INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY ENVIRONMENTAL HEALTH CRITERIA 156 HEXACHLOROBUTADIENE This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the United Nations Environment Programme, the International Labour Organisation, or the World Health Organization. First draft prepared by Dr. T. Vermeire, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization World Health Orgnization Geneva, 1994 The International Programme on Chemical Safety (IPCS) is a joint 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 Hexachlorobutadiene. (Environmental health criteria: 156) 1. Butadienes - toxicity 2. Environmental exposure I.Series ISBN 92 4 157126 X (NLM Classification QV 305.H7) ISSN 0250-863X 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 1994 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 impression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of every 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 HEXACHLOROBUTADIENE 1. SUMMARY 1.1. Identity, physical and chemical properties, analytical methods 1.2. Sources of human and environmental exposure 1.3. Environmental transport, distribution and transformation 1.4. Environmental levels and human exposure 1.5. Kinetics and metabolism 1.6. Effects on organisms in the environment 1.7. Effects on experimental animals and in vitro test systems 1.7.1. General toxicity 1.7.2. Reproduction, embryotoxicity and teratogenicity 1.7.3. Genotoxicity and carcinogenicity 1.7.4. Mechanisms of toxicity 1.8. Effects on humans 1.9. Evaluation of human health risks and effects on the environment 1.9.1. Evaluation of human health risks 1.9.2. Evaluation of effects on the environment 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS 2.1. Identity 2.2. Physical and chemical properties 2.3. Conversion factors 2.4. Analytical methods 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1. Natural occurrence 3.2. Anthropogenic sources 3.2.1. Production levels and processes 3.2.2. Uses 3.2.3. Waste disposal 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION 4.1. Transport and distribution between media 4.2. Abiotic degradation 4.2.1. Photolysis 4.2.2. Photooxidation 4.2.3. Hydrolysis 4.3. Biodegradation 4.4. Bioaccumulation 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1. Environmental levels 5.1.1. Air 5.1.2. Water 5.1.3. Soil and sediment 5.1.4. Biota 5.2. General population exposure 5.3. Occupational exposure 6. KINETICS AND METABOLISM 6.1. Absorption and distribution 6.2. Metabolism 6.2.1. In vitro studies 6.2.2. In vivo studies 6.3. Reaction with body components 6.4. Excretion 7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT 7.1. Aquatic organisms 7.1.1. Short-term toxicity 7.1.2. Long-term toxicity 7.2. Terrestrial organisms 7.2.1. Short-term toxicity 8. EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO TEST SYSTEMS 8.1. Single exposure 8.1.1. Inhalation exposure 184.108.40.206 Mortality 220.127.116.11 Systemic effects 8.1.2. Oral exposure 18.104.22.168 Mortality 22.214.171.124 Systemic effects 8.1.3. Dermal exposure 126.96.36.199 Mortality 188.8.131.52 Systemic effects 8.1.4. Other routes of exposure 8.2. Short-term exposure 8.2.1. Inhalation exposure 8.2.2. Oral exposure 184.108.40.206 Rats 220.127.116.11 Mice 8.3. Long-term exposure 8.4. Skin and eye irritation; sensitization 8.4.1. Irritation 8.4.2. Sensitization 8.5. Reproduction, embryotoxicity and teratogenicity 8.5.1. Reproduction 8.5.2. Embryotoxicity and teratogenicity 8.6. Mutagenicity and related end-points 8.6.1. In vitro effects 8.6.2. In vivo effects 8.7. Carcinogenicity/long-term toxicity 8.7.1. Inhalation exposure 8.7.2. Oral exposure 8.7.3. Dermal exposure 8.7.4. Exposure by other routes 8.8. Other special studies 8.8.1. Effects on the nervous system 8.8.2. Effects on the liver 18.104.22.168 Acute effects 22.214.171.124 Short-term effects 8.8.3. Effects on the kidneys 126.96.36.199 Acute effects 188.8.131.52 Short- and long-term effects 8.9. Factors modifying toxicity; toxicity of metabolites 8.9.1. Factors modifying toxicity 184.108.40.206 Surgery 220.127.116.11 Inhibitors and inducers of mixed-function oxidases (MFO) 18.104.22.168 Inhibitors of gamma-glutamyltrans- peptidase (EC 22.214.171.124) 126.96.36.199 Inhibitors of cysteine conjugate ß-lyase 188.8.131.52 Inhibitors of organic anion transport 184.108.40.206 Non-protein sylfhydryl scavengers 8.9.2. Toxicity of metabolites 220.127.116.11 In vitro studies 18.104.22.168 In vivo studies 8.10. Mechanisms of toxicity - mode of action 8.10.1. Mechanisms of toxicity 8.10.2. Mode of action 9. EFFECTS ON HUMANS 9.1. General population exposure 9.2. Occupational exposure 9.3. In vitro metabolism studies 9.4. Extrapolation of NOAEL from animals to humans 10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT 10.1. Evaluation of human health risks 10.1.1. Hazard identification 10.1.2. Exposure 10.1.3. Hazard evaluation 10.2. Evaluation of effects on the environment 10.2.1. Hazard identification 10.2.2. Exposure 10.2.3. Hazard evaluation 11. FURTHER RESEARCH 12. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES REFERENCES RESUME RESUMEN WHO TASK GROUP MEETING ON ENVIRONMENTAL HEALTH CRITERIA FOR HEXACHLOROBUTADIENE Members Dr T.M. Crisp, Reproductive and Development Toxicology Branch, Human Health Assessment Group, Office of Health and Environmental Assessment, Environmental Protection Agency, Washington, DC, USA (Joint Rapporteur) Professor W. Dekant, Toxicology Institute, Würzburg University, Würzburg, Germany Dr I.V. German, Ukrainian Institute for Ecohygiene and Toxicology of Chemicals, Kiev, Ukraine Dr B. Gilbert, Fundaçao Oswaldo Cruz, Ministry of Health, Manguinhos, Rio de Janeiro, Brazil (Joint Rapporteur) Ms E. Kuempel, Document Development Branch, National Institute for Occupational Safety and Health, Robert A. Taft Laboratories, Cincinnati, Ohio, USA Dr E.A. Lock, Biochemical Toxicology Section, Imperial Chemical Industries, Central Toxicological Laboratory, Alderly Park, Macclesfield, Cheshire, United Kingdom Professor M.H. Noweir, Industrial Engineering Department, College of Engineering, King Abdul Aziz University, Jeddah, Saudi Arabia (Chairman) Dr A. Smith, Toxicology Unit, Health and Safety Executive, Bootle, Merseyside, United Kingdom Secretariat Professor F. Valic, IPCS Consultant, World Health Organization, Geneva, Switzerland, also Vice-Rector, University of Zagreb, Zagreb, Croatia (Responsible Officer and Secretary) Dr T. Vermeire, National Institute of Public Health and Environmental Protection, Toxicology Advisory Centre, Bilthoven, The Netherlands NOTE TO READERS OF THE CRITERIA MONOGRAPHS Every effort has been made to present information in the criteria monographs as accurately as possible without unduly delaying their publication. In the interest of all users of the Environmental Health Criteria monographs, readers are kindly requested to communicate any errors that may have occurred to the Director of the International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland, in order that they may be included in corrigenda. * * * A detailed data profile and a legal file can be obtained from the International Register of Potentially Toxic Chemicals, Case postale 356, 1219 Châtelaine, Geneva, Switzerland (Telephone No. 9799111). * * * This publication was made possible by grant number 5 U01 ES02617-14 from the National Institute of Environmental Health Sciences, National Institutes of Health, USA. ENVIRONMENTAL HEALTH CRITERIA FOR HEXACHLOROBUTADIENE A Task Group on Environmental Health Criteria for Hexachlorobutadiene met at the Institute of Hygiene and Epidemiology, Brussels, Belgium, from 10 to 15 December 1992. Dr C. Vleminckx welcomed the participants on behalf of the host institution and Professor F. Valic opened the meeting on behalf of the three cooperating organizations of the IPCS (UNEP/ILO/WHO). The Task Group reviewed and revised the draft monograph and made an evaluation of the risks for human health and the environment from exposure to hexachlorobutadiene. The first draft of this monograph was prepared by Dr T. Vermeire, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands. Professor F. Valic was responsible for the overall scientific content of the monograph and for the organization of the meeting, and Dr P.G. Jenkins, IPCS, for the technical editing of the monograph. The efforts of all who helped in the preparation and finalization of the monograph are gratefully acknowledged. ABBREVIATIONS ACPB 1-( N-acetylcystein- S-yl)-1,2,3,4,4-pentachloro-1,3- butadiene BCTB 1,4-(bis-cystein- S-yl)-1,2,3,4-tetrachloro-1,3- butadiene BGTB 1,4-(bis-glutathion- S-yl)-1,2,3,4- tetrachloro-1,3-butadiene CMTPB 1-carboxymethylthio-1,2,3,4,4-pentachloro-1,3- butadiene CPB 1-(cystein- S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene GPB 1-(glutathion- S-yl)-1,2,3,4,4-pentachloro-1,3- butadiene GSH reduced glutathione HCBD hexachlorobutadiene ip intraperitoneal iv intravenous MTPB 1-methylthio-1,2,3,4,4-pentachloro-1,3-butadiene NIOSH National Institute of Occupational Safety and Health NOAEL no-observed-adverse-effect level OECD Organisation for Economic Co-operation and Development PATPB 1-(pyruvic acid thiol)-1,2,3,4,4-pentachloro-1,3- butadiene PBSA 1,2,3,4,4-pentachloro-1,3-butadienyl sulfenic acid TBA 2,3,4,4-tetrachloro-1,3-butenoic acid TPA 12- o-tetradecanoylphorbol-13-acetate TPB 1-thiol-1,2,3,4,4-pentachloro-1,3-butadiene UDS unscheduled DNA synthesis 1. SUMMARY 1.1 Identity, physical and chemical properties, analytical methods Hexachlorobutadiene is a non-flammable, incombustible, clear, oily and colourless liquid at ordinary temperature and pressure. It is poorly soluble in water but miscible with ether and ethanol. The substance can be detected and determined quantitatively by gas chromatographic methods. The detection limits are 0.03 µg/m3 in air, 0.001 µg/litre in water, 0.7 µg/kg wet weight in soil or sediment, and 0.02 µg/litre in blood. A level of 0.47 µg/kg wet weight has been determined in tissue. 1.2 Sources of human and environmental exposure Hexachlorobutadiene is not reported to occur as a natural product. It is chiefly produced as a by-product of the manufacture of chlorinated hydrocarbons where it occurs in the heavy fractions (Hex-waste). The world annual production of the compound in heavy fractions was estimated in 1982 to be 10 000 tonnes. Hexachlorobutadiene can be used for recovery of chlorine-containing gas in chlorine plants and as a wash liquor for removing certain volatile organic compounds from gas streams. It has further been used as a fluid in gyroscopes, as heat transfer, transformer, insulating and hydraulic fluids, as a solvent for elastomers, and as an intermediate and fumigant. 1.3 Environmental transport, distribution and transformation The main pathways of entry into the environment are emissions from waste and dispersive use. Intercompartmental transport will chiefly occur by volatilization, adsorption to particulate matter, and subsequent deposition or sedimentation. Hexachlorobutadiene does not migrate rapidly in soil and accumulates in sediment. In water, it is considered persistent unless there is high turbulence. Hydrolysis does not occur. The substance seems to be readily biodegradable aerobically, though biodegradability has not been investigated thoroughly. Hexachlorobutadiene photolyses on surfaces. In addition to deposition, reaction with hydroxyl radicals is assumed to be an important sink of hexachlorobutadiene in the troposphere, and the estimated atmospheric half-life is up to 2.3 years. The substance has a high bioaccumulating potential as has been confirmed by both laboratory and field observations. Average steady-state bioconcentration factors of 5800 and 17 000, based on wet weight, have been determined experimentally in rainbow trout. Biomagnification has not been observed either in the laboratory or in the field. 1.4 Environmental levels and human exposure Hexachlorobutadiene has been measured in urban air: in all cases levels were below 0.5 µg/m3. Concentrations in remote areas are less than 1 pg/m3. In lake and river water in Europe concentrations of up to 2 µg/litre have been recorded, but mean levels are usually below 100 ng/litre. In the Great Lakes area of Canada, much lower levels (around 1 ng/litre) were measured. Bottom sediment levels here can be as high as 120 µg/kg dry weight. Older sediment layers from around 1960 contained higher concen-trations (up to 550 µg/kg wet weight). The sediment concentration was demonstrated to increase with particle size in the sediment. Concentrations of hexachlorobutadiene in aquatic organisms, birds and mammals indicate bioaccumulation but not biomagnification. In polluted waters, levels of over 1000 µg/kg wet weight have been measured in several species and 120 mg/kg (lipid base) in one species. Present levels generally remain below 100 µg/kg wet weight away from industrial outflows. The compound has been detected in human urine, blood and tissues. Certain food items containing a high lipid fraction have been found to contain up to about 40 µg/kg and, in one case, over 1000 µg/kg. One study reported occupational exposures of 1.6-12.2 mg/m3 and urine levels of up to 20 mg/litre. 1.5 Kinetics and metabolism Hexachlorobutadiene is rapidly absorbed following oral administration to experimental animals, but the rate of absorption following inhalation or dermal exposure has not been investigated. In rats and mice, the compound distributes mainly to the liver, kidneys and adipose tissue. It is rapidly excreted. Binding to liver and kidney protein and nucleic acids has been demonstrated. The biotransformation of the compound in experimental animals appears to be a saturable process. This process proceeds mainly through a glutathione-mediated pathway in which hexachlorobutadiene is initially converted to S-glutathione conjugates. These conjugates can be metabolized further, especially in the brush-border membrane of renal tubular cells, to a reactive sulfur metabolite, which probably accounts for the observed nephrotoxicity, genotoxicity and carcinogenicity. 1.6 Effects on organisms in the environment Hexachlorobutadiene is moderately to very toxic to aquatic organisms. Fish species and crustaceans were found to be the most sensitive, 96-h LC50 values ranging from 0.032 to 1.2 and 0.09 to approximately 1.7 mg/litre for crustaceans and fish, respectively. The kidney was demonstrated to be an important target organ in fish. Based on several long-term tests with algae and fish species, a no-observed-effect level (NOEL) of 0.003 mg/litre was established; this classifies the compound as very toxic to aquatic species. End-points investigated include general toxicity, neurotoxicity, biochemistry, haematology, pathology, and reproductive parameters. In one 28-day early-lifestage test with fathead minnows, reproduction was unaffected at concentrations of up to 0.017 mg/litre, whereas increased mortality and a decreased body weight were observed at 0.013 and 0.017 mg/litre. The NOEL was 0.0065 mg/litre. Only one reliable test with terrestrial organisms has been described. In a 90-day test with Japanese quail, receiving a diet containing the compound at concentrations from 0.3 to 30 mg/kg diet, the survival of chicks was decreased at 10 mg/kg diet only. 1.7 Effects on experimental animals and in vitro test systems 1.7.1 General toxicity Hexachlorobutadiene is slightly to moderately toxic to adult rats, moderately toxic to male weanling rats, and highly toxic to female weanling rats following a single oral dose. The major target organs are the kidney and, to a much lesser extent, the liver. Based on animal data, the vapour of hexachlorobutadiene is irritating to mucous membranes and the liquid is corrosive. The substance should be regarded as a sensitizing agent. In the kidneys of rats, mice and rabbits, hexachlorobutadiene causes a dose-dependent necrosis of the renal proximal tubules. Adult male rats are less sensitive to renal toxicity than adult females or young males. Young mice are more susceptible than adults, no sex difference being apparent. In adult female rats the lowest single intraperitoneal dose at which renal necrosis was observed was 25 mg/kg body weight, and in adult male and female mice it was 6.3 mg/kg body weight. Biochemical changes and distinct functional alterations in the kidneys occurred at doses similar to or higher than those at which necrosis occurred. In six short-term oral tests, two reproductive studies and one long-term diet study with rats, the kidney was also the major target organ. Dose-related effects included a decreased relative kidney weight and tubular epithelial degeneration. The no-observed- adverse-effect level (NOAEL) for renal toxicity in rats in a 2-year study was 0.2 mg/kg body weight per day. In mice the NOAEL in a 13-week study was 0.2 mg/kg body weight per day. In both species, adult females were more susceptible than adult males. In one short-term inhalation test (6 h/day for 12 days), similar effects on the kidneys were observed with a nominal vapour concentration of 267 mg/m3, at which concentration respiratory difficulties and cortical degeneration in the adrenal glands were also observed. 1.7.2 Reproduction, embryotoxicity and teratogenicity Two reproduction diet studies in rats at doses up to 20 and 75 mg/kg body weight per day, respectively, revealed reduced birth weight and neonatal weight gain at maternally toxic doses of 20 and 7.5 mg/kg body weight, respectively. The highly toxic dose of 75 mg/kg body weight per day was sufficient to prevent conception and uterine implantation. Skeletal abnormalities were not observed. In two teratogenicity tests, where rats were exposed either to hexachlorobutadiene vapour at concentrations between 21 and 160 mg/m3 for 6 h/day (from days 6 to 20 of pregnancy) or intraperitoneally to 10 mg/kg body weight per day (from days 1 to 15 of pregnancy), fetuses demonstrated developmental toxicity, including reduced birth weight, delay in heart development and dilated ureters, but no gross malformations. The retarded development was observed at levels which were also toxic to the dams. 1.7.3 Genotoxicity and carcinogenicity Hexachlorobutadiene induces gene mutations in the Ames Salmonella test under special conditions favouring the formation of glutathione conjugation products. It induced chromosomal aberrations in one in vivo study but not in two in vitro studies. In one in vitro test the frequency of sister chromatid exchanges was increased in Chinese hamster ovary cells. High mutagenic potency by sulfur metabolites of hexachlorobutadiene was reported. In in vitro studies, the compound induced unscheduled DNA synthesis in Syrian hamster embryo fibroblast cultures but not in rat hepatocyte cultures. It induced unscheduled DNA synthesis in rats in vivo, but did not induce sex-linked recessive lethal mutations in Drosophila melanogaster. In the only long-term (2 years) study, in which rats received a diet containing hexachlorobutadiene at doses of 0.2, 2 or 20 mg/kg body weight per day, an increased incidence of renal tubular neoplasms was observed only at the highest dose level. 1.7.4 Mechanisms of toxicity The nephrotoxicity, mutagenicity and carcinogenicity of hexachlorobutadiene is dependent on the biosynthesis of the toxic sulfur conjugate 1-(glutathion- S-yl)-1,2,3,4,4-pentachloro- 1,3-butadiene (GPB). This conjugate is mainly synthesised in the liver and is further metabolized in the bile, gut and kidneys to 1-(cystein- S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene (CPB). The activation of CPB, dependent on cysteine conjugate ß-lyase, to a reactive thioketene in the proximal tubular cells finally results in covalent binding to cellular macromolecules. 1.8 Effects on humans No pathogenic effects in the general population have been described. There have been two reports of disorders among agricultural workers using hexachlorobutadiene as a fumigant, but they were also exposed to other substances. An increased frequency of chromosomal aberrations was found in the lymphocytes of peripheral blood of workers engaged in the production of hexachlorobutadiene and reported to be exposed to concentrations of 1.6-12.2 mg/m3. 1.9 Evaluation of human health risks and effects on the environment 1.9.1 Evaluation of human health risks As there have been very few human studies, the evaluation is mainly based on studies in experimental animals. However, limited human in vitro data suggest that the metabolism of hexachlorobutadiene in humans is similar to that observed in animals. Hexachlorobutadiene vapour is considered to be irritating to the mucous membranes of humans, and the liquid is corrosive. The compound should also be regarded a sensitizing agent. The main target organs for toxicity are the kidney and, to a much lesser extent, the liver. On the basis of short- and long-term oral studies in rats and mice, the NOAEL is 0.2 mg/kg body weight per day. In one short-term inhalation study in rats (12 days, 6 h/day), the NOAEL was 53 mg/m3. Reduced birth weight and neonatal weight gain was observed only at maternally toxic doses, as was developmental toxicity. Hexachlorobutadiene has been found to induce gene mutations, chromosomal aberrations, increased sister chromatid exchanges and unscheduled DNA synthesis, although some studies have reported negative results. There is limited evidence for the genotoxicity of hexachlorobutadiene in animals, and insufficient evidence in humans. Long-term oral administration of hexachlorobutadiene to rats was found to induce an increased frequency of renal tubular neoplasms, but only at a high dose level causing marked nephrotoxicity. There is limited evidence for carcinogenicity in animals and insufficient evidence in humans. On the basis of the NOAEL for mice or rats of 0.2 mg/kg body weight per day, a NOAEL of 0.03-0.05 mg/kg body weight per day has been estimated for humans. There is a margin of safety of 150 between the estimated NOAEL and the estimated maximum total daily intake assuming absorption of the compound via contaminated drinking-water and food of high lipid content. 1.9.2 Evaluation of effects on the environment Hexachlorobutadiene is moderately to highly toxic to aquatic organisms; crustaceans and fish are the most sensitive species. An environmental concern level of 0.1 µg/litre has been established. It is estimated that the maximum predicted environmental concentration away from point sources is twice the extrapolated environmental concern level and, consequently, aquatic organisms may be at risk in polluted surface waters. Adverse effects on benthic organisms cannot be excluded. Considering the toxicity of hexachlorobutadiene to mammals, consumption of benthic or aquatic organisms by other species may cause concern. 