INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY ENVIRONMENTAL HEALTH CRITERIA 173 Tris(2,3-dibromopropyl) phosphate and Bis(2,3-dibromopropyl) phosphate. 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. G.J. van Esch, Bilthoven, Netherlands Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization World Health Organization Geneva, 1995 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 Tris(2,3-dibromopropyl) phosphate and Bis(2,3-dibromopropyl) phosphate. (Environmental health criteria ; 173) 1.Phosphoric acid esters 2.Environmental exposure 3.Flame retardants I.Series ISBN 92 4 157173 X (NLM Classification: QP 981.P49) 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 1995 Publications of the World Health Organization enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Convention. All rights reserved. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. CONTENTS ENVIRONMENTAL HEALTH CRITERIA FOR TRIS(2,3-DIBROMOPROPYL) PHOSPHATE AND BIS(2,3-DIBROMOPROPYL) PHOSPHATE INTRODUCTION TRIS(2,3-DIBROMOPROPYL) PHOSPHATE 1. SUMMARY AND EVALUATION; CONCLUSIONS AND RECOMMENDATIONS 1.1. Summary and evaluation 1.1.1. Production and use 1.1.2. Physical and chemical properties 1.1.3. Environmental transport, distribution, and transformation 1.1.4. Environmental levels and human exposure 1.1.5. Kinetics and metabolism in laboratory animals and humans 1.1.6. Effects on laboratory mammals and in vitro test systems 1.1.7. Effects on humans 1.1.8. Effects on other organisms in the laboratory and field 1.2. Conclusions 1.3. Recommendations 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS 2.1. Identity 2.1.1. Technical product 2.2. Physical and chemical properties 2.3. Analytical methods 2.3.1. General 2.3.2. Urine 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. Sources of human and environmental exposure 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION 4.1. Transport and distribution between media 4.2. Transformation 4.2.1. Biodegradation 4.2.2. Abiotic degradation 4.2.3. Bioaccumulation 4.3. Interaction with other physical, chemical, or biological factors 4.4. Ultimate fate following use 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1. Environmental levels 5.1.1. Air 5.1.2. Water 5.1.3. Soil 5.1.4. Fish 5.2. General population exposure 5.2.1. Subpopulation at special risk 5.3. Occupational exposure 6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS 6.1. Absorption 6.2. Elimination 6.2.1. Different routes (rat and rabbit) 6.2.2. Dermal exposure (rat and rabbit) 126.96.36.199 TBPP 188.8.131.52 TBPP-treated fibres 6.2.3. Dermal exposure (human) 6.3. Distribution 6.3.1. Rat 184.108.40.206 Oral 220.127.116.11 Intravenous 6.3.2. Dermal (rabbit) 6.4. Metabolic transformation 6.4.1. In vivo studies 18.104.22.168 Oral (rat) 6.4.2. In vitro studies 6.5. Covalent binding to macromolecules 7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS 7.1. Single exposure 7.2. Short-term exposure 7.2.1. Oral exposure (rat) 22.214.171.124 TBPP 126.96.36.199 TBPP-treated fibres 7.2.2. Oral exposure (dog) 188.8.131.52 TBPP 184.108.40.206 TBPP-treated fibres 7.2.3. Dermal exposure 220.127.116.11 Rabbit 18.104.22.168 Dog 7.3. Long-term exposure 7.4. Skin and eye irritation; sensitization 7.4.1. Skin irritation 7.4.2. Eye irritation 7.4.3. Sensitization 7.5. Reproductive toxicity, embryotoxicity, and teratogenicity 7.5.1. Reproductive toxicity 7.5.2. Teratogenicity 7.6. Mutagenicity and related end-points 7.6.1. DNA damage 22.214.171.124 In vivo 126.96.36.199 In vitro 7.6.2. Mutation assay with Salmonella typhimurium strains 7.6.3. Mutations by urine of rats treated with TBPP65 7.6.4. Other mutation assays 7.6.5. Chromosomal effects 7.6.6. Cell transformation 7.6.7. Miscellaneous tests 7.6.8. Mechanisms of TBPP genotoxicity 7.7. Carcinogenicity 7.7.1. Oral 188.8.131.52 Mouse 184.108.40.206 Rat 7.7.2. Dermal 220.127.116.11 Mouse 7.8. Special studies 7.8.1. Kidneys 7.9. Factors modifying toxicity; toxicity of metabolites 7.9.1. Toxicity of metabolites 7.9.2. Mutagenicity of metabolites 8. EFFECTS ON HUMANS 8.1. General population exposure 8.2. Occupational exposure 9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD 9.1. Laboratory studies 9.1.1. Microorganisms 9.1.2. Aquatic organisms 18.104.22.168 Invertebrates 22.214.171.124 Vertebrates 9.1.3. Terrestrial organisms 126.96.36.199 Plants 13. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES BIS(2,3-DIBROMOPROPYL) PHOSPHATE AND SALTS A1. SUMMARY AND EVALUATION; CONCLUSIONS AND RECOMMENDATIONS A2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS A2.1 Identity A2.2 Physical and chemical properties A2.3 Analytical methods A3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE A3.1 Natural occurrence A3.2 Anthropogenic sources A3.2.1 Production levels and processes A3.2.2 Uses A3.3 Contamination of the environment A3.4 Environmental transport, distribution, transformation, and exposure levels A4. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS A4.1 Absorption, distribution, elimination, and biotransformation A5. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS A5.1 Single exposure A5.2 Short-term exposure A5.3 Long-term exposure A5.3.1 Mutagenicity and related end-points A5.3.2 Carcinogenicity A5.4 Special studies A5.4.1 Kidneys A5.5 Effects on humans and other organisms in the laboratory and field REFERENCES RESUME ET EVALUATION, CONCLUSIONS ET RECOMMANDATIONS RESUMEN 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 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 ES02617-15 from the National Institute of Environmental Health Sciences, National Institutes of Health, USA, and by financial support from the European Commission. Environmental Health Criteria PREAMBLE Objectives In 1973 the WHO Environmental Health Criteria Programme was initiated with the following objectives: (i) to assess information on the relationship between exposure to environmental pollutants and human health, and to provide guidelines for setting exposure limits; (ii) to identify new or potential pollutants; (iii) to identify gaps in knowledge concerning the health effects of pollutants; (iv) to promote the harmonization of toxicological and epidemiological methods in order to have internationally comparable results. The first Environmental Health Criteria (EHC) monograph, on mercury, was published in 1976 and since that time an everincreasing number of assessments of chemicals and of physical effects have been produced. In addition, many EHC monographs have been devoted to evaluating toxicological methodology, e.g., for genetic, neurotoxic, teratogenic and nephrotoxic effects. Other publications have been concerned with epidemiological guidelines, evaluation of short-term tests for carcinogens, biomarkers, effects on the elderly and so forth. Since its inauguration the EHC Programme has widened its scope, and the importance of environmental effects, in addition to health effects, has been increasingly emphasized in the total evaluation of chemicals. The original impetus for the Programme came from World Health Assembly resolutions and the recommendations of the 1972 UN Conference on the Human Environment. Subsequently the work became an integral part of the International Programme on Chemical Safety (IPCS), a cooperative programme of UNEP, ILO and WHO. In this manner, with the strong support of the new 14 partners, the importance of occupational health and environmental effects was fully recognized. The EHC monographs have become widely established, used and recognized throughout the world. 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It is accepted that the following criteria should initiate the updating of an EHC monograph: new data are available that would substantially change the evaluation; there is public concern for health or environmental effects of the agent because of greater exposure; an appreciable time period has elapsed since the last evaluation. All Participating Institutions are informed, through the EHC progress report, of the authors and institutions proposed for the drafting of the documents. A comprehensive file of all comments received on drafts of each EHC monograph is maintained and is available on request. The Chairpersons of Task Groups are briefed before each meeting on their role and responsibility in ensuring that these rules are followed. WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR TRIS- AND BIS(2,3-DIBROMOPROPYL)PHOSPHATE Members Dr D. Anderson, BIBRA Toxicology International, Carshalton, United Kingdom Dr D. Osborn, Institute of Terrestrial Ecology, Monks Wood, Huntingdon, United Kingdom Dr E. Soderlund, National Institute of Public Health, Oslo, Norway (Rapporteur) Dr B. Jansson, Institute of Applied Environmental Research, Stockholm University, Solna, Sweden Dr J. Kielhorn, Fraunhofer Institute for Toxicology and Aerosol Research, Hannover, Germany Dr R.D. Kimbrough, Institute for Evaluating Health Risks, Washington DC, USA (Vice-chairman) Dr Wai-On Phoon, Department of Occupational Health, University of Sydney, Sydney, Australia (Chairman) Dr R. Benson, Drinking Water Branch, US EPA, Denver, USA Dr J. Sekizawa, National Institute of Health Sciences, Tokyo, Japan (Rapporteur) Observers Dr M.L. Hardy, Toxicology Advisor, Albemarle Corporation, Baton Rouge, USA Dr D.L. McAllister, Director, Quality, Environment, Health and Safety, and Research Support, Great Lakes Chemical Corporation, West Lafayette, USA Secretariat Dr K.W. Jager, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland (Secretary) ENVIRONMENTAL HEALTH CRITERIA FOR TRIS- AND BIS(2,3-DIBROMOPROPYL) PHOSPHATE A WHO Task Group on Environmental Health Criteria for tris- and bis(2,3-dibromopropyl) phosphate met at BIBRA Toxicology International, Carshalton, United Kingdom, from 6 to 11 June 1994. Dr K.W. Jager, IPCS, welcomed the participants on behalf of Dr M. Mercier, Director of the IPCS, and the three IPCS cooperating organizations (UNEP/ILO/WHO). The Group reviewed and revised the draft and made an evaluation of the risks for human health and the environment from exposure to tris- and bis(2,3-dibromopropyl) phosphate. The first draft was prepared by Dr G.J. van Esch, the Netherlands, who also prepared the second draft, incorporating comments received following circulation of the first drafts to the IPCS Contact Points for Environmental Health Criteria monographs. Dr K.W. Jager of the IPCS Central Unit was responsible for the scientific content of the monograph and Mrs M.O. Head of Oxford for the technical editing. The efforts of all who helped in the preparation and finalization of the monograph are gratefully acknowledged. INTRODUCTION The IPCS is preparing several EHC monographs on Flame Retardants, which will provide additional information relevant to TBPP. There will be one monograph, "Flame Retardants - A General Introduction" (in preparation), giving a general introduction to the use, the mode of action, and the potential risks of flame retardants, and listing the substances used as flame retardants with a general indication of the data available on them. Flame retardants in wide use are discussed in separate monographs, e.g., EHC 162: Brominated Diphenyl Ethers (IPCS, 1994a) and EHC 172: Tetrabromobisphenol-A (IPCS, 1995). Some flame retardants considered hazardous for humans and the environment have been reviewed in separate monographs including EHC 152: Polybrominated Biphenyls (IPCS, 1994b), and EHC 173: Tris- and Bis(2,3-dibromopropyl) phosphate (this monograph). Because of the possibility of the formation of halogenated dibenzodioxins and dibenzofurans under certain circumstances, such as pyrolysis, the following monographs have been developed: EHC 88: Polychlorinated Dibenzodioxins and Dibenzofurans (IPCS, 1989) and Polybrominated Dibenzodioxins and Dibenzofurans (in preparation). The reader should consult these monographs for further information. Tris(2,3-dibromopropyl) phosphate was an important commercial flame retardant ("TRIS"), especially for children's sleepwear. In 1977, the US Consumer Product Safety Commission banned children's clothing treated with tris(2,3-dibromopropyl) phosphate. Since then, in several other countries, the use of this compound as a flame retardant has been severely restricted in consumer products and prohibited in textiles. Because tris(2,3-dibromopropyl) phosphate can also be used for other applications, the information available on physical and chemical properties, behaviour in the environment, occurrence in the environment and humans, kinetics and metabolism, toxicity for laboratory animals and in the field, and the exposure of the general population and workers, is summarized in this Environmental Health Criteria monograph. General properties and uses of brominated flame retardants are given in "Flame Retardants - A General Introduction" (in preparation). ABBREVIATIONS BA 2-bromoacrolein BBPP bis(2,3-dibromopropyl) phosphate DBCP 1,2-dibromo-3-chloropropane DBP 2,3-dibromopropanol DMBA dimethylbenzanthracene mono-BPP mono(2,3-dibromopropyl) phosphate TBPP tris(2,3-dibromopropyl) phosphate TPA tetradecanoyl phorbolacetate TRIS-(2,3-DIBROMOPROPYL) PHOSPHATE 1. SUMMARY AND EVALUATION; CONCLUSIONS AND RECOMMENDATIONS 1.1 Summary and evaluation 1.1.1 Production and use Tris(2,3-dibromopropyl) phosphate (TBPP) was first produced in about 1950; commercial production was reported in 1959. Production of TBPP, in the USA, in 1975, was estimated to be between 4100 and 5400 tonnes. As far as is known, TBPP is not produced or used currently in the world as a flame retardant in textiles, but may be added to polymers used for other purposes. TBPP was an important flame retardant for cellulose and tri-acetate and polyester fabrics, especially in children's sleepwear, but has been banned for these applications in several countries in Europe, the USA (1977), and Japan (1978). TBPP exists both in, and on, the fabric. When it is in the fabric, it is not extractable with solvents and, therefore, probably not available for dermal absorption. However, when it is on the fibre surface, it can be extracted during laundering, and by acetic acid, other solvents, and saliva, and is available for dermal absorption. In this case, substantial losses of surface TBPP from fabrics during use and/or laundering of the finished products, will occur, and will contaminate the environment. Furthermore, release of TBPP into the environment has been reported from textile-finishing plants and the ultimate disposal of solid wastes, containing TBPP. 1.1.2 Physical and chemical properties TBPP is available in at least two grades. The high-purity grade is a clear, pale-yellow, viscous liquid, with up to 1.5% volatiles. The low-purity grade may contain up to 10% volatiles. TBPP (purity > 97%), has a boiling point of 390°C, a melting point of 5.5°C, and a vapour pressure of 1.9 × 10-4mmHg at 25°C. The solubility of TBPP in water is low (8 mg/litre). When heated to decomposition, above 260-300°C, TBPP emits compounds containing bromine and phosphorus. The n-octanol/water partition coefficient (log Pow) is 3.02. Analytical methods to determine TBPP and its metabolites in biological samples and other matrices are available. 1.1.3 Environmental transport, distribution, and transformation The limited information available suggests that TBPP is relatively persistent in the environment. Oxidation and photodegradation are not likely to be significant fate processes. However, hydrolysis involving the bromine atoms on the propyl group may occur, especially under basic conditions. Volatilization from water may occur, but no actual data are available. Although biodegradation of TBPP (half-life 19.7 h) in activated sewage is reported to occur, it is not thought to be an important process in natural soils and waters. In sterilized sludge, almost no breakdown takes place. Bis(2,3-dibromopropyl) phosphate (BBPP) was found as a major breakdown product. Because TBPP is virtually insoluble in water, adsorption on particulate matter and sediment may be an important process. An estimated log Koc (3.29) suggests strong adsorption on soil. On the basis of this Koc value and the low measured water solubility, TBPP is expected to leach only slowly to groundwater. TBPP will tend to accumulate in rubbish dumps and other disposal sites, which may result in biological accumulation. A bioaccumulation study with fathead minnow showed a bioconcentration factor of 2.7, which is low, while the n-octanol/water partition coefficient (Log Pow) was 3.02. Because of its low vapour pressure, TBPP is expected to be mostly sorbed on particulate matter in air. Thermal oxidative degradation of TBPP at 370°C showed that hydrogen bromide and C3-brominated compounds, such as bromopropenes, dibromopropenes, and diand tribromopropanes, are formed. 