Blowers SD, & Davies MJ (1988) Genotoxic effects in peripheral blood and urine of workers exposed to low level benzene. Br J Ind Med, 45: 694-700. Yardley-Jones A, Anderson D, Lovell DP, & Jenkinson PC (1990) Analysis of chromosomal aberrations in workers exposed to low level benzene. Br J Ind Med, 47: 48-51. Yin S-N, Li G-L, Tain F-D, Fu Z-I, Jin C, Chen Y-J, Luo S-J, Ye P-Z, Zhang J-Z, Wang G-C, Zhang X-C, Wu H-N, & Zhong Q-C (1987) Leukaemia in benzene workers: a retrospective cohort study. Br J Ind Med, 44: 124-128. Yin S-N, Li G-L, Tain F-D, Fu Z-I, Jin C, Chen Y-J, Luo S-J, Ye P-Z, Zhang J-Z, Wang G-C, Zhang X-C, Wu H-N, & Zhong Q-C (1989) A retrospective cohort study of leukemia and other cancers in benzene workers. Environ Health Perspect, 82: 207-213. Young RJ, Rinsky RA, Infante PF, & Wagoner JK (1978) Benzene in consumer products. Science, 199: 248. Zhurkov VS, Fel'dt EG, & Kosyakov VV (1983) Dependence of the frequency of chromosomal aberrations in mouse bone marrow cells on concentration (dose) and mode of administration of benzene. Bull Exp Biol Med, 96: 1741-1743. 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: Benzene (CHEMINFO) Benzene (IARC Summary & Evaluation, Supplement 7, 1987) Benzene (IARC Summary & Evaluation, Volume 29, 1982) Benzene (IARC Summary & Evaluation, Volume 7, 1974) Benzene (ICSC) Benzene (PIM 063)