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS 2.1 Identity Chemical formula: C4Cl6 Chemical structure: Common name: hexachlorobutadiene Common synonyms: 1,3-hexachlorobutadiene, 1,1,2,3,4,4- hexachloro-1,3-butadiene, perchloro- butadiene Common trade names: C-46, Dolen-pur, GP40-66: 120, UN2279 Common abbreviation: HCBD CAS registry number: 87-68-3 RTECS registry number: EJ 0700000 Relative molecular mass: 260.8 2.2 Physical and chemical properties Hexachlorobutadiene is a non-flammable, incombustible, clear, colourless and oily liquid at ordinary temperature and pressure. Its odour is described as turpentine-like. The odour threshold for the compound in air is reported to be 12 mg/m3 (Ruth, 1986). In water an odour threshold of 0.006 mg/litre has been reported (US EPA, 1980). The compound is poorly soluble in water but is miscible with ether and ethanol. Hexachlorobutadiene is very stable to acid and alkali in the absence of an appropriate solvent and has no tendency to polymerize even under high pressure. It reacts with chlorine under severe reaction conditions, often with cleavage of the carbon skeleton (Ullmann, 1986). Some physical and chemical data on hexachlorobutadiene are presented in Table 1. Table 1. Some physical and chemical properties of hexachlorobutadienea Physical state liquid Colour clear, colourless Melting point -18 °C Boiling point 212 °C at 101.3 kPa Water solubility 3.2 mg/litre at 25 °Cb Log n-octanol-water partition coefficient (Kow) 4.78b, 4.90c Density 1.68 g/cm3 at 20 °C Relative vapour density 9.0 Vapour pressure 20 Pa (0.15 mmHg) at 20 °Cd Autoignition temperature 610 °C a Unless otherwise stated, the data are selected from secondary sources. b Experimentally derived by Banerjee et al. (1980) c Experimentally derived by Chiou (1985) d McConnell et al. (1975) 2.3 Conversion factors 1 ppm = 10.67 mg/m3 air at 25 °C and 101.3 kPa (760 mmHg) 1 mg/m3 air = 0.094 ppm. 2.4 Analytical methods A summary of relevant methods of sampling and gas chromatographic analysis is presented in Table 2. The analytical method for air, reported by Dillon (1979) and Boyd et al. (1981) has been approved by NIOSH and was published in the NIOSH Manual of Analytical Methods (NIOSH, 1979, 1990). Table 2. Sampling, preparation and analysis of hexachlorobutadiene Medium Sampling method Analytical method Detection limit Sample size Comments Reference Air adsorption on Chromosorb gas chromatography 360 litre developed for personal Mann et al. 101; extraction by hexane with electron capture sampling in industry (1974) detection Air adsorption on Amberlite gas chromatography 10 µg/m3 3 litre suitable for personal Boyd et al. XAD-2; extraction by with electron capture and area monitoring; (1981); Dillon hexane detection validation range (1979) 10-2000 µg/m3 Air adsorption on Tenax-GC; gas chromatography 11 µg/m3 2 litre suitable for Melcher & purging of water vapour, with flame ionization continuous area Caldecourt oxygen, etc., by nitrogen; detection monitoring (1980) desorption by heating Air adsorption on Tenax-GC; gas chromatography 0.03 µg/m3 a developed for the Krost et al. desorption by heating (capillary column) analysis of ambient (1982); Pellizari under a helium flow; with mass air (1982); Barkley cryofocussing spectro-metric et al. (1980) detection Water extraction by hexane; gas chromatography 0.05 µg/litre 16 litre developed for the Oliver & Nicol concentration; drying with (capillary column) analysis of surface (1982) Na2SO4; clean-up by silica with electron water gel chromatography capture detection Table 2 (contd). Medium Sampling method Analytical method Detection limit Sample size Comments Reference Water extraction by gas chromatography 0.0014 µg/litre 0.8-1 litre US EPA Method Lopez-Avila dichloro-methane-acetone; with electron capture 8120 et al. (1989) drying; concentration detection by N2 stream Water extraction by gas chromatography 0.001 µg/litre 12 litre developed for Zogorski (1984) dichloro-methane; with electron capture monitoring of drying; concentration detection domestic and process waters Water extraction by gas chromatography 0.34 µg/litre 1 litre US EPA Method 612; US EPA (1984a) dichloro-methane; with electron capture developed for the drying; concentration detection analysis of municipal and exchange to and industrial hexane; clean-up by discharges fluorisil chromatography Water extraction by gas chromatography 0.9 µg/litre 1 litre US EPA Method 625; US EPA (1984b) dichloro-methane at pH developed for the >11, then at pH <2; analysis of municipal drying; concentration and industrial discharges Water purging by helium; gas chromatography 0.4 µg/litre 0.1 litre developed for the Otson & Chan trapping; desorption by (capillary column) analysis of volatile (1987); heating with mass organics in waters Eichelberger spectro-metric et al. (1990) detection Table 2 (contd). Medium Sampling method Analytical method Detection limit Sample size Comments Reference Soil, extraction by gas chromatography 0.7 µg/kg Laseter et sediment acetone-benzene with electron capture wet weight al. (1976) detection Soil add water; adjust to pH gas chromatography developed for Kiang & Grob >12; extraction by (capillary column) screening of soil (1986) dichloromethane; with flame ionization for priority centrifugation; drying; and mass pollutants concentration spectro-metric detection Sediment add water; adjust to pH gas chromatography developed for Lopez-Avila et > 11; extraction by (capillary column) screening of al. (1983) dichloromethane; with flame/electron sediment for centrifugation; drying; capture/mass priority pollutants concentration; spectro-metric clean-up by silica detection gel chromatography Sediment extraction by gas chromatography 13 µg/kga 10-15 g dry Oliver & Nicol hexane-acetone; removal (capillary column) weight (1982) of acetone by with electron capture water extraction; drying; detection concentration; clean-up by silica gel chromatography and agitation with mercury Table 2 (contd). Medium Sampling method Analytical method Detection limit Sample size Comments Reference Biota homogenization; filtration; gas chromatography 0.7 µg/kg method applied to Laseter et separation; extraction by with electron capture analysis of fish al. (1976) hexane; clean-up by detection fluorisil chromatography Biota grind and mix edible gas chromatography 0.005 mg/kg 25 g (eggs) wet weight Yurawecz et tissue; extraction; with electron capture wet weight 50 g (fish) wet weight al. (1976) clean-up by fluorisil detection or 0.04 3 g (milk fat) chromatography mg/kg fat 100 g (vegetables) wet weight Biota grinding with Na2SO4; gas chomatography 0.47 µg/kga 15 g method applied to Oliver & Nicol extraction by (capillary column) analysis of fish (1982) hexane-acetone; with electron capture back-extraction of acetone detection by water; concentration; clean-up by silica gel chromatography Biota extraction by gas chromatography 1 µg/kg 2 g method applied to Mes et al. benzene-acetone; with electron capture wet weighta analysis of (1982; 1985; filtration; concentration; detection chlorinated 1986) redissolution in hexane; hydrocarbon residues clean-up including in human adipose fluorisil-silicic tissue and human milk acid chromatography Table 2 (contd). Medium Sampling method Analytical method Detection limit Sample size Comments Reference Biota extraction by hexane gas chromatography 0.0182 µg/litre 100 mg method applied to Kastl & Hermann containing an internal with electron capture whole (rat) blood (1983) standard; centrifugation; detection analysis direct injection a lowest reported level measured The method was validated for the concentration range of 10-2000µg/m3 in 3 litre air samples. The lowest detectable quantity for this method was reported to be 20 ng, the desorption efficiency 98%, and the relative standard deviation 9%. Melcher & Caldecourt (1980) described a gas chromatographic method for the direct determination of organic compounds in air using a collection precolumn from which the compounds are directly injected into the analytical column by rapid heating of the precolumn. The method was reported to be suitable for the analysis of aqueous samples by purging the precolumn following injection of the sample (0.01-0.2 cm3). The analytical method developed for volatile halogenated compounds by Krost et al. (1982) was applied by Pellizari (1982) and Barkley et al. (1980). Barkley et al. (1980) also described the analysis of volatile halogenated compounds in water, blood and urine using a modification of this method: the substances are recovered from water by heating and from biological matrices by heating and purging and are subsequently trapped on a Tenax column. A spectrophotometric method for the determination of hexachlorobutadiene in blood and urine has been reported. The method involves extraction by heptane and determination by either UV spectroscopy or colorimetry after derivatization with pyridine. Reported detection limits were 0.05 mg/litre for the UV method and 5 mg/litre for the colorimetric method (Gauntley et al., 1975). Interference by other chlorinated hydrocarbons can be expected. 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1 Natural occurrence Hexachlorobutadiene has not been reported to occur as a natural product. 3.2 Anthropogenic sources 3.2.1 Production levels and processes The available data are in general of poor quality and not up-to-date. Commercial production of hexachlorobutadiene was reported to occur in Germany and Austria (SRI, 1984). In the USA, commercial production was apparently terminated around 1970 (Mumma & Lawless, 1975). The compound was and is chiefly produced as by-product of the manufacture of chlorinated hydrocarbons, often in association with hexachlorobenzene. In the USA, the manufacture of tetrachloroethene, trichloroethene and carbon tetrachloride accounted in 1972 for over 99% of this production of hexachlorobutadiene in heavy fractions, the so-called Hex-waste, and amounted to 3310-6580 tonnes (Brown et al., 1975; Mumma & Lawless, 1975; Yurawecz et al., 1976; see also section 3.2.3). It was also reported to be a by-product of the manufacture of vinyl chloride, allyl chloride and epichlorohydrin by chlorinolysis processes (Kusz et al., 1984). Hexachlorobutadiene has been identified in the effluents of sewage treatment plants (section 5.2) and as a by-product of the pyrolysis of trichloro-ethene (Yasuhara & Morita, 1990) and plastics (Singh et al., 1982). The annual world production of hexachlorobutadiene in heavy fractions was estimated in 1982 to be 10 000 tonnes (Hutzinger, 1982). No data have been found regarding the amount of hexachlorobutadiene, if any, which is now recovered from this waste. Apart from the possible commercial production of hexachloro-butadiene by recovery from Hex-waste, three pathways for chemical synthesis are known: the chlorination and dehydro-chlorination of hexachlorobutene; the chlorination of polychlorobutanes; and the catalytic chlorination of butadiene (Mumma & Lawless, 1975; CESARS, 1981). There is no evidence, however, that the latter reactions have ever been used commercially. The fraction of hexachlorobutadiene released to the environment during its industrial life cycle (not defined) has been estimated to be between 1 and 3% (SRI, 1984). The fraction of hexachlorobutadiene lost to the environment during its production at a tetrachloroethene manufacturing plant in the USA was estimated to be 1.5% (Brown et al., 1975). Using a simple model describing the troposphere, the global annual emission rate was calculated to be 3000 tonnes of hexachlorobutadiene based on air sampling data of 1985 (Class & Ballschmiter, 1987; see also section 4.2.2). 3.2.2 Uses Hexachlorobutadiene can be used for the recovery of "snift", which is chlorine-containing gas in chlorine plants, and as a wash liquor for removing volatile organic compounds from gas streams. It can be used as a fluid in gyroscopes, as heat transfer, transformer, insulating and hydraulic fluids, and as solvent for elastomers. It can be an intermediate in the manufacture of lubricants and rubber compounds. In the ex-USSR, the substance was reported to find widespread application as a fumigant for treating Phylloxera on grapes, and 600-800 tonnes was used for this purpose in 1975 (Brown et al., 1975; Mumma & Lawless, 1975). 3.2.3 Waste disposal Hex-waste containing hexachlorobutadiene may be destroyed by incineration, placed in landfill, or simply stored. Another procedure involves recycling the compound by catalytic chlorination and subsequent high temperature chlorinolysis to carbon tetrachloride and tetrachloroethene (Markovec & Magee, 1984). 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION 4.1 Transport and distribution between media The main pathways for entry of hexachlorobutadiene into the environment are its emission via industrial waste (section 3.2.3) and following dispersive use (section 3.2.2). The compound may enter surface and ground water, soil and air. In view of its physical properties, intercompartmental transport of hexachloro-butadiene is expected to occur by volatilization and adsorption to suspended particulate matter. Considering the vapour pressure of the compound, i.e. 20 Pa at 20 °C (McConnell et al., 1975), transfer across soil-air boundaries may be significant. Depending on the soil type, adsorption will hinder this transport (see below). In a field study in the ex-USSR, concentrations of hexachlorobutadiene in air above a vineyard were found to be 0.08 and 0.003 mg/m3 at 1 day and 3 months, respectively, following a spring application of 250 kg/ha. The method of analysis was not reported. Volatilization of the compound from light soils was more rapid than from heavy soils (Litvinov & Gorenshtein, 1982). The Henry coefficient of hexachlorobutadiene is 0.43 (1040 Pa.m3.mol-1) at 25 °C (Shen, 1982) and 0.3 at 22 °C Hellmann, 1987a). These values are comparable to those of other chlorinated aliphatic alkenes. They indicate possible transfer of the compound across water-air boundaries leading to a wide distribution, with aerial transport playing a major role (McConnell et al., 1975). In a model experiment, hexachlorobutadiene was allowed to evaporate from a 20-mg/litre aqueous-methanolic solution, containing 10% methanol, in a porcelain basin with slow magnetic stirring at 22 °C. UV spectrophotometry recorded a 25% loss within 28 min. It was shown that methanol decreased the disappearance time. For the transfer of this and other model results to flowing waters, a reduction factor of 30 was proposed for the rate of evaporation on the basis of limited data for two compounds (Hellmann, 1987a). In a model experiment, UV spectrophotometric analysis of solutions of hexachlorobutadiene in deionized water to which 1 g/litre of clay mineral (Fuller's earth) was added revealed a clay-water partition coefficient of 500 litre/kg, showing limited adsorption to pure clay minerals comparable to that of other chlorinated alkenes (Hellmann, 1987b). Based on the log octanol-water partition coefficient (log Kow) of 4.78-4.90 (Table 1), hexa-chlorobutadiene is expected to adsorb strongly to organic matter. The organic carbon-water partition coefficient (Koc) can be estimated to be 25 120 litre/kg on the basis of a log Kow of 4.8 using the semi-empirical equation of Karickhoff (1981). Oliver & Charlton (1984) determined a Koc value of 158 500 litre/kg on the basis of sediment and water concentrations in the Niagara River, USA. Partition coefficients of approximately 200-260 litre/kg were found for two unspecified types of soil in model experiments employing gas chromatographic analysis of solutions of hexa-chlorobutadiene in water (Leeuwangh et al., 1975; Laseter et al., 1976). In field experiments conducted along the Mississippi river in the USA in 1974-1975, some water samples were found to contain 1.0-1.5 µg/litre, whereas levee soil samples at the same sites contained 62-1001 µg/kg dry weight. At a more polluted site near a Hex-waste landfill, water samples contained 0.04-4.6 µg/litre and mud samples 270-2370 µg/kg dry weight. These studies show that soil-water partition coefficients can range over 2 to 4 orders of magnitude assuming equilibrium (Laseter et al., 1976). It can be concluded that the compound does not migrate rapidly in soils and will accumulate in sediment. It should be noted that the micro-particles onto which hexachlorobutadiene is absorbed may themselves migrate in the sub-surface resulting in facilitated transport. The degree of adsorption to soil is highly dependent on the content of organic matter and is less pronounced in sandy soils. On the basis of data for Dutch surface waters, the half-lives of hexachlorobutadiene were estimated to be 3-30 days in rivers and 30-300 days in lakes and ground water. This suggests that turbulence, and therefore increased aerobic biodegradation, volatilization and adsorption, account for the shorter half-lives in river water, that the compound is difficult to degrade both biologically and chemically (see below), and that, overall, the compound is persistent in water (Zoeteman et al., 1980). 4.2 Abiotic degradation 4.2.1 Photolysis Hexachlorobutadiene absorbs light within the solar spectrum. Irradiation of a solution of hexachlorobutadiene in benzene at 254 nm for 15 min resulted in the formation of numerous products having a relative molecular mass greater than that of hexachloro-butadiene itself (Laseter et al., 1976). The extent of mineralization of the compound adsorbed to silica gel and exposed to oxygen was examined following irradiation with ultraviolet light filtered by quartz (wavelength < 290 nm) or by pyrex (simulating tropospheric UV with a wavelength > 290 nm). After 6 days, 50-90% mineralization to hydrogen chloride and/or chlorine, and carbon dioxide was observed (Gb et al., 1977). These experiments indicate that hexachlorobutadiene present as a virtual monolayer on silica gel undergoes quite rapid photolysis. 4.2.2 Photooxidation Using a steady-state mathematical model for the troposphere (describing it as 2 boxes one north one south of the equator) and on the basis of gas chromatographic analysis of air samples from sites far away from anthropogenic sources, the tropospheric lifetime of hexachlorobutadiene was estimated to be 2.3 years for the northern hemisphere and 0.8 years for the southern hemisphere. It was assumed that the reaction with hydroxyl radicals in the troposphere is the main sink for hexachloro-butadiene, by analogy with other halocarbons. The calculated lifetimes at -8 °C correspond to a pseudo-first order rate constant of (2 ± 1) x 10-14 cm3.molecules-1.sec-1 at estimated hydroxyl radical concentrations of 7 x 105 molecules.cm-3 for the northern hemisphere and 17 x 105 for the southern hemisphere (Class & Ballschmiter, 1987). Experimentally, a half-life of 1 week was determined when hexachlorobutadiene was exposed to air in flasks outdoors. This relatively short disappearance time was possibly due to heterogeneous reactions on the vessel walls, as suggested by the authors of the report. Hydrogen chloride was found to be the main degradation product after exposure of samples to xenon arc radiations (wavelength > 290 nm) (Pearson & McConnell, 1975). 4.2.3 Hydrolysis Hexachlorobutadiene is highly resistant to chemical degradation by strong acids and alkalis in the absence of appropriate solvents, although it is readily degraded by ethanolic alkali (Roedig & Bernemann, 1956). Based on the measured hydrolysis rate of the compound in a 1:1 acetone-water mixture, a half-life of over 1800 h was calculated (Hermens et al., 1985). 4.3 Biodegradation Hexachlorobutadiene, at concentrations of 5 or 10 mg/litre, was completely degraded by adapted aerobic microorganisms within 7 days in a static-culture flask screening procedure at 25 °C, as shown by gas chromatography and by determination of total and dissolved organic carbon. The inoculum was taken from settled domestic waste water (Tabak et al., 1981). Approximately 70% adsorption to sludge and 10% degradation was found to occur within 8 days in a pilot low-loaded biological sewage treatment plant (Schröder, 1987). Anaerobic degradation of hexachlorobutadiene at 100 mg/litre was not observed in 48-h batch assays at 37 °C using an inoculum from a laboratory digester (Johnson & Young, 1983). 4.4 Bioaccumulation Considering the low water solubility of 3.2 mg/litre and the high log Kow of 4.78-4.90 (Table 1), a strong bioaccumulating potential would be expected. Both laboratory and field data support this prediction. In flow-through laboratory tests with algae, crustaceans, molluscs and fish in fresh or marine waters, bioconcentration factors (on a wet weight basis) were between 71 and 17 000. The results appear to be highly dependent on the exposure period and there is great variability between organisms (Leeuwangh et al., 1975; Pearson & McConnell, 1975; Laseter et al., 1976; Oliver & Niimi, 1983). Steady state was clearly demonstrated to be reached in only one of these tests. Oliver & Niimi (1983) exposed rainbow trout (Salmo gairdnerii) to aqueous solutions of hexachlorobutadiene at 0.10 and 3.4 ng/litre and found average bioconcentration factors of 5800 and 17 000, steady states having been reached after 69 and 7 days, respectively. When Oligochaete worms were exposed via spiked Lake Ontario sediments to a pore water concentration of 32 ng/litre in a flow-through system, steady state was reached within 4 to 11 days and the average bioconcentration factor was 29 000, based on dry weight of which about 8% is lipid (Oliver, 1987). Biomagnifi-cation, the concentrating of a substance through a food chain, was not observed for hexachlorobutadiene in two limited laboratory experiments with fish fed contaminated food (Pearson & McConnell, 1975; Laseter et al., 1976). The bioaccumulation factors found in plankton, crustaceans, molluscs, insects and fish in surface waters are comparable to those observed in the laboratory: available bioaccumulation factors based on wet weight range between 33 and 11 700 (Goldbach et al., 1976; Laseter et al., 1976). No biomagnification was observed when levels in fish were compared with those of detritus and several invertebrates (Goldbach et al., 1976). The latter was confirmed by a trophodynamic analysis in the Lake Ontario ecosystem (Oliver & Niimi, 1988). Limited bioaccumulation of hexachlorobutadiene was observed in the fat of rats following exposure for 4 to 12 weeks to a mixture of this compound and 1,2,3,4-tetrachlorobenzene, hexachloroben-zene, 1,3,5-trichlorobenzene, o-dichlorobenzene and gamma-hexa-chlorocyclohexane in food (each compound at 2 or 4 mg/kg body weight per day). Fat concentrations of up to 8 mg/kg were observed at the higher dose rates (Jacobs et al., 1974). 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1 Environmental levels 5.1.1 Air Concentrations of hexachlorobutadiene measured in air at different locations are summarized in Table 3. 5.1.2 Water Concentrations of hexachlorobutadiene measured in water at different locations are summarized in Table 4. 5.1.3 Soil and sediment Concentrations of hexachlorobutadiene measured in soil and sediment at different locations are summarized in Table 5. 5.1.4 Biota Concentrations of hexachlorobutadiene measured in aquatic organisms, birds and mammals are summarized in Table 6. 5.2 General population exposure Levels of hexachlorobutadiene encountered in the food and drinking-water of the general population are summarized in Table 7. Hexachlorobutadiene was not detected in the urine or blood of nine individuals living near Old Love Canal, USA, whereas trace levels were found in the breath of one of them (Barkley et al., 1980). In another investigation the compound could not be detected in the blood of 36 Love Canal area residents (Bristol et al., 1982). Hexachlorobutadiene was found at levels of 0.8-4 µg/kg wet weight (fat) and 1.2-13.7 µg/kg wet weight (liver) in postmortem tissues from 6 out of 8 United Kingdom residents in 1970 (McConnell et al., 1975). In the adipose tissue of accident victims in Canada (1976), levels of 1 to 8 µg/kg wet weight were measured in 128 out of 135 samples (Mes et al., 1982, 1985). In Canada (1982), hexachlorobutadiene could not be detected in any of 210 samples of breast milk (Mes et al., 1986). When 15 samples of hazardous waste from incineration facilities in the USA were analysed, 4 sites were found to contain hexa-chlorobutadiene, but the levels were reported to be below 10 mg/kg (Demarini et al., 1987). In sewage sludge, Alberti & Ploger (1986) measured levels of below 1 µg/kg dry weight (3 samples of municipal or municipal/industrial sludge), up to 0.6 µg/kg dry weight (1 sample of municipal/industrial sludge), and 15 µg/kg dry weight (1 sample of industrial sludge). 