1.1.4 Environmental levels and human exposure Data on environmental levels and human exposure are limited. Studies carried out in Japan in 1975 showed that 20 samples of water, soil, and fish did not contain TBPP. TBPP was identified, but not quantified, in air particulates in the surroundings of an industry. Children wearing TBPP-treated sleepwear were the group of the general population particularly exposed to this flame retardant. The estimated intake via the skin of children wearing TBPPtreated sleepwear in the USA was calculated to be 9 µg/kg body weight per day. The Consumer Product Safety Commission of the USA calculated that, over a 6-year period, a child wearing TBPP-treated clothing could absorb a total of 2-77 mg TBPP/kg body weight or more. 1.1.5 Kinetics and metabolism in laboratory animals and humans TBPP is absorbed readily from the gastrointestinal tract and at a moderate rate via the skin in rats and rabbits. In rats, TBPP or its metabolites are eliminated within 5 days. Approximately 50% is eliminated via the urine, 10% via the faeces, and 10-20% is exhaled as CO2. One day after oral administration of labelled TBPP to rats, radioactivity was found in the blood, liver, kidneys, muscles, fat, and skin, in a range of 0.2-6.6%. The half-life of clearance of radioactivity from these organs was approximately 2-4 days. After 8 h, only bis(2,3-dibromopropyl) (BBPP) phosphate was still present in substantial concentrations in most tissues. After oral administration of TBPP to rats, six metabolites were identified in the urine and bile. The main metabolite in the urine, faeces, bile, and tissues was BBPP. The metabolite 2,3-dibromopropanol (DBP) was also identified in rats and in children wearing TBPP-treated clothing. Liver microsomes metabolize TBPP in the presence of NADPH and oxygen. The main metabolites are BBPP and 2,3-dibromopropanol (DBP). It has been shown that BBPP is formed by oxidation at the C3 and, possibly, also at the C2 position of TBPP. In addition to BBPP, 2-bromoacrolein, 2-bromoacrylic acid, and propyl-hydroxylated compounds and metabolites conjugated with glutathione have been found. S-(2,3-dihydroxypropyl) glutathione was identified in the bile of rats, and, it was suggested that TBPP and/or BBPP are conjugated directly with glutathione by glutathione S-transferase with the formation of episulfonium ion metabolites. TBPP has been shown to be activated to form products that bind covalently to proteins and DNA in vivo and in vitro. After intraperitoneal injections of tritiated-TBPP, male mice, hamsters, and guinea-pigs, which are less sensitive to TBPP-induced nephrotoxicity than rats, showed similar levels of covalent binding to proteins in the liver and kidneys. In the male rat, which is highly susceptible to TBPP-induced nephrotoxicity, much higher amounts of radiolabel were bound to kidney proteins than to liver proteins. Liver microsomes from mice, guinea-pigs, hamsters, and humans all metabolized TBPP to genotoxic intermediates. However, the rate of formation of reactive TBPP metabolites with human liver microsomes was lower than with liver microsomes from the rodents. The binding of labelled TBPP and analogues in rats at a nephrotoxic dose showed that the covalent protein binding was highest in the kidneys followed by the liver and testes. The results of comparative in vitro and in vivo studies on renal DNA damage suggested that BBPP is formed in the liver by P450-mediated oxidation at either C2 or C3 of TBPP. BBPP is transported to the kidneys, where it is metabolized to reactive intermediates that cause DNA damage and bind to kidney proteins. The activation occurring in the kidney appears not to involve P450 but seems to be mediated by GSH-dependent metabolism. In vitro studies with labelled TBPP and analogues showed that oxidation of TBPP incorporates one atom of oxygen from water. This implies that oxidation at C2 of the propyl moiety yields a reactive alphabromoketone that can alkylate protein directly or hydrolyse to BBPP and a reactive bromo-alpha-hydroxyketone. 1.1.6 Effects on laboratory mammals and in vitro test systems The acute and short-term oral, and the acute dermal, toxicities of TBPP are low. The oral LD50 for the rat > 2 g/kg and the dermal LD50 for the rabbit > 8 g/kg body weight. Extensive kidney damage (necrosis of renal proximal tubular cells) was noted in male rats following a single ip injection of 100 mg TBPP/kg body weight. Four-week, and 90-day, oral toxicity tests with TBPP (by gavage or in the diet) in rats showed a dose-related increase in the incidence and severity of chronic nephritis at dose levels of 25 mg/kg body weight or more. In rabbits, daily dermal applications of 2.2 g TBPP/kg body weight or more, for 4 weeks, resulted in degenerative changes in the liver and kidneys. All rabbits died within four weeks. No deaths occurred in another study with dose levels of up to 250 mg/kg body weight. In a 90-day test on rabbits, weekly application of 2.27 g/kg body weight to the skin resulted in kidney changes, testicular atrophy, and aspermatogenesis. No skin or eye irritation was observed in rabbits with dose levels of 1.1 g or 0.22 g TBPP and no skin sensitization was observed in guinea-pigs. Two teratogenicity studies were carried out on rats. In one study with dose levels of up to 125 mg/kg body weight, no teratogenicity was observed. In another study with a dose level of 200 mg/kg body weight, a significant increase in skeletal variations in the fetuses was observed, and, with 50 and 100 mg/kg body weight, a significantly lower viability index was found. The authors concluded that the observed effect resulted from maternal toxicity. Extensive DNA damage was found in various organs of rats administered TBPP. In vitro, TBPP has been shown to induce DNA strand breaks in human KB cells. It induced unscheduled DNA synthesis in rat liver hepatocytes, but not in human foreskin epithelial cells. TBPP was mutagenic in several studies on Salmonella typhimurium, especially in base-pair substituting strains with, and without, metabolic activation. Forward gene mutation assays using Chinese hamster V79 cells, with, and without, metabolic activation were negative. However, a positive effect in the presence of liver microsomes of rats pretreated with phenobarbital was obtained. A weak positive effect was obtained with mouse lymphoma cells (L5178YTK locus). TBPP increased the number of sister chromatid exchanges (SCEs) in Chinese hamster V 79 cells, but no chromosomal aberrations were induced in Chinese hamster cells, mouse bone marrow cells, or in cultured human lymphoid cells. SCEs but no chromosomal aberrations were found with diploid human fibroblastic cells (line HE 2144) without metabolic activation. However, in an in vitro chromosome aberration test with the Chinese hamster cell line (CHL), TBPP was positive. A positive result was obtained with TBPP in a micronucleus test on Chinese hamster bone marrow cells. Another micronucleus study with mice showed a weak positive effect. Studies with Drosophila melanogaster showed that TBPP increased sex-linked recessive lethals in male germ cells and in adult males, reciprocal translocations were induced. TBPP showed a strong positive response in the w/w+ eye mosaic assay. Several studies have been directed towards the elucidation of the mechanisms involved in TBPP-induced mutagenicity and/or genotoxicity. Bacterial mutagenicity of TBPP is mediated by the microsomal monooxygenase system. TBPP is activated by cytochrome P450 in a reaction depending on NADPH and oxygen. Microsomes prepared from livers of animals treated with phenobarbital or PCBs give increased mutagenicity. The mono-and bis(2,3-dibromopropyl) phosphates are less mutagenic than TBPP. In vitro studies have shown that oxidation at C3 of the TBPP molecule yields the potent direct acting mutagen 2bromoacrolein that also binds to DNA. Species differences in the bioactivation of TBPP to metabolites mutagenic to Salmonella typhimurium TA 100 have been reported. Liver microsomes from mice were more effective than those from guinea-pigs, hamsters, and rats. Three studies in which C3H/10T1/2 cells were used to study cell transformation were carried out. In one study, a positive effect was noted, but, in the other two studies, the results were negative. TBPP was tested on mice and rats by oral administration and on female mice by skin application in long-term studies. In mice, following oral administration, TBPP produced tumours of the fore-stomach and lung in the animals of both sexes, benign and malignant liver tumours in females, and benign and malignant tumours of the kidneys in males. In rats, TBPP produced benign and malignant tumours of the kidneys in males and benign kidney tumours in females. After skin application to female mice, TBPP produced tumours of the skin, lung, fore-stomach, and oral cavity. From these studies, it can be concluded that TBPP has carcinogenic potential in mice and rats. When the TBPP metabolite BBPP was administered to rats orally, it caused tumours in both sexes in the digestive system. The tumours found included papillomas and adenocarcinomas of the tongue, oesophagus, and forestomach, adenocarcinomas of the intestine, and hepatocellular adenomas and carcinomas. Another metabolite of TBPP, DBP, was tested on rats and mice by dermal application. In male rats, there was an increased incidence of neoplasms in skin, nose, oral mucosa, oesophagus, forestomach, small and large intestine, Zymbal's gland, liver, kidney, tunica vaginalis, and spleen. In female rats, there was an increased incidence of neoplasms of the skin, nose, oral mucosa, oesophagus, forestomach, small and large intestine, Zymbal's gland, liver, kidney, clitoral gland, and mammary gland. In male mice, there was an increased incidence of neoplasms in the skin, forestomach, liver, and lung, and in female mice, there was an increased incidence of neoplasms of the skin and the forestomach. 1.1.7 Effects on humans Limited data are available regarding the effects of TBPP on humans. TBPP has been tested for skin sensitization potential in a few studies on humans. The results of these studies indicate that TBPP has a low sensitization potential and no skin irritation was reported. However, persons who showed a positive sensitization response to pure TBPP also reacted when exposed to fabrics treated with TBPP. 1.1.8 Effects on other organisms in the laboratory and field There are very few data on the effects of TBPP on other organisms. All 6 goldfish (Carassius auratus), exposed to 1 mg TBPP/litre, died within 5 days. The EC50 for growth inhibition in oat seed was 1000 mg/kg soil. This concentration caused a 100% inhibition of growth in turnip seed (Brassica rapa sp.). 1.2 Conclusions TBPP has been used as a flame retardant in fabrics, particularly in children's sleepwear, but there is inadequate information on its use in other applications. Exposure of the general population was primarily through contact with fabrics treated with TBPP. There is little information on the exposure of, and hazards to, workers from the commercial production of TBPP and its use in a variety of products. Because of the paucity of data, no firm conclusions can be drawn as to the exposure levels and hazards of TBPP for organisms in the environment, other than humans. Animal studies have shown that TBPP can be absorbed from the gastrointestinal tract and, to a lesser extent, from the skin. TBPP can also be absorbed through the skin of humans. In the rat, TBPP appears to be extensively metabolized in the liver to BBPP, which is the major metabolite detected in the urine and, to a lesser extent, to DBP. In addition, other brominated metabolites of TBPP have been found in small amounts. DBP has also been detected in humans wearing TBPP-treated fabrics. The main route of elimination is the urine and very little is excreted as the parent compound. The main metabolic pathway seems to be through metabolism by cytochrome P450 and glutathione S-transferases. From the available data, it can be concluded that TBPP has a low acute toxicity for experimental animals. Repeated dose studies with relatively high doses of TBPP have revealed kidney and liver damage in rats and also testicular toxicity in rabbits. TBPP has elicited a clear genotoxic effect in several test systems, both in vitro and in vivo. Carcinogenic effects were found in rats and mice. The metabolites BBPP and DBP have also been shown to produce carcinogenic effects in experimental animals. No irritation effects were found in animals and a low sensitization potential in humans was noted. In 1977, the US Consumer Product Safety Commission banned children's clothing treated with TBPP, because of concerns that the chemical might be a human carcinogen, and, because of the possibility of significant human exposure through contact with treated fabrics. Since then, the use of this substance as a flame retardant in consumer products has been severely restricted in several other countries and it has been prohibited in textiles. 1.3 Recommendations Because of its toxic effects, TBPP should no longer be used commercially. If uses are identified for which there are no less hazardous alternatives to TBPP, studies to demonstrate the absence of exposure of, and hazards for, humans and the environment should be conducted. 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS 2.1 Identity Chemical formula C9H15Br6O4P Chemical structure BrCH2-CHBr-CH2O \ BrCH2-CHBr-CH2O - P = O / BrCH2-CHBr-CH2O Relative molecular mass 697.7 Synonyms tris(2,3-dibromopropyl) phosphate; tris(2,3-dibromopropyl) phosphoric acid ester; phosphoric acid, tris(2,3- dibromo-propyl) ester; tris(dibromopropyl) phosphate CAS registry number 126-72-7 CAS chemical name 2,3-dibromo-1-propanol-phosphate (3:1) RTECS registry number UB0350000 Trade names T 23 P; TP-69; DBP-TP; Apex (emulsion) 462-5; Hamcogard FR; Fyrol 59; Tanotard PN-2; Cav Gard FR 1811 and FR 1812; Pyrosan 497; Firemaster LV-T23P and T23P-LV; Firemaster 200; Glotard PE-2; PE 10; Anfram 3PB; Bromkal P 67-6HP; ES 685; Firemaster T23 and T23P; Flacavon R; Flamex T23P; Flammex AP; Zetofex ZN; Fyrol HB-32; NCI- CO3270; Phoscon PE60; Phoscon UF- S; RCRA waste number U 235; USAF-DO-41 (LeBlanc, 1976; IARC, 1979; Ulsamer et al., 1980; IRPTC, 1987). FR 2406; Berkflam T23 P; Flammex LVT 23P; 3PBR; TDBP; TDBPP; TRIS; TRIS-BP; Zetifex ZN; (Andersen, 1977). 2.1.1 Technical product Commercial TBPP contains up to 0.2% of the following impurities: 2,3-dibromopropanol, 1,2,3-tribromopropane, and 1,2-dibromo- 3-chloropropane (DBCP) (Blum & Ames, 1977; Van Duuren et al., 1978; Ulsamer et al., 1980). 2.2 Physical and chemical properties Two grades of TBPP were available in the USA. The highpurity grade had the following typical properties: a clear, pale-yellow, viscous liquid; relative density at 25°C, 2.20-2.26; refractive index at 25°C, 1.576-1.577; viscosity at 25°C, 3900-4200 centistokes; acid number (mg KOH/g), 0.05 max; volatiles, 1.5% max; bromine content, 68.7%, and phosphorus content, 4,0%. Typical properties for a lower grade are as follows: density at 25°C, 2.2-2.3; viscosity at 25°C, 1400-1700 centistokes; acid number (mg KOH/g), 0.05 max; and volatiles, 10% max. (US EPA, 1976; IARC, 1979). Osterberg et al. (1977) reported a viscosity of 9200 cP (25°C) for TBPP of a purity of 99.76%. Firemaster LVT 23P has a viscosity of 9200 cP (Kerst, 1974). Specific gravity 2.27 (2.2-2.3) g/ml at 25°C (density) (Kerst, 1974) Boiling point: 390°C (Dybing et al., 1989) Melting point: 5.5°C (Dybing et al., 1989) Vapour pressure: 1.9 × 10-4 mmHg at 25°C 1.2 × 10-3 mmHg at 45°C 4.8 × 10-3 mmHg at 65°C (Kerst, 1974) Solubility: Virtually insoluble in water (6.3 mg/litre at 20°C) and hexane; miscible in organic solvents, such as carbon tetrachloride, acetone, chloroform, methylene chloride, dimethyl formamide, methanol, xylene, benzene, toluene, and ethyl acetate (Kerst, 1974) Stability: Heat stability: Major decomposition begins at about 260-300°C; when heated to decomposition, TBPP emits toxic fumes of Br- and POx (Sax, 1984) Light stability: Stable in sunlight Hydrolytic stability: Hydrolysed by acids and bases (IRPTC, 1987) n-Octanol/water partition coefficient (log Pow): 3.02 (IARC, 1979) 2.3 Analytical methods 2.3.1 General TBPP is determined using a gas chromatograph equipped with a flame photometric detector with possible cleaning processes. Direct mass spectrometry, GC-MS, and HPLC are also used for the analysis of biological samples containing TBPP and its metabolites (Cope, 1973; Lynn et al., 1980, 1982; Pearson et al., 1993a). Recovery and limits of determination vary, depending on sampling procedures and matrices. GC analysis shows that TBPP can be determined at the 10 ng level by using a column packed with a high liquid loaded support. In an indirect analytical method, TBPP is determined by spectrophotometry, by complexing phosphor with molybdenum blue after hydrolysis of the TBPP by hydrobromic acid (Nakamura, 1980; Gutenmann & Lisk, 1975). Gardner (1979) described a densitometric method using thin-layer chromatography. TBPP was chromatographed on silicagel thin-layer plates, using ethyl acetate hexane (30:70) as a developing solvent. TBPP was visualized by spraying the chromatograms with 1% aqueous silver nitrate followed by exposure to UVR for 40 min. The spots were quantified by densitometry at 600 nm. The lower level of sensitivity was 50 ng; calibration plots were linear from 50 to 800 ng. The recovery of TBPP from sewage sludge samples fortified at the 1.0 ppm level was 97%. Techniques for the qualitative detection of TBPP in textiles have been described, including thin-layer chromatography, HPLC, and NMR (Iliano et al., 1982). 