5.3 Occupational exposure Hexachlorobutadiene levels of 1.6-12.2 mg/m3 air have been measured in the workplace, resulting in reported urine levels of up to 20 mg/litre in workers at the end of the day (German & Viter, 1985). Table 3. Levels of hexachlorobutadiene in environmental air Type of Year Location Detection Levels determineda Reference air limit (ng/m3) (ng/m3) Ambient 1985 Atlantic Ocean, lower 0.0001-0.0004 (r) Class & Ballschmiter troposphere in a south-north 0.0003 (m, north) (1987) cross section, 8 sites 0.0001 (m, south) Urban 1978 USA, Niagara Falls, inside nd (n=9) Barkley et al. (1980) homes near dump site USA, Niagara Falls, outside nd (n=6) homes near dump site trace (n=3) USA, Niagara Falls area nd (n=3) trace (n=1) 50-390 (r, n=2) Urban 1980- USA, 7 cities nd-117 (r,m) Singh et al. (1982) 1981 nd-251 (r) Table 3 (contd). Type of Year Location Detection Levels determineda Reference air limit (ng/m3) (ng/m3) Polluted 1975 USA, 9 sites with chemical nd-460 000 (r) Li et al. (1976)b industries, on plant property USA, 9 sites with chemical nd-22 000 (r) industries, off plant property Polluted 1978 USA, Niagara Falls, household < 45 (n=1) Barkley et al. basement near dump site (1980) Polluted 1978 idem 30-410 (r, n=4) Pellizari (1982) Polluted 1982 USA, liquid waste lagoon 2 nd (n=2) Guzewich et al. 3-160 (n=4) (1983) a nd = not detectable; r = range of individual values; r,m = range of mean values; m = mean; n = number of samples b The highest levels were associated with the production of tetrachloroethene and trichloroethene. At other plants, levels of hexachlorobutadiene remained below 3 ng/m3. Waste holding areas (especially when involving open storage) were often the most significant sources of hexachlorobutadiene, contaminated soil being a secondary source. The total number of samples examined was 405. Table 4. Levels of hexachlorobutadiene in environmental water Type of Year Location Detection limit Levels determineda Reference water (ng/litre) (ng/litre) Surface Canada, Niagara River 50 1.5 Oliver & Nicol (1982) Surface 1982 Canada, Niagara River 0.82 (m, n=5) Oliver & Charlton (1984) Surface 1981-1983 Canada, Niagara River 0.01 0.78 (m, n=104) Oliver & Nicol (1984) 0.67 (median) 0.27-3.2 (r) Surface 1981 Canada, Niagara River nd-0.6 (n=1) Fox et al. (1983) Surface 1972-1973 Netherlands, River IJssel, 50-130 (r, n=5) Goldbach et al. (1976) Ketelmeer, IJsselmeer Surface 1976-1978 Netherlands, River Rhine 1000-2000 Zoeteman et al. (1980) Surface 1975 USA, 9 sites with chemical nd-240 000 (r) Li et al. (1976) industries, on plant property idem, off plant property nd-23 000 (r) Table 4 (contd). Type of Year Location Detection limit Levels determineda Reference water (ng/litre) (ng/litre) Surface 1976 Germany, River Rhine, 865 km 10 10 (50-percentile) Alberti (1983) 180 (90-percentile) 1978 Germany, idem 10 20 (50-percentile) 60 (90-percentile) 1981 Germany, idem 10 < 10 (50-percentile) 40 (90-percentile) 1980-1981 Germany, 4 River Rhine 10 nd tributaries Germany, River Lippe 10 40-200 Surface 1979-1981 Germany, River Rhine, < 50 Haberer et al. (1988) 1979-1981 Germany, River Main < 1000 Surface 1983 Netherlands, River Rhine, River Lek < 100 (m, n=52) Meijers (1988) idem, before dune infiltration 70 (m, n=13) Table 4 (contd). Type of Year Location Detection limit Levels determineda Reference water (ng/litre) (ng/litre) Surface 1984-1985 Germany, River Rhine 10-20 Petersen (1986) Germany, River Elbe 10-150 Estuarine USA, Calcasieu River estuary, 1298 Pereira et al. (1988) vicinity of industrial outfall Sea 1972-1973 United Kingdom, Liverpool Bay 1 4 (m, n=150) Pearson & McConnell nd-30 (r) (1975) Sea 1977 USA, Gulf of Mexico, Sauer (1981) open ocean 1 nd (n=4) coast 1 nd-15 (n=4) Ground Switzerland, aquifer contaminated 200-300 (r) Giger & Schaffner (1981) water by leachate from a chemical waste disposal site a nd = not detectable; r = range of individual values; r,m = range of mean values; m = mean; n = number of samples; x percentile = x percent of samples with values up to that given Table 5. Levels of hexachlorobutadiene in soil and sediment Type of soil Year Location Levels determineda Reference or sediment (µg/kg) Soil, vineyards infected with Phylloxera < 7300 (8 mo) Vorobyeva (1980) agricultural and treated at 250 kg/ha < 2990 (32 mo) Soil 1975 USA, 9 sites with chemical nd-980 000 (r)b Li et al. (1976) industries, on plant property idem, off plant property nd-110 (r)b Sediment 1975 idem, on plant property nd-33 000 (r)b Li et al. (1976) idem, off plant property nd-40 (r)b Sediment, United Kingdom, Liverpool Bay < 1 (n=110) Pearson & McConnell marine > 1 (n=30) (1975) Sediment, Canada, Niagara Falls 18 Oliver & Nicol (1982) river/lake Sediment, 1980 Canada, Lake Ontario nd (n=9) Kaminsky et al. (1983) lake trace (n=3) 8.7 (n=1) Table 5 (contd). Type of soil Year Location Levels determineda Reference or sediment (µg/kg) Sediment, 1981 Canada, Niagara River, downstream 9.6-37 (n=5, dwt)c Fox et al. (1983) river idem, upstream nd (n=1, dwt) Sediment, 1982 Germany, River Rhine, 707 km 0.002 (dwt) Alberti (1983) river idem, 815 km 0.005 (dwt) Sediment, 1981 Canada, Lake Ontario 12-120 (n=5, dwt) lake Sediment, 1968-1978 Canada, Niagara Falls sediment nd Durham & Oliver (1983) lake 1959-1962 core near Niagara River 550 1980-1981 18 1868-1981 nd-550 Sediment, 1980-1982 Canada, lakes 0.04-9.3 (r, n=57) Oliver & Bourbonniere lake 1980 Canada, Lake Huron 0.08 (m, n=9, dwt) (1985) 1982 Canada, Lake St. Clair 7.3 (m, n=2, dwt) 1982 Canada, Lake Erie 0.2-1.6 (r,m, n=46, dwt) Sediment, 1982 Canada, Niagara Falls, settling nd (n=1) Oliver & Charlton (1984) lake particulates at 20 m depth 2.9-11 (r, n=5), 5.9 (m) idem, settling particulates at 68 m depth 7.4 (m) bottom sediment 32 (m, n=12) Table 5 (contd). Type of soil Year Location Levels determineda Reference or sediment (µg/kg) Sediment, lake Canada, Lake Ontario 0.1-75 (r, n=3) Oliver (1984) Sediment, USA, Eagle Harbour, creosote < 0.79 (m, n=15, dwt) Malins et al. (1985) sea harbour contaminated sediment, 3 sites Sediment, USA, President Point, 1 < 2.0 (n=1, dwt) sea harbour reference site Sediment USA, Calcasieu River estuary, 85 (bottom) Pereira et al. (1988) estuarine vicinity of industrial outfall 1.7 (suspended) a dwt = dry weight; nd = not detectable; r = range of individual values; r,m = range of mean values; m = mean; mo = months after treatment; n = number of samples b The highest levels were associated with the production of tetrachloroethene and trichloroethene. Waste holding areas (especially when involving open storage) were often the most significant sources of hexachlorobutadiene, contaminated soil being a secondary source. c surficial sediment; the sediment concentration increased with fraction size d surficial sediment Table 6. Concentrations of hexachlorobutadiene in aquatic organisms, birds and mammals Type of biota Year Location Levels determineda Reference (µg/kg wwt) Detritus (bottom) 1972-1976 Netherlands, surface water 200 Goldbach et al. (1976) Detritus (floating) 220 Invertebrates Plankton 1972-1973 United Kingdom, sea water nd-2.0 Pearson & McConnell (1975) Ragworm, Nereis diversicolor 0.06 Mussel, Mytilus edulis nd-3.8 Crab, Cancer pagarus nd-1.1 Others nd Cerastoderma edule Ostrea edulis Buccinum undatum Crepidula fornicata Carcinus maenus Eupagurus bernhardus Crangon crangon Asterias rubens Solaster sp. Echinus esculentus Table 6 (contd). Type of biota Year Location Levels determineda Reference (µg/kg wwt) Snail 1972-1976 Netherlands, surface water Goldbach et al. (1976) Lymnaea peregra 30, 1670 Clam, Sphaerium sp. 2410 Oligochaetes 0.3 (m, n=3) Oligochaetes 1981 Canada, Lake Ontario nd-37 (dwt) Fox et al. (1983) Amphipods 7.5-62 (dwt) Mysids 6 (dwt) Benthic organisms in 1983-1984 USA, sea water < 5 (dwt) Malins et al. (1985) stomachs of fish Clam, 1982-1983 Canada, Great Lakes area Kauss & Hamdy (1985) E. complanatus nd-83 (r, n=34) Marine algae 1972-1973 United Kingdom, sea water Pearson & McConnell (1975) Enteromorpha compressa nd Ulva lactuca nd Fucus vesiculosis 8.9 Fucus serratus 0.6 Fucus spiralis 0.6 Table 6 (contd). Type of biota Year Location Levels determineda Reference (µg/kg wwt) Fish Ray, 1972-1973 United Kingdom, sea water Pearson & McConnell (1975) Raja clavata (flesh) 0.1-0.4 Raja clavata (liver) 0.2-1.5 Plaice, Pleuronectes platessa (flesh) nd-0.4 Pleuronectes platessa (liver) 0.2-1.2 Dab, Limanda limanda (flesh) < 0.1 Limanda limanda (liver) nd Mackerel, Scomber scombrus (flesh) nd-2.6 Cod, Gadus morrhua (flesh) < 0.1 Gadus morrhua (air bladder) 0.35 Others (liver and/or flesh), nd Platycthus flesus Solea solea Aspitrigla cuculus Trachurus trachurus Trisopterus luscus Table 6 (contd). Type of biota Year Location Levels determineda Reference (µg/kg wwt) Trout, USA, Niagara River, Lake 0.47 Oliver & Nicol (1982) Salmo gairdneri Ontario Trout 1981 Canada, Lake Ontario 1.3 (dwt) Fox et al. (1983) Catfish (flesh) 1973 USA, surface water near trace-4600 Yurawecz et al. (1976) Gaspergoo (flesh) chemical plants manufacturing 200 Buffalo fish (flesh) tetrachloroethene 100 Mullet (flesh) trace Sea trout (flesh) trace Sheepshead minnow (flesh) trace Catfish 1973 USA, < 40 km from tetrachloro- 10-1200 Yip (1976) Carp ethene or trichloroethene 62 Gaspergoo manufacturing plants 12-30 Buffalo fish 120 Whiting 20 Drum 10 Table 6 (contd). Type of biota Year Location Levels determineda Reference (µg/kg wwt) Pike perch, Goldbach et al. (1976) Stizostedion lucioperca 1972-1976 Netherlands, Ketelmeer (lake) 440 (m, n=8) Netherlands, IJsselmeer (lake) 23 (m, n=4) Perch, Perca fluviatilis Netherlands, Ketelmeer 130, 400 (n=2) Pike, Esox lucius Netherlands, Ketelmeer 260 Tench, Tinca tinca Netherlands, Ketelmeer 950 Common bream, Abramis brama Netherlands, Ketelmeer 1520 (m, n=5) Netherlands, IJsselmeer 33 (m, n=5) White bream Blicca bjoerkna Netherlands, Ketelmeer 360 (m, n=3) Roach, Rutilis rutilis Netherlands, Ketelmeer 910 (m, n=10) Netherlands, IJsselmeer 61 (m, n=4) Eel, Anguilla anguilla Netherlands, IJsselmeer 33 (m, n=4) Smelt Osmerus eperlanus Netherlands, IJsselmeer 43 (m, n=3) Table 6 (contd). Type of biota Year Location Levels determineda Reference (µg/kg wwt) English sole (liver) 1983-1984 USA, sea water < 9 (dwt) Malins et al. (1985) English sole (muscle) < 0.2 (dwt) Catfish USA, vicinity of industrial 46 000-120 000 Pereira et al. (1988) outfall in Calcasieu River (lipid base) estuary Atlantic croaker idem, in Calcasieu River 41 000 (lipid base) Blue crab 12 000 (lipid base) Spotted sea trout 15 000 (lipid base) Blue catfish 46 000 (lipid base) Coho salmon 1980 USA, Great Lakes nd (n=31) Clark et al. (1984) trace-10 (r, n=5) Several species 1983 USA, 14 Lake Michigan nd Camanzo et al. (1987) tributaries and embayments Birds Guillemot, 1972-1973 United Kingdom Pearson & McConnell (1975) Uria aalge (eggs) 1.6-9.9 Table 6 (contd). Type of biota Year Location Levels determineda Reference (µg/kg wwt) Swan, Pearson & McConnell (1975) Cygnus olor (liver) 5.2 Cygnus olor (kidney) nd Moorhen, 1972-1973 United Kingdom Pearson & McConnell (1975) Gallinula chloropus (liver) 0.8 Gallinula chloropus (muscle) 2.6 Gallinula chloropus (eggs) nd Others nd Sula bassana (liver, eggs) Phalacrocerax aristotelis (eggs) Alca torda (eggs) Rissa tridactyla (eggs) Anas platyrhyncos (eggs) Mammals 1972-1973 United Kingdom Pearson & McConnell (1975) Grey seal, Halichoerus grypus (blubber) 0.4-3.6 Halichoerus grypus (liver) nd-0.8 Common shrew, Sorex araneus nd a dwt = dry weight; r = range of individual values; m = mean of individueal values; n = number of samples; nd = not detectable; wwt = wet weight Table 7. Levels of hexachlorobutadiene in food and drinking-water Type of food Year Location Levels determineda Reference or drinking-water (µg/kg wwt or µg/litre) Tap water 1978 USA, houses bordering Old nd-trace (r, n=3) Barkley et al. (1980) Love Canal, Niagara Falls 0.06-0.17 (r, n=6) Well water 1978 USA, Tennessee, contaminated nd-2.53 (r, n=28) Clark et al. (1982) by leachate from waste dump 0.15 (m, n=22) Fresh milk United Kingdom 0.08 McConnell et al. (1975) Butter 2 Cheese, eggs nd Meat (3 types) nd Oils/fats (4 out of 5 types) nd Vegetable cooking oil 0.2 Beverages (5 out of 6 types) nd Light ale 0.2 Fruits/vegetables (5 out of 7 types) nd Tomatoes United Kingdom, reclaimed lagoon 0.8 Black grapes United Kingdom, import 3.7 Table 7 (contd). Type of food Year Location Levels determineda Reference or drinking-water (µg/kg wwt or µg/litre) Fresh bread United Kingdom nd Eggs 1973 USA, < 40 km from tetrachloro- nd (n=15) Yip (1976) Milk ethylene or trichloroethylene nd (n=19) manufacturing plants, 6-7 sites 1320 (n=1, fat basis) Vegetables (7 types) nd (n=20) Condensed milk 1975 Germany, Bonn 4 Kotzias et al. (1975) Milk (products) (2 types) nd Eggs (white) nd Eggs (yolk) 42 Meats (4 types) nd Tinned fish (2 types) nd Onion bread nd Chicken feed 39 Chicken meal 2 a nd = not detected; m = mean of individual values; n = number of samples; r = range; wwt = wet weight 6. KINETICS AND METABOLISM 6.1 Absorption and distribution Whole body autoradiography of longitudinal sagittal sections of male rats after administration of a single oral dose of 200 mg uniformly labelled hexachlorobutadiene/kg body weight in corn oil demonstrated that intestinal absorption of the parent compound was virtually complete by 16 h. The radioactivity in the gastrointestinal tract at this point in time was mainly due to water-soluble metabolites, whereas 85% of the radioactivity in the small intestine was still present as unchanged hexachlorobutadiene 4 h after the administration. At all points in time radioactivity levels in the stomach were low compared to those in the intestines. The autoradiogram showed a specific distribution of radioactivity, especially in the outer medulla of the kidney (Nash et al., 1984). Reichert et al. (1985) orally administered 1 or 50 mg of labelled hexachlorobutadiene/kg body weight in tricaprylin to female rats and recovered, at 72 h, approximately 7% of the label in carcass and tissues, mainly liver, brain and kidneys. Most of the label was excreted via urine or faeces within this time period (section 6.4). In mice given 30 mg of labelled hexachlorobutadiene per kg body weight in corn oil, over 85% of the label was excreted within 72 h (section 6.4); 6.7-13.6% was found in the carcass, especially in adipose tissue (Dekant et al., 1988a). This report on mice supports the study by Reichert et al. (1985) on rats with respect to the amount of labelled hexachlorobutadiene absorbed. 6.2 Metabolism The extent of metabolic transformation and the identity of excretion products found in studies with rodents are summarized in Table 8. The available evidence suggests that hexachloro-butadiene is metabolized in a glutathione-dependent reaction to toxic sulfur metabolites. The glutathione- S-conjugate 1-(glutathion-S-yl)- 1,2,3,4,4-pentachloro-1,3-butadiene (GPB) is formed in the liver and excreted with bile. GPB is reabsorbed from the gut both intact and after degradation to 1-(cystein- S-yl)-1,2,3,4,4-pentachloro- 1,3-butadiene (CPB). Finally, these sulfur conjugates and the corresponding mercapturic acid 1-( N-acetylcystein- S-yl)- 1,2,3,4,4-pentachloro-1,3-butadiene (ACPB) are delivered to the kidney. In the kidney, high concentrations of CPB are present due to renal accumulation, enzymes with acylase activity and gamma-glutamyltranspeptidase. CPB is finally cleaved by renal cysteine conjugate ß-lyase to the electrophile trichlorovinyl-chlorothioketene. The renal accumulation of sulfur conjugates and the location of ß-lyase along the nephron (MacFarlane et al., 1989) explain the organ- and site-specific toxicity of hexachlorobutadiene (Lock, 1987a,b; Anders et al., 1987; Dekant et al., 1990a,b; Koob & Dekant, 1991). Table 8. Tracer studies with [14C] hexachlorobutadiene Species Route Dose (mg/kg Medium Metabolitea Fraction of Time after Reference body weight) dose (%) dosing (h) Rat ip 0.1 urine total 29 48 Davis et al. (1980) water-soluble 25 48 faeces total 40 48 300.1 urine total 7 48 water-soluble 6 48 faeces total 7 48 Rat oral 200 urine total 11 120 Nash et al. (1984) PBSA 1 120 non-ether soluble 7 120 faeces total 39 120 Rat oral 1 expired air total 8.9 72 Reichert et al. (1985) HCBD 5.3 72 CO2 3.6 72 urine total 30.6 72 faeces total 42.1 72 50 expired air total 6.6 72 HCBD 5.4 72 Table 8 (contd). Species Route Dose (mg/kg Medium Metabolitea Fraction of Time after Reference body weight) dose (%) dosing (h) CO2 1.2 72 urine total 11.0 72 faeces total 69 72 Rat oral 100 urine total 5.4 24 Reichert et al. (1985); S-containing ca 4.3 24 Reichert & Schutz (1986) ACPB } MTPB } 0.5 24 CMTPB} faeces total 60 72 oral 1 expired air total 7.45 72 C2 2.2 urine total 17.5 faeces & gitb total 61.8 carcass total 10.5 100 expired air total 7.57 72 CO2 0.7 urine total 9.0 faeces & gitb total 72.1 carcass total 5.8 Table 8 (contd). Species Route Dose (mg/kg Medium Metabolitea Fraction of Time after Reference body weight) dose (%) dosing (h) Rat iv 1 expired air total 8.54 72 Payan et al. (1991) CO2 2.6 urine total 21.1 faeces & gitb total 59.3 carcass total 12.9 100 expired air total 8.11 72 CO2 0.9 urine total 9.2 faeces & gitb total 71.5 carcass total 11.1 Mouse oral 30 expired air total = HCBD 4.5 72 Dekant et al. (1988a) urine total 7.2 72 faeces total 72.0 72 HCBD > 57 72 GPB 7.2 72 a For abbreviations see Fig. 1; "total" indicates that no individual chemicals were specified b git = gastrointestinal tract 6.2.1 In vitro studies Incubation of hexachlorobutadiene with rat or mouse liver or kidney subcellular fractions caused a depletion of non-protein sulfhydryl groups, which was not due to oxidation (Kluwe et al., 1981). The formation of GPB and of 1,4-(bis-glutathion- S-yl)- 1,2,3,4-tetrachloro-1,3-butadiene (BGTB) is catalysed by glutathione- S-transferase in rat and mouse liver microsomes and cytosol (Wolf et al., 1984; Wallin et al., 1988; Dekant et al., 1988a,b). GPB formation has also been observed in human liver microsomes and those from several other species (Oesch & Wolf, 1989; McLellen et al., 1989). Conjugation in mouse liver microsomes, but not in those from rat liver, is significantly faster in females than in males (Wolf et al., 1984; Dekant et al., 1988a). GPB formation has also been demonstrated in the isolated perfused rat liver; in this system, GPB formed in the liver was almost exclusively excreted with bile by a carrier-mediated active transport mechanism; only after infusing very high concentrations of hexachlorobutadiene was sinusoidal excretion of GPB into the perfusate observed (Gietl & Anders, 1991). A large number of studies have used GPB, CPB and ACPB to further delineate the fate of hexachlorobutadiene in the organism. These studies have demonstrated that CPB is the penultimate intermediate in hexachlorobutadiene metabolism. CPB is a substrate for renal cysteine conjugate ß-lyase and is metabolized by this enzyme to 2,3,4,4-tetrachlorobutenoic acid and 2,3,4,4-tetrachlorothionobutenoic acid (Dekant et al., 1988a). Trichloro-vinyl-chlorothioketene has been identified as the ultimate reactive intermediate in hexachlorobutadiene metabolism catalysed by ß-lyase (Dekant et al., 1991). ACPB accumulated by the renal organic anion transporter is cleaved to CPB by renal acylases (Vamvakas et al., 1987; Pratt & Lock, 1988). 6.2.2 In vivo studies In in vivo studies, hexachlorobutadiene caused a marked, dose-related depletion of renal nonprotein sulfhydryl (NP-SH) in mice at single intraperitoneal doses of 33-50 mg/kg body weight but little or no decrease in hepatic NP-SH (Kluwe et al., 1981; Lock et al., 1984). This pattern was also observed in female rats at single intraperitoneal doses from 300 mg/kg body weight (Hook et al., 1983). Conversely, the compound caused a marked, dose-related depletion of hepatic NP-SH in male rats from 300 mg/kg body weight intraperitoneally, but no decrease (or even an increase) in renal NP-SH (Kluwe et al., 1981, 1982; Lock & Ishmael, 1981; Baggett & Berndt, 1984). When cannulated male rats were given intravenously either a tracer dose of 0.071 mg radiolabelled hexachlorobutadiene/kg body weight or the same dose at 24 h after an intraperitoneal nephrotoxic dose of 300 mg/kg body weight in corn oil, 13 and 10% of the label was recovered in the bile, respectively, within the 3 h following the tracer dose. The labelled material was completely water soluble (Davis et al., 1980). In a study by Payan et al. (1991), rats with cannulated bile ducts received once, either orally or intravenously, 1 or 100 mg of radiolabelled hexachlorobutadiene/kg body weight. At 72 h after exposure, fractional urinary excretion (7.5% of the dose) was independent of the dose and route of administration, in contrast to the situation in intact rats (see section 6.4). Fractional biliary excretion decreased with increasing dose following oral administration (66.8% versus 58%) and intravenous injection (88.7% versus 72%). Fractional faecal excretion was minimal following intravenous injection (3.1% following the low oral dose and 16.2% following the high oral dose). In a group of bile duct-duodenum cannula-linked rats given one dose of 100 mg/kg body weight, all tissue concentrations (kidney, liver, plasma, carcass) and the urinary excretions at 30 h after dosing were higher in bile donor rats than in recipient rats. The biliary contribution to both urinary and tissue concentrations was calculated to be 40%. Of the biliary metabolites entering the recipients, 80% was found to be reabsorbed. Nash et al. (1984) administered 200 mg labelled hexachloro-butadiene in corn oil/kg body weight to male rats with exteriorized bile flow. They recovered 35% of the label in the bile during the 48 h following treatment, 40% of which was identified as GPB (Fig. 1) and 12% as CPB. In another investigation into the identity of biliary excretion products, male rats were given intravenously an aqueous suspension of 0.026 mg of labelled hexachlorobutadiene. During the next two hours over 30% of the label was recovered in bile; 35% of this radioactivity was identified as GPB and 6% as BGTB (Fig. 1), but the remaining labelled material was not identified. Since some of the unidentified peaks disappeared after treatment of bile with inhibitors of gamma-glutamyltranspeptidase, they probably represent degradation products of GPB and BGTP (Jones et al., 1985). The intestinal absorption of GPB and CPB was studied in rats by infusing the compounds into the intestine via a biliary cannula. When GPB was infused, both GPB and CPB were found in the blood in approximately equal concentrations. Higher blood CPB concentrations were found after CPB infusion than after GPB infusion (Gietl et al., 1991). In studies with radiolabelled hexachlorobutadiene, several urinary metabolites were identified. The structure of these metabolites further supported the hypothesis that hexachloro-butadiene is bioactivated by glutathione conjugation. ACPB was found to be the main metabolite (representing approximately 80% of the radioactivity present in urine) excreted after the administration of [14C] hexachlorobutadiene (200 mg/kg) in female rats (Reichert & Schütz, 1986). The same authors also identified 1-carboxymethylthion-1,2,3,4,4-pentachlorobuta-1,3-diene and 1-methylthio-1,2,3,4,4-pentachloro-1,3-butadiene (MTPB) as urinary metabolites (Reichert et al., 1985). It is the opinion of the Task Group that the identification of MTPB by diazomethane treatment of the urinary extract is questionable. In male rats, 1,2,3,4,4-pentachloro-1,3-butadienyl sulfenic acid (PBSA) is the only metabolite excreted in urine that has so far been identified. The data presented suggest that ACPB is not a major urinary metabolite of hexachlorobutadiene in male rats (Nash et al., 1984). In urine of mice exposed to radiolabelled hexachlorobutadiene (30 mg/kg), CPB, ACPB and 2,3,4,4-tetra-chlorobutenoic acid were identified as urinary metabolites (Dekant et al., 1988a). It is probable that 2,3,4,4-tetrachlorobutenoic acid is formed by reaction of the intermediate thioketene with water and further hydrolysis of the thionol acid thus formed (Dekant et al., 1988a). The weight of evidence suggests that oxidative reactions involving cytochrome P-450 have little role in the metabolism of hexachlorobutadiene (Wolf et al., 1984; Dekant et al., 1988a). 