2.3.2 Urine In mammalian species, organophosphates undergo enzymatic or chemical hydrolysis to form the corresponding acids and alcohols. The alcohols are often excreted in the urine as soluble conjugates. Since the hydrolysis of TBPP yields 2,3-dibromopropanol (DBP), an analytical method has been developed to determine free, and conjugated, DBP. Extraction of urine by diethylether/hydrochloric acid, followed by methylation with diazomethane gives the methylether of DBP. Determination is by electron affinity gas chromatography. The limits of determination in rat and human urine were 0.4 and 0.2 mg/litre, respectively (St. John et al., 1976). 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1 Natural occurrence TBPP is not known to occur naturally. 3.2 Anthropogenic sources 3.2.1 Production levels and processes It is estimated that TBPP was first produced in 1950, when it was prepared by the addition of bromine to a solution of triallyl phosphate in benzene. However, it is synthesized in the USA by a two-step process in which bromine is added to allyl alcohol to give 2,3-dibromopropanol (DBP). This is then reacted with phosphorus oxychloride, in the presence of a Lewis acid such as, aluminum chloride or stannium chloride as a catalyst (Overbeek & Nametz, 1962). The commercial production of TBPP was reported in 1959 and US production in 1975 has been estimated to have been between 4100-5400 tonnes (US EPA, 1976). Prior to 1977, 4500 tonnes of TBPP were produced annually in the USA by 6 manufacturers. There was no evidence of production of TBPP in the USA in 1986. Production of TBPP in Japan in 1976 and 1977 is estimated to have been 100 and 300 tonnes per year, respectively, made by one manufacturer. No TBPP is produced in Japan at present. It has not been possible to assess whether TBPP is currently produced. However, no reports are available that describe any production of TBPP. 3.2.2 Uses TBPP has been used as a flame retardant for cellulose and triacetate and polyester fabrics, which are widely used in children's sleepwear. It has also been used as a flame retardant in other materials, such as urethane foam and acrylic carpets and sheets, polyvinyl- and phenolic resins, polystyrene foam, paints, lacquers, paper coatings and styrene-butadiene rubber, latexes, and cured unsaturated polyesters products. Rigid foams containing TBPP were used in insulation, furniture, automobile interior parts, and water flotation devices. About 65% of the 4500 tonnes of TBPP that were produced annually in the USA by 6 manufacturers was applied to fabrics used for children's clothing. TBPP was added to these children's garments to an extent of 5-10% by weight (US EPA, 1976; Kirk-Othmer, 19781984). TBPP was applied to cellulose acetate and triacetate by addition to the melt prior to spinning. The process involved the thermal diffusion of TBPP by driving it into the fibre under pressure dying. For materials such as, polyesters, nylons, and acrylics, the TBPP was either "padded on" at 5-10% by weight with heat fixation to the woven or knitted material or applied via emulsion from conventional batch dying equipment (Prival, 1975). Fire-retarded polyurethane required about 0.5% phosphor and 4-7% bromine; being equivalent to about 10% TBPP by weight in the product (US EPA, 1976). By actions taken on 8 April and 1 June 1977, on the basis of the genotoxic and possible carcinogenic effects of TBPP, the US Consumer Product Safety Commission banned children's clothing treated with TBPP, the chemical itself when used or intended to be used in children's clothing, and fabric, yarn, or fibre containing it, when intended for use in such clothing (US Consumer Product Safety Commission, 1977a,b; US Consumer Product Safety Commission, 1977a,b). In March 1978, The Consumer Product Safety Commission listed 22 products that contained TBPP and were available to USA consumers. These included children's clothing, industrial uniforms, draperies, tent fabric, automobile headliners, epoxy resins for the electronics industry, Christmas decorations, and polyester thread (IARC, 1979). In Japan, the use of TBPP as a fire-retardant in textile products was banned in 1981, because the chemical might be a human carcinogen and genotoxicant. As from December 1987, TBPP could not be used in the EC in textile articles such as, garments, under-garments, and linen intended to come into contact with the skin (EEC, 1976, 1979). Several other countries including Finland, New Zealand, and Sweden have also banned, or severely restricted, the use of TBPP in textiles and textile articles (UN, 1991). 3.2.3 Sources of human and environmental exposure Potential sources of human exposure and environmental contamination include: the manufacturing of the flame retardant, its application to materials, leaching out of the flame retardant during use and/or washing, and ultimate disposal of the material. Studies indicated substantial losses of surface TBPP from fabrics after laundering, but TBPP was not completely removed after repeated laundering. For example, acetate fabrics (65-600 mg TBPP/kg) showed up to 85% reduction in surface concentration after one laundering, and, polyester fabrics (260-37 500 mg TBPP/kg), from 21 to 82% reduction after one laundering. A significant portion, approximately 10% of the total production reached the environment from textile-finishing plants and laundries. Most of the rest will find its way into solid wastes (US EPA, 1976). Surface TBPP can be extracted from treated fabric by saliva (up to 3%) as well as by water, acetic acid, sodium bicarbonate, and salt (Ulsamer et al., 1980). Gutenmann & Lisk (1975) heated polyester flannel material, treated with TBPP, in distilled water at 60°C for 20 min, simulating a laundering operation. It was calculated from the extraction rate that laundering of flame-retarded sheets could result in a concentration of 6 mg/litre in combined washing and rinsing water. This release was maintained during several subsequent launderings. The presence of detergents may increase the extraction rate. TBPP exists both in, and on, the fabric. In the fabric fibres, it is not extractable with a benzene/hexane mixture and, therefore, is probably not available for dermal absorption. However, when it is on the fibre surface, it is extractable and is available for dermal absorption (Morrow et al., 1976; Ulsamer et al., 1980). While most of the TBPP is within the fabric in both polyester and acetate, polyester contains considerably more surface TBPP as a result of differences in methods of addition. Concentrations of surface bromine in polyester fabric ranged from 2000 to 37 500 mg/kg with the actual TBPP content ranging from 20 to 90% of the bromine value. The non-TBPP organic bromides have not yet been identified (Ulsamer et al., 1980). 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION 4.1 Transport and distribution between media An estimated log Koc (3.29) suggests strong adsorption on soil. On the basis of this Koc value and the low measured water solubility of the technical chemical (8.0 mg/litre), TBPP is expected to leach only slowly into groundwater. The water solubility of pure TBPP may be lower than the solubility of the technical grade chemical and so the extent of leaching of the pure chemical may be even lower than the Koc above suggests (Kenaga, 1980; Lyman, 1982; Verschueren, 1983; US EPA, 1985). Although hydrolysis of the phosphate ester is not expected to be significant, hydrolysis involving the bromine atoms on the propyl groups may occur, especially under basic conditions. Direct photolysis is not expected to be a major process, since TBPP should not absorb light of wavelengths found in sunlight (> 290 nm) (Mabey & Mill, 1978). No data on volatilization from water or soil are available. Using measured water solubility (8.0 mg/litre) and vapour pressure of 1.9 × 10-4 mmHg, volatilization half-life values were estimated. The half-life values for TBPP volatilization from streams, rivers, and lakes were 3.64, 4.66, and 392 days, respectively, assuming current velocities of 3, 1, and 0.01 m/second, respectively. The river and stream depths were assumed to be 1 m, while the lake was assumed to be 50 m deep (Verschueren, 1983). 4.2 Transformation 4.2.1 Biodegradation The biodegradability of TBPP was determined following a shake-flask test. TBPP was incubated with a microbial inoculum of raw sewage. Samples of the test solutions were taken at 0, 5, 10, and 15 days for final analysis using neutron activation to determine the bromine content of the liquid. Assuming the increased bromide content of the inoculated samples relative to the blank samples is due to biodegradation, and the solubility of TBPP is 1.6 mg/litre, an amount of TBPP equal to 2.4 times the dissolved TBPP was degraded in 5 days (Kerst, 1974). Activated return sludge (at 21°C), used within 1 h of collection, diluted with a basal medium, with an added 2 mg 14C-labelled TBPP/kg, showed that 6% of the added radio-activity was evolved as 14CO2. A major metabolite bis(2,3-dibromopropyl) phosphate (BBPP) was identified, but neither dibromopropanol (DBP) nor dibromopropionic acid was detected. The half-life of TBPP was 19.7 h (by least squares regression analysis). In a sterilized sludge control study, 93% of the added TBPP was found and metabolites were not identified (Alvarez et al., 1982). A biodegradation study on TBPP (100 mg/litre) was carried out under sewage treatment condition with sludge (30 mg/litre). The degree of biodegradation, as measured by BOD, was 1.8% of TBPP after a 2-week incubation period (Chemicals Inspection & Testing Institute, 1992). 4.2.2 Abiotic degradation No data available. 4.2.3 Bioaccumulation Tissue residue analysis of rats fed TBPP for a period of 28 days at levels of 100 or 1000 mg/kg diet has shown dose-related residue levels (measured as total bromine) in the muscle, liver, and body fat, of the treated animals (see section 7.2.1.). Groups of 30 adult fathead minnow (Pimephales promelas) (six months old), were exposed to 47.7 µg TBPP/litre for 2-32 days in a flow-through system. The temperature of the water was 25°C, pH 7.49, dissolved oxygen > 5 mg/litre, and hardness 45.5 mg/litre. The bioconcentration factor determined was 2.7 (Veith et al., 1979). Bioconcentration of TBPP (0.1 mg/litre, 0.03 mg/litre) from water to carp was estimated to be between < 0.7 to 1.9, and < 2.2 to 4.3, respectively, after 6 weeks of exposure (Chemicals Inspection & Testing Institute, 1992). 4.3 Interaction with other physical, chemical, or biological factors The thermal oxidative degradation at 370°C of TBPP produced hydrogen bromide and the C3-brominated species - bromopropenes, dibromopropenes, dibromopropanes and tribromopropanes, accounting for 87% of the volatiles. The detection of chlorinated species can only be explained by the presence of chlorinated impurities in the original ester. The residue (ether soluble aliquot) was composed mainly of 1,2,3-tribromopropane, whereas the aqueous layer contained the phosphoric acid produced. The gas chromatographic analyses of the volatiles showed a number of isomeric dibromopropenes. It was established that 1,3-dibromopropene was the major dibromopropene formed (Paciorek et al., 1978). 4.4 Ultimate fate following use It is to be expected that TBPP would be released into the environment in wastewater after laundering articles coated with TBPP flame retardant. With regard to disposal, it must be assumed that clothes and other products containing TBPP ultimately end up in landfills, which may result in some biological accumulation. Incineration should be carried out at high temperature with scrubbers or the equivalent. 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1 Environmental levels 5.1.1 Air TBPP was identified, but not quantified, in Arkansas air particulates (DeCarlo, 1979). 5.1.2 Water In 1975, 20 water samples were collected at different places in Japan and analysed for the presence of TBPP. None of the samples contained the compound (limit of determination 1 µg/litre) (Environment Agency Japan, 1978, 1987). 5.1.3 Soil In 1975, 20 sediment samples were collected at different places in Japan and analysed for the presence of TBPP. None of the samples contained TBPP (limit of determination 0.4-10 mg/kg) (Environment Agency Japan, 1978, 1987). TBPP was identified, but not quantified, in Arkansas soil (DeCarlo, 1979). 5.1.4 Fish In 1975, 20 fish samples, collected at different places in Japan, were analysed for the presence of TBPP. None of the samples contained TBPP (limit of determination 1 mg/kg) (Environment Agency Japan, 1978, 1987). 5.2 General population exposure 5.2.1 Subpopulation at special risk Tests for the extraction of TBPP from fabrics by water at various pH values and by a simulated saliva solution failed to reveal any TBPP in the extracts, but sodium bromide and hydrobromic acid were detected (limits of determination not mentioned) (Prival, 1975). However, surface TBPP can be extracted from treated fabric by saliva (up to 3%) as well as by water, acetic acid, sodium bicarbonate, and salt (Ulsamer et al., 1980). In the USA, the estimated intake via the skin of children, wearing sleepwear treated with the compound, was estimated to be 9 µg/kg body weight (Blum et al., 1978). The Consumer Product Safety Commission of the USA stated that, over a 6-year period, a child wearing TBPP-treated clothing could absorb a total of 2-77 mg TBPP/kg body weight and there are indications that this may be even higher (IRPTC, 1987). 5.3 Occupational exposure There are no data on levels of exposure to TBPP during manufacture or further processing. 6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS 6.1 Absorption TBPP is absorbed readily by the gastrointestinal tract and at a moderate rate via the skin in rats and rabbits. Studies on children revealed that TBPP is dermally absorbed from TBPP-treated sleepwear (Kerst, 1974; Blum et al., 1978; Ulsamer et al., 1978, 1980). Following the dermal application of 14C-TBPP to the clipped backs of New Zealand White rabbits (2-3 kg), 3.5-3.8% of the 0.9 ml/kg dose and 15.2% of the 0.05 ml/kg dose were absorbed over 96 h. Osborne Mendel rats (200-250 g) absorbed approximately 1/6 as much 14C-TBPP at each dose, when TBPP was applied to an equivalent area of skin/kg. The dermal uptake of 14C-TBPP by rats and rabbits showed that the primary elimination was via the kidneys (Ulsamer et al., 1980). 6.2 Elimination 6.2.1 Different routes (rat and rabbit) Four male Sprague-Dawley rats (290-310 g) were administered 14C-TBPP (98%) intravenously. The animals were housed in metabolism cages for 5 days. Urine, faeces, and air samples were collected for 5 days, and bile for 1 day. In 5 days, 58% of the administered radioactivity was found in the urine; 9% in the faeces and 19% in the air as CO2. In 24 h, bile contained 34% of the radioactivity while 9% was found in the bodies of the rats. In three additional rats, it was found that biliary excretion and enterohepatic recirculation was a major route in the disposition of TBPP. Bis(2,3-dibromopropyl) phosphate (BBPP) was detected in the urine of male rats (290-310 g) dosed iv with 25 mg 14C-TBPP (98%)/animal (in Emulphor) in amounts of 7.8% of the dose during 5 days following administration. BBPP was identified in the urine, faeces, bile, and tissues. 2,3-Dibromopropanol (DBP) was found in tissues and DBP and a few other metabolites were found in urine, but TBPP was not detected (Lynn et al., 1980, 1982). An adult male Sprague-Dawley rat (150-200 g) was administered (iv or orally) 1.39 mg 14C(propyl)-TBPP (99%)/kg body weight. One day after iv administration, 17% of the administered radioactivity was found in the urine, 7.4% in the faeces and 20% in the air (as CO2). One day after oral administration of TBPP, the concentrations were 24% in the urine and 11.5% in the faeces, but no radioactivity was detected in the air. Mainly metabolites were excreted in the urine and bile (Nomeir & Matthews, 1983). Small amounts of DBP and conjugates appeared in urine, when the rat was allowed to chew on TBPP-finished polyester fabric (St. John et al., 1976). Radiolabel from 14C-TBPP, applied to the skin, was excreted primarily in the urine (70% for rabbits and 50% for rats) with lesser amounts appearing in the faeces and 12 and 18% exhaled as CO2, respectively. TBPP itself did not appear in the urine, but a number of metabolites including DBP were found (section 6.4) (Ulsamer et al., 1980). 6.2.2 Dermal exposure (rat and rabbit) 188.8.131.52 TBPP One hundred mg of TBPP was spread over the surface of a gauze pad (one square inch) bandage and pressed tightly against the shaved skin of a rat. Urine was assayed for free and conjugated (released by acid hydrolysis) DBP. By day 7, the total concentrations of free and conjugated DBP in the urine were 17.61 and 23.58 mg/litre, respectively (St. John et al., 1976). 