6.3 Reaction with body components The covalent binding of [14C]-hexachlorobutadiene-related radioactivity to tissue proteins has been shown to be time dependent, with the highest level occurring during the first 6 h after treatment. The half-life of hexachlorobutadiene binding was 22 h in both liver and kidney (Reichert, 1983; Reichert et al., 1985). In a DNA binding study, rats received a single oral dose of 20 mg [14C]-hexachlorobutadiene, and DNA was isolated from the kidneys of these rats at 6, 18.5 and 30 h after dose administration. Although the results have been reported only in summary form, various levels of radioactivity were recovered with the DNA, but there was a marked variation in the level of radioactivity between samples. Furthermore complete analysis of the DNA was not performed and protein may have been associated with the DNA (Stott et al., 1981). Covalent binding to mouse liver and kidney DNA was demonstrated after the oral administration of radiolabelled hexachlorobutadiene (30 mg/kg body weight) in corn oil (Schrenk & Dekant, 1989). In the liver and kidneys, the binding capacity of mitochondrial DNA was significantly higher than that of nuclear DNA. The level of binding to nuclear DNA in the liver was indistinguishable from that of controls. HPLC separation of the hydrolysed DNA indicated the presence of three distinct peaks of radioactivity. 6.4 Excretion Following oral administration in rats and mice of single doses of hexachlorobutadiene up to 100 mg/kg body weight, the total excretion within 72 h was at least 65% of the dose. In mice, less than half of a dose of 30 mg/kg body weight was metabolized (Dekant et al., 1988a). In rats, assuming that the faeces mostly contain unchanged compound and no non-resorbed conjugates, 44% of an orally administered low dose of hexachlorobutadiene (1 mg/kg body weight) was metabolized (Reichert et al., 1985). At higher doses the percentage of hexachlorobutadiene metabolized decreased dramatically. The biotransformation of hexachloro-butadiene in rats appears to be a saturable process considering the reduced excretion of carbon dioxide and renal metabolites at increasing doses (Davis et al., 1980; Reichert et al., 1985; Reichert & Scuhtz, 1986; Payan et al., 1991). This could be explained by saturation of the gastrointestinal absorption, which was observed by Reichert et al. (1985). It should be noted, however, that the observed increase in the amount of unchanged hexachloro-butadiene in faeces with increasing dose applies only up to 100 mg/kg body weight. At higher dose levels, the amount of unchanged hexachlorobutadiene in faeces decreases, probably due to a decrease in faecal output (Davis et al., 1980; Nash et al., 1984). The results of studies of Payan et al. (1991) on bile-duct cannulated (see section 6.2.2) and intact (see Table 8) rats show that saturation of gastrointestinal absorption indeed occurs following oral administration. Pharmacokinetic data concerning the fate of hexachloro-butadiene in organisms were not available to the Task Group. 7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT 7.1 Aquatic organisms 7.1.1 Short-term toxicity A summary of short-term aquatic toxicity data is presented in Table 9. In most of these studies the concentration of hexachloro-butadiene was not reported. Therefore, the actual effect concentrations may be lower than the nominal ones. In several cases these nominal values far exceed the solubility limits. Based on these data the substance is moderately to highly toxic to aquatic organisms (Canton et al., 1990). Adverse effects reported in some of these acute tests included loss of equilibrium, erratic swimming (Leeuwangh et al., 1975; Laseter et al., 1976), decreased activity, increased rate of opercular movement, jumping (Leeuwangh et al., 1975), inverted positions, fin fibrillation and muscle tetany (Laseter et al., 1976). In a special investigation into kidney pathology, groups of five goldfish (Carassius auratus) received one intraperitoneal injection of hexachlorobutadiene at a dose level of 500 mg/kg body weight in corn oil. They were subsequently fasted for up to 6 days and sacrificed at different points in time up to day 7. Controls received corn oil only. The temperature was kept between 18 and 21 °C. By 24 h the fish showed decreased activity, swam with dorsal fins down, and had difficulty in following food. By day 4 exophthalmos, distended abdomen and ascites were observed. These signs of toxicity were all reversible. Relative kidney weights were elevated on day 4 only. From 12 h after exposure, P2 and P3 renal epithelial cells exhibited marked vacuolation and necrosis, which persisted up to day 7. Increased gamma-glutamyl-transferase (EC 22.214.171.124) staining was seen in P2 and P3 segments (Reimschüssel et al., 1989). In a report of this experiment, the fish were sacrificed at different points in time up to day 70 after exposure. In one of the two experiments the fish also received 5-bromo-2'-deoxyuridine 4 h prior to sacrifice. Morphometric analysis of developing nephrons showed an increase in the percentage of volume occupied by basophilia clusters and developing nephrons from day 14 onwards. The apparent number of basophilic clusters and developing nephrons per unit surface area was also increased from day 14 (Reimschüssel et al., 1990). Table 9. Short-term aquatic toxicity of hexachlorobutadiene Organisms Species Temperature pH Dissolved Hardness Stat/flowa Exposure Parameter Concentration Reference (°C) oxygen (mg CaCO3 open/closed period (mg/litre) (mg/litre) per litre) (h) Fresh water Algae Haematococcus 20 stat, closed 24 EC10b > 2 Knie et al. pluvialis (1983) Bacteria Pseudomonas putida 25 7.0 stat, closed 16 TTc > 25 Bringmann & Kuhn (1977) Bacteria Pseudomonas putida 20 7.2 stat, open 0.5 EC10b > 0.9 Knie et al. (1983) Protozoans Chilomonas 20 6.9 stat, closed 48 TTc > 10 Bringmann et al. paramecium (1980) Molluscs great pond snail, 19 stat,d closed 24 LC50 0.21 Leeuwangh et Lymnaea stagnalis 96 LC50 0.21 al. (1975)e Crustaceans water flea, 20 7 250 stat, open 24 EC50 0.5 Knie et al. Daphnia magna (1983) aquatic sowbug, 19 stat,d closed 96 LC50 0.13 Leeuwangh et Asellus aquaticus al. (1975)e Table 9 (contd). Organisms Species Temperature pH Dissolved Hardness Stat/flowa Exposure Parameter Concentration Reference (°C) oxygen (mg CaCO3 open/closed period (mg/litre) (mg/litre) per litre) (h) Fish goldfish, 17.5 stat,d open 96 LC50 0.09 Leeuwangh et Carassius auratus al. (1975)e Fish zebrafish, 20 8.0 9.0 180 flow, closed 48 LC50 1 Slooff (1979) Brachydanio rerio Fish rainbow trout, LC50 0.320 US EPA (1980) Salmo gairdnerii Fish bluegill sunfish, LC50 0.326 US EPA (1980) Lepomis macrochirus Fish sheepshead minnow, LC50 0.557 US EPA (1980) Cyprinodon variegatus Fish golden orfe, 20 8 270 open 48 LC50 3 Knie et al. Leuciscus idus (1983)e Fish fathead minnow, 25 6.7-7.6 8.0 45 flow, open 96 LC50 0.10 Walbridge et Pimephales promelas al. (1983)e Table 9 (contd). Organisms Species Temperature pH Dissolved Hardness Stat/flowa Exposure Parameter Concentration Reference (°C) oxygen (mg CaCO3 open/closed period (mg/litre) (mg/litre) per litre) (h) Marine Crustaceans harpacticoid 21 7.9 > 5 stat, open 96 LC50 1.2 Bengtsson & copepod Tarkpea (1983) grass shrimp, LC50 0.032 US EPA (1980) Palaemonetes pugio Mysid shrimp, LC50 0.059 US EPA (1980) Mysidopsis bahia Fish sailfin molly, 22-24 6.6-7.9 8-9 flow, open 26 LC50 4.2 Laseter et Poecilia latipinna 30 LC50 4.5 al. (1976)e,f 77 LC50 1.4-1.9 115 LC50 1.7 138 LC50 1.2 pinfish, Lagodon LC50 0.399 US EPA (1980) rhomboides a static or flow-through test, open or closed system d semi-static (daily renewal) test b effect is 10% reduction in oxygen consumption e analysis for hexachlorobutadiene was reported c TT = toxic threshold for inhibition of cell multiplication f salinity was 0.25%, 96-h LC50 was calculated to be 1.6 mg/litre 7.1.2 Long-term toxicity The cell multiplication of green algae (Scenedesmus quadricauda) was not inhibited after 8 days of static exposure to a nominal concentration of 25 mg/litre (well above pure water solubility) in a closed system at 27 °C and a pH of 7 (Bringmann & Kühn, 1977). A 14-day LC50 of 0.4 mg/litre was determined for 2- to 3-month old guppies (Poecilia reticulata) in a semi-static test using an open system at 22 °C, a water hardness of 25 mg CaCO3/litre, and a dissolved oxygen concentration of > 5 mg/litre. No analysis for hexachlorobutadiene was reported (Könemann, 1981). In the same test under the same conditions, but with analysis for the compound, the 14-day LC50 was 0.16 mg/litre (Hermens et al., 1985). In a study by Leeuwangh et al. (1975), groups of six goldfish (Carassius auratus) were each exposed to hexachlorobutadiene in tap water at measured concentrations of 0, 0.0003, 0.003, 0.0096 or 0.03 mg/litre for 49 and 67 days. The static test in an open system was conducted at 19 °C, a pH of 7.6, and a dissolved oxygen concentration between 3.2 and 6.3 mg/litre. Body weights were decreased after 49 days at 0.03 mg/litre, and body weight gain was still reduced at 67 days. Abnormal behaviour, jumping, incoordination, increased opercular movement and overall immobility were noted at 0.0096 mg/litre. After 49 days at 0.0096 mg/litre (no data at 0.03 mg/litre), relative liver weights were increased, and the activity of liver glucose-6-phosphatase (EC 126.96.36.199) was decreased, whereas the activity of liver glucose-6-phosphate dehydrogenase (EC 188.8.131.52) was increased. After 67 days the activity of liver phenylalanine hydroxylase (EC 184.108.40.206) showed a concentration-related increase. No effects were found on haemoglobin concentration and haematocrit after 49 days or on the activity of serum alanine aminotransferase (EC 220.127.116.11) and serum alkaline phosphatase (EC 18.104.22.168) after 67 days. Groups of 12 largemouth bass (Micropterus salmoides) were each exposed to hexachlorobutadiene for 10 days at measured concentrations of 0.00343 and 0.03195 mg/litre in filtered tap water with a salinity of 0.08-0.1%, at 22-24 °C, a pH of 6.6-7.9 and a dissolved oxygen concentration of 7.6-8.5 mg/litre. A control group comprised 12 water and 12 vehicle (acetone) controls. Plasma cortisol levels were increased at both concentrations, but haematocrit values were not affected. At the higher concentration there was leukocytic infiltration in the kidneys of one of the fish and paleness and accentuated lobulation of parenchyma in the livers (Laseter et al., 1976). In an early lifestage test, four replicate groups, each of 30 fathead minnow (Pimephales promelas) eggs, 2-4 h after spawning, were exposed to measured hexachlorobutadiene concentrations in sand-filtered and sterilized lake water of 0.0017, 0.0032, 0.0065, 0.013 and 0.017 mg/litre in an open system. Following hatching (4-5 days after spawning), four replicate groups of 15 larvae continued to be exposed for 28 days. Control groups of equal size were exposed to slightly contaminated water containing 0.00008 mg/litre. The temperature was 25 °C, pH was 7.4, dissolved oxygen concentration 7 mg/litre, and water hardness 45 mg CaCO3/litre. The hatchability of embryos and the percentage of normal larvae at hatch were not affected. An increased fish mortality and a concentration-related decrease of body weight were observed at the two highest concentrations at the end of the exposure period (Benoit et al., 1982). 7.2 Terrestrial organisms 7.2.1 Short-term toxicity Except for one test with birds, reliable tests with terrestrial organisms have not been reported. Groups of 12 female and four male Japanese quails (Coturnix coturnix japonica) were exposed to a diet containing hexachloro-butadiene at levels of 0, 0.3, 3, 10 or 30 mg/kg diet for 90 days. Each cage contained three females and one male of the same dose group. Feed analysis indicated levels close to the nominal values. Adults were all histopathologically examined. Eggs were collected on days 37-46, 64-73, and 81-90. Six adults died during the study: 4 at 0.3 mg/kg, 1 at 10 mg/kg, and 1 at 30 mg/kg, but this was not considered to be related to treatment. The survival of chicks from eggs collected on days 81-90 was decreased at 10 mg/kg only. Egg production, the percentage of fertile eggs, the percentage of hatchable eggs, and eggshell thickness were unaffected compared to controls (Schwetz et al., 1974). 8. EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO TEST SYSTEMS 8.1 Single exposure The available mortality data are summarized in Table 10. 8.1.1 Inhalation exposure 22.214.171.124 Mortality In the only reported study of the mortality of hexachloro-butadiene following inhalation, groups of 20 SPF mice of the OF1 strain were exposed for 6 h (Gage, 1970). The vapour concentrations were measured by gas chromatography, and the concentrations were within 10% of the nominal value. Results are shown in Table 10. 126.96.36.199 Systemic effects The decrease in respiratory rate (reflex bradypnoea) in groups of six male Swiss OF1 mice was measured following exposure for 15 min to hexachlorobutadiene vapour at concentrations between 886 and 2625 mg/m3. The vapour concentrations were checked by gas chromatography. The mice were restrained in a body plethysmograph, while the heads were extended into an inhalation chamber. The 50% effect level, calculated from the concentration-effect curve, was 2250 mg/m3 (De Ceaurriz et al., 1988). 8.1.2 Oral exposure 188.8.131.52 Mortality Based on acute mortality data, hexachlorobutadiene is slightly to moderately toxic to adult rats, moderately toxic to male weanling rats, and highly toxic to female weanling rats following ingestion. Young rats are far more sensitive than adult rats (Kociba et al., 1977a). Gradiski et al. (1975) observed delayed mortality (after 24 h) in oral LD50 studies on rats and mice. 184.108.40.206 Systemic effects Hexachlorobutadiene mainly affects the kidneys and, to a lesser extent, the liver. The effects on these organs and the related biochemical findings are discussed extensively in sections 8.8.2 (liver) and 8.8.3 (kidney). In oral LD50 studies on rats and mice, Gradiski et al. (1975) observed hyper-reactivity just after exposure, followed by decreased activity and staggering. Table 10. Mortality of hexachlorobutadiene from single exposure Species/strain Age Sex Route of exposure Observation LD50 (mg/kg Reference period (days) body weight) or LC50 (mg/m3)a Mouse oral 87 (78.1-95.9) Murzakayev (1963)b OF1 adult female inhalation (6 h) 14 107 (102-113) Gage (1970) OF1 adult female oral 14 65 (60-70) Gradiski et al. (1975)c OF1 adult male oral 14 80 (75-85) Gradiski et al. (1975)c Alderley Park adult male intraperitoneal 14 67 (53-85) Lock et al. (1984)e Alderley Park adult female intraperitoneal 14 85 (65-111) C57BL/10J adult male intraperitoneal 14 57 (41-81) C3H adult male intraperitoneal 14 25-75 BALB/c adult male intraperitoneal 14 32-40 DBA/2J adult male intraperitoneal 14 53 (36-76) Rat oral 350 (323-377) Murzakayev (1963)b OF2 rat adult female oral 14 270 (250-290) Gradiski et al. (1975)c OF2 rat adult male oral 14 250 (220-280) Gradiski et al. (1975)c Sprague-Dawley adult female oral 200-400 Kociba et al. (1977a)d Sprague-Dawley adult male oral 580 (504-667) Kociba et al. (1977a)d Sprague-Dawley weanling female oral 46 (26-81) Kociba et al. (1977a)d Sprague-Dawley weanling male oral 65 (46-91) Kociba et al. (1977a)d Table 10 (contd). Species/strain Age Sex Route of exposure Observation LD50 (mg/kg Reference period (days) body weight) or LC50 (mg/m3)a Alderley Park weanling male intraperitoneal 7 57 (38-87) Hook et al. (1983)e Alderley Park 29 days male intraperitoneal 7 96 (72-128) Alderley Park adult male intraperitoneal 7 360 (325-396) Guinea-pig oral 90 (81.5-98.5) Murzakayev (1963)b Rabbit New Zealand adult female dermal (8 h) 14 1120 (890-1400) Duprat & Gradiski (1978)f a When available, 95% confidence limits are reported between brackets. b Observation period, strain, sex, age (or body weight) and vehicle were not reported. c Vehicle was olive oil. d Confidence limits not calculable; observation until no toxicity was observed any longer; vehicle was corn oil. e Vehicle was corn oil. f Application undiluted using glass vials (3.6 cm2). 8.1.3 Dermal exposure 220.127.116.11 Mortality Hexachlorobutadiene was harmful to rabbits following acute dermal exposure (LD50 = 1120 mg/kg body weight; (range, 890-1400 mg/kg; Table 10). After a dose of 780 mg/kg body weight, death occurred within 24 h from respiratory and cardiac failure (Duprat & Gradiski, 1978). 18.104.22.168 Systemic effects New Zealand rabbits dermally exposed to undiluted hexa-chlorobutadiene at doses of 1170 and 1550 mg/kg body weight exhibited stupor, dyspnoea and cyanosis (Duprat & Gradiski, 1978). 8.1.4 Other routes of exposure Hexachlorobutadiene has been shown to be toxic to various strains of mice after intraperitoneal injection (Lock et al., 1984) and harmful to adult rats (Hook et al., 1983). The compound was considerably more toxic to young and weanling rats (Table 10). Rats intraperitoneally exposed to single lethal doses of hexachlorobutadiene from 500 to 1000 mg/kg in corn oil exhibited piloerection, sedation, hunching, incoordination, loss of muscle tone and hypothermia (Lock & Ishmael, 1979). A macroscopic and haematological investigation of rats intraperitoneally exposed to doses between 121 and 336 mg/kg body weight in olive oil did not reveal any damage to the gastrointestinal tract, spleen, heart or gonads. In the lungs, congestion, haemorrhage, and oedema were observed, but these were attributed by the authors to ether anaesthesia. At dose levels of 213 mg/kg body weight or more, lymphopenia and related neutrophilia were induced (Gradiski et al., 1975). 8.2 Short-term exposure 8.2.1 Inhalation exposure In the only inhalation study published, groups of four adult Alderley Park SPF rats of each sex were dynamically exposed to nominal concentrations of 53, 107 or 267 mg/m3 6 h/day for 15 days, 1067 mg/m3 6 h/day for 12 days, or 2668 mg/m3 4 h/day for 2 days. Petroleum ether was used as a solvent for concentrations below 1067 mg/m3. Many other chemicals were tested similarly, and batches of control rats of unknown size were maintained at intervals of 2 months during the whole experimental period. No analysis for hexachlorobutadiene was carried out. Two of the four female rats exposed to 1067 mg/m3 died, and autopsy revealed pale, enlarged kidneys, adrenal regeneration and renal cortical tubular degeneration with epithelial regeneration. Rats of both sexes lost weight at 1067 mg/m3 and the weight gain of females was reduced at 107 and 267 mg/m3. Irritation of eyes and nose was observed at the two highest levels. At 267 and 1067 mg/m3 rats were in a poor condition, females being more affected than males. Respiratory difficulties were seen at and above 267 mg/m3. Haematological examination at the end of the exposure period showed slight anaemia in females at 1067 mg/m3. Urinalysis did not reveal abnormalities at any of the exposure levels. Macroscopically enlarged, pale kidneys were found at 267 and 1067 mg/m3 and enlarged adrenals at 1067 mg/m3. Histopatho-logical investigations revealed proximal tubular degeneration in the kidneys and cortical degeneration in adrenals at concentrations of 267 mg/m3 or more. No toxic signs were observed at the lowest exposure level and autopsy revealed no gross abnormalities (Gage, 1970). 8.2.2 Oral exposure 22.214.171.124 Rats Groups of five adult male Sprague-Dawley rats were exposed to daily oral doses of hexachlorobutadiene (0, 0.2 or 20 mg/kg body weight) in corn oil for 3 weeks. Only the kidneys were examined histologically. At the higher dose level, body weight gain was decreased and relative kidney weight increased. Histopathological examination of the kidneys revealed damage in the middle and inner cortical region, including loss of cytoplasm, nuclear pyknosis, increased basophilia and mitotic activity, and increased cellular debris. No toxic signs were observed at a dosage of 0.2 mg/kg per day (Stott et al., 1981). In a study by Kociba et al. (1971), groups of four female Sprague-Dawley rats consumed for 30 days a diet containing hexachlorobutadiene, which resulted in ingested nominal daily doses of 0, 1, 3, 10, 30, 65 and 100 mg/kg body weight. Analysis of the compound in the feed was not reported. Body weights were decreased at the two highest dose levels. At 10 mg/kg or more, a dose-related decrease in body weight gain was observed along with a decrease in food consumption. There was also an increase in haemoglobin concentrations, which, although significant, was not clearly dose related. There was no effect on serum alanine aminotransferase activity (EC 126.96.36.199). A dose-related increase in relative kidney weight was observed at dose levels of 3 mg/kg or more. Histopathological examination, which was restricted to liver and kidneys, showed proximal tubular degeneration, individual cell necrosis, and regenerative changes in the kidneys at doses of 10 mg/kg or more. Hepatocellular swelling was seen at 100 mg/kg. The no-observed-adverse-effect level (NOAEL) was 1 mg/kg body weight per day. In a 2-week experiment, groups of six weanling Wistar-derived rats of each sex were exposed to measured dietary hexachlorobutadiene levels of 0, 73, 182 or 447 mg/kg (the Task Group considered this equivalent to doses of 0, 7.3, 18.2 and 44.7 mg/kg body weight per day, respectively). At all dose levels, body weight and food conversion efficiency (g of weight gain/g of food) were decreased in a dose-related manner. Food consumption per g of body weight was decreased at 44.7 mg/kg body weight. Relative kidney weights were increased at the two highest dose levels. At all dose levels a dose-related degeneration of kidney tubular cells was observed, especially in the straight limbs of the proximal tubules located in the outer medulla. No toxic signs were observed in the liver. A NOAEL was not found (Harleman & Seinen, 1979). In a follow-up to the dietary study, groups of 10 weanling Wistar-derived rats of each sex received daily doses by gavage of 0, 0.4, 1, 2.5, 6.3 or 15.6 mg/kg body weight in peanut oil for 13 weeks (Harleman & Seinen, 1979). Body weight gain, food consumption and food utilization efficiency were decreased at 6.3 and 15.6 mg/kg. Polyuria was observed in females at these dose levels after week 10, while a dose-related decrease in urine osmolarity occurred at dose levels of 2.5 mg/kg or more. In males, the latter effect was observed at 15.6 mg/kg. No other changes were observed in urinalysis (after week 10) and haematological investigations (after week 8). A dose-related increase in relative kidney weight was measured in males of all treatment groups but only at 6.3 mg/kg or more in females. Dose-related increases in the relative weight of liver and spleen were measured at 6.3 mg/kg or more. Histopathological examinations revealed changes in liver and kidneys. In the livers of males dosed with 6.3 mg/kg or more, an increased basophilic, flocky granulation was observed. At dose levels of 6.3 mg/kg or more in males and 2.5 mg/kg or more in females, there was a dose-related degeneration of the renal proximal tubules, as shown by hyperchromatic nuclei, hypercellularity, vacuolation and focal necrosis of epithelial cells and a diminished brush border. No adverse effects were observed at daily doses of 1 mg/kg in females or 2.5 mg/kg in males (Harleman & Seinen, 1979). 