184.108.40.206 TBPP-treated fibres TBPP has been shown to penetrate rabbit skin from 14C-TBPP labelled polyester cloth containing 15 000 mg TBPP/kg of surface (4.3% of the radioactivity in 96 h) (Ulsamer et al., 1980). A shaved rat wore a garment made of 100% polyester flannel (4 × 6 inches), treated with TBPP, for 9 days. No DBP could be detected in the urine (limit of determination 0.4 mg/litre) (St. John et al., 1976). 6.2.3 Dermal exposure (human) The skin of a 7-year-old child was exposed on days 1, 2, and 8-12, by wearing repeatedly washed sleepwear that may have been TBPP-treated. On days 3-7, she wore new TBPP-treated pyjamas. Urine samples were collected daily from the child. In the urine, a maximum concentration of DBP of 29 µg/litre was found 2 days after wearing the new treated pyjamas. DBP at a concentration of 0.4 µg/litre was present in the urine, prior to wearing the new treated pyjamas. DBP was still excreted 5 days after the child stopped wearing the new TBPP-treated pyjamas. Urine samples were collected from 10 other children and one adult. All samples were analysed for DBP; it was not found in the urine of one child and one adult (who had never used washed TBPP-treated sleepwear). Seven children had levels of about 0.5 µg DBP/litre in the urine and one child had a level of 5 µg/litre. Approximately 180 µg/day (9 µg/kg body weight) was absorbed through the skin of children wearing pyjamas treated with TBPP (Blum et al., (1978). No DBP could be detected in the urine of an adult or in the urine of a 5-year-old boy who wore 100% polyester knit pyjamas, treated with TBPP, for 7 nights. Morning urine samples were collected daily throughout this period and up to 8 days thereafter (limit of determination 0.2 mg/litre) (St. John et al., 1976). 6.3 Distribution 6.3.1 Rat 220.127.116.11 Oral Male adult Sprague-Dawley rats (150-200 g) were administered 1.39 mg 14C(propyl)-TBPP (99%) orally. The percentages of the total dose of radioactivity, found after one day, in the blood, liver, kidneys, lung, muscles, fat, and skin, were 6.6, 3.4, 0.7, 0.2, 5.5, 1.3, and 3.4%; 24 and 11.5% of the total dose were found in the urine and faeces, respectively. The terminal clearance of TBPP-derived radioactivity from most of the tissues was described by a single component exponential decay with a half-life of 2.5 days. The half-life of TBPP in the liver and kidneys was 3.8 days (Nomeir & Matthews, 1983). Dose-related bromine concentrations were detected by neutron activation analysis in the muscles, liver, and fat of male rats fed TBPP for 28 days. The levels decreased to control levels during the six-week withdrawal period (Kerst, 1974). 18.104.22.168 Intravenous Eight male Sprague-Dawley rats (290-310 g) were administered 14C-TBPP (98%) by the iv route and the distribution was studied. All tissues contained TBPP-derived radioactivity. The concentrations of TBPP-derived radioactivity declined rapidly in most tissues, but the concentration of radioactivity in kidneys was 11 times the average body concentration, five days after dosing. No TBPP was detected, though bis(2,3-dibromopropyl) phosphate (BBPP) was still present in substantial concentrations. By day five, only small quantities of this metabolite were detected. The concentration of TBPP increased in the fat during the first 5-30 min, but, after 8 h, TBPP was no longer detectable. In contrast to the rapid disappearance of TBPP, the half-life of BBPP was relatively long in most tissues. BBPP represented a major portion of the radioactivity in several tissues including the lung, muscles, fat, and blood. In blood, it accounted for 90% of the radioactivity at 30 min and 8 h. By 5 min, 75% of the radioactivity in plasma was BBPP. The initial plasma half-life of this metabolite was 6 h. For 5 days it was 36 h. TBPP was not detectable in plasma after 1 h (Lynn et al., 1982). 6.3.2 Dermal (rabbit) Substantially more TBPP-derived radiolabel was detected in the kidneys and liver than in other organs of New Zealand rabbits, dermally treated with polyester fabrics containing 14C-TBPP (Ulsamer et al., 1978). 6.4 Metabolic transformation 6.4.1 In vivo studies 22.214.171.124 Oral (rat) TBPP was readily metabolized in rats. The main metabolite found in the urine, faeces, bile, and tissues of rats was BBPP. 2,3-Dibromopropanol (DBP) was also identified in tissues and urine. Only small amounts of unchanged TBPP were found in the excreta (Lynn et al., 1982; Nomeir & Matthews, 1983). Male adult rats (150-200 g) were administered 1.39 mg 14C(propyl)-TBPP (99%) orally (by intubation), and the urine and bile were analysed for metabolites. Six metabolites were identified in urine and bile, respectively: - 2,3-dibromopropanol; 1.0 and 1.1%; - bis(2,3-dibromopropyl) phosphate; 2.8 and 25.8%; - 2-bromo-2-propenyl 2,3-dibromopropyl phosphate; 4.8 and 13.8%; - bis(2-bromo-2-propenyl) phosphate; 10.3 and 5.2%; - 2,3-dibromopropyl phosphate; 4.1 and 2.6%; - 2-bromo-2-propenyl phosphate; 9.5 and 2.4% and TBPP was found in concentrations of 0.8 and 2.0%, respectively. These data are expressed as a percentage of total radioactivity excreted in the urine in 24 h, and, bile in 3 h. The total quantity of metabolites eliminated in the urine and bile were, in these periods, 33.3 and 52.9% of the radioactivity administered, respectively (Nomeir & Matthews, 1983). The formation of BBPP has been studied using selectively deuterated analogues of TBPP. Plasma concentrations of BBPP in rats dosed with either C2-D1- or C3-D2-TBPP were substantially lower than levels obtained with TBPP up to 4-6 h after administration. This indicates that oxidative metabolism of TBPP to form BBPP is important in vivo. Furthermore, in addition to oxidation at C3, BBPP formation may result from oxidation at C2. This latter reaction may be of particular importance with phenobarbital-pretreated microsomes (Pearson et al., 1993a; Dybing et al., 1989). In addition to these TBPP metabolites, 2-bromoacrolein, 2-bromoacrylic acid, bis(2,3-dibromopropyl)-3-hydroxypropyl phosphate, S-(2,3-dihydroxypropyl) glutathione, S-(3hydroxypropyl) glutathione and S-(2-carboxyethyl) glutathione have been detected in vitro and/or in vivo (Marsden & Casida, 1982; Nelson et al., 1984). 2-Bromoacrylic acid has been detected in the urine of rats administered TBPP. It was suggested that 2-bromoacrylic acid is an oxidation product of 2-bromoacrolein and that 2-bromoacrolein is formed spontaneously from DBP generated via initial cytochrome P450-mediated oxidation of TBPP (Marsden & Casida, 1982; Soderlund et al., 1984). Recent data indicate that the formation of 2-bromoacrolein occurs mainly from oxidative dehalogenation at the C3 position (Pearson et al., 1993a). Although glutathione acts as a detoxifying agent for reactive TBPP metabolites (Soderlund et al., 1984), conjugation could also result in the formation of reactive episulfonium ion intermediates (Pearson et al., 1993b). Van Beerendonk (1994) noted that there is S-(2,3-dihydroxypropyl) glutathione in the bile of Sprague-Dawley rats. They suggested that TBPP and/or BBPP are conjugated directly with glutathione by glutathione S-transferases, with subsequent formation of episulfonium ions. 6.4.2 In vitro studies TBPP is readily metabolized by microsomal and cytosolic rat liver fractions. Liver microsomes metabolized TBPP in the presence of NADPH and oxygen, as evidenced by the release of bromine and the formation of BBPP (Kerst, 1974; Nomeir & Matthews, 1983). The role of debromination in the formation of reactive metabolites was demonstrated in a series of TBPP analogues (Soderlund et al., 1984). The rate of NADPH-dependent metabolism was increased 5-10 times with microsomes from phenobarbital-pretreated rats compared with control microsomes and was reduced in the presence of cytochrome P450 inhibitors, indicating that cytochrome P450 is responsible for microsomal TBPP biotransformation (Soderlund et al., 1979, 1981, 1984; Nomeir & Matthews, 1983). Liver microsomes from mice, guinea-pigs, hamsters, and humans all metabolized TBPP to reactive intermediates. However, the rate of formation of reactive TBPP metabolites with human liver microsomes was lower than with liver microsomes from rodents (Soderlund et al., 1982a). In addition, a 1.5 to 2-fold increase in the rate of TBPP metabolism occurred when phenobarbital-pretreated microsomes were fortified with GSH, indicating that microsomal GSH- S-tranferases are able to conjugate TBPP with GSH. Dialysed rat liver cytosolic fractions, supplemented with GSH, metabolized TBPP at rates that were 3 times higher than those observed with control microsomes and NADPH (Nomeir & Matthews, 1983; Soderlund et al., 1981, 1984). Thus, in animals, GSH-dependent metabolism may be an important route in the in vivo biotransformation of TBPP to more water-soluble products. Soderlund et al. (1984) detected the in vitro formation of 2-bromoacrolein, by a reaction catalysed by cytochrome P450, in a process liberating bromide ions with subsequent formation of BBPP using rat liver microsomes (Soderlund et al., 1984). Mass spectral analysis of 2-bromoacrolein, formed from selectively deuterated analogues of TBPP, revealed that the primary mechanism for the formation of 2-bromoacrolein involves the initial oxidative dehalogenation at C-3 followed by a betaelimination reaction (Nelson et al., 1984). In vitro studies were carried out with deuterated analogues of TBPP, or, analogues labelled at specific positions with carbon-14, phosphorus-32, or oxygen-18, or dual-labelled with both deuterium and tritium. These were used as metabolic probes to study the chemical and metabolic events in the bioactivation of TBPP to chemically reactive metabolites in the liver microsomal preparations of male Sprague-Dawley rats. Studies with deuterated analogues of TBPP implicated oxidation at C-2 of the propyl moiety as a major pathway that leads to protein binding, which is enhanced by phenobarbital pretreatment of rats. Investigations with 18O-TBPP and H218O showed that the BBPP that is formed from the oxidation of TBPP incorporates one atom of oxygen from water. These results imply that oxidation at C-2 yields a reactive alpha-bromoketone that can alkylate proteins directly, or, hydrolyse to BBPP and a reactive alpha- bromoalpha'-hydroxyketone that alkylates microsomal proteins (Pearson et al., 1993a). These studies also showed that TBPP is oxidized at C-3, yielding the direct acting mutagen 2-bromoacrolein as the major metabolite that binds to DNA. This is consistent with earlier studies that indicate that 2-bromoacrolein is the major reactive metabolite formed in in vitro microsomal incubations (Nelson et al., 1984; Dybing et al., 1989). 6.5 Covalent binding to macromolecules TBPP has been shown to be activated to products that bind covalently to proteins (total macromolecules) and DNA in vitro and in vivo (Soderlund et al., 1981, 1984; Pearson et al., 1993a,b). The covalent binding of radiolabel TBPP to macromolecules was dependent on microsomes and NADPH, and was reduced by carbon monoxide, inhibitors of P450, and glutathione (Soderlund et al., 1981). The extent of TBPP covalent binding in vivo was five times higher in the kidneys than in the liver, whereas the rate of in vitro covalent binding was much higher with liver microsomes than with kidney microsomes. The low levels of TBPP binding in the liver in vivo may be the result of an extensive detoxification of TBPP to non-reactive metabolites or to low tissue concentrations of the proximate metabolite(s). Male NMRI and female B6C3F1 mice (20-25 g), male F344 rats (200-250 g), and guinea-pigs (80-100 g) were injected ip once with 250 mg 3H-TBPP/kg body weight in DMSO. The animals were killed 9 h after injection. All species showed similar levels of covalent binding to proteins in the liver and kidneys except for the rat which had much higher amounts of radiolabel bound to kidney proteins (Soderlund et al., 1982a). The binding of TBPP and analogues has also been studied in vivo. Analogues of TBPP either labelled at specific positions with carbon-14, and phosphorus-32 or dual-labelled with both deuterium and tritium were administered to male Wistar rats at a nephrotoxic dose of 360 µmol/kg body weight. The covalent binding of TBPP metabolites to rat hepatic, renal, and testicular proteins was determined after 9 and 24 h. The covalent protein binding was 5 times higher in the kidneys than in the liver and approximately 25 times higher than that in the testes. The results of comparative studies on renal DNA damage induced by TBPP and BBPP labelled with deuterium at C-2 or C-3 suggested that BBPP is formed in the liver by P450-mediated oxidation at either C-2 or C-3 of TBPP. BBPP is then transported to the kidneys, where it is subsequently metabolized to reactive intermediates that cause DNA damage and bind to kidney proteins in a process, independent of cytochrome P450, involving activation by conjugation with glutathione (Pearson et al., 1993b). Van Beerendonk et al. (1992) studied the formation of thymidine adducts and the cross-linking potential of 2-bromoacrolein (BA), a reactive metabolite of TBPP. In this study, [3-3H]BA was reacted with single-stranded (ss) DNA or double-stranded (ds) DNA and subsequently incubated with methoxylamine to covert the reaction product to an unstable BA:thymidine adduct. Because the unstable BA:thymidine adduct may have the potential to form cross-links, the reaction with various nucleophiles in vitro was studied. A reaction occurred between the adduct and cystein, but not with lysine or desoxynucleosides. Reaction of BA with ssDNA in the presence of [3H]glutathione also resulted in the binding of radiolabelled GSH to DNA. The results indicated that the reactive aldehyde group of the adduct can react with thiol groups in proteins to form protein-DNA cross-links. When the possibility that tris- and bis-(2,3- dibromopropyl) phosphates form such cross-links was examined in vivo in Drosophila, it was found that TBPP was a cross-linking agent, whereas BBPP was not. 7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS 7.1 Single exposure The oral LD50 for TBPP was calculated to be 5.24 g/kg body weight, when administered as a suspension in propyleneglycol to male albino Spartan rats with a weight of 202-250 g. The observation period was 14 days (Kerst, 1974). In another study, TBPP dissolved in propyleneglycol or in ethanol was given to Osborn-Mendel rats. Oral LD50s of 1.88 and 3.12 g/kg body weight, respectively, were obtained (Ulsamer et al., 1980). A dermal toxicity study showed an acute LD50 for rabbits of 17.6 g/kg body weight (Ulsamer et al., 1980). In another study, TBPP was applied once to the back of four groups of two male and two female New Zealand white rabbits (2.56-2.96 kg) in concentrations of 1, 2, 4, or 8 g/kg body weight. The application area was wrapped with a gauze bandage and occluded; after 24 h, the bandages were removed and the skin washed with water. The observation time was 14 days. An LD50 of > 8 g/kg body weight was found (Kerst, 1974). A dose of 2 g TBPP was applied to the intact and abraded skin of 10 albino rabbits. No deaths were observed during a 14-day observation period (Moldovan, 1972). 7.2 Short-term exposure 7.2.1 Oral exposure (rat) 126.96.36.199 TBPP Groups of male rats received daily doses of 250 mg TBPP/kg in either propyleneglycol or saline, by gavage, and were sacrificed after 1, 2, 4, 6, 8, or 10 days. Liver and testes were unaffected by any treatment, but nephrotic changes were observed to commence on day 2 and to become progressively more severe with time. In addition to the tubular lesions, the glomeruli were adversely affected, an observation not seen in the 13-week study (Osterberg et al., 1979). In a pilot study, groups of 10 non-pregnant rats were administered TBPP for 10 days at dose levels of 100, 150, 500, or 1000 mg/kg body weight per day. Mortality rates were 0, 0, 70, and 100%, respectively (Seabaugh et al., 1981). Male weanling rats were fed TBPP at concentrations of 100 or 1000 mg/kg diet for 28 days. The animals were then sacrificed immediately or after 2 or 6 additional weeks of recovery. The results showed a decrease in food efficiency (approximately 10% at the highest dose), decreased body weight gain (approximately 20% at the highest dose), and decreased organ to body weight ratios for heart, liver, spleen, kidney, and gonads (approximately 20% for each organ at the highest dose). Haematology, blood chemistry, urinalysis, and histopathology did not differ from the control values. In the recovery period, the body weight gain became normal. The authors suggested that the effect might be because of the palatability of the substance. Tissue residues (measured as bromine) increased 40-50 times in the first 4 weeks of treatment in the fat, liver, and muscles. By the end of the 6-week withdrawal period, the residues were at control levels (Kerst, 1974). Groups of rats were gavaged with TBPP in corn oil at 10, 50, or 100 mg/kg per day for 4 weeks. One half of each group was sacrificed at 4 weeks and the remainder at 6 weeks. While no adverse responses were observed, elevated bromine levels in blood were reported (Brieger et al., 1968). A 90-day study was carried out on rats administered TBPP in propylene glycol, daily (by gavage), at 25, 100, or 250 mg/kg body weight. The control groups received either the vehicle, normal saline, or no treatment. Weight gain for males was 34-50% less and, for females, 40% less in the test groups and vehicle group compared with the control values. Liver/body weight ratios were lower for both sexes in the low TBPP group, but higher in females in the highest dose group, compared with those in the control group. Kidney/body weight ratios were 18% lower than in controls. Testes/body weight ratios in the TBPP groups were 25% lower. There was an increased incidence and severity of chronic nephritis associated with regenerative epithelium, hypertrophy, and dysplasia of renal tubular epithelial cells in all TBPP-treated rats. The complex of changes was more severe with higher dose, and among males (Osterberg et al., 1978). 188.8.131.52 TBPP-treated fibres The results of a 2-week study on rats fed 15% shredded TBPP-treated acetate fibres in their food (3 times/week) showed no changes in blood-bromine levels and no adverse effects (Ulsamer et al., 1980). 7.2.2 Oral exposure (dog) 184.108.40.206 TBPP In a study on dogs, doses of 50 or 100 mg TBPP/kg body weight were given in the diet for four weeks. A decrease in body weight was noted in the treated dogs as well as increased blood-bromine levels. Cholinesterase activity was reported to be unaffected (Brieger et al., 1968). No further details were available for this study. 220.127.116.11 TBPP-treated fibres In a 2-week study on dogs fed 15% shredded TBPP-treated acetate fibres in their food (3 times/week), no changes in blood-bromine levels or adverse effects were seen. Two additional, 3-week studies on dogs using TBPP-treated shredded rayon and acetate fibres added to foods did not show any detectable changes in health or in blood-bromine levels (Brieger et al., 1968). 7.2.3 Dermal exposure 18.104.22.168 Rabbit Short-term dermal studies have been performed using groups of clipped rabbits dosed with 2.2, 4.4, or 8.8 g TBPP/kg body weight, daily, for 4 weeks. A dose-related increase in bromine was found in the blood and urine. All rabbits died within 4 weeks. Significant degenerative changes in the kidneys and the liver were found. Slight decreases in cholinesterase activity were recorded (Brieger et al., 1968). In another study in which the animals were administered dose levels of 50 and 250 mg/kg body weight, bromide levels in the blood and urine were increased, but no deaths occurred (Ulsamer et al., 1980). A 13-week study was carried out on 12 young (3 months old) New Zealand white rabbits, 6 with intact, and 6 with abraded, dorsal skin. They were treated with a weekly application of 2.27 g TBPP (99.76%)/kg body weight for 13 weeks. In a third group, 6 rabbits were initially clipped and maintained untreated as controls. The TBPP-treated sites were not occluded with a patch, but the animals were fitted with a collar. Besides a statistically significant increase in relative liver weights in the rabbits with intact and abraded skin (53% and 59%, respectively), a significant decrease in testes weight (54% and 40%, respectively) was observed. Microscopically, chronic interstitial nephritis (in 6/8 males) with tubule involvement and bizarre nuclei as well as testicular atrophy and aspermatogenesis (spermatogonia were present in seminiferous tubules, and also secondary spermatocytes, but no spermatozoa) were observed in 7/8 males of the test groups. Female rabbits did not exhibit any adverse responses. No histopathological changes were seen in the liver (Osterberg et al., 1977, 1978). In a study in which TBPP-treated rayon cloth was applied to the clipped skin of rabbits for 4 weeks, no significant effects were found (bromine levels were not increased) in treated animals (Ulsamer et al., 1980). No further details were available for this study. 22.214.171.124 Dog When TBPP-treated rayon cloth was applied to the clipped skin of dogs for 4 weeks, no significant effects (no increased bromine levels) were found in the treated animals (Brieger et al., 1968). No further details were available for this study. 7.3 Long-term exposure Apart from carcinogenicity studies, no long-term toxicity studies are available (see section 7.7). 7.4 Skin and eye irritation; sensitization 7.4.1 Skin irritation TBPP (1.1 g) was applied to the abraded or intact skin of six albino rabbits. The animals were fitted with collars for 24 h. After this period, the coverings were removed and the test material washed off. The extent of erythema and oedema was determined after 24 and 72 h. No signs of irritation were observed (Kerst, 1974). 7.4.2 Eye irritation Administration of 0.22 g TBPP to the eyes of 6 adult rabbits did not cause noticeable irritation or damage to the cornea, iris, or palpebral conjunctiva during a 72-h observation period (Kerst, 1974; US EPA, 1976). 7.4.3 Sensitization TBPP was tested for skin sensitization in groups of 5-10 guinea-pigs using a modified Landsteiner method and the footpad technique. No sensitization was noted in either test (no details given) (Morrow et al., 1976). 7.5 Reproductive toxicity, embryotoxicity, and teratogenicity 7.5.1 Reproductive system Groups of 6 adult male Sprague-Dawley rats (56-60 days of age) were used in a study to investigate the effects of TBPP on the reproductive system. Six rats were injected with 0.1 ml propyleneglycol intraperitoneally, three times/week, and, six rats were untreated controls. Nine groups of 6 rats were given (ip injection), three times/week, 0.4, 0.9, 1.8, 3.5, 7.1, 14.2, 28.4, 56.8, or 113.5 mg TBPP in propyleneglycol for a period of 72 days. The four highest dose levels of TBPP did not dissolve completely and were injected as an emulsion. The rats were treated for a minimum of 72 days (6 cycles of the germinal epithelium) before being killed. The three highest dose levels (28.4-113.5 mg/injection) caused significant dose-related declines in the weights of the testes and prostate, epididymides, and seminal vesicles. Sperm production of testes and sperm storage in the epididymides were reduced, and the percentage of the motile sperm and the motility index were decreased. Histological examination of the testes revealed that the seminiferous tubules were affected. The affected tubules contained very few germinal cells and the macrophages in the interstitium of the affected testes appeared to be phagocytically active. The Leydig cells were normal. TBPP did not have any significant effects on the serum concentration of testosterone or on the in vitro testicular capacity for testosterone secretion (Cochran & Wiedow, 1986). The effects on the testes were also reported in a 13-week study on New Zealand white rabbits, treated with weekly dermal applications of 2.27 g TBPP on the intact or abraded skin. Decreased testes weights and, microscopically, testicular atrophy and aspermatogenesis were found in male rabbits (Osterberg et al., 1977). B6C3F1 mice (15 weeks old) were administered (ip) TBPP in corn oil at dose levels of 0, approximately 200, 400, 600, 800, and 1000 mg/kg body weight daily, for 5 days. The mice were killed 35 days after the fifth treatment. Their epididymides were removed and abnormal sperm heads determined. The frequency of abnormal sperm heads in TBPP-treated mice was significantly greater than in controls, predominantly at dose levels of 800 mg/kg body weight or more (Salamone & Katz, 1981). 7.5.2 Teratogenicity In a pilot study on groups of ten pregnant Sprague-Dawley rats, orally intubated with 0, 250, or 1000 mg TBPP/kg body weight on days 6-15 of gestation, an increase in maternal mortality was observed. The mortality rates were 0, 10, and 100% respectively. The rats given 1000 mg/kg died on days 9-11 of gestation (Seabaugh et al., 1981). Sexually mature, timed-pregnant Sprague-Dawley rats, 30 animals per group, were intubated on days 6-15 of gestation with TBPP (99.7% TBPP, 0.14% 1,2,3-tribromopropane, and 0.17% 2,3-dibromopropanol) in undiluted propyleneglycol at levels of 0, 5, 25, or 125 mg/kg body weight per day. Maternal body weight gain was decreased at the highest dose level. No effects of treatment were apparent on the number of corpora lutea, implantations, or early or late deaths. Furthermore, the percentage of females with resorptions, the number of viable fetuses, the percentage of resorptions, and the percentage of pre-implantation losses, did not show compound-related changes. Fetal body weight and crown-rump length were not affected. Some fetal soft tissue and skeletal variations found were not dose-related or statistically significant. It was concluded that TBPP was not teratogenic in this study (Seabaugh et al., 1981). Female Wistar rats were exposed orally to 25, 50, 100, or 200 mg TBPP in olive oil/kg body weight on days 7-15 of gestation. A significant increase in skeletal variation was found in the fetuses at 200 mg/kg. A significantly lower viability index was observed in the 50 and 100 mg/kg groups. The authors concluded that TBPP did not produce teratogenic effects in rats. A dose of 200 mg/kg elicited maternal toxicity (Kawashima et al., 1983). 7.6 Mutagenicity and related end-points 7.6.1 DNA damage 126.96.36.199 In vivo When male Wistar rats (250-320 g) were given a single ip injection of 350 µmol TBPP/kg (250 mg/kg) body weight and assayed for DNA damage 2 h later, single strand breaks/alkali labile sites were found in the DNA from nuclei isolated from several organs. DNA damage was detected using an automated alkaline elution system. Extensive DNA damage was detected in the liver, kidneys, and small intestines. In addition, substantial DNA damage was found in the brain and lungs; less DNA damage was detected in the testes, spleen, and large intestines (Holme et al., 1983; Soderlund et al., 1992). DNA damage was clearly detectable in the kidneys 20 min after a single ip dose of 36 µmol TBPP/kg (25 mg/kg) body weight (Pearson et al., 1993b). 188.8.131.52 In vitro Monolayer cultures of human (KB) cells were grown with [3H]-thymidine for 30 h, and without, for another 17 h. The cells were then exposed to TBPP (2 µl/ml of growth medium devoid of serum) for 4.5 h and processed for analysis of the DNA on alkaline-sucrose gradients. They were re-incubated for various intervals to permit DNA repair. TBPP was shown to have induced DNA repair, which indicated a specific action on human cellular DNA. TBPP was found to damage human DNA in vitro and to cause unscheduled DNA synthesis in human cells in tissue culture (Gutter & Rosenkranz, 1977; Blum & Ames, 1977). A semiquantitative, in vitro method for measuring unscheduled DNA synthesis (UDS) was developed by Lake et al. (1978). Normal foreskin epithelial cells from a cryopreserved skin pool were grown from explants and replanted in replicate culture wells. Cultures were then treated for 3 days in an arginine-deficient medium and further inhibited in S-phase DNA-synthesis by a 2-h (10 mmol/litre) hydroxyurea treatment. 3H-Thymidine and TBPP were added simultaneously and the UDS, accumulated over a 24-h incubation period, was determined by direct scintillation counting of acid-precipitable whole-cell radioactivity. TBPP did not induce an UDS response in this assay, with input dose ranges of 10-99 and 100-400 µg/ml. UDS was detected in rat liver hepatocytes, grown as monolayer cultures, exposed to 0.01-0.1 mmol TBPP/litre for 18-19 h in the presence of [3H]-thymidine and hydroxyurea. UDS was determined by scintillation counting (Holme et al., 1983; Holme & Soderlund, 1984; Gordon et al., 1985; Soderlund et al., 1985). In in vitro test systems, DNA damage was detected in isolated rat hepatocytes exposed to concentrations as low as 5 µmol TBPP/litre, while a 10-fold higher concentration was necessary to induce DNA damage in testicular cells (Soderlund et al., 1992). No DNA damage was found in cultured Reuber rat hepatoma cells, without the addition of an exogenous metabolism system (Gordon et al., 1985). 7.6.2 Mutation assay with Salmonella typhimurium strains Species differences in the bioactivation of TBPP to metabolites, mutagenic to Salmonella typhimurium TA 100, have been reported. Liver microsomes from mice (NMRI strain) were more effective in activating TBPP to mutagenic intermediates than those from guinea-pigs, hamsters, and rats. Phenobarbitalinduced liver microsomes from NMRI mice were especially effective (Soderlund et al., 1982a). TBPP was activated to mutagens in the Salmonella/microsome test. S9-fractions from rats pretreated with phenobarbital increased the mutagenicity of 0.05 mmol TBPP/litre in TA 100 strain compared with liver microsomes from untreated rats (Holme et al., 1983). It was demonstrated that the metabolic activation is dependent on the presence of NADPH and oxygen, which indicates that TBPP is metabolized by cytochrome P450 enzymes to mutagenic products. In studies conducted in an anaerobic atmosphere or in the presence of GSH, the mutagenicity of TBPP was significantly decreased (Soderlund et al., 1979, 1984). TBPP (97%) in DMSO was tested in concentrations of 0.0110 µlitre on Salmonella typhimurium TA 100, TA 1535, TA 1537, and TA 1538, using the plate assay, in the absence, and presence, of a metabolic activation system from rat liver. A mutagenic effect was found with TA 100 and TA 1535 with, and without, metabolic activation. TA 1537 and TA 1538 gave negative results (Blum & Ames, 1977; Brusick et al., 1978; Prival et al., 1977). TBPP was tested on Salmonella typhimurium tester strains TA 1535 and TA 1538 in the absence, and presence, of metabolic activation derived from Aroclor-induced rat liver. Dose levels of 0, 0.1, and 1.0 µlitre/plate were used. Weak mutagenic activity was observed in TA 1535 without activation, but a strong effect was seen with microsomal activation. TA 1538 gave negative results (Carr & Rosenkranz, 1978). MacGregor et al. (1980) confirmed the mutagenicity of TBPP in the Salmonella typhimurium strains TA 100, TA 98, and TA 1535, with dose levels ranging from 10 to 1000 µg/plate, with metabolic activation. Without activation, no mutagenicity was found. A negative result was obtained in strain TA 1537 with, and without, activation. Nakamura et al. (1979) tested TBPP on Salmonella typhimurium strains TA 100 and TA 1535 with, and without, metabolic activation at dose levels of 0.3-100 µmol/plate. A positive effect was seen in both strains, without and with S9 mix. McCann & Ames (1977) found a mutagenic effect in Salmonella typhimurium TA 100 with dose levels up to 100 µg/plate, in the presence of liver S9 fraction of rats treated with Aroclor. TBPP at dose levels of 0, 112, 224, 2240, 4480, and 11 200 µg/plate was tested on Salmonella typhimurium strain TA100 with, and without, metabolic activation by Aroclor 1254-induced rat liver S9 fraction. With the S9 fraction, all dose levels showed a mutagenic effect. Without the S9 fraction, TBPP showed direct-acting properties only at dose levels of 2240 µg/plate or more (Salamone & Katz, 1981). In an interlaboratory study, TBPP and 62 other chemicals were tested for mutagenic activity. TBPP was tested on the Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, and on Escherichia coli WP2uvrA. The dose levels were between 0.3 and 10 000 µg/plate. TBPP was tested without metabolic activation and with liver S9 fractions from uninduced and Aroclor 1254-induced F344 rats, B6C3F1 mice, and Syrian hamsters. TBPP tested positive in all four laboratories involved in this study (Dunkel et al., 1985). Results obtained by Prival et al. (1977) indicated that TBPP induces mutations of the base-pair substitution type in Salmonella typhimurium TA100. Although, at higher concentrations (> 1 µl/plate), TBPP behaves as a direct acting mutagen not requiring metabolic activation, at a much lower concentration (0.01 µl/plate) it demonstrates significant genetic activity only with metabolic activation. Brusick and coworkers demonstrated that amounts of 50 µg/plate or more were clearly mutagenic for Salmonella typhimurium TA 100 (Brusick et al., 1980). When tested for bacterial mutagenicity in Salmonella typhimurium TA 100, a 4-fold interindividual variation in the capability to activate TBPP was noted with human liver microsomes prepared from 5 liver donors (Soderlund et al., 1982a). The CASE structure-activity method was applied to a Gene-Tox derived Salmonella mutagenicity data base. Strains TA 97, TA 98, TA 100, TA 1535, TA 1537, and TA 1538 with, or without, exogenous metabolic activation, were used. TBPP was found to be positive (Klopman et al., 1990). 