188.8.131.52 Mice In a two-week study, groups of five B6C3F1 mice of each sex were fed a diet containing hexachlorobutadiene at nominal doses of 0, 30, 100, 300, 1000 or 3000 mg/kg feed for 15 days (calculated by the Task Group to be equivalent to 0, 4.3, 14.3, 43, 143 and 430 mg/kg body weight per day, respectively, using standard values for average body weight and food consumption in mice). Analysis of the feed was carried out by gas chromatography, and no more than 9% loss of the chemical was observed in one day (feed was replaced every 2 days). All mice given 143 or 430 mg/kg body weight died or were sacrificed in a moribund condition within 7 days. A dose-related growth retardation was observed. At the two highest doses, the observed toxic effects included renal tubular necrosis, hepatic cytoplasmic vacuolization, and testicular degeneration characterized by the presence of syncytial giant cell formation of spermatocytes. At dose levels of 43 mg/kg body weight or more, minimal to mild depletion of bone marrow (characterized by a decrease in the haematopoietic cells) was seen in two out of five mice of both sexes per dose group. At dose levels of 4.3, 14.3 and 43 mg/kg body weight, at which all animals survived to the end of the study, renal tubular cell regeneration was observed (Yang et al., 1989; Yang, 1991). In a 13-week study, groups of 10 B6C3F1 mice of each sex were fed a diet containing hexachlorobutadiene at concentrations of 0, 1, 3, 10, 30 or 100 mg/kg feed. Using measurements of food consumption and body weight, the authors determined doses of 0, 0.1, 0.4, 1.5, 4.9 or 16.8 mg/kg body weight per day for males, and 0, 0.2, 0.5, 1.8, 4.5 or 19.2 mg/kg body weight per day for females. Analysis of the compound was carried out as in the two-week study. No treatment-related clinical signs or deaths were observed. The motility of sperm from treated mice was significantly lower than in controls, although this effect was not dose related (Yang et al., 1989; Yang, 1991). Body weight gain was reduced at dose levels of 4.5 and 19.2 mg/kg body weight in females. Reductions in kidney weight occurred at dose levels of 1.5 mg/kg or more in males and at 19.2 mg/kg body weight in females, and reductions in heart weight occurred at 19.2 mg/kg body weight in males. Necropsy revealed a treatment-related increase in renal tubular regeneration (prominent in the outer stripe of the medulla) at dose levels of 0.2 mg/kg body weight or more in females (Table 11). Although the author concluded that a NOAEL was not observed for females, the Task Group noted that the occurrence of renal tubular regeneration in one out of ten female mice in the 0.2-mg/kg body weight group is insufficient evidence of an adverse effect at this dose level in females. Table 11. Incidences of renal tubular regeneration in 13-week feed studies on B6C3F1 micea Concentration Dose (mg/kg body Number of mice with (mg/kg feed) weight per day) lesions/number examined Male Female Male Female 0 0 0 0/10 0/10 1 0.1 0.2 0/10 1/10 3 0.4 0.5 0/10 9/10 10 1.5 1.8 0/9 10/10 30 4.9 4.5 10/10 10/10 100 16.8 19.2 10/10 10/10 a Modified from: Yang (1991) 8.3 Long-term exposure No long-term inhalation studies have been reported. A study describing long-term exposure of mice by the dermal route is presented in section 8.7.3. An oral toxicity/carcinogenicity test in rats has been reported (see section 8.7.2). 8.4 Skin and eye irritation; sensitization 8.4.1 Irritation The vapour of hexachlorobutadiene has been found to be irritating to the eyes and nose of rats (Gage, 1970; see section 8.2.1). Groups of six New Zealand rabbits received either 0.78 g (0.5 ml) of undiluted hexachlorobutadiene on the intact or abraded skin for 24 h, or 0.15 g (0.1 ml) in the conjuctival sac of the left eye. Assessment of the degree of irritation was conducted according to Draize et al. (1944) and by calculating the primary irritation index. Hexachlorobutadiene was moderately irritating for the skin (primary irritation index 4) but not irritating for the eyes (primary irritating index 1.5). Moderate conjunctivitis, epithelial abrasion and, at day 7, epithelial keratitis were observed in the eyes (Duprat et al., 1976). Duprat & Gradiski (1978) applied undiluted hexachloro-butadiene to New Zealand rabbits at doses of 0.39, 0.78, 1.17 and 1.55 mg/kg body weight (0.25, 0.50, 0.75 and 1.00 ml, respectively) under occluded conditions, using glass vials, for 8 h. The observation period was 14 days. The skin was histopathologically examined in all dead animals, in half the survivors at day 15, and in the remaining survivors at day 36. After 12 h of exposure to the two highest doses, epidermis and subcutaneous tissue revealed oedema and polymorphonuclear leukocyte infiltration. In the epidermal cells, degeneration with pyknosis of nuclei and perinuclear oedema, and focal separation from the corium with vesicle formation were seen. After 3 to 5 days of exposure to the three highest doses, dermal necrosis was observed, leading to eschar formation and partial destruction of hair follicles. The effects increased with time, not with dose. Two to five weeks after application, repair was apparent at all dose levels, with scarring and upper dermis fibrosis, and epidermal acanthosis with focal dyskeratosis. Diffuse mononuclear infiltrate was seen in the dermis. 8.4.2 Sensitization A group of 20 Hartley guinea-pigs were treated according to the Magnusson-Kligman protocol by intradermal injections of 5% hexachlorobutadiene in peanut oil and, after one week and subsequent treatment by sodium lauryl sulfate, by a 48-h dermal application of a 25% suspension of the chemical in vaseline. The challenge was performed by dermal application of a 20% suspension in vaseline. A group of five controls was induced similarly and challenged by vaseline only. All exposed animals, but none of the controls, showed a positive reaction. The test was repeated in the same fashion without adjuvant in five guinea-pigs: all animals showed positive reactions (Gradiski et al., 1975). 8.5 Reproduction, embryotoxicity and teratogenicity 8.5.1 Reproduction A group of female albino rats was exposed to one dose of hexachlorobutadiene (20 mg/kg body weight) administered subcutaneously before mating. Within 90 days after exposure, all 86 newborn rats had died, compared with 13 of the 61 controls. The offspring from exposed dams were reported to show excitation, disturbances of motor coordination, a decrease in appetite and a loss of weight, lymphocytosis, neutropenia, myelocytes, Jolly's and Cabeau's bodies, pneumonia, bronchitis, granular dystrophy of renal cells, glomerulonephritis, inflammatory destructive lesions of the gastrointestinal tract and vascular hyperaemia (Poteryayeva, 1966). The Task Group noted major deficiencies and incomplete reporting of the experiment, the unusual route of administration, and the high percentage of mortality in control rats. Groups of 10-12 male and 20-24 female Sprague-Dawley rats received a diet containing hexachlorobutadiene at dose levels of 0.2, 2.0 or 20 mg/kg body weight per day for 90 days prior to mating, 15 days during mating, and subsequently throughout gestation and lactation. In the mating period, two females were placed with one male of the same dose group. The control group consisted of 17 males and 34 females. The diets were reportedly analysed for the test compound. No mortality was observed. At 20 mg/kg, adults showed decreased food consumption and body weight gain. Blood urea nitrogen, serum alanine aminotransferase (EC 184.108.40.206) and serum creatinine were unchanged compared to controls. The dams had an increased relative brain weight and the male rats had an increased relative liver weight at 20 mg/kg. The relative kidney weights were increased in both sexes at 20 mg/kg. At 2 and 20 mg/kg the kidneys of adult rats revealed dose-related tubular dilatation and hypertrophy with foci of epithelial degeneration and regeneration; however, there was no effect at 0.2 mg/kg. The only adverse reproductive effect in neonates was a decreased weanling weight at 20 mg/kg. There was no detectable effect on the percentage pregnancy, the period from first cohabitation to delivery, survival indices, sex ratio, histopathology of weanlings, and the incidence of skeletal alterations and abnormalities in neonates (Schwetz et al., 1977). In a third reproductive study, groups of six female SPF Wistar-derived rats, 10 weeks of age, received a diet containing hexa-chlorobutadiene at levels of 0, 150 and 1500 mg/kg diet (estimated by the Task Group to be equivalent to 0, 7.5 and 75 mg/kg body weight per day) for 3 weeks prior to mating, 3 weeks during mating, and subsequently throughout gestation and lactation. In the mating period, two untreated males were placed with the females, after which the females were housed individually. The 75-mg/kg female adults were killed in week 10, while those given 0 or 7.5 mg/kg were killed in week 18. Food analysis at the low dose level revealed hexachlorobutadiene levels within 96% of nominal values after 1 week and within 81% of nominal values after 2 weeks. Diets were prepared weekly. There was a reduced body weight gain by female rats in the two groups receiving hexachlorobutadiene. Weakness of hind legs, unsteady gait, incoordination and ataxia were seen at 75 mg/kg. The relative kidney weight was increased at both dose levels. Histopathological investigations revealed hypercellularity of epithelial cells, hydropic degeneration, and necrosis of proximal tubules in the kidneys at 7.5 mg/kg. At 75 mg/kg, slight proliferation of bile duct epithelial cells, fragmentation and demyelination of single fibres of the femoral nerve, and extensive renal degeneration were observed. Again at 75 mg/kg, no conceptions occurred, the ovaries showing little follicular activity, and there was no uterine implantation sites. At 7.5 mg/kg fertility and litter size were reduced, but not significantly. In both the control and 7.5-mg/kg groups, the resorption quotient was low. Compared to controls, pup weights were reduced significantly on days 0, 10 and 20 in the 7.5-mg/kg group. No gross abnormalities were observed (Harleman & Seinen, 1979). 8.5.2 Embryotoxicity and teratogenicity In a teratology study, groups of 24-25 female rats were exposed to hexachlorobutadiene vapour at measured concentrations of 0, 21, 53, 107 or 160 mg/m3 for 6 h per day from days 6 to 20 of pregnancy. The breathing zone atmosphere was analysed by gas chromatography. Maternal weight gain decreased at 53 and 160 mg/m3. At the other two exposure levels, the slight decrease in maternal weight was not significant. The mean number of implantation sites, total fetal losses, resorptions, live fetuses, incidences of pregnancy, and sex ratio were not affected by exposure to hexachlorobutadiene, compared to controls. Fetal body weight was reduced in both sexes at 160 mg/m3. The incidences of external, visceral, and skeletal alterations were not significantly increased (Saillenfait et al., 1989). In a study by Hardin et al. (1981), groups of 10-15 mated Sprague-Dawley rats received hexachlorobutadiene in corn oil by intraperitoneal injection at a dose level of 10 mg/kg body weight per day from days 1 to 15 of gestation. It was reported (without further details) that at least two maternal organ weights were changed and that pre- or postimplantation survival was reduced. Maternal tissues did not reveal histopathological effects. Fetuses had a reduced weight or length, a 1-2 day delay in heart development, and dilated ureters. No grossly visible external or internal malformations were observed (Hardin et al., 1981). It was reported briefly by Badaeva et al. (1985) that daily oral administration of hexachlorobutadiene to pregnant rats at a dose level of 8.1 mg/kg body weight per day resulted in histopathologi-cal changes of nerve cells and myelin fibres of the spinal cord in the dams and their offspring. 8.6 Mutagenicity and related end-points 8.6.1 In vitro effects Purified hexachlorobutadiene induces gene mutations in the Ames Salmonella test when specific incubation conditions are employed. In preincubation assays adapted to include rat liver microsomes and additional reduced glutathione, hexachlorobutadiene induced point mutations in Salmonella typhimurium TA100 (Vamvakas et al., 1988a). Assays lacking specialized metabolic activation conditions have generally yielded negative results (Table 12). Data from bacterial mutagenicity assays are consistent with the proposed scheme for the biotransformation of hexachlorobutadiene in animals (section 6.3; Fig. 1). Activity in S. typhimurium TA100, mediated by subcellular fractions of rat kidney, was inhibited by the addition of the ß-lyase inhibitor, AOAA (Vamvakas et al., 1988a, 1989a) and the gamma-glutamyltranspeptidase inhibitor, acivicin (Vamvakas et al., 1989a). Several of the proposed metabolites of hexachlorobutadiene have been assayed for mutagenic activity in S. typhimurium TA100 (Table 13). The mono-glutathione (GPB) and mono-cysteine (CPB) conjugates were mutagenic in the presence or absence of rat kidney S9. Rat liver microsomes and mitochondria that exhibit high gamma-glutamyltranspeptidase activities strongly enhanced the mutagenic potency of GPB in the presence of additional glutathione, in contrast to liver microsomes that exhibit lower gamma-glutamyltranspeptidase activity. Furthermore, AOAA and acivicin both inhibit the activation of GPB mediated by kidney fractions. The di-glutathione (BGTB) and di-cysteine (BCTB) conjugates of hexachlorobutadiene were not mutagenic either in the presence or absence of rat kidney S9 (Green & Odum, 1985; Dekant et al., 1986; Vamvakas et al., 1988a, 1989a). The mercapturic acid, ACPB, was mutagenic both in the presence and absence of rat liver S9. It has been suggested that the metabolism of ACPB in animals is catalysed by an N-deacetylase and by ß-lyase (section 6.3) (Reichert et al., 1984). The Task Group considered that S. typhimurium possesses both of these enzymes activities. Both MTPB and CMTPB gave negative results in tests with S. typhimurium TA100 and are considered to be detoxified metabolites of hexachlorobutadiene (Wild et al., 1986). Table 12. Studies on mutagenicity of hexachlorobutadiene Test description Species/strain/cell type Conditionsa Resultc Reference Reverse mutations Salmonella typhimurium TA98, +/- rat liver S9, purity 98%, - De Meester et al. TA100, TA1530, TA1535, TA1538 plate incorporation (1981) S. typhimurium TA100 +/- rat liver S9, purity > 99%, - Stott et al. (1981) plate incorporation S. typhimurium TA98, TA100 +/- rat liver S9, purity not - Reichert et al. (1983) reported, suspension testb S. typhimurium TA98, TA100, +/- rat liver S9, purity not - Haworth et al. (1983) TA1535, TA1537 reported, preincubation test + rat liver S9*, purity > 99.5%, + preincubation test S. typhimurium TA100, TA1535 +/- rat liver S9, purity not - Chudin et al. (1985) TA1538 reported, plate incorporation S. typhimurium TA100 +/- rat liver S9, - Reichert et al. (1984) - rat liver S9* + S. typhimurium TA100 + rat liver S9*, purity >99.5%, +d Wild et al. (1986) preincubation test S. typhimurium TA100 no activation, purity 98% + Vamvakas et al. (1988a) no activation, purity > 99.5%, - preincubation test Table 12 (contd). Test description Species/strain/cell type Conditionsa Resultc Reference S. typhimurium TA100 + rat liver microsomes - Vamvakas et al. (1988a) without additional GSH + rat liver microsomes and +e additional GSH, purity > 99.5%, plate incorporation Sex-linked lethals Drosophila melanogaster feeding or injection - Woodruff et al. (1985) Chromosome aberrations CHO cells +/- rat liver S9 - Galloway et al. (1987) Chromosome aberrations human lymphocytes - rat liver S9 - German (1988) Sister chromatid exchanges CHO cells +/- rat liver S9 + Galloway et al. (1987) Chromosome aberrations mouse bone marrow cells inhalation, 4 h + German (1988) Chromosome aberrations mouse bone marrow cells oral gavage + German (1988) a S9* = a fortified S9 mix containing 3 times the normal protein concentration; GSH = reduced glutathione b The extreme toxicity of the compound without S9 was supposed to exclude testing in this system c + = > twice the background rate or, in the case of bacterial studies, a reproducible dose-related increase in the number of revertants per plate; - = negative d 0.23 revertants per nmol e Addition of rat kidney microsomes further increased the number of revertants; positive results were inhibited by the ß-lyase inhibitor aminooxyacetic acid Chinese hamster ovary (CHO) cells were exposed to between 5 and 24 mg hexachlorobutadiene/litre for 2 h in the presence of rat liver S9 and throughout the incubation period (8-26 h, depending on cell cycle delay) in the absence of rat liver S9. In comparison with concurrent controls, no significant increase in chromosome aberration frequency was observed (Galloway et al., 1987). In a further study, in which human lymphocyte cultures were exposed to between 0.01 and 0.001 mg hexachlorobutadiene per litre in the absence of S9 for 27 h, there was also no clastogenic effect. At the highest dose level there was a reduction of approximately 60% in the mitotic index of human lymphocyte cultures (German 1988). However, hexachlorobutadiene at a dose level of at least 4 mg/litre did cause a significant increase in the frequency of sister chromatid exchange in CHO cells in both the presence and absence of rat liver S9 (Galloway et al., 1987). Hexachlorobutadiene was found to induce unscheduled DNA synthesis (UDS) in Syrian hamster embryo fibroblast cultures. Moreover, the magnitude of the response was increased when a preincubation period with rat liver S15 was employed (Schiffman et al., 1984). However, there was no induction of UDS in a study using rat hepatocyte cultures (Stott et al., 1981). In summary, the Task Group concluded that hexachloro-butadiene was genotoxic in vitro and that the negative results reported in some studies may have resulted from the use of inappropriate conditions for metabolic activation. 8.6.2 In vivo effects Hexachlorobutadiene induced a significant increase in the frequency of chromosomal aberrations in mouse bone marrow cells following the administration of acute oral doses of 2 or 10 mg/kg body weight or acute inhalation exposure to 10 mg/m3 for 4 h. Both experiments used six mice per dose group, and the animals were sacrificed after 24 h (German, 1988). Six hours after the administration of a single oral dose of 20 mg hexachlorobutadiene/kg body weight to two groups of five male Sprague-Dawley rats, there were statistically significant increases in kidney UDS of 27% and 54% above concurrent control levels. Administration of a positive control substance, dimethyl-nitrosamine, resulted in an increase of 187% over controls (Stott et al., 1981). As described in section 6.3, radiolabelled nucleotides were recovered from the kidneys of rats and mice administered 14C-labelled hexachlorobutadiene by gavage (Stott et al., 1981; Schrenk & Dekant, 1989). The Task Group concluded that these studies indicated covalent binding of hexachlorobutadiene or its metabolites Table 13. Tests for reverse mutations in Salmonella typhimurium TA100 by proposed metabolites of hexachlorobutadiene Metabolite and abbreviationa Conditionsb Resultc Reference 1-(glutathion- S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene (GPB) no activation - Green & Odum (1985) + rat kidney S9 + +/- rat kidney fractions +d Vamvakas et al. (1988a) 1,4-(bis-glutathion- S-yl)-1,2,3,4-tetrachloro-1,3-butadiene +/- rat kidney fractions - Vamvakas et al. (1988a) (BGTB) 1,4-(bis-cystein- S-yl)-1,2,3,4-tetrachloro-1,3-butadiene +/- rat kidney fractions - Vamvakas et al. (1988a) (BCTB) 1-(cystein- S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene (CPB) +/- rat kidney S9 +e Green & Odum (1985) no activation +f Dekant et al. (1986) 1-( N-acetylcystein- S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene - rat liver S9 - Wild et al. (1986) (ACPB) + rat liver S9 +g 1-carboxymethylthio-1,2,3,4,4-pentachloro-1,3-butadiene + rat liver S9* - Wild et al. (1986) (CMTPB) Table 13 (contd). Metabolite and abbreviationa Conditionsb Resultc Reference 1-methylthio-1,2,3,4,4-pentachloro-1,3-butadiene (MTPB) + rat liver S9 - Wild et al. (1986) 2,2,3,4,4-pentachloro-3-butenoic acid (PBA) +/- rat liver S9 + Reichert et al. (1984) 2,2,3,4,4-pentachloro-3-butenoic acid chloride (PBAC) +/- rat liver S9 + Reichert et al. (1984) a See also Figure 1 b Plate incorporation assays with, except in the case of the tests by Green & Odum, preincubation; S9* = a fortified S9 mix containing 3 times the normal protein concentration c + = > twice background rate; - = negative d Mutagenic potency enhanced by rat kidney microsomes or mitochondria and less so by cytosol; positive results were inhibited by the ß-lyase inhibitor aminooxyacetic acid e Mutagenic potency enhanced by rat kidney S9 f Positive results were inhibited by the ß-lyase inhibitor aminooxyacetic acid g 18.7 revertants per nmol; mutagenic potency decreased by addition of pyridoxal phospate; activation by cytosol, with and without cofactors, had the same results as S9 mix; microsome mix was inactive to kidney DNA in vivo. The study with mice showed that the level of binding to mitochondrial DNA was greater than that to nuclear DNA. In addition, radioactivity was recovered in mitochondrial DNA, but not nuclear DNA, from mouse liver (Schrenk & Dekant, 1989). Hexachlorobutadiene did not induce sex-linked recessive lethal mutations in Drosophila melanogaster following treatment of adults either via the diet or by injection (Woodruff et al., 1985). 8.7 Carcinogenicity/long-term toxicity 8.7.1 Inhalation exposure No long-term carcinogenicity studies, where inhalation was the route of exposure, have been reported. 8.7.2 Oral exposure In a study by Kociba et al. (1977a,b), groups of 39-40 adult Sprague-Dawley rats of each sex received a diet containing hexachlorobutadiene at 0.2, 2 or 20 mg/kg body weight per day for 22 (males) or 24 (females) months. Control groups comprised 90 rats of each sex. Analysis for the compound was not reported. An increased mortality was observed in males at 20 mg/kg. Hexachlorobutadiene caused a depression of the body weight gain in both sexes at the highest dose level without any effect on food consumption. Haematological investigations performed at 12-14 and 22-24 months, revealed a slight, but statistically significant, depression in the red blood cell count of males at 20 mg/kg (22 months). Urinalysis at 12-14 months and 22-24 months did not reveal effects except for a small increase in coproporphyrin excretion. The analysis of the clinical chemistry parameters of blood urea nitrogen, serum alanine aminotransferase (EC 220.127.116.11) and serum alkaline phosphatase (EC 18.104.22.168) at 12 months revealed no treatment-related effects, except for statistically significant decreases in serum alanine aminotransferase in males of the 20-mg/kg dose group and females of the 0.2- or 20-mg/kg dose groups. These changes were considered by the authors to be of questionable toxicological significance. The relative kidney weights were elevated at 20 mg/kg for both sexes, as were the relative weights of the brain in females and of the testes in males. In both sexes, an extensive histopathological examination revealed tubular epithelial hyperplasia at 2 and 20 mg/kg, but not at 0.2 mg/kg, and an increased incidence of renal tubular neoplasms at 20 mg/kg (see Table 14) (Kociba et al., 1977a,b). 