7.6.3 Mutations by urine of rats treated with TBPP The urine was collected of rats exposed to TBPP directly by either the oral or dermal route, or from treated fabric. Salmonella typhimurium TA 1535 was used as indicator organism. TBPP was dermally applied at doses of 5, 50, 500, or 5000 mg/kg body weight or given orally at 5, 50, or 500 mg/kg body weight. In the oral study, only 500 mg/kg produced a positive response. In the dermal studies, a dose of 500 mg/kg produced a weak positive response, while 5000 mg/kg produced a definitive positive response. When fabrics with surface TBPP levels of 3000, 28 000, and 67 000 mg/kg product were applied dermally, no mutagenic responses were detected in the urine of the rats over the 5-day period (data were lacking on whether or not metabolic activation was used) (Brusick et al., 1978; Ulsamer et al., 1980). TBPP at 500 mg/kg body weight in corn oil was applied dermally to CD-1 mice. Urine was collected over approximately 16 h and the bacterial mutagenicity of 0.3 ml urine samples was assayed in Salmonella typhimurium TA1535, TA1537, and TA100. A positive response was found only with TA100 (Brusick et al., 1982). 7.6.4 Other mutation assays TBPP was tested in the forward mutation assay with mouse-lymphoma cells (L5178YTK locus). While the results at lower doses were inconclusive, a 2 to 3-fold increase in mutations was consistently produced at 5 mg/litre (Brusick et al., 1978; Ulsamer et al., 1980). TBPP has been reported to induce increased mutation frequencies (6-TG resistance) in V79 Chinese hamster cells incubated with 0.02 mmol TBPP/litre in the presence of liver microsomes of rats pretreated with phenobarbital as an exogenous metabolism system (Holme et al., 1983; Soderlund et al., 1985). However, in a similar study, concentrations of TBPP up to 150 µg/ml did not increase the frequency of 6-TG resistance, both with, and without, an exogenous metabolism system (Sala et al., 1982). 7.6.5 Chromosomal effects Using Chinese hamster V79 cells, TBPP severely inhibited the colony-forming activity and significantly increased sisterchromatid exchanges, but no significant increase in chromosome aberrations was found (Furukawa et al., 1978). Interestingly, chromosomal aberrations were not significantly increased in Chinese hamster cells, in mouse bone-marrow cells, or in cultured human lymphoid cells. The lack of a TBPP effect on rat bone-marrow chromosomes was also observed after rats received 25, 250, or 2500 mg TBPP/kg body weight, by gavage, after either a single dose, or, 5 daily doses/week for 13 weeks (Osterberg, 1977; Nakanishi & Schneider, 1979). TBPP was tested for the induction of chromosome aberrations, and sister chromatid exchanges in the diploid human fibroblastic cell line HE 2144 (from a 10-week-old male embryo) without metabolic activation (Sasaki et al., 1980). The dose levels used were 0.349, 0.070, and 0.035 mg/ml. Sister chromatid exchanges were induced with 0.070 mg TBPP/ml in the human HE 2144 cell line. No chromosomal aberrations were found. In a comparative study, Brusick and coworkers found that TBPP gave a positive response in tests for sister chromatid exchanges and chromosomal aberrations in the mouse lymphoma L5178Y cell line at concentrations of 0.005 and 0.01 µlitre/ml, respectively (Brusick et al., 1980). TBPP was tested in an in-vitro test for sister chromatid exchanges in Chinese hamster V79 cells with, and without, S9 fraction of livers of Wistar rats administered (ip) methylcholanthrene. Acetone was used as solvent. The dose levels 17.2, 35, 100, and 200 µg/ml were tested only without S9 fraction, while levels of 24.5 and 50 µg/ml were tested with, and without, metabolic activation. A significant increase in sister chromatid exchanges was found at dose levels of more than 35 µg/ml (Sala et al., 1982). Two male and two female Chinese hamsters per group were used in a micronucleus test. The dose levels were 200, 400, and 800 mg TBPP/kg body weight administered by ip injection. The solvent was DMSO. Bone-marrow samples were obtained after 24 h. Two thousand polychromatic erythrocytes/animal were analyzed for the presence of micronuclei. Levels of 400 and 800 mg/kg body weight showed a positive effect (Sala et al., 1982). Salamone & Katz (1981) studied the clastogenic effect of TBPP in a bone marrow micronucleus test. B6C3F1 mice (15 weeks old) were given two ip treatments of TBPP in corn oil. Dose levels of 0, 204, 408, 612, 816, 1020, 1275, and 1530 mg/kg body weight were tested. In this test, TBPP showed a weak clastogenic effect. An in vitro chromosome aberration test was carried out with TBPP, using a Chinese hamster CHL cell line of lung fibroblast origin. CHL cells cultured in plates were exposed to different dose levels of TBPP including the 50% growth inhibition dose. The number of polyploid cells and cells with structural aberrations, such as chromatid-type gaps, breaks, exchanges, and rings, were scored. A microsome fraction (S9-mix) from the liver of Wistar rats, pretreated with the PCB; KC-400, was used. TBPP was positive in this test. A dose level of 0.25 mg/ml showed chromosomal aberrations in 20% of the metaphases (Ishidate et al., 1981). Vogel & Nivard (1993) studied the effects of TBPP in the (white/white+) (w/w+) eye mosaic assay, and an in vivo, short-term test measuring genetic damage in the somatic cells of Drosophila melanogaster, after treatment of the larvae. The genetic principle of this system is the loss of heterozygosity for the wild-type reporter gene w+, an event predominantly resulting from homologous, interchromosomal, mitotic recombination between the two X-chromosomes of female genotypes. The w/w+ eye mosaic test detects a broad spectrum of DNA modifications. Between 12 and 15 pairs of flies were permitted to lay eggs for three days on food supplemented with 0.25, 0.5, or 1.0 mmol TBPP/litre (dissolved in 3% ethanol). TBPP gave a strong positive response in the w/w+ bioassay. 7.6.6 Cell transformation TBPP was tested for its ability to induce malignant transformation in vitro using mouse BALB/3T3 cells. The results of this test showed that TBPP can transform mammalian cells in vitro, perhaps indicating a potential for the induction of carcinogenic responses (Brusick et al., 1978; Ulsamer et al., 1980). C3H/10T1/2 cells were treated with TBPP, with or without S9 mix from the liver of Wistar rats administered methylchloanthrene intraperitoneally. Some cell samples were additionally treated several times with tetradecanoyl phorbolacetate (TPA) (0.1 µg/ml). The TBPP concentrations tested were 40 µg/ml (with and without S9 fraction) and 80 µg/ml (without S9 fraction). A very low frequency of transformed type 3 foci was obtained and the authors considered the results of this study to be negative (Sala et al., 1982). Dunkel et al. (1988) also found a negative result for TBPP in the C3H/10T1/2 cell transformation assay. The dose levels tested were between 0.16 and 20 µg/ml. 7.6.7 Miscellaneous tests TBPP induced a significant increase in sex-linked recessive lethal mutations in male germ-cell stages of Drosophila melanogaster at a dose of 1000 mg/kg. The spermatids were the most sensitive (Valencia, 1978). Adult Canto-S male Drosophila flies aged 7 days were fed for 48 h on a 1% solution of glucose containing 1000 or 10 000 mg TBPP/litre. The TBPP-exposed males were mated immediately after treatment to brown ebony virgin females. The results showed that TBPP caused reciprocal translocations in Drosophila. There was no difference between the translocation recoveries at the two dose levels (Berkowitz, 1978). 7.6.8 Mechanisms of TBPP genotoxicity Following the initial mutagenicity reports, several studies have been directed towards the elucidation of the mechanisms involved in TBPP-induced genotoxicity. Such studies include investigations into the enzyme systems involved in the bioactivation of TBPP to genotoxic metabolites, delineating structural requirements for genotoxicity, and the characterization and identification of the genotoxicity of possible TBPP metabolites. Some of these investigations have recently been reviewed (Dybing et al., 1989). The bacterial mutagenicity of TBPP is mediated by the microsomal monooxygenase system. Several studies have shown that TBPP is activated to metabolites, mutagenic to Salmonella typhimurium TA100, by cytochrome P450 in a reaction depending on NADPH and oxygen (Soderlund et al., 1979; Dybing et al., 1989). The mutagenicity of TBPP was increased in the presence of microsomes prepared from livers of rodents pretreated with phenobarbital or PCBs, but not from those pretreated with 3-methylcholanthrene or beta-naphthoflavone (Soderlund et al., 1979; 1982a). The metabolism of TBPP by soluble enzymes (e.g., glutathione S-transferase) appears to be of minor importance in the bioactivation of TBPP to mutagenic species. The available data indicate that reductive metabolism and episulfonium ion formation are not major activation pathways in TBPP-induced mutagenicity (Dybing et al., 1989). However, it has recently been suggested that human glutathione transferases may further metabolize P-450-generated TBPP intermediates to more potent mutagenic species (Simula et al., 1993). Interestingly, singlet oxygen, obtained from the illumination of riboflavin, may also activate TBPP to mutagenic metabolites (McCoy et al., 1980). Studies investigating the bacterial mutagenicity of known and postulated metabolites of TBPP have given little information regarding the mechanisms of its bioactivation. With the exception of 2-bromoacrolein (BA) and DBP, other postulated or identified TBPP metabolites were less mutagenic than TBPP when tested with, or without, an exogenous metabolism system (Prival et al., 1977; Soderlund et al., 1979; Zeiger et al., 1982; Holme et al., 1983; Gordon et al., 1985). Such metabolites include the two major TBPP metabolites 2,3-dibromopropanol (DBP) and bis(2,3-dibromopropyl) phosphate (BBPP) as well as mono(2,3-dibromopropyl) phosphate (mono- BP) (McCann & Ames, 1977; Prival et al., 1977; Soderlund et al., 1982b; Holme et al., 1983). These metabolites were also less mutagenic than TBPP in V79 Chinese hamster lung cells with microsomal activation (Holme et al., 1983). Eight coded samples of different lots of TBPP from different manufacturers were tested in TA 1535. All were positive (Prival, 1975). Several commercial TBPP samples and a few known contaminants (1,2,3-tribromopropane, DBP, and 1,2-dibromo-3-chloropropane) were tested in amounts of 0.01 up to 10 µl/plate on Salmonella strains TA 100, TA 1535, and TA 1538 with, and without, Aroclor 1254-activated, or non-activated, rat liver S9 fractions. The results indicated that the highest levels of mutagenicity obtained with commercial TBPP were probably not due to the presence of the contaminants (Prival et al., 1977). The bacterial mutagenicity of compounds structurally related to TBPP, including monobrominated and chlorinated analogues, unsaturated and saturated methyl esters, and halogenated propanols, indicate the following structural effects on mutagenic activity: (a) a decrease in the number of alkyl chains decreases mutagenicity; (b) a decrease in the number of halogens in the alkyl chain decreases mutagenicity; (c) compounds with vicinal halogens are more mutagenic than 1,3-dihalo- isopropyl analogues; (d) brominated compounds are more mutagenic than the corresponding chlorinated ones; and (e) salts of mono- and diphosphate esters are more mutagenic than the free base (Carr & Rosenkranz, 1978; Nakamura et al., 1979, 1983; Zeiger et al., 1982; Holme et al., 1983). Mutagenicity testing of TBPP metabolites has yielded little information on the mechanisms of activation of TBPP to mutagenic metabolites, since none of the metabolites were more mutagenic than the parent compound. The first evidence for the possible formation of a potent bacterial mutagen from TBPP came when 2-bromoacrylic acid was found in the urine of rats, administered large doses of TBPP (Marsden & Casida, 1982). These authors proposed that TBPP is initially oxidized at the C-1 position to yield 2,3-dibromopropanal, which then spontaneously dehydrobrominates to give BA. BA and 2,3- dibromopropanal, which are potent, direct-acting, bacterial mutagens in Salmonella typhimurium TA100, caused DNA damage in Reuber rat hepatoma cells, and transformation of Syrian hamster embryo cells (Rosen et al., 1980; Gordon et al., 1985). In 1984, BA was detected in incubations of TBPP with rat liver microsomes, using GC/MS. In these experiments, substitution of deuterium atoms for hydrogen at the C-3 position decreased the mutagenicity of TBPP by approximately 80%, whereas only a small deuterium isotope effect was noted at C-2 and C-1. These data indicate that an initial oxidation at the terminal carbon atom is the key step in the formation of mutagenic metabolites from TBPP (Nelson et al., 1984; Soderlund et al., 1984). Subsequent experiments with variously deuterated TBPP analogues revealed that, according to the number of deuteriums retained in the BA formed, oxidation at C-3 is the major pathway for BA formation (Nelson et al., 1984). The results of the studies with deuterated TBPP analogues were later substantiated with selectively methylated analogues. These findings demonstrated that the initial oxidation of TBPP at C-3 was followed by spontaneous dehydrohalogenation and dehydrophosphorylation, with the subsequent formation of BA and BBPP (Omichinski et al., 1987). Although BA is the most potent TBPP metabolite with regard to bacterial mutagenicity in vitro, additional TBPP metabolites appear to be involved in TBPP-induced DNA damage in mammalian cells. The formation of these metabolites has not yet been elucidated, but is likely to involve conjugation with glutathione (Soderlund et al., 1992). All the known metabolites, including the two major metabolites, DBP and BBPP, are considerably less mutagenic than the parent compound, when tested directly or in the presence of an activation system (Blum et al., 1978; Soderlund et al., 1979, 1982b; Zeiger et al., 1982). However, BA is a more potent mutagen than TBPP (Rosen et al., 1980; Nelson et al., 1984; Gordon et al., 1985). 7.7 Carcinogenicity 7.7.1 Oral 184.108.40.206 Mouse Groups of 50 male, and 50 female, B6C3F1 hybrid mice, 6 weeks old, were fed technical TBPP (containing no detectable 1,2-dibromo-3- chloropropane) at concentrations of 500 or 1000 mg/kg diet in the diet for 103 weeks followed by a 1-week observation period. The experimental design of the study is shown in Table 1. Of the males, 44/55 matched controls, 38/50 low-dose mice and 43/50 high-dose mice survived until the end of the study; of the females 44/55 controls, 37/50 low-dose mice, and 38/50 high-dose mice survived. TBPP increased the incidence of squamous-cell carcinomas and papillomas of the fore-stomach and of adenomas and carcinomas of the lungs in both male and female treated animals compared with the controls. There was also an increased incidence of renal tubular cell adenomas and adenocarcinomas in treated male mice and liver cell adenomas and carcinomas in treated female mice. Neoplastic lesions associated with the administration of TBPP are summarized in Table 1. The incidence of "preneoplastic" kidney changes, dysplasia, and hyperplasia, were: controls (males and females) 0/109, low-dose females 20/50, low-dose males 46/50, high-dose females 40/46 and high-dose males 49/50 (US NCI, 1978; Reznik et al., 1979; IARC, 1979). 220.127.116.11 Rat Groups of 55 male, and 55 female Fischer 344 rats, 6 weeks old, were fed diets containing concentrations of 50 or 100 mg technical TBPP/kg for 103 weeks, followed by a 1- or 2-week observation period. The experimental design of the study is shown in Table 1. Of the males 39/55 control, 35/55 low-dose, and 40/55 high-dose rats survived until the end of the study; of the females 36/55 control, 44/55 low-dose, and 36/55 high-dose rats survived. The compound increased the incidences of both renal tubular cell adenomas in rats of both sexes and tubular cell adenocarcinomas in high-dose males. Neoplastic lesions associated with the administration of TBPP are summarized in Table 1. The incidence of "preneoplastic" kidney changes, dysplasia, and hyperplasia, were; controls (males and females) 0/105, low-dose females 25/54, low-dose males 53/54, high-dose females 46/54, and high-dose males 39/54 (US NCI, 1978; IARC, 1979; Reznik et al., 1979). Table 1. Tumour incidences in mice and rats fed tris(2,3-dibromopropyl) phosphate (TBPP) Species Sex Number Concentration Duration Number of tumour-bearing animals/number of animals examined of animals (mg/kg (weeks) treated diet) Forestomach** Lung** Kidneys* Liver** (squamous-cell (adenomas or (tubular-cell (adenomas or carcinomas or carcinomas) adenomas or carcinomas) papillomas) adenocarcinomas) Mouse male 55 0 105 0/51 12/54 0/54 28/54 male 50 500 103 10/47a 18/44c 6/50 31/49 mate 50 1000 103 13/48b 25/50d 17/49e 23/49 female 55 0 105 2/53 4/55 0/55 11/54 female 50 500 103 14/48b 9/50 3/50 23/50f female 50 1000 103 22/44b 17/50d 3/46 35/49f Rat male 55 0 107 - 0/54 0/53 0/54 male 55 50 103 - 3/55 30/54e 1/55 male 55 100 103 - 0/55 30/54a 4/54 female 55 0 107 - - 0/52 - female 55 50 103 - - 4/54 - female 55 100 103 - - 13/54a - From: * Reznik et al. (1979); ** US-NCI (1978). Fisher analysis of treated group versus control: a Squamous-cell papillomas; P < 0.01; b Squamous-cell carcinomas and papillomas; P < 0.01; c Alveolar/bronchiolar adenomas and carcinomas; P < 0.05; d Alveolar/bronchiolar adenomas and carcinomas: P < 0.01: e Tubular-cell adenomas and adenocarcinomas: P < 0.01; f Hepatocellular adenomas and carcinomas; P < 0.01. Male F344 rats, 4 weeks old, were administered (by gavage) 0 (untreated), and 0 (vegetable oil), or 100 mg TBPP in vegetable oil/kg body weight per day. Treatment was given for 5 days per week and continued for 4 or 52 weeks. Selected rats of the treated and control groups were killed at various time intervals. Control and treated animals (2-9 animals) were killed after 1, 5, 10, 20, 50, 75, and 260 treatments. After 4 weeks (20 treatments), the TBPP group was divided into two subgroups; TBPP administration was continued in one subgroup of 15 rats and, in the second subgroup, 15 other rats (treated for four weeks) received only vegetable oil for the remainder of the study. The histomorphology and ultrastructure of the kidneys were studied. Twenty-four hours after the first TBPP treatment, the epithelial cells at the corticomedullary junction developed increased nucleus/cytoplasm ratios, cytomegaly, and nuclear vacuolization and pleomorphism. These changes increased in severity to a toxic tubular nephrosis as treatment continued, and extended to the peripheral cortex by 52 weeks. After 52 weeks of TBPP treatment, small tubular papillary hyperplasia had developed in three animals and an adenocarcinoma was observed in one of the five animals killed at the end of the study. Electron microscopy showed loss of microvilli and polarity in the epithelium of the proximal convoluted tubules. At the ultrastructural level, the cytoplasm of the neoplastic cells was poorly differentiated and, in many areas, the surfaces of the cells were covered by microvilli. In the animals in which treatment was discontinued after 4 weeks, a gradual, but incomplete, restoration of the tubular epithelia to nearly normal morphology was observed. Nuclear changes persisted after cytoplasmic abnormalities had disappeared. At 52 weeks, three out of five surviving rats, administered TBPP throughout the study, were found to have polyploid adenomas of the descending colon (Reznik et al., 1981). Cunningham et al. (1992, 1993) examined the mechanisms whereby chemicals produce mutagenicity in short-term in vitro assays, yet fail to produce carcinogenicity in long-term rodent bioassays. Previous studies indicated that mutagenic carcinogens increased the amount of cell turnover in the target organ, but that mutagenic noncarcinogens failed to do so. An association of cell-proliferation, as determined by labelling with bromodeoxyuridine, and tumour development was investigated in groups of five male Fischer 344 rats (150 g). Administration of TBPP in the diet at dose levels of 0, 50, or 100 mg/kg, for 14 days, resulted in increased incidences of cell proliferation in the kidneys. A further association of cell proliferation with tumour development in the kidneys was suggested by their similar location in the kidneys, i.e., the renal outer medulla. 7.7.2 Dermal 18.104.22.168 Mouse Female ICR/Ha Swiss mice (29 or 30 per group) (6-8 weeks old) were treated with 10 or 30 mg TBPP (97%) in 0.2 ml acetone, 3 times weekly, on the shaved skin, for 496 and 474 days, respectively. Two control groups were used; 30 mice received the acetone and 249 mice were untreated. Besides a significant increase in skin tumours (papillomas and/or carcinomas), a substantial number of tumours were also found at distant sites, such as squamous cell carcinomas of the tongue and in the gingival area; papillomas and carcinomas were observed in the forestomach (Table 2) (Van Duuren et al., 1978). TBPP was tested in an in vivo, short-term skin test for sebaceous gland suppression and the induction of epidermal hyperplasia. Groups of 25 Swiss mice (aged 45 days) received dorsal applications of TBPP in acetone on days 1, 3, and 5. The dose levels applied on the skin were 49.5, 82.5, and 115.5 mg (total dose applied in three applications in acetone). TBPP did not suppress the sebaceous gland and did not induce hyperplasia (Sala et al., 1982). Groups of 28-34 female (60 days old) Swiss mice were given a single application of dimethylbenzanthracene (DMBA) (50 µg) or TBPP (110 µg) in acetone, on the dorsal skin. In the tumour promotion studies, the mice received, for 78 weeks, twice weekly applications to the same area of the dorsal skin of an acetone solution of tetradecanoyl phorbolacetate (TPA) (1 µg) or TBPP (33 mg), started one week after the initiation with DMBA or TBPP. In order to test TBPP for its ability to act as a complete carcinogen, a second series of mice received the same twice weekly applications as the promoted mice, but without any initiation treatment. The total TPA dose applied, was 156 µg/mouse and that of TBPP, 5.1 g/mouse. TBPP did not have any effect as a complete carcinogen on the skin, with a total dose of 5.1 g/mouse. The same dose did not have a promoting activity after DMBA initiation. However, TBPP initiated a significant number of skin tumours, when TPA was used as promoter. Furthermore, the number of lung adenomas increased significantly (Table 3) (Sala et al., 1982). Table 2. Tumour incidences in female Swiss mice after dermal application of tris(2,3-dibromopropyl) phosphate (TBPP)a Number of Dose Number of mice with tumours/number necropsiedb animals (mg/animal) treated Forestomach Lung Skin Oral cavity 29 0 1/29 7/29 0/29 0/29 29 10 10/29 26/29 2/29 2/29 30 30 20/30 28/30 5/30 4/30 a From: Van Duuren et al. (1978). b Increases in incidences of tumours of the forestomach, lung, skin, and oral cavity in treated animals were statistically significant compared with those in controls ( P < 0.05). 7.8 Special studies 7.8.1 Kidneys Osterberg et al. (1978) found an increased incidence and severity of chronic nephritis in a 90-day gavage study on rats, administered TBPP at dose levels of 25, 100, or 250 mg/kg body weight in propyleneglycol. The renal changes were associated with regenerative epithelium, hypertrophy, and dysplasia of renal tubular epithelial cells and were found at all three dose levels (section 22.214.171.124). TBPP caused proximal tubular damage and acute renal failure in rats, with elevation of serum creatinine and urea, and depression of organic anion and cation transport (Soderlund et al., 1980; Elliott et al., 1982; Lynn et al., 1982). It has been demonstrated that TBPP caused urinary excretion of renal cytoplasmic enzymes associated with the initial damage (characterized by increased membrane permeability), followed by the excretion of cell organelle-linked enzymes with necrosis of the renal tubular epithelium (Nomiyama et al., 1974; Emanuelli et al., 1979; Fukuoka et al., 1987, 1988a,b). TBPP also produced impairment of the tubular reabsorption capacity for metabolic fuels such as, lactate, glucose, and citrate, maintained across the brush border membrane by the sodium co-transport system (Kurokawa et al., 1985; Pitts, 1987). Male Wistar rats (56 rats in test group and 15 as controls; 7 weeks of age) were given a single oral dose in olive oil of 286.8 µmol TBPP (98%)/kg, to study TBPP nephrotoxicity. TBPP caused tubular necrosis. The animals received a single dose and some were killed daily for 10 days. The following effects were observed: on day 1, pyknosis of the renal tubular epithelial cells, necrosis on day 2, regeneration from day 3 and large nuclei formation from day 4. 13C-NMR spectra were applied to clarify changes of the renal low-molecular weight components in the kidneys injured by TBPP; sialic acid and inositol were found to be the desired marker components. The lesions produced by TBPP were characterized by changes in the renal components and enzyme activities. Increases in the sialic acid content of the kidneys were observed on day 1, suggesting destruction of the epithelial cell membrane. On day 5, regeneration accompanied by an increase in inositol contents was found. Renal activity of the cytoplasmic enzyme, alanine aminopeptidase, was increased on days 2, 5, 6, and 7 (Fukuoka et al., 1988a). There are large species differences in TBPP nephrotoxicity, since neither hamsters, guinea-pigs, nor mice developed acute kidney damage at doses of 500-1000 mg/kg body weight (Soderlund et al., 1982a). Table 3. Development of tumours in female Swiss mice in initiation-promotion studies carried out with TBPPa Treatment Number of mice with tumours Group Initiation Promotion or Number Skin Lung Number Other tumours repeated treatment of mice tumours adenomas Type 1 - TBPPb 33 0 14 3 2: lymphosarcoma, hepatoma 2 TBPP TPAc 34 26d 7 0 3 DMBA TBPPa 33 3 11 2 mammary tumour, perianal carcinoma 7 - TPAc 28 12 5 0 a From: Sala et al. (1982). b TBPP: Total dose, 5.1 g/animal (170 g/kg body weight). c TPA: Total dose, 156 µg/animal. d Two squamous cell carcinomas in each group. BBPP was clearly more nephrotoxic for Wistar and Sprague-Dawley rats than TBPP, whereas mono(2,3-dibrompropyl) phosphate (mono-BPP) was less nephrotoxic (Elliott et al., 1982; Soderlund et al., 1982b). However, female Sprague-Dawley rats appeared to be resistant to BBPP nephrotoxicity (Elliott et al., 1982), paralleling the carcinogenicity of TBPP in female animals. The nephrotoxicity of TBPP has been compared to that of BBPP, using equimolar doses. Both chemicals caused reversible acute renal failure, accompanied by tubular necrosis. Polyuria, high urinary glucose, lactate, and enzyme levels, and increased serum creatinine levels were observed. It was suggested, therefore, that BBPP or a metabolite of this compound, mediated the nephrotoxicity associated with TBPP (Takada et al., 1991a). The finding that BBPP was a major urinary metabolite of TBPP and that this compound was at least as nephrotoxic as TBPP, indicates that it is a proximate nephrotoxic metabolite of TBPP (Lynn et al., 1980; Soderlund et al., 1982b). Generally, attempts to modulate TBPP nephrotoxicity by various pretreatments have not been very successful. Pretreatment of Wistar rats with the cytochrome P-450 inducers, phenobarbital and polychlorinated biphenyls, known to increase the metabolism of TBPP, had no effect on its nephrotoxicity. However, pretreatment with cobaltous chloride resulted in a moderate reduction in TBPP nephrotoxicity. Interestingly, depletion of glutathione in vivo with diethyl maleate did not alter TBPP nephrotoxicity (Soderlund et al., 1980). The indication that the oxidative metabolism of TBPP or BBPP plays only a minor role in its nephrotoxicity, is confirmed by the findings that none of the selectively deuterated analogues (see below) significantly altered morphological evidence of nephrotoxicity compared with the protio compounds. It appears that C-H bond cleavage was not the rate-limiting step in the overall process leading to nephrotoxicity (Soderlund et al., 1988). However, recent studies have revealed that deuterium substitution at C-2 and C-3 of TBPP results in a considerable decrease in the plasma levels of BBPP at earlier time points compared with those after protio TBPP. The time-integrated plasma concentrations of the resulting deuterated BBPP analogues at later time points are not significantly different from that of BBPP, indicating that the target organ exposures to BBPP and deuterated BBPP analogues are similar (Pearson et al., 1993b). This may explain the lack of deuterium isotope effect on nephrotoxicity. Thus, a role of oxidative metabolism cannot be completely ruled out. Activation of nephrotoxic alkyl halides with glutathione by the beta-lyase pathway is documented. However, known inhibitors of the beta-lyase pathway (e.g., AT-125 and aminooxyacetic acid) did not affect the extent of nephrotoxicity in rats caused by a single ip dose of TBPP (Soderlund et al., 1988). Because of its acidic nature, BBPP may be the species transported into the renal tubular cells. Probenecid, an inhibitor of the organic anion transport system in the kidneys, reduces the nephrotoxicity of BBPP (Soderlund et al., 1988). The nature of the toxic metabolites formed from BBPP has not been identified. One possible candidate is an episulfonium ion generated by the conjugation of BBPP with glutathione. At present, the mechanisms involved in TBPP organ toxicity are not known with certainty. DNA damage, as measured by alkaline elution, was detected after 20 min in the kidneys of Wistar rats given a single ip dose of 25 mg/kg body weight. Thus, DNA appears to be an early target in TBPP nephrotoxicity, leading to cell death (Pearson et al., 1993b). 7.9 Factors modifying toxicity; toxicity of metabolites 7.9.1 Toxicity of metabolites 2,3-Dibromo-1-propanol (DBP), a metabolite of TBPP, was tested in 2-year dermal carcinogenicity studies on F344/N rats and B6C3F1 mice. The doses used were 0, 188, or 375 mg/kg body weight in rats and 0, 88, or 177 mg/kg body weight in mice. Under the conditions of these studies, there was clear evidence of carcinogenic activity in both sexes of both species in a variety of organs (US NTP, 1992). In male rats, there was an increased incidence of neoplasms of the skin, nose, oral mucosa, oesophagus, forestomach, small and large intestine, Zymbal's gland, liver, kidney, tunica vaginalis, and spleen. In female rats, there was an increased incidence of neoplasms of the skin, nose, oral mucosa, oesophagus, forestomach, small and large intestine, Zymbal's gland, liver, kidney, clitoral gland, and mammary gland. In male mice, there was an increased incidence of neoplasms of the skin, forestomach, liver, and lung. In female mice, there was an increased incidence of neoplasms of the skin and forestomach. BBPP, DBP, as well as TBPP, are acute nephrotoxins, BBPP being the most potent (Lynn et al., 1982). In three groups of rats, the 24-h urine volume was measured after ip injection of equimolar amounts (in 1 ml Emulphor) of TBPP (50 mg), BBPP (36 mg), or DBP (39 mg). The single dose of TBPP caused a 5-fold increase in urine volume after two days. After one week, urine volume had returned to normal. In contrast, the single dose of BBPP resulted in a 7 to 8-fold increase in urine volume from days 2-5. Urine volume had not returned to normal after 10 days. The single dose of DBP produced a 2 to 3-fold increase in urine volume, which rapidly returned to normal. In another study, rats received a single ip injection of TBPP, BBPP, or mono-BPP (in dimethyl-sulfoxide, 0.25 ml per 100 g) of 0, 10, 25, 50, 100, or 200 mg/kg body weight; the animals were killed 48 h later (except the high dose group which was killed 40 h later). A significant increase in kidney/body weight ratio occurred with all three compounds at the 200 mg/kg dose. Enlarged kidneys were pale, oedematous, and showed a prominent necrotic band in the inner part of the cortex. Histologically, tubular kidney necrosis was demonstrated in rats receiving 100 mg TBPP or mono-BP/kg or more and, in animals receiving 50 mg BBPP/kg or more. Plasma creatinine was significantly elevated at doses from 10 mg BBPP/kg, 25 mg mono-BP/kg, and 50 mg TBPP/kg upwards. Plasma urea was significantly elevated after doses of 100 mg mono-BP/kg or more and 200 mg BBPP/kg. Plasma GDT was also significantly increased at the highest doses of BBPP and mono-BP (Soderlund et al., 1982b). Comparable results were reported by Fukuoka et al. (1988b). Rats received a single oral dose of 0, 71.7, 143.4, or 286.8 µmol BBPP/kg and were evaluated for seven days after the dose. In addition to polyuria, changes in the excretion of lactate, uric acid, and glucose, and changes in the activities of urinary enzymes (alkaline phosphatase, aspartate aminotransferase, and gamma- glutamyltransferase) at various times after dosing, histopatho-logical changes occurred in the kidney. The histopathological changes included pyknosis, necrosis, and desquamation. 7.9.2 Mutagenicity of metabolites The following TBPP metabolites have been identified in in vivo and in vitro test systems: BBPP, mono(2,3-dibromopropyl) phosphate (mono-BPP), 2-bromoacrolein (BA), 2,3-dibromo-1propanol (DBP), 2,3-dibromopropyl-2-bromopropen-2-yl, bis(2-bromopropen-2-yl) phosphate and 3,3-dibromopropyl phosphate (Lynn et al., 1980, 1982; Zeiger et al., 1982; Nelson et al., 1984). Several of these TBPP metabolites have been tested for their mutagenic potential. All the identified TBPP metabolites were mutagenic in Salmonella typhimurium TA 100 (Prival et al., 1977; Zeiger et al., 1982; Holme et al 1983; Nakamura et al., 1983; Gordon et al., 1985). DBP was mutagenic in Salmonella typhimurium TA 100 and TA 1535, but not in TA 1538 (Carr & Rosenkranz, 1978). However, only BA was more mutagenic than the parent compound (Gordon et al., 1985). Unscheduled DNA synthesis was induced in monolayer cultures of rat hepatocytes by DBP and, to a lesser extent, by BBPP and mono(2,3-dibromopropyl) phosphate (mono-BPP). A similar relative response of DBP, BBPP, and mono-BPP was found with respect to mutagenicity in V79 Chinese hamster lung cells with liver microsomal activation. The concentration of BBPP that was tested was 0.05 mmol/litre (Holme et al., 1983). BBPP was less potent than TBPP in causing DNA damage in both the liver and testicular cells. DNA damage, as measured by alkaline elution, was found in isolated rat liver cells exposed to 100 micromolar BBPP and to a lesser extent in testicular cells. DNA damage caused by BBPP phosphate could be decreased by diethyl maleate- pretreatment in testicular cells, but not in the liver cells (Soderlund et al., 1992). 