8.7.3 Dermal exposure In a study by Van Duuren et al. (1979), groups of 30 female Ha:ICR Swiss mice received 6.0 mg hexachlorobutadiene in acetone applied 3 times per week to the shaven dorsal skin for between 144 and 594 days. A group of 100 untreated females were included in the study, together with 30 controls which received acetone only. The study duration was described as being between 440 and 594 days. Sections of skin, liver, stomach and kidney were sampled at autopsy, but no increase in the number of distant tumours was observed. In a two-stage initiation-promotion experiment, each of 20 female Swiss mice received one application of 15.0 mg hexa-chlorobutadiene in acetone to the dorsal skin. After 14 days, the mice similarly received 5 µg of the tumour promoter 12- o-tetra-decanoylphorbol-13-acetate (TPA) three times weekly for between 428 and 576 days. Hexachlorobutadiene administration did not induce a significant increase in the fraction of mice developing skin papillomas in this study (Van Duuren et al., 1979). Table 14. Renal tubular neoplasms in rats after long-term exposure to hexachlorobutadienea Dose (mg/kg Sex Incidence of renal tubular neoplasms body weight per day) adenoma adenocarcinoma total 0 males 1/90 0/90 1/90 0.2 0/40 0/40 0/40 2.0 0/40 0/40 0/40 20 2/39 7/39 9/39 (P < 0.05) 0 females 0/90 0/90 0/90 0.2 0/40 0/40 0/40 2.0 0/40 0/40 0/40 20 3/40 3/40b 6/40 (P < 0.05) a From: Kociba et al. (1977a) b One of these was an undifferentiated carcinoma 8.7.4 Exposure by other routes In a study of repeated exposure to hexachlorobutadiene by ip injection, groups of 20 A/St strain male mice (from 6 to 8 weeks of age) received 12 or 13 ip injections of hexachlorobutadiene (4 or 8 mg/kg body weight) in tricaprylin, respectively. The purity of the hexachlorobutadiene was stated to exceed 99.9%. Urethane was used as a positive control for carcinogenesis, and a negative control group of 50 mice receiving tricaprylin only. Survival was 95% for mice receiving 4 mg/kg and 70% for mice receiving 8 mg/kg, compared to 92% for controls. Mice were sacrificed at 24 weeks after the first injection, and the number of surface adenomas in the lungs was counted. No significant increase in adenomas, compared to the vehicle-treated control, was observed (Theiss et al., 1977). The Task Group noted major deficiencies of this study; including the choice of a sensitive strain of mice, the short duration of both the exposure period (4 weeks) and the follow-up period (24 weeks), the small group sizes of the experimental animals, the unusual route of administration, and the limited histopathology. The strain A mice used in this study are highly predisposed to spontaneous lung cancer, which is likely to have further compromised the value of the study. 8.8 Other special studies 8.8.1 Effects on the nervous system Acute high exposure to hexachlorobutadiene has a depressant effect on the central nervous system (see sections 22.214.171.124 and 8.1.2). Subchronic exposure of rats at high dose levels (1500 mg/kg diet for 13 weeks) also produced some signs of neurotoxicity, which was associated with demyelinization and fragmentation of femoral nerve fibres (Harleman & Seinén, 1979; see also Badaeva et al., 1985, section 8.5.2). 8.8.2 Effects on the liver 126.96.36.199 Acute effects Hexachlorobutadiene causes hydropic changes in the liver of rats (Gradiski et al., 1975; Lock & Ishmael, 1981; Lock et al., 1982), mice (Lock et al., 1985), and rabbits (Duprat & Gradiski, 1978), sometimes accompanied by fat accumulation (Duprat & Gradiski, 1978; Lock & Ishmael, 1981; Lock et al., 1982). Male rats, exposed to a single intraperitoneal dose of hexachlorobutadiene (200 or 300 mg/kg body weight) in corn oil showed increased relative liver weights, mitochondrial swelling in liver and bile duct, proliferation of smooth endoplasmic reticulum, lipid accumulation, and increased water content in the liver. Biochemical changes in the liver were a decrease, followed after 1 day by an increase, in non-protein sulfhydryl (NP-SH) concentration, and an increase in potassium content. All effects were reversible within 10 days. Increases in plasma urea and alkaline phosphatase (EC 188.8.131.52.) were also reported. In a separate experiment, the highest dose administered by ip injection which did not cause an increased water content in the liver was 25 mg/kg body weight (Lock et al., 1982). Male rats exposed to single intraperitoneal doses up to 100 mg/kg body weight showed an increase in serum bile acids and bilirubin (Bai et al., 1992). Male mice, exposed to single intraperitoneal doses of 50, 100 and 200 mg/kg body weight in corn oil, showed a dose-related increase in relative liver weight at 100 and 200 mg/kg, and, at all dose levels, dose-related, reversible changes in the liver (mitochondrial swelling, proliferation of smooth endoplasmic reticulum, and an increased water content). Reversible biochemical changes included increases in sodium and potassium content, NP-SH concentration in the liver, and serum alanine aminotransferase activity (EC 184.108.40.206) at 50 mg/kg (Lock et al., 1985). 220.127.116.11 Short-term effects As discussed in section 18.104.22.168, slight hepatotoxic effects have been observed following oral exposure of rats (Kociba et al., 1971; Harleman & Seinen, 1979). 8.8.3 Effects on the kidneys This section will describe the main features of the renal toxicity induced by hexachlorobutadiene. For more detail the reader is referred to the reviews of Rush et al. (1984), Lock (1988), Yang (1988) and Dekant et al. (1990a). 22.214.171.124 Acute effects Inhalation exposure of rats produces renal tubular necrosis (Gage, 1970; see section 8.2.1). Enzyme histochemical investi-gations were performed on groups of 10 male Swiss OF1 mice 24 h after whole-body inhalation exposure for 4 h to hexachloro-butadiene at measured concentrations of 29.3, 53.4, 106.7 or 266.8 mg/m3. A concentration-related increase in the percentage of damaged kidney tubules, which had been stained for alkaline phosphatase (EC 126.96.36.199), was observed at all exposure levels. The EC50 was calculated to be 76.8 mg/m3 (De Ceaurriz et al., 1988). A single oral dose of hexachlorobutadiene (200 mg/kg body weight) in polyethylene glycol caused an increase in plasma urea concentration, a decrease in plasma alanine aminotransferase activity, and, in urine, increases in the levels of glucose, protein, alanine aminotransferase, N-acetyl-ß-D-glucosaminidase, gamma-glutamyltranspeptidase (EC 188.8.131.52) and alanine aminopeptidase (EC 184.108.40.206) (Nash et al., 1984). Following in vivo administration, hexachlorobutadiene caused dose-dependent necrosis of the renal proximal tubules in rats (Gradiski et al., 1975; Lock & Ishmael, 1979, 1981; Kluwe et al., 1982; Hook et al., 1982, 1983; Ishmael et al., 1982; Ishmael & Lock, 1986), mice (Ishmael et al., 1984) and rabbits (Duprat & Gradiski, 1978). In rats, the lesions were restricted to the pars recta (S3-segment) and were macroscopically observed as a distinct band of damage in the outer stripe of the medulla (Lock & Ishmael, 1979; Ishmael et al., 1982). In mice and rabbits both the pars recta and the pars convoluta of the proximal convoluted tubules were damaged (Duprat & Gradiski, 1978; Ishmael et al., 1984). The lesion is characterized microscopically by necrotic epithelial cells, most of which are devoid of nuclei. The few remaining nuclei show karyorrhexis, and the cytoplasm is strongly eosinophilic. Many renal tubules contained cellular debris (Duprat & Gradiski, 1978; Lock & Ishmael, 1979; Lock et al., 1984; Ishmael et al., 1984). Vacuolation of the pars convoluta was observed (Duprat & Gradiski, 1978; Ishmael et al., 1982, 1984). Mitochondrial swelling and loss of brush-borders were prominent ultrastructural findings (Ishmael et al., 1982, 1984). Adult male rats have been found to be less sensitive to the renal toxicity induced by hexachlorobutadiene than adult females and young males (Hook et al., 1983; Kuo & Hook, 1983). When male rats were dosed intraperitoneally with a single dose of 300 mg/kg in corn oil, the earliest pathological change was mitochondrial swelling in proximal tubular cells observed after 1-2 h. Extensive necrosis was evident between days 1 and 4, and active regeneration by day 5 (Ishmael et al., 1982). Similar renal toxicity was seen at a dose level of 25 or 50 mg/kg body weight in female rats and young males, respectively. A similar pattern of pathological changes with comparable intensity was observed in mice at an intraperitoneal dose of 50 mg/kg body weight (Ishmael et al., 1984). In a study by Lock et al. (1984), young mice were found to be more susceptible than adults, but no sex difference was apparent. In both rats and mice, differences in strain susceptibility were observed (Hook et al., 1983; Lock et al., 1984). The lowest intraperitoneal dose at which renal necrosis was observed in adult female rats was 25 mg/kg body weight (Lock & Ishmael, 1985) and in adult male and female mice was 6.3 mg/kg body weight (Lock et al., 1984). Biochemical changes found following intraperitoneal exposure in both rats and mice were increases in renal water content (Kluwe et al., 1982; Ishmael et al., 1982, 1984; Gartland et al., 1989), plasma urea (Lock & Ishmael, 1979, 1981; Ishmael et al., 1982, 1984; Hook et al., 1983; Lock et al., 1984; Ishmael & Lock, 1986; Stonard et al., 1987; Gartland et al., 1989), plasma alkaline phosphatase (EC 220.127.116.11.) (Lock & Ishmael, 1981), serum alanine aminotransferase (EC 18.104.22.168) (Gradiski et al., 1975; Kuo & Hook, 1983) and serum aspartate aminotransferase (Gradiski et al., 1975; Davis et al., 1980). In the urine of rats, increases in urinary protein, glucose and ketones have been measured (Lock & Ishmael, 1979; Berndt & Mehendale, 1979; Davis et al., 1980; Stonard et al., 1987), as well as increases in the activities of alkaline phosphatase and N-acetyl-ß-D-glucosaminidase (EC 22.214.171.124) (Lock & Ishmael, 1979; Stonard et al., 1987) and in lactic acid level (Gartland et al., 1989). In the kidneys of rats, increased sodium concentrations were accompanied by equally decreased potassium concentrations (Davis et al., 1980). All these changes occurred at similar or higher intraperitoneal doses than those at which renal necrosis was observed. Distinct renal functional changes in adult rats have been observed at intraperitoneal doses of 100-400 mg/kg body weight. These include a decrease in urine-concentrating ability (polyuria) (Lock & Ishmael, 1979; Berndt & Mehendale, 1979; Davis et al., 1980; Stonard et al., 1987), a reduced glomerular filtration rate (Davis et al., 1980) and a reduction of in vivo renal clearance of inulin, urea, p-aminohippuric acid (PAH), tetraethyl-ammonium bromide (TEA) (Lock & Ishmael, 1979) and imipramine (Davis et al., 1980). When organic ion transport was assessed in vitro in renal cortical slices of rats and mice that had been exposed to an intraperitoneal dose of 100 mg/kg, 200 mg/kg body weight or more, the transport of anions (PAH) was found to be reduced, but the transport of the cation (TEA), aminoisobutyrate was not (or was only slightly reduced) in rats (Lock & Ishmael, 1979; Berndt & Mehendale, 1979; Kluwe et al., 1982; Hook et al., 1982, 1983). In male adult mice, transport of PAH and TEA was reduced from intraperitoneal doses of 12.5 and 25.0 mg/kg body weight, respectively. The anion transport was reduced in adult females (Lock et al., 1984). 126.96.36.199 Short- and long-term effects The short- and long-term effects of hexachlorobutadiene on the kidneys of experimental animals have already been discussed in sections 8.2 , 8.5 and 8.7. Based on the studies of Kociba et al. (1971, 1977a,b), Schwetz et al. (1977), Harleman & Seinen (1979), Stott et al. (1981) and Yang et al. (1989), the oral NOAEL for renal toxicity is 0.2 mg/kg body weight per day. Results of the studies are summarized in Table 15. Female rats and mice were found to be distinctly more susceptible than males upon oral exposure for 13 weeks (Yang et al., 1989; Yang, 1991); this was also observed in the single-exposure mortality studies (section 8.1). 8.9 Factors modifying toxicity; toxicity of metabolites 8.9.1 Factors modifying toxicity 188.8.131.52 Surgery Complete protection from the nephrotoxic effects of hexachlorobutadiene was observed in rats that had been fitted with a biliary cannula before being given a single oral dose of 200 mg/kg body weight. Administration of bile, collected from rats dosed orally with the compound, to naive rats produced marked renal toxicity but no liver toxicity (Nash et al., 1984). 184.108.40.206 Inhibitors and inducers of mixed-function oxidases (MFO) In the majority of studies, the effects of MFO inhibitors (piperonyl butoxide, SKF 525A) and MFO inducers (Aroclor 1254, isosafrole, ß-naphthoflavone, phenobarbitone) on the nephro-toxicity induced by hexachlorobutadiene in rats and mice were absent or negligible (Lock & Ishmael, 1981; Hook et al., 1982; Lock et al., 1984; Davis, 1984). Furthermore, phenobarbitone pretreatment for 7 days at 0.05% in drinking-water enhanced the renal toxicity induced by intraperitoneal doses of hexachloro-butadiene in weanling rats (Hook et al., 1983). 220.127.116.11 Inhibitors of gamma-glutamyltranspeptidase (EC 18.104.22.168) Male rats pretreated with Acivicin (L-(alphaS, 5S)- alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid), an inhibitor of gamma-glutamyltranspeptidase (down to 3% of control activity in this study), and subsequently exposed intraperitoneally to hexachlorobutadiene, did not show a decrease in nephrotoxicity compared to rats treated with hexachlorobutadiene alone. It was concluded that gamma-glutamyltranspeptidase inhibition did not limit the formation of nephrotoxic metabolites (Davis, 1988). Male Swiss OF1 mice, pretreated with Acivicin and subsequently exposed to a single oral dose of hexachlorobutadiene (80 mg/kg body weight), showed a decrease in nephrotoxicity compared to mice treated with hexachlorobutadiene alone, as measured by alkaline phosphatase staining (De Ceaurriz & Ban, 1990). The Task Group noted that only one marker for nephrotoxicity was employed in this study. Table 15. No-observed-adverse-effect level (NOAEL) calculated from short-term and long-term studies of exposure to hexachlorobutadiene by oral administration Species (Strain) Age Sex Number of Duration of Dose (mg/kg NOAEL (mg/kg References animals study body weight body weight per group per day) per day) Rat (Wistar-derived) weanling male 6 2 weeks 7.3, 18.2, 44.7 < 7.3 Harleman & Seinen female 6 (1979) Rat (Sprague-Dawley) adult male 5 3 weeks 0.2, 20 0.2 Stott et al. (1981) Rat (Sprague-Dawley) adult female 4 30 days 1, 3, 10, 30, 65, 100 3 Kociba et al. (1971) Rat (Sprague-Dawley) adult male 10-12 3 months 0.2, 2.0, 20 0.2 Schwetz et al. female 20-24 (1977) Rat (Sprague-Dawley) adult male 39-40 24 months 0.2, 2.0, 20 0.2 Kociba et al. female 39-40 (1977a,b) Mouse (B6C3F1) adult male 5 2 weeks 4.3, 14.3, 43, 143, 430 < 4.3 Yang et al. female 5 (1989); Yang (1991) Mouse (B6C3F1) adult male 10 13 weeks 0.1, 0.4, 1.5, 4.9, 18.8 1.5 Yang et al. female 10 0.2, 0.5, 1.8, 4.5, 19.2 < 0.2 (1989); Yang (1991) 22.214.171.124 Inhibitors of cysteine conjugate ß-lyase Male Swiss OF1 mice, pretreated with the two ß-lyase inhibitors amino-oxyacetic acid (AOAA) and DL-propargylglycine (PPG) and subsequently exposed to a single oral dose of hexachlorobutadiene (80 mg/kg body weight), showed a decrease in nephrotoxicity compared to mice treated with hexachlorobutadiene alone, as measured by alkaline phosphatase staining (De Ceaurriz & Ban, 1990). The Task Group again noted that only one marker for nephrotoxicity was employed in this study. 126.96.36.199 Inhibitors of organic anion transport Pre-treatment of male rats with probenecid [(4-(dipropyl-amino)sulfonyl)] benzoic acid (105 µmol/kg body weight), an inhibitor of organic anion transport, did not alter the increase in plasma urea or decrease in renal clearance of p-aminohippuric acid induced by hexachlorobutadiene (Hook et al., 1982). However, in female rats, a higher dose (500 µmol/kg body weight) of probenecid totally protected against the renal toxicity, both functional and morphological, produced by ACPB (Lock & Ishmael, 1985). In addition this dose of probenecid protected female rats against the toxic effects produced by CPB and GPBN as well as the parent chemical (Lock & Ishmael, 1985). Male mice pre-treated with probenecid were also protected against the nephrotoxicity produced by hexachlorobutadiene (Ban & De Ceaurriz, 1988). The Task Group noted that this latter study used only one marker for nephrotoxicity. 188.8.131.52 Non-protein sulfhydryl scavengers Depletion of hepatic and renal non-protein sulfhydryl content (glutathione) by diethylmaleate in rats appears to potentiate the nephrotoxicity of hexachlorobutadiene as measured by a number of functional markers such as plasma urea (Hook et al., 1982; Baggett & Berndt, 1984, Davis et al., 1986). However, the Task Group noted that no information was available on the metabolism of hexachlorobutadiene to help interpret these studies. 8.9.2 Toxicity of metabolites This section discusses the renal toxicity of some metabolites of hexachlorobutadiene, the formation of which was discussed in section 6.2. These metabolites are 1-(glutathion- S-yl)-1,2,3,4,4- pentachloro-1,3-butadiene (GPB), 1-(cystein- S-yl)-1,2,3,4,4- pentachloro-1,3-butadiene (CPB), and 1-( N-acetylcystein- S-yl)- 1,2,3,4,4-pentachloro-1,3-butadiene (ACPB). Their mutagenic activity was described along with that of hexachlorobutadiene in section 8.6. 184.108.40.206 In vitro studies GPB decreased the viability of isolated renal epithelial cells of male rats, as measured by leakage of lactate dehydrogenase (EC 220.127.116.11), with a very steep dose-response curve and a lag period of 30 min. No cytotoxicity was observed when the GPB metabolism was blocked by anthglutin, an inhibitor of gamma-glutamyl-transpeptidase (EC 18.104.22.168) or amino-oxyacetic acid (AOAA), an inhibitor of renal cysteine conjugate ß-lyase (EC 22.214.171.124). The cytotoxicity of GPB was related to an impairment of mitochondrial function, as shown by loss of mitochondrial Ca2+ and ATP and inhibition of respiration and thiol depletion (Jones et al., 1986b). Likewise, GPB produced a concentration-dependent nephro-toxicity in the isolated perfused rat kidney, as indicated by the appearance in the urine of alkaline phosphatase, gamma-glutamyl-transpeptidase and glucose (Jones et al., 1986a). These changes were prevented by Acivicin and by AOAA (Schrenk et al., 1988a). In the study of Schrenk et al. (1988a), CPB also caused a marked nephrotoxicity in the isolated perfused kidney, which could be prevented by AOAA. In isolated rabbit renal tubules, CPB was observed to decrease the accumulation of p-amino-hippuric acid and tetraethylammonium (Jaffe et al., 1983), to affect mitochondrial function as shown by effects on cell respiration, and to decrease the glutathione content and, after a lag period of 60 min, cell viability (Schnellmann et al., 1987). The effects on respiration resulted initially from the uncoupling of oxidative phosphorylation, followed later by inhibition of state 3 respiration (Schnellmann et al., 1987). Impaired mitochondrial function was observed in CPB-exposed isolated rat renal cortical mitochondria as an inability to retain Ca2+, collapse of the membrane potential, impaired state 3 respiration with succinate as substrate, and nonenzymatic depletion of thiol content. The latter effect was blocked by AOAA. From these results it was concluded that the reactive intermediate formed from CPB interacts with the inner mitochondrial membrane (Wallin et al., 1987). CPB also inhibited rat kidney mitochondrial DNA, RNA and protein synthesis, and AOAA blocked this effect. Moreover, CPB converted supercoiled DNA to relaxed circular DNA and shorter linear fragments (Banki & Anders, 1989). Chen et al. (1990) observed a decreased viability of isolated human renal proximal tubular cells upon exposure to CPB, which was again blocked by AOAA. Using radiolabelled ACPB and rat renal cortical slices, it was established that ACPB is transported by the same renal mechanisms involved in the movement of many organic anions into tubular fluid. This carrier-mediated transport is reduced by specific inhibitors like probenecid and sulfinpyrazone, a competitive and metabolic inhibitor like 2,4-dinitrophenol, and the transport substrate p-aminohippuric acid (Lock et al., 1986). This was confirmed by recent studies on the mechanism of uptake of GPB and CPB in the isolated perfused rat kidney (Schrenk et al., 1988b). Probenecid has also been reported to protect renal proximal tubular cells against ACPB-induced cytotoxicity, as determined by monitoring proline incorporation into renal proteins (Bach et al., 1986). 126.96.36.199 In vivo studies A single oral dose of 138 mg/kg body weight (0.27 mmol) of GPB or a single equimolar oral dose of 100 mg ACPB/kg body weight in polyethylene glycol to male rats caused marked nephrotoxicity similar in both biochemical and histopathological aspects to that observed with an oral dose of 200 mg/kg body weight (0.97 mmol) of hexachlorobutadiene (Nash et al., 1984). When rats received intraperitoneally GPB, CPB or ACPB in polyethylene glycol at single doses between 6.25 and 100 mg/kg body weight, increases in plasma urea level and renal proximal tubular necrosis were observed at dose levels of > 6.25 mg/kg body weight in females and 10 or 12.5 mg/kg body weight in males. The conjugates exhibited a similar pattern of nephrotoxicity at equimolar doses and were more nephrotoxic than the parent compound (Lock & Ishmael, 1985; Ishmael & Lock, 1986). All compounds tested were more toxic to female rats than males (Ishmael & Lock, 1986). Probenecid pretreatment protected the rats against the nephrotoxicity of these metabolites. Probenecid was shown to block the active tubular secretion of ACPB and to reduce the extent of covalent binding to renal protein (Lock & Ishmael, 1985). In mice, GPB and ACPB were also shown to be more toxic than the parent compound: renal necrosis was found following single intraperitoneal doses of 5.0 mg hexachlorobutadiene/kg body weight, 3.1 mg GPB/kg body weight and 3.0 mg ACPB/kg body weight in corn oil, which were the lowest doses tested (Lock et al., 1984). A single intraperitoneal dose of 10 mg CPB/kg body weight in DMSO and water caused dose-related damage in the pars recta of renal proximal tubules in male mice (Jaffe et al., 1983). The nephrotoxicity of some structural analogues of the above-mentioned conjugates, e.g. S-(1,2-dichlorovinyl)-L-cysteine, has been investigated extensively and has revealed a remarkable similarity (Anders et al., 1987; Lock, 1988). 8.10 Mechanisms of toxicity - mode of action 8.10.1 Mechanisms of toxicity The following evidence supports the hypothesis that the nephrotoxicity, mutagenicity and carcinogenicity of hexachloro-butadiene is dependent on the biosynthesis of the toxic sulfur conjugate GPB. This conjugate is mainly synthetized in the liver and further metabolized in the bile, gut, and kidneys to the CPB. Cysteine conjugate ß-lyase-dependent activation of CPB to a reactive thioketene in the proximal tubular cells finally results in covalent binding to cellular macromolecules. 1. The nephrotoxicity of hexachlorobutadiene in rats was prevented by the implantation of a biliary cannula; administration of bile from hexachlorobutadiene-treated rats to naive rats resulted in nephrotoxicity identical to the nephrotoxicity caused by hexachlorobutadiene (see section 188.8.131.52). 2. Inhibitors of renal organic anion transport protected rats against the nephrotoxicity of hexachlorobutadiene and its sulfur conjugates. Inhibition of the organic anion transport also protected isolated kidney cells against the nephrotoxicity of hexachlorobutadiene-derived sulfur-conjugates (see sections 184.108.40.206 and 220.127.116.11). 3. Anthglutin, Acivicin and aminooxyacetic acid, specific inhibitors of gamma-glutamyltranspeptidase and cysteine conjugate ß-lyase protected against the cytotoxicity of hexachloro-butadiene-derived sulfur-conjugates in freshly isolated rat renal proximal tubular cells (see section 18.104.22.168). 4. Synthetic sulfur-conjugates of hexachlorobutadiene show a higher nephrotoxicity than the parent compounds in rats and mice and produce renal damage identical to the renal damage induced by hexachlorobutadiene, based on clinical chemistry and histopathological examination (see section 22.214.171.124). 5. Hexachlorobutadiene and its sulfur-conjugates are genotoxic in bacteria; bioactivation by glutathione conjugation is required for hexachlorobutadiene genotoxicity. The ultimate mutagen is formed by cysteine conjugate ß-lyase-dependent cleavage of CPB (see section 8.6). 6. Hexachlorobutadiene induces renal tumours in rats only at doses that produce marked nephrotoxicity (see sections 8.7 and 126.96.36.199). 8.10.2 Mode of action The in vitro studies of Jones et al. (1986b), Wallin et al. (1987) and Schnellmann et al. (1987) on the cytotoxicity of sulfur-conjugates to renal tubular cells (section 188.8.131.52) point to renal cortical mitochondria as the major target for sulfur-conjugates of hexachlorobutadiene, analogous to that established for close structural analogues (Dekant et al., 1990b). The hypothesis proposes an interaction of the reactive metabolite with the inner mitochondrial membrane, which ultimately causes respiratory insufficiency. 9. EFFECTS ON HUMANS 9.1 General population exposure Hexachlorobutadiene has been found in postmortem examinations, but not in living persons. No pathogenic effects have been recorded (see section 5.2). 9.2 Occupational exposure Two reports on certain disorders among agricultural workers in vineyards where hexachlorobutadiene has been used as a fumigant (Krasniuk et al., 1969; Burkatskaya et al., 1982) cannot be evaluated, since such workers are known to be occupationally exposed to additional substances. In two cytogenetic studies of occupationally exposed workers from the same plant engaged in the production of hexachloro-butadiene, an increase in the frequency of chromosomal aberrations in peripheral blood lymphocytes was observed (German, 1986). The workers were exposed to hexachloro-butadiene concentrations that ranged from 1.6 to 16.9 mg/m3. The Task Group noted that exposure concentrations were determined by the factory and that the frequency of chromosome aberrations was not associated with the period of employment. 9.3 In vitro metabolism studies The following studies have been reported: a) Purified human liver microsomal glutathione- S-transferase and human liver cytosol metabolize hexachlorobutadiene to form GPB (McLellan et al., 1989; Oesch & Wolf, 1989). b) The enzyme cysteine conjugate ß-lyase has been isolated and purified from human kidney cytosol (Lash et al., 1990). The activity of the human cytosolic enzymes with a structurally related compound (1,2,2-trichlorovinyl-L-cysteine) is about 10-fold lower than that of rat renal cytosol (Green et al., 1990). c) Studies in isolated human proximal tubular cells have shown that CPB causes a ß-lyase-dependant cytotoxicity (Chen et al., 1990). These limited studies suggest that humans have the ability to metabolize hexachlorobutadiene to toxic metabolites. 9.4 Extrapolation of NOAEL from animals to humans Conversion of equivalent doses across species can utilize allometric relationships that relate physiological and anatomical variables across species. Physiological and metabolic rates have been shown to relate closely to body weight to the power 0.75 (Boxenbaum, 1982). The equivalent NOAEL in humans (mg/kg body weight per day) can be determined from the following equation: Wa dh = da (--)0.25 Wh where d = dose rate (mg/kg body weight per day) in humans (dh) or animals (da) wa = weight (kg) of animals (mice 30 g; rats 400 g) wh = weight of humans (70 kg) The NOAEL for humans, based on the NOAEL in mice, is: dh= (0.2 mg/kg body weight per day) (0.03)0.25 70 = 0.03 mg/kg body weight per day 10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT 10.1 Evaluation of human health risks 10.1.1 Hazard identification The following evaluation is based on toxicity studies in experimental animals; however, there are some human in vitro data which indicate that hexachlorobutadiene metabolism can occur by a similar route to that shown in experimental animals. Hexachlorobutadiene is slightly to moderately toxic based on acute oral experiments with adult rats, and moderately to highly toxic based on acute oral experiments with weanling rats (based on the WHO pesticide toxicity classification). The specific toxicity of the compound to the kidneys is higher for females than males. Following acute dermal exposure of rabbits, the compound was found to be weakly toxic. Regardless of species studied and the route of exposure (ip, oral, inhalation, dermal) in both short- and long-term studies, the target organ for toxicity is the kidney. Bioactivation to produce a reactive sulfur metabolite occurs following conjugation with glutathione. The monoglutathione conjugate of hexachloro-butadiene is processed to the cysteine- S-conjugate, which is then a substrate for renal cysteine conjugate ß-lyase. Hexachloro-butadiene produces a dose-dependent necrosis of the renal proximal tubules, which is followed by regenerative and/or proliferative changes. On the basis of both short- and long-term studies in rats and mice orally exposed to hexachlorobutadiene, the no-observed-adverse- effect level (NOAEL) is 0.2 mg/kg body weight per day. In one short-term inhalation study (12 days, 6 h/day), the NOAEL was 53 mg/m3. The vapour of hexachlorobutadiene was found to be irritating to the eyes and nose of rats in one short-term inhalation study. The undiluted compound appeared corrosive in an experiment with rabbits. Based on these limited data, the vapour should be regarded as irritating to human mucous membranes and the liquid should be regarded as corrosive. In a well-conducted Magnusson-Kligman test hexachloro-butadiene was a sensitizing agent both with and without adjuvant. Therefore, the compound should be regarded as a sensitizing agent for humans. In reproductive studies, reduced birth weight and neonatal weight gain in rats were observed, but these effects may be attributed to maternal toxicity. Developmental toxicity to rat fetuses was observed in two teratogenicity tests, but again only at levels that were also toxic to the dams. This developmental toxicity included reduced birth weight, a 1- to 2-day delay in heart development, and dilated ureters, but no gross abnormalities were observed. In vitro studies have shown that hexachlorobutadiene and, to a much greater extent, its sulfur metabolites induce mutations in Salmonella typhimurium. In one study of exposure to hexachloro-butadiene by inhalation or oral administration, an increased frequency of chromosomal aberrations was observed in mouse bone marrow cells. There is limited evidence for the genotoxicity of hexachlorobutadiene in animals, and insufficient evidence in humans. The long-term oral administration of hexachlorobutadiene to rats induced an increased frequency of renal tubular neoplasms, but only at doses that caused marked nephrotoxicity; at the lowest dose, no adverse effects were observed. The Task Group concluded that there is limited evidence for the carcinogenicity of hexachlorobutadiene in animals (one study in one rodent strain) and insufficient evidence in humans. 10.1.2 Exposure Hexachlorobutadiene is mainly a waste product. As such, it can be encountered in different environmental compartments, but predominantly in sediment and biota (see also 10.2.1). Exposure of the general public therefore mainly occurs indirectly via drinking-water and food of high lipid content. Assuming a maximum concentration of 2.5 µg/litre in contaminated drinking-water and 10 µg/kg wet weight in contaminated fatty food items (meat, fish, milk) and daily intakes of 2 litres drinking-water, 0.3 kg meat, 0.2 kg fish and 0.5 kg milk, a maximum total daily intake of 0.2 µg/kg body weight can be calculated for a 70-kg person. 10.1.3 Hazard evaluation The NOAEL for mice or rats exposed to hexachlorobutadiene is 0.2 mg/kg body weight per day (see Table 15), from which a NOAEL of 0.03-0.05 mg/kg body weight day has been derived for humans (see section 9.4). The Task Group considered the margin of safety of 150 between the estimated NOAEL in humans and the maximum total daily intake (see section 10.1.2) to be sufficient to protect the general population against the adverse effects of hexachlorobutadiene. 10.2 Evaluation of effects on the environment 10.2.1 Hazard identification Hexachlorobutadiene is a chemically stable compound. Complete aerobic biodegradation has been observed following adaptation of the inoculum. Partial biodegradation was found to occur in a pilot sewage treatment plant. Based on these observations and the chemical structure, it can be concluded that hexachlorobutadiene is not readily biodegradable, but can be considered to be inherently biodegradable. Experimental photolysis of hexachlorobutadiene in the presence of a surface was rapid, but in the absence of a surface the compound is believed to be persistent. Degradation in the atmosphere is assumed to occur by a rather slow reaction with hydroxyl radicals. A half-life of up to 2.3 years has been calculated. Once hexachlorobutadiene is released into the environment, intercompartmental transport will occur chiefly by volatilization from water and soil, adsorption to particulate matter in water and air, and subsequent sedimentation or deposition. In view of a strong adsorption potential to organic matter, the compound accumulates in sediment and will not migrate rapidly in soils. Both field and laboratory exposure data support these conclusions. Field and laboratory data also support the high bioaccumu-lation potential in aquatic and benthic organisms which can be expected on the basis of the lipophilic nature of the compound. However, no evidence has been obtained for biomagnification. Hexachlorobutadiene is moderately to highly toxic to aquatic organisms; crustaceans and fish are the most sensitive species. The lowest E(L)C50 for freshwater organisms is 0.09 mg/litre (goldfish). The lowest chronic NOEC is 3 µg/litre (goldfish). Applying the preliminary effect assessment extrapolation procedure, as adopted in the OECD Workshop on Aquatic Effect Assessment (OECD, 1990), an Environmental Concern Level of 0.1 µg/litre can be established. The toxicity data on terrestrial organisms are insufficient to establish any toxicity threshold. 10.2.2 Exposure Current environmental levels in surface waters are generally below 0.2 µg/litre, rising to 1.3 µg/litre in highly polluted rivers. Levels in the upper sediment can be as high as 120 µg/kg in heavily polluted rivers or estuaries. In older sediment layers much higher concentrations can be measured. The concentrations in freshwater biota measured since 1980 generally do not exceed 100 µg/kg fresh weight, but in a polluted area can reach 120 mg/kg in the lipid of fish. 10.2.3 Hazard evaluation It can be concluded that away from point sources the maximum predicted environmental concentration (PEC) is twice the extrapolated Environmental Concern Level of 0.1 µg/litre. Aquatic organisms therefore may be at risk in polluted surface waters. In view of the rather high concentrations of the compound measured in some sediments, adverse effects on benthic organisms cannot be excluded. Considering the toxicity of the substance to mammals (the NOAEL for rats or mice is 0.2 mg/kg body weight per day) and its high bioaccumulating potential, the consumption of benthic or aquatic organisms in polluted surface water by other species may give cause for concern. For example, an otter weighing 10 kg and consuming 1 kg fish per day in waters containing 0.2 µg hexachlorobutadiene/litre could ingest 1200 µg/day (assuming a bioconcentration factor for fish of 6000, leading to a concentration of 1200 µg/kg wet weight) or 120 µg/kg body weight per day, which is above the calculated NOAEL value for the otter (calculated as in section 9.4). 11. FURTHER RESEARCH Hexachlorobutadiene is primarily a waste product and hence an environmental contaminant having only limited use as a fumigant in some parts of the world. The Task Group identified the following areas for which additional information is needed: a) the degradation of hexachlorobutadiene in the environment focusing on photodegradation and biodegradation; b) the terrestrial toxicity of hexachlorobutadiene including tests on benthic organisms; c) the genotoxic activity of hexachlorobutadiene in vivo. A further test for micronucleus or chromosome aberration induction in mouse bone marrow cells would strengthen the available data; d) the metabolism of hexachlorobutadiene and its glutathione- derived conjugates by human liver and renal enzymes and inter-individual variability. 12. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES The carcinogenic risk of hexachlorobutadiene was evaluated by the International Agency for Research on Cancer in 1979 (IARC, 1979). The summary of data reported and the evaluation of the IARC monograph on hexachlorobutadiene is reproduced here. 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Yurawecz MP, Dreifuss PA, & Kamps LR (1976) Determination of hexachloro-1,3-butadiene in spinach, eggs, fish, and milk by electron capture gas-liquid chromatography. J Assoc Anal Chem, 59: 552-558. Zoeteman BCJ, Harmsen K, Linders JBHJ, Morra CFH, & Slooff W (1980) Persistent organic pollutants in river water and ground water of The Netherlands. Chemosphere, 9: 231-249. Zogorski JS (1984) Experience in monitoring domestic water sources and process waters for trace organics. J Environ Sci Health, A19: 233-249. RESUME 1. Identité, propriétés physiques et chimiques, méthodes d'analyse L'hexachlorobutadiène est un liquide ininflammable, incombustible, limpide et huileux à la température et la pression ordinaires. Il est peu soluble dans l'eau mais miscible à l'éther et à l'éthanol. On peut le mettre en évidence et le doser par chromatographie en phase gazeuse. Les limites de détection sont de 0,03 µg/m3 dans l'air, 0,001 µg/litre dans l'eau, de 0,7 µg/kg de matière humide dans le sol ou les sédiments et de 0,02 µg/litre dans le sang. Dans les tissus, cette limite est de 0,47 µg/kg de tissus frais. 2. Sources d'exposition humaine et environnementale L'hexachlorobutadiène n'existe pas à l'état naturel. C'est essentiellement un sous-produit de la fabrication des hydro-carbures chlorés que l'on retrouve dans les fractions lourdes. La production annuelle mondiale (dans les fractions lourdes) a été estimée à 10 000 tonnes en 1982. L'hexachlorobutadiène peut être utilisé pour la récupération des gaz contenant du chlore dans les ateliers de fabrication du chlore et comme liquide de lavage pour éliminer du courant gazeux certains composés organiques volatils. On l'utilise également dans les gyroscopes, comme fluide calo-porteur, dans les transformateurs, comme liquide isolant ou liquide hydraulique ainsi que comme solvant des élastomères, comme intermédiaire et comme fumigant. 3. Transport, distribution et transformation dans l'environnement Les principales voies de pénétration dans l'environnement sont les émissions résultant des déchets et les utilisations qui entraînent la dispersion du produit. Le transport inter-compartimental s'effectue principalement par volatilisation, adsorption sur les matières particulaires puis dépôt ou sédimentation. L'hexachloro-butadiène ne migre pas facilement dans le sol et s'accumule dans les sédiments. Dans l'eau, on le considère comme persistant, sauf turbulences importantes. Il n'y a pas d'hydrolyse. Le produit semble être facilement biodégradable par voie aérobie, encore que le phénomène n'ait pas été étudié à fond. L'hexachlorobutadiène présent sur les surfaces subit une photolyse. Outre le dépôt, on estime que la réaction de l'hexachlorobutadiène avec les radicaux hydroxyles constitue un mode de piégeage important de ce composé dans la troposphère, la demi-vie atmosphérique estimative de l'hexachlorobutadiène pouvant aller jusqu'à 2,3 ans. Le produit a un potentiel élevé de bioaccumulation, comme l'ont confirmé les observations en laboratoire et sur le terrain. Ainsi, on a trouvé des facteurs de bioconcentration à l'état stationnaire (obtenus expérimentalement par rapport au poids de tissus frais) respectivement égaux en moyenne à 5800 et 17 000 chez la truite arc-en-ciel. On n'a pas observé d'amplification biologique au laboratoire ou sur le terrain. 4. Niveaux dans l'environnement et exposition humaine Le dosage de l'hexachlorobutadiène dans l'air des villes a donné dans tous les cas des valeurs inférieures à 0,5 µg/m3. Dans les régions écartées, les concentrations sont inférieures à 1 pg/m3. Dans les lacs et les cours d'eau d'Europe, on a enregistré des concentrations pouvant aller jusqu'à 2 µg/litre mais les valeurs moyennes sont généralement inférieures à 100 ng/litre. Dans la région des grands lacs au Canada, on a obtenu des valeurs beaucoup plus faibles (autour de 1 ng/litre). En revanche la teneur des sédiments du fond peut, dans cette zone, atteindre 120 µg/kg de poids sec. Les couches sédimentaires plus anciennes, remontant aux environs de 1960, présentaient des teneurs plus élevées (jusqu'à 550 µg/kg de matière humide). On a montré que la concentration dans les sédiments augmentait avec la granulométrie des particules. A en juger par la concentration de l'hexachlorobutadiène dans les organismes aquatiques, les oiseaux et les mammifères, le composé s'accumule mais ne subit pas d'amplification biologique. Dans les eaux polluées, on a relevé des concentrations dépassant 1000 µg/kg de tissus frais chez plusieurs espèces et même 120 mg/kg (par rapport aux lipides) chez une espèce. Les concentrations actuelles restent généralement inférieures à 1000 µg/kg de poids frais à distance des points de décharge industrielle. On a décelé la présence du composé dans l'urine, le sang et les tissus humains. Dans certaines denrées alimentaires ayant une fraction lipidique importante, on en a relevé jusqu'à 40 µg/kg et dans un cas, plus de 1000 µg/kg. D'après une étude, le niveau d'exposition pourrait atteindre 1,6 à 12,2 mg/m3 et les concentrations urinaires, 20 mg/litre. 5. Cinétique et métabolisme Après administration par voie orale, l'hexachlorobutadiène est rapidement absorbé chez l'animal de laboratoire mais le taux de résorption après inhalation ou exposition par voie cutanée n'a pas été étudié. Chez le rat et la souris, le composé se répartit principalement dans le foie, les reins et les tissus adipeux. Il est rapidement excrété. On a mis en évidence une fixation aux protéines et aux acides nucléiques dans le foie et les reins. La biotransformation du composé chez l'animal de laboratoire se révèle être un processus saturable. Elle s'effectue principalement par l'intermédiaire du glutathion, l'hexachlorobutadiène étant d'abord transformé en conjugué du S-glutathion. La métabolisation de ce conjugué se poursuit ensuite, en particulier au niveau de la membrane constituant la bordure en brosse des cellules des tubules rénaux, pour donner un métabolite sulfuré réactif qui est probablement responsable des effets néphrotoxiques, génotoxiques et cancérogènes observés. 6. Effets sur les êtres vivants dans leur milieu naturel L'hexachlorobutadiène est modérément à très toxique pour les organismes aquatiques. Certaines espèces de poissons et de crustacés se sont révélées être les plus sensibles, les valeurs de la CL50 à 96 h. allant de 0,032 à 1,2 et de 0,09 à environ 1,7 mg/litre, respectivement pour les crustacés et les poissons. Chez les poissons, le rein est organe-cible important. On a établi la valeur de la dose sans effets observables à 0,003 mg/litre, à partir des résultats d'un certain nombre d'épreuves à long terme sur certaines espèces d'algues et de poissons; cela permet de considérer ce composé comme très toxique pour les organismes aquatiques. Parmi les points d'aboutissement biologiques étudiés figuraient la toxicité générale, la neurotoxicité, la biochimie, l'hématologie, l'anatomopathologie et la reproduction. Lors d'une étude de 28 jours portant sur les premiers stades de la vie de Pimephales promelas, une espèce de vairon, on a observé que la reproduction n'était pas affectée à des concentrations allant jusqu'à 0,017 mg/litre, alors qu'à 0,013 et 0,017 mg/litre il y avait accroissement de la mortalité et réduction du poids du corps. La dose sans effets observables était de 0,0065 mg/litre. On n'a décrit qu'une seule épreuve fiable portant sur des organismes terrestres. Lors d'une épreuve de 90 jours sur des cailles japonaises qui recevaient une alimentation contenant ce composé à des concentrations allant de 0,3 à 30 mg/kg de nourriture, on a constaté que la survie des oisillons n'était réduite qu'à partir de 10 mg/kg de nourriture. 7. Effets sur les animaux de laboratoire et les systèmes d'épreuves in vitro 7.1 Toxicité générale L'hexachlorobutadiène est légèrement à modérément toxique pour le rat adulte, modérément toxique pour le raton juste sevré et extrêmement toxique pour les rattes juste sevrées après administration d'une seule dose par voie buccale. Les principaux organes-cibles sont le rein et dans une bien moindre mesure, le foie. D'après les données obtenues sur l'animal d'expérience, les vapeurs d'hexachlorobutadiène sont irritantes pour les muqueuses et le liquide est corrosif. On peut considérer ce composé comme un agent sensibilisateur. Chez le rat, la souris et le lapin, l'hexachlorobutadiène provoque une nécrose, liée à la dose, des tubules proximaux du rein. Les rats mâles adultes sont moins sensibles à la néphrotoxicité que les femelles ou les jeunes mâles. Les souriceaux sont plus sensibles que les souris adultes sans qu'on puisse observer de différences entre les deux sexes. Chez la ratte adulte, la dose intrapéritonéale unique la plus faible à laquelle on ait observé une nécrose rénale était de 25 mg/kg de poids corporel; elle était de 6,3 mg/kg de poids corporel chez les souris adultes mâles et femelles. A des doses égales ou supérieures à celles qui entraînaient une nécrose, on a observé des modifications biochimiques et une nette amélioration de la fonction rénale. Lors de six épreuves à court terme où le composé a été administré par la voie orale, deux études de reproduction et une étude d'alimentation à long terme portant sur des rats, c'est également le rein qui s'est révélé être l'organe-cible. Parmi les effets liés à la dose, on notait une diminution du poids relatif des reins et une dégénérescence de l'épithélium des tubules. La dose sans effets nocifs observables au niveau des reins, tirée d'une étude de deux ans sur le rat, était de 0,2 mg/kg de poids corporel et par jour. Une étude de 13 semaines sur des souris a montré que cette dose était de 0,2 mg/kg de poids corporel et par jour pour cet animal. Chez les deux espèces, les femelles adultes étaient plus sensibles que les mâles adultes. Lors d'une étude d'inhalation à court terme (six heures par jour pendant 12 jours) on a observé des effets analogues au niveau des reins avec une concentration nominale de vapeur d'hexa-chlorobutadiène égale à 267 mg/m3; cette concentration a également entraîné des difficultés respiratoires, ainsi qu'une dégénérescence des corticosurrénales. 7.2 Reproduction, embryotoxicité et teratogétogénicité Deux études d'alimentation portant sur la reproduction ont été effectuée sur des rats à des doses quotidiennes allant jusqu'à 20 et 75 mg/kg de poids corporel respectivement; elles ont fait ressortir une réduction du poids de naissance et du gain de poids néonatal aux doses toxiques pour la mère. La dose quotidienne de 75 mg/kg de poids corporel, qui était hautement toxique, s'est révélée suffisante pour empêcher la conception et la nidation intra-utérine. On n'a pas observé d'anomalies du squelette. Lors de deux études de tératogénicité, des rats ont été exposés soit à des vapeurs d'hexachlorobutadiène à des concentrations allant de 21 à 160 mg/m3, six heures par jour du sixième au vingtième jour de la gestation, soit par voie intrapéritonéale à une dose quotidienne de 10 mg/kg de poids corporel (du premier au quinzième jour de la gestation). Des effets nocifs ont été notés sur le développement des foetus, qui consistaient en une réduction du poids de naissance, un retard dans le développement cardiaque, une dilatation des uretères, mais pas de malformations macroscopiques. Le retard de développement a été observé à des doses qui étaient également toxiques pour les mères. 7.3 Génotoxicité et cancérogénicité L'hexachlorobutadiène provoque des mutations géniques dans l'épreuve d'Ames sur salmonelle dans des conditions particulières qui favorisent la formation de produits de conjugaison avec le glutathion. Lors d'une étude in vivo on a observé des aberrations chromosomiques qui n'ont en revanche pas été constatées lors de deux autres études in vitro. Une étude in vitro portant sur des cellules ovariennes de hamster chinois a révélé une augmentation de la fréquence des échanges entre chromatides soeurs. On a fait état de la très forte mutagénicité des métabolites sulfurés de l'hexachlorobutadiène. D'autres études in vitro ont montré que ce composé provoquait une synthèse non programmée de l'ADN dans des cultures de fibroblastes embryonnaires de hamsters de Syrie, effets qui n'étaient pas observés dans des cultures d'hépatocytes de rats. Le composé provoque également une synthèse non programmée de l'ADN in vivo mais n'induit pas de mutations létales récessives liées au sexe chez Drosophila melanogaster. Lors de la seule étude à long terme (deux ans) qui ait été effectuée, des rats ont reçu une alimentation contenant de l'hexachlorobutadiène à des doses quotidiennes respectives de 0,2, 2 ou 20 mg/kg de poids corporel et seule la dose la plus élevée a provoqué un accroissement de l'incidence des tumeurs malignes au niveau des tubules rénaux. 7.4 Mécanismes de la toxicité La néphrotoxicité, la mutagénicité et la cancérogénicité de l'hexachlorobutadiène sont liées à la biosynthèse d'un conjugué sulfuré toxique, le 1-glutathion- S-yl-1,2,3,4,4-pentachloro- butadiène. Ce conjugué est principalement synthétisé dans le foie et métabolisé ensuite dans la bile, l'intestin et les reins en 1-cystéine- S-yl-1,2,3,4,4-pentachlorobutadiène (CPB). L'activation du CPB en thiocétène réactif (qui dépend de la cystéine-conjuguée- béta lyase) au niveau des cellules des tubules proximaux, aboutit en définitive à la formation de liaisons covalentes avec les macromolécules cellulaires. 8. Effets sur l'homme On n'a pas décrit d'effets pathogènes sur la population dans son ensemble. On possède deux rapports faisant état de troubles chez des ouvriers agricoles qui utilisaient de l'hexachlorobutadiène comme fumigant mais il est vrai qu'ils avaient également été exposés à d'autres substances. On a également observé un accroissement de la fréquence des aberrations chromosomiques dans les lymphocytes du sang périphériques de travailleurs employés à la production d'hexachlorobutadiène et qui avaient été exposés à des concentrations de 1,6 à 12,2 mg/m3. 9. Evaluation des risques pour la santé humaine et des effets sur l'environnement 9.1 Evaluation des risques pour la santé humaine Comme très peu d'études ont été effectuées sur l'homme, l'évaluation repose essentiellement sur les animaux de laboratoire. Toutefois les données in vitro limitées dont on dispose au sujet de l'homme incitent à penser que le métabolisme de l'hexachloro-butadiène est analogue chez l'homme et l'animal. On estime que les vapeurs d'hexachlorobutadiène sont irritantes pour les muqueuses et que le liquide est corrosif. Ce composé doit également être considéré comme un agent sensibilisateur. Les principaux organes-cibles de son action toxique sont les reins et dans une mesure bien moindre, le foie. Sur la base des études à court et à long terme effectuées sur des rats et des souris, la dose quotidienne sans effets nocifs observables est évaluée à 0,2 mg/kg de poids corporel. On l'a estimée à 53 mg/m3 lors d'une étude d'inhalation à court terme chez le rat (12 jours, six heures par jour). L'action toxique sur le développement, de même que la réduction du poids de naissance et du gain de poids néonatal n'ont été observés qu'à des doses toxiques pour la mère. On a observé que l'hexachlorobutadiène produisait des mutations géniques, des aberrations chromosomiques, un accroissement des échanges entre chromatides soeurs et une synthèse non programmée de l'ADN, encore que certaines études aient donné des résultats négatifs. On ne possède que des indices limités en faveur d'une génotoxicité de l'hexachlorobutadiène chez l'animal, indices qui sont insuffisants en ce qui concerne l'homme. On a constaté que l'administration d'hexachlorobutadiène par voie orale pendant une longue période à des rats accroissait la fréquence des tumeurs malignes au niveau des tubules rénaux, mais il s'agissait uniquement de doses élevées fortement néphrotoxiques. En ce qui concerne la cancérogénicité de cette substance, les indices sont limités chez l'animal et insuffisants chez l'homme. En se basant sur la dose quotidienne sans effets nocifs observables estimée à 0,2 mg/kg de poids corporel chez la souris ou le rat, on a fixé à 0,03-0,05 mg/kg de poids corporel la dose quotidienne sans effets nocifs observables chez l'homme. La marge de sécurité entre la dose estimative sans effets nocifs observables et la dose journalière maximale totale ingérée estimée en se basant sur une absorption du composé par l'intermédiaire d'une eau de boisson et de produits alimentaires contaminés à forte teneur en lipides, est égale à 150. 9.2 Evaluation des effets sur l'environnement L'hexachlorobutadiène est modérément à fortement toxique pour les organismes aquatiques: les crustacés et les poissons sont les espèces les plus sensibles. On a fixé à 0,1 g/litre la concentration écologiquement préoccupante. On estime que la concentration maximale prévisible dans l'environnement à distance des sources ponctuelles de pollution est égale à deux fois la dose écologique-ment préoccupante extrapolée et, par voie de conséquence, que les organismes aquatiques peuvent être menacés dans les eaux de surface polluées. On ne peut exclure des effets nocifs sur le benthos. Compte tenu de la toxicité de l'hexachlorobutadiène pour les mammifères, la consommation de benthos ou d'organismes aquatiques par d'autres espèces pourrait être préoccupante. RESUMEN 1. Identidad, propiedades físicas y químicas, métodos de análisis El hexaclorobutadieno es un líquido no inflamable, incombus-tible, claro, oleoso e incoloro a temperatura y presión ordinarias. Es poco soluble en el agua, pero miscible con éter y etanol. La sustancia puede detectarse y determinarse cuantitativamente por métodos de cromatografía de gases. Los límites de detección son de 0,03 µg/m3 de aire, 0,001 µg/litro de agua, 0,7 µg/kg de peso húmedo en el suelo o en sedimentos y de 0,02 µg/litro de sangre. Se ha determinado un nivel de 0,47 µg/kg de peso húmedo de tejido. 2. Fuentes de exposición humana y ambiental No hay indicaciones de que el hexaclorobutadieno exista como producto natural. Es principalmente un subproducto de la fabricación de hidrocarburos clorados y se presenta en las fracciones pesadas (como residuo). La producción anual mundial del compuesto en las fracciones pesadas en 1982 se estimó en 10 000 toneladas. El hexaclorobutadieno puede utilizarse para recuperar gas que contiene cloro en plantas productoras de cloro y como líquido de lavado para eliminar ciertos compuestos orgánicos volátiles de las corrientes de gases. También se ha utilizado como fluido en giróscopos, como transmisor de calor, transformador, fluido aislante y fluido hidráulico, disolvente para elastómeros y como intermediario y sustancia para fumigar. 3. Transporte, distribución y transformación en el medio ambiente Las principales vías de ingreso en el medio ambiente son las emisiones de residuos y el uso dispersivo. El paso de un entorno a otro ocurre principalmente por volatilización, adsorción a corpúsculos de materia y subsiguiente deposición o sedimentación. El hexaclorabutadieno no migra rápidamente en el suelo y se acumula en el sedimento. Se considera persistente en el agua a menos que haya mucha turbulencia. No produce hidrólisis. La sustancia parece ser fácilmente biodegradable aeróbicamente, aunque su biodegradabilidad no se ha investigado a fondo. El hexaclorobutadieno se fotoliza en las superficies. Se supone que, además de la deposición, la reacción con radicales hidroxilo es un importante sumidero de hexaclorobutadieno en la troposfera y su semivida atmosférica estimada es de hasta 2,3 años. La sustancia tiene un elevado potencial de bioacumulación, que se ha comprobado mediante observaciones en laboratorio y sobre el terreno. En la trucha arco iris se han determinado experimentalmente factores de bioconcentración en estado estacionario de 5800 y 17 000 como promedio, sobre la base del peso húmedo. No se ha observado biomagnificación en laboratorio ni sobre el terreno. 4. Niveles ambientales y exposición humana Se ha determinado la presencia de hexaclorubutadieno en el aire urbano; en todos los casos, los niveles eran inferiores a 0,5 µg/m3. Las concentraciones en lugares aislados son inferiores a 7 pg/m3. En las aguas de lagos y ríos de Europa se han registrado concentraciones de hasta 2 µg/litro, pero los niveles medios son generalmente inferiores a 100 ng/litro. En la región de los Grandes Lagos del Canadá se han detectado niveles muy inferiores (de aproximadamente 1 ng/litro). Allí los niveles en el sedimento del fondo pueden ser de 120 µg/kg de peso en seco. En capas más antiguas de sedimento, de 1960 aproximadamente, se encontraron concentraciones más elevadas (de hasta 550 µg/kg de peso húmedo). Se ha demostrado que la concentración en el sedimento aumenta con el tamaño de la partícula de sedimento. Las concentraciones de hexaclorobutadieno en organismos acuáticos, aves y mamíferos indican bioacumulación pero no biomagnificación. En las aguas contaminadas se han detectado niveles de más de 1000 µg/kg de peso húmedo en varias especies y de 120 mg/kg (base grasa) en una especie. Lejos de los efluentes industriales, los niveles actuales se mantienen en general por debajo de 100 µg/kg de peso húmedo. Se ha detectado la presencia del compuesto en la orina, en la sangre y en tejidos humanos. En ciertos alimentos que contienen una elevada fracción lipídica se han encontrado hasta unos 40 µg/kg y, en un caso, más de 1000 µg/kg. Un estudio señala exposiciones ocupacionales de 1,6-12,2 mg/m3 y en la orina niveles de hasta 20 mg/litro. 5. Cinética y metabolismo Se ha observado que los animales de laboratorio absorben rápidamente el hexaclorobutadieno después de la administración oral, pero no se ha investigado la velocidad de absorción después de la inhalación o de la exposición dérmica. En ratas y ratones, el compuesto se distribuye principalmente al hígado, a los riñones y al tejido adiposo. Se excreta rápidamente. Se ha demostrado que se fija a las proteínas y ácidos nucleicos del hígado y de los riñones. La biotransformación del compuesto en animales de experimentación parece ser un proceso saturable. Se produce principalmente a través de una vía mediada por el glutatión, en la cual el hexaclorobutadieno se convierte inicialmente en conjugados de S-glutatión. Estos conjugados pueden seguir metabolizándose, especialmente en el ribete en cepillo de las membranas de las células de los tubos renales, produciendo un metabolito sulfuroso reactivo que probablemente explique la nefrotoxicidad, genotoxicidad y carcinogenicidad observadas. 6. Efectos en organismos presentes en el medio ambiente El hexaclorobutadieno es de moderadamente a muy tóxico para los organismos acuáticos. Los más sensibles que se hayan observado han sido especies de peces y crustáceos; los valores de la CL50 en 96 horas oscilan entre 0,032 y 1,2 mg/litro en crustáceos y entre 0,09 y 1,7 mg/litro en peces. Se ha demostrado que el riñón es un órgano muy afectado en los peces. Sobre la base de varias pruebas a largo plazo con especies de algas y de peces, se ha establecido un nivel sin efectos observados de 0,003 mg/litro; así pues, el compuesto se clasifica como muy tóxico para las especies acuáticas. Los valores extremos investigados comprenden parámetros de toxicidad general, neurotoxicidad, bioquímicos, hematológicos, patológicos y relacionados con la reproducción. En una prueba de 28 días de duración en la que se examinaron las primeras fases de la vida de carpas se observó que la reproducción no se veía afectada con concentraciones de hasta 0,017 mg/litro, mientras que con concentraciones de 0,013 y 0,017 mg/litro se observaron un aumento de la mortalidad y una disminución del peso corporal. El nivel sin efectos observados era de 0,0065 mg por litro. Se ha descrito una sola prueba fiable con organismos terrestres. En una prueba de 90 días con codornices japonesas alimentadas con una dieta que contenía el compuesto en concentraciones de 0,3 a 30 mg/kg de dieta se observó que la supervivencia de los polluelos disminuía a partir de 10 mg/kg de dieta. 7. Efectos en animales de experimentación y en sistemas de prueba in vitro 7.1 Toxicidad general Después de la ingestión de una dosis oral única, el hexaclorobutadieno es de levemente a moderadamente tóxico para las ratas adultas, moderadamente tóxico para las ratas macho destetadas y muy tóxico para las ratas hembras destetadas. Los principales órganos afectados son el riñón y, en grado mucho menor, el hígado. Los datos obtenidos con animales indican que el vapor de hexaclorobutadieno es irritante para las membranas mucosas y el líquido es corrosivo. La sustancia debe considerarse como un agente sensibilizador. En los riñones de ratas, ratones y conejos, el hexacloro-butadieno causa en los tubos proximales del riñón una necrosis que depende de la dosis. Las ratas macho adultas son menos vulnerables a la toxicidad renal que las hembras adultas y que los machos jóvenes. Los ratones jóvenes son más vulnerables que los adultos y no se observaron diferencias entre un sexo y otro. En las ratas hembra adultas la dosis intraperitoneal única más baja con la cual se observó necrosis renal fue de 25 mg/kg de peso corporal y en ratones adultos, machos y hembras, fue de 6,3 mg/kg de peso corporal. Se observaron cambios bioquímicos y alteraciones funcionales marcados en los riñones con dosis iguales o mayores que las asociadas con necrosis. Asimismo, en seis pruebas orales de corto plazo, dos estudios sobre reproducción y un estudio de largo plazo sobre la dieta realizados con ratas, el riñón fue el principal órgano afectado. Los efectos relacionados con la dosis comprenden una reducción del peso relativo del riñón y una degeneración del epitelio de los tubos. El nivel sin efectos nocivos observados de toxicidad renal en ratas en un estudio de dos años fue de 0,2 mg/kg de peso corporal por día. En un estudio de 13 semanas efectuado en ratones se obtuvo un nivel sin efectos nocivos observados de 0,2 mg/kg de peso corporal por día. Las hembras adultas de ambas especies eran más vulnerables que los machos adultos. En una prueba de inhalación de corto plazo (6 horas por día durante 12 días) se observaron efectos semejantes en los riñones con una concentración de vapor nominal de 267 mg/m3, con la cual también se observaron trastornos respiratorios y degeneración de la corteza suprarrenal. 7.2 Reproducción, embriotoxicidad y teratogenicidad Dos estudios sobre dieta y reproducción en ratas con dosis de hasta 20 y 75 mg/kg de peso corporal por día, respectivamente, mostraron una reducción del peso al nacer y un aumento del peso neonatal cuando se administraban a la madre dosis tóxicas de 20 y 7,5 mg/kg de peso corporal, respectivamente. La dosis altamente tóxica de 75 mg/kg de peso corporal por día fue suficiente para impedir la concepción y la implantación uterina. No se observaron anormalidades del esqueleto. En dos pruebas de teratogenicidad en las que se expuso a las ratas o bien a vapor de hexaclorobutadieno en concentraciones que oscilaban entre 21 y 160 mg/m3 durante 6 horas diarias (desde el 6° hasta el 20° día del embarazo) o bien a la administración intraperitoneal de 10 mg/kg de peso corporal por día (desde el 1° al 15° día de embarazo) se observaron en el desarrollo del feto efectos tóxicos tales como una reducción del peso al nacer, un retraso del desarrollo del corazón y uréteres dilatados pero sin grandes malformaciones. El retraso del desarrollo se observó en niveles que también eran tóxicos para las madres. 7.3 Genotoxicidad y carcinogenicidad En la prueba de Ames Salmonella se ha observado que el hexaclorobutadieno induce mutaciones genéticas en condiciones especiales que favorecen la formación de productos de conjugación con el glutatión. En un estudio in vivo se observó que había inducido aberraciones cromosómicas, pero no se observaron tales aberraciones en dos estudios in vitro. En una prueba in vitro se observó que la frecuencia de los intercambios entre cromátidas hermanas había aumentado en las células ováricas de hámsters de China. Se ha señalado el gran potencial mutagénico de los metabolitos sulfurosos del hexaclorobutadieno. En estudios in vitro, el compuesto indujo síntesis imprevistas de ADN en cultivos de fibroblastos de embriones de hámsters de Siria, pero no en cultivos de hepatocitos. Indujo síntesis imprevistas de ADN en ratas in vivo, pero no indujo mutaciones letales recesivas ligadas al sexo en Drosophila melanogaster. En el único estudio de largo plazo (dos años), en el cual las ratas recibieron una dieta que contenía hexaclorobutadieno en dosis de 0,2, 2 ó 20 mg/kg de peso corporal por día, se observó una mayor incidencia de neoplasias de los tubos renales únicamente con la dosis más elevada. 7.4 Mecanismos de toxicidad La nefrotoxicidad, mutagenicidad y carcinogenicidad del hexaclorobutadieno depende de la biosíntesis del conjugado sulfuroso tóxico 1-glutatión- S-yl-1,2,3,4,4-pentaclorobutadieno. Este conjugado se sintetiza principalmente en el hígado y se metaboliza luego en la bilis, el intestino y los riñones convirtiéndose en 1-cisteína- S-yl-1,2,3,4,4-pentaclorobutadieno (CPB). La activación de CPB, que depende del conjugado de cisteína beta-lyasa, en una tiocetena reactiva en las células de los tubos proximales finalmente da lugar a un enlace covalente con macromoléculas celulares. 8. Efectos en el ser humano No se han descrito efectos patogénicos en la población en general. Se conocen dos casos de trastornos padecidos por trabajadores agrícolas que utilizaban el hexaclorobutadieno como fumigante, pero esas personas también habían estado expuestas a otras sustancias. En los linfocitos de la sangre periférica de operarios que trabajaban en la producción de hexaclorobutadieno y estaban expuestos, según se informa, a concentraciones de 1,6 a 12,2 mg/m3 se observó una frecuencia mayor de aberraciones cromosómicas. 9. Evaluación de los riesgos para la salud humana y de los efectos en el medio ambiente 9.1 Evaluación de los riesgos para la salud humana Como se han hecho muy pocos estudios en el ser humano, la evaluación se basa principalmente en estudios efectuados en animales de laboratorio. Sin embargo, los limitados datos existentes sobre el ser humano, obtenidos in vitro, sugieren que el metabolismo del hexaclorobutadieno en el ser humano es semejante al observado en animales. Se considera que el vapor de hexaclorobutadieno irrita las membranas mucosas del ser humano y que en estado líquido es corrosivo. El compuesto también debe considerarse como un agente sensibilizador. Los principales órganos afectados por la toxicidad son los riñones y, en mucho menor grado, el hígado. En base a estudios de corto y largo plazo de ingestión por vía oral por ratas y ratones, se determinó un nivel sin efectos adversos observados de 0,2 mg/kg de peso corporal por día. En un estudio de inhalación en el corto plazo en ratas (12 días, a razón de 6 horas por día), el nivel sin efectos adversos observados fue de 53 mg/m3. Se observaron una reducción del peso al nacer y un aumento de peso neonatal únicamente con dosis tóxicas para la madre; lo mismo puede decirse de los efectos tóxicos para el desarrollo. Se ha observado que el hexaclorobutadieno induce mutaciones genéticas, aberraciones cromosómicas, aumento de los intercambios entre cromátidas hermanas y síntesis imprevistas de ADN, aunque algunos estudios han dado resultados negativos. Con respecto a la genotoxicidad del hexaclorubutadieno, las observaciones realizadas en animales son limitadas y las efectuadas en el ser humano insuficientes. Tras la administración oral a largo plazo del hexacloro-butadieno a ratas, se ha observado una mayor frecuencia de neoplasias de los tubos renales, pero solamente con dosis elevadas causantes de nefrotoxicidad notable. Hay indicios limitados de carcinogenicidad en animales e indicios insuficientes en el ser humano. En base al nivel sin efectos adversos observados en ratones y ratas, que es de 0,2 mg/kg de peso corporal por día, se ha estimado un nivel sin efectos adversos observados en el ser humano, que es de 0,03 a 0,05 mg/kg de peso corporal por día. Hay un margen de seguridad de 150 entre el nivel sin efectos adversos observados estimado y la ingesta diaria total máxima estimada, suponiendo que el compuesto se absorba a través del agua de bebida contaminada y de alimentos con elevado contenido de lípidos. 9.2 Evaluación de los efectos en el medio ambiente El hexaclorobutadieno es de moderadamente a muy tóxico para los organismos acuáticos; los crustáceos y peces son los más vulnerables. Se ha establecido un nivel de riesgo para el medio ambiente de 0,1 µg/litro. Se estima que la concentración ambiental prevista máxima lejos de las fuentes equivale al nivel de riesgo ambiental extrapolado multiplicado por dos y, por consiguiente, los organismos acuáticos tal vez estén en peligro en las aguas de superficie contaminadas. No pueden excluirse efectos adversos en organismos bentónicos. En vista de la toxicidad del hexaclorobutadieno para los mamíferos, el consumo de organismos bentónicos o acuáticos por otras especies tal vez sea motivo de inquietud.
See Also: Hexachlorobutadiene (IARC Summary & Evaluation, Volume 20, 1979) Hexachlorobutadiene (IARC Summary & Evaluation, Volume 73, 1999)