8. EFFECTS ON HUMANS 8.1 General population exposure Fifty-two out of 61 (22 males and 39 females) human volunteers completed a repeated insult patch study in which TBPP (1.1 g) was applied 10 times over a 24-day period, followed by a single challenge patch 14 to 21 days later. Fifty persons showed no reaction. After the 6th or 7th application 2 showed itching (or pruritis), and urticaria. The study was stopped for a month in these 2 subjects, and then restarted. No adverse effects were seen the second time. The conclusion of the author was that TBPP did not produce primary skin irritation, skin fatigue, or skin sensitization (Kerst, 1974; US EPA, 1976). A maximization test was carried out and 8 out of 24 human volunteers, exposed to a 100% TBPP solution, showed a sensitization reaction; while a 20% solution in a petroleum ether sensitized 2 out of 25 subjects. The TBPP was considered to be a mild skin sensitizer in humans. Subjects who had been sensitized by the 20% solution also showed reactions when tested with TBPP-treated fabrics. The response varied with the type of fabric substrate. The degree of sensitization appeared to depend upon the availability of the agent at the surface of the fibre. This is different for different types of fibres and the methods in which the flame retardant is applied. Washing the fibre reduced surface concentrations (Morrow et al., 1976). Andersen (1977) reported the incidence of sensitization to TBPP in human subjects from seven European countries, patch-tested with TBPP (5% in petrolatum), and found two positives among 1103 patients. Since TBPP-treated fabrics can be expected to contact the skin for long exposure periods, patches from both rayon and acetate fabrics, previously treated with TBPP were applied to humans, but skin reactions were not elicited (Brieger et al., 1968). 8.2 Occupational exposure No case reports or epidemiological studies are available. 9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD 9.1 Laboratory studies 9.1.1 Microorganisms Nitrifying return activated sludge diluted with fresh settled sewage, filtered, aerated and containing Nitromonas and Nitrobacter was used to test nitrification. No inhibition was found with 300 mg TBPP/litre and no depression of the BOD was noticed at 170 mg/litre (Wood et al., 1981). 9.1.2 Aquatic organisms 126.96.36.199 Invertebrates A 57% inhibition of southern armyworm (Spodoptera eridania) microsomal p-chloro- N-methylanaline N-demethylase was measured at 1 mg TBPP/ml, in an in vitro incubation mixture (Eldefrawi et al., 1977). 188.8.131.52 Vertebrates Six goldfish (Carassius auratus) were exposed to 1 mg TBPP (dissolved in acetone) per litre water. All died within 5 days. The fish appeared to swim in a disoriented manner prior to death. The fish showed necrosis of the kidneys (Gutenmann & Lisk, 1975). TBPP (1 mmol/litre) inhibited by 19% the acetylcholinesterase (AChE) activity in the electric organ of the electric ray (Torpedo ocellata). The binding of acetylcholine to its electric organ receptor was not inhibited (Eldefrawi et al. 1977). 9.1.3 Terrestrial organisms 184.108.40.206 Plants Seed of oat (Avena sativa) was added to loamy sand soil (1.5% organic carbon) and exposed to 1, 10, 100 or 1000 mg TBPP/kg soil for 14 days. The temperature of the soil was 20°C, the pH 6.0 and 16 h light/8 h dark cycle was used. The study was performed according to a modified OECD terrestrial plant-growth test. The EC50 for growth inhibition was at 1000 mg/kg soil (Pestemer, 1988). In a comparable study, turnip seed (Brassica rapa sp.) was tested under the same conditions as the oat seed. With 1000 mg TBPP/kg soil, 100% inhibition of growth was obtained (Pestemer, 1988). 10. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES IARC concluded in 1979 that there is sufficient evidence that TBPP is carcinogenic in mice and rats. In the absence of adequate data on humans, it is reasonable, for practical purposes, to regard TBPP as if it presented a carcinogenic risk to humans (IARC, 1979). In 1987, colon tumours were reported in a short-term study on male rats. The overall evaluation made by IARC (1987) was that TBPP is probably carcinogenic to humans (Group 2A) (IARC, 1987). BIS(2,3-DIBROMOPROPYL) PHOSPHATE AND SALTS A1. SUMMARY AND EVALUATION; CONCLUSIONS AND RECOMMENDATIONS The data base on bis(2,3-dibromopropyl) phosphate (BBPP) and its salts is inadequate for an evaluation, and to support its commercial use. From the available data, there is some indication that the substance may be mutagenic and carcinogenic. The substance cannot be evaluated unless additional data become available on physical and chemical properties, production and use, environmental transport, distribution, and transformation, environmental levels and human exposure, kinetics and metabolism in animals and humans, effects on laboratory mammals, humans, and in vitro test systems, and effects on other organisms in the laboratory and field. More mutagenicity data on at least two end-points are also needed. A2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS A2.1 Identity Chemical formula C6H11Br4O4P Chemical structure (BrCH2-BrCH-CH2O)2P - OH ' (BrCH2-BrCH-CH2O)2P - O - P = O H ' OH Relative molecular 497.8 mass CAS Chemical name 2,3-dibromo-1-propanol-hydrogen phosphate Common name bis(2,3-dibromopropyl) phosphate; Synonyms bis(2,3-dibromopropyl)hydrogen phosphate; bis(2,3-dibromopropyl) phosphoric acid CAS registry number 5412-25-9 The ammonium, magnesium, potassium, and sodium salts have also been proposed. A2.2 Physical and chemical properties No data are available on this subject. A2.3 Analytical methods See section 2.3, TBPP. A3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE A3.1 Natural occurrence BBPP and its salts are not known to occur as natural products. A3.2 Anthropogenic sources A3.2.1 Production levels and processes Bis(2,3-dibromopropyl)ammonium phosphate has been prepared by a reaction of bis(2,3-dibromopropyl) phosphate with NH4OH (Mischutin, 1972). A3.2.2 Uses In the 1960s and 1970s, BBPP and its magnesium and ammonium salts were proposed for use as fire-proofing agents for textiles and plastics. No evidence was found that BBPP or its salts are currently used for commercial applications. A3.3 Contamination of the environment BBPP has been identified as a major biodegradation product of TBPP, in a laboratory activated sludge system (Alvarez et al., 1982). A3.4 Environmental transport, distribution, transformation, and exposure levels No data are available. A4. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS A4.1 Absorption, distribution, elimination, and biotransformation BBPP has been identified as a metabolite of TBPP (Lynn et al., 1982). Following intravenous administration of [14C]-tris(2,3- dibromopropyl) phosphate to male Sprague-Dawley rats, approximately half of the label appeared in the urine within 120 h and 7.8% of the recovered urinary label was BBPP. BBPP also constituted 21.5% of the biliary label (33.9% of administered dose in 24 h). The tissue distribution of BBPP was studied at different intervals (5 and 30 min, 8, 24, and 120 h, and 5 days) after administration of the tris compound. BBPP was identified in nearly all organs within 5 min of administration. Tissue levels declined after 5 or 30 min at all sites except the large intestines and carcass. Five min after administration, 75% of the plasma label was BBPP. The plasma concentration of BBPP increased between 0 and 1 h and declined biphasically thereafter, with an initial plasma half-life of 6 h, declining to approximately 36 h, by one to five days. A5. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS A5.1 Single exposure Adult male Wistar rats, 5 per group, were administered a single ip injection of 0, 10, 25, 50, 100, or 200 mg BBPP/kg body weight in DMSO (2.5 ml/kg). All rats were killed 40-48 h after dosing. One rat of the highest dose group died. Kidneys and body weight ratios were increased in 200 mg/kg rats. The kidneys were pale and oedematous with necrosis of the inner cortex. Microscopically, tubular cell necrosis was observed in rats with 50 mg/kg or more. However, plasma creatinine was significantly elevated at doses of 10 mg/kg body weight or more and plasma urea and plasma-GOT were elevated at 200 mg/kg body weight (Soderlund et al., 1982b). Elliot et al. (1982) carried out a comparable study with only one dose level of 120 mg/kg body weight, administered to male Sprague-Dawley rats intraperitoneally. Rats were killed after 48 h. Serum creatinine was elevated, and renal cortical slices showed decreased uptake of para-aminohippuric acid and N-[14C]- methylnicotinamide. Microscopically, tubular cell necrosis of the loops of Henle was found. When the Mg salt of BBPP was administered by oral intubation to Wistar rats, eyelid closure, crouching, shivering, and staggering gait were observed. LD50 values in male and female rats were 283 and 261 mg/kg, respectively (Takada et al., 1991b). A5.2 Short-term exposure The Mg salt of BBPP was given to Wistar rats (5/sex per group) in the diet at levels of 0, 30, 100, 300, or 1000 mg/kg for 45 days. There was no difference between treated and control animals in body weight and food consumption. At 1000 mg/kg, a significant increase in liver and kidney weights was observed in the males. Desquamation, swelling and large nuclei formation of the tubular epithelium, and tubular dilation of kidney were observed. It was concluded that BBPP-Mg has apparent renal toxicity (Takada et al., 1991b). A5.3 Long-term exposure No data were available on the following subjects: * Skin and eye irritation; sensitization * Reproductive toxicity, embryotoxicity, and teratogenicity A5.3.1 Mutagenicity and related end-points 2,3-Dibromo-1-propanol (DBP) was mutagenic in Salmonella typhimurium TA 100 and TA 1535, but not in TA 1538 (Carr & Rosenkranz, 1978). Magnesium BBPP at doses of 3-100 µmol/plate was more mutagenic to Salmonella typhimurium TA 1535 and TA 100 in the presence of metabolic activation by Kanechlor 500-induced rat-liver S9 than in its absence; it was weakly mutagenic to TA 98 with S9, but showed no mutagenic activity in TA 1537 and TA 1538 (Nakamura et al., 1979). The ammonium salt of BBPP was more mutagenic than the magnesium salt, which itself was more mutagenic than the free acid (Nakamura et al., 1983). BBPP purified as a urinary metabolite from rats treated with TBPP was mutagenic to Salmonella typhimurium TA1535 and TA100 in the presence of metabolic activation (Aroclor 1254-induced rat liver S9) at doses ranging from 0.05 to 1.0 µmol (Lynn et al., 1982). Mutagenic activity in Salmonella typhimurium TA100 was detected when BBPP at concentrations of 50 and 100 µmol/litre was incubated for 30 min with hepatic microsomal fractions from untreated rats or rats pretreated with phenobarbital (Soderlund et al., 1982b). A5.3.2 Carcinogenicity Four groups of 40 Wistar rats (5 weeks old) of each sex per dose level were fed diets containing 0, 80, 400, or 2000 mg/kg of the magnesium salt of bis(2,3-dibromopropyl) phosphate (BBPP), for 24 months. Food consumption and body weight gain were measured immediately prior to the beginning of the study, and then weekly for 6 weeks, biweekly for 6 months, and monthly, thereafter. Blood samples of 8-13 rats/sex per dose were taken at 12, 18, and 24 months. Body weight gain was reduced significantly at a level of 2000 mg/kg. Male and female rats receiving 2000 mg/kg and female rats receiving 400 mg/kg showed a significant increase in absolute and relative liver and kidney weights. A high incidence of tumours was observed in both sexes. In the digestive system, papillomas and adenocarcinomas were found in the tongue, oesophagus, and forestomach, and adenocarcinomas of the intestines. In the liver, hepatocellular adenomas (neoplastic nodules) and carcinomas were found (Table 4). Pre-terminal mortalities were associated with an increased incidence of forestomach papillomas in both sexes, adenocarcinomas of the small intestines in male rats, and hepatocellular carcinomas in females. Non-neoplastic lesions were mainly found in the kidneys of the rats of the 2000 mg/kg group and, in a few instances, also in the 400 mg/kg group. The changes were epithelial swelling and desquamation, large bizarre nuclei, pyknosis, and basement membrane thickening. Serum biochemistry was performed using commercially available assay kits for the diagnosis of liver and kidney function and of disorders of the digestive system. Eighteen parameters were studied. Significant increases or decreases in the parameters were mainly observed in the 2000 mg/kg group with a few in the 400 mg/kg group. Statistically significant decreases were seen in the serum, in total protein, albumin, and cholinesterase; and significant increases were seen in blood urea nitrogen, total cholesterol, alkaline phosphatase, gamma-glutamyl transferase, magnesium, GOT, and GPT (Takada et al., 1991a). A5.4 Special studies A5.4.1 Kidneys Many nephrotoxic agents exert their effects primarily on the cells of the proximal tubules. Isolated tubular cells were used to study the uptake of alpha-methylglucose as indicator of effects on the functional integrity of the cells. BBPP, which is acutely nephrotoxic in vivo, inhibited the uptake of alpha-methylglucose at low concentrations (Boogaard et al., 1989). See also TBPP section 7.8.1 and 7.9.1 on the nephrotoxicity of BBPP. A5.5 Effects on humans and other organisms in the laboratory and field No data are available. Table 4. Neoplastic lesions and tumour incidence in Wistar rats fed diets containing BBP magnesium salta Organ Sex Dose level Papillomas Squamous cell carcinomas Tongue male 400 1/40 1/40 female 400 1/40 1/40 2000 5/40 0/40 Oesophagus male 400 6/40 1/40 2000 2/40 0/40 female 2000 6/40b 0/40 Forestomach male 400 8/40b 1/40 2000 17/40c 2/40 female 400 4/40 2/40 2000 20/40c 4/40 Adenoma Adenocarcinoma Small intestines male 400 0/40 2/40 2000 2/40 14/40c female 2000 0/40 9/40c Table 4 (contd). 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New York, Van Nostrand Reinhold Company, 1173 pp. Vogel EW & Nivard MJM (1993) Performance of 181 chemicals in a Drosophila assay predominantly monitoring interchromosomal mitotic recombination. Mutagenesis, 8(1): 57-81. Wood LB, Hurley BJE, & Matthews PJ (1981) Some observations on the biochemistry and inhibition of nitrification. Water Res, 15: 543-551. Zeiger E, Pagano DA, & Nomeir AA (1982) Structure-activity studies on the mutagenicity of tris(2,3-dibromopropyl) phosphate (Tris-BP) and its metabolites in Salmonella. Environ Mutagen, 4: 271-277. RESUME ET EVALUATION, CONCLUSIONS ET RECOMMANDATIONS 1. Tris-2,3-dibromopropyle 1.1 Résumé et évaluation 1.1.1 Production et usage La production de phosphate de tris-2,3-dibromopropyle (TBPP) a commencé pour la première fois vers 1950 et sa production commerciale est documentée à partir de 1959. Aux Etats-Unis, en 1975, la production de TBPP se situait, selon les estimations, entre 4100 et 5400 tonnes. Autant qu'on sache, le TBPP n'est ni produit ni utilisé actuellement dans le monde comme retardateur de flamme dans les textiles, mais il peut être incorporé à des polymères utilisés à d'autres fins. Le TBPP a constitué un important retardateur de flamme, que l'on ajoutait aux tissus de cellulose, de triacétate et de polyester, en particulier pour les vêtements de nuit destinés aux enfants, mais il a été depuis interdit pour cet usage dans plusieurs pays d'Europe, aux Etats-Unis d'Amérique (1977) ainsi qu'au Japon (1978). Le TBPP peut se trouver à l'intérieur ou à la surface du tissu. Lorsqu'il se trouve à l'intérieur, on ne peut pas l'extraire par solvants et il est donc probable qu'il ne peut pas non plus être absorbé par voie percutanée. Toutefois, lorsqu'il se trouve à la surface de la fibre, il peut être extrait lors de la lessive ainsi qu'au moyen d'acide acétique ou d'autres solvants ou encore par la salive, et peut être alors absorbé par voie percutanée. Dans ce cas, il peut y avoir en cours d'utilisation ou pendant la lessive des produits finis une perte importante du TBPP qui se trouve à leur surface, d'où contamination de l'environnement. En outre, on a signalé la décharge de TBPP dans l'environnement par des ateliers de finissage et le rejet final de déchets solides. 1.1.2 Propriétés physiques et chimiques Le TBPP existe en au moins deux qualités. Le produit de haute qualité est un liquide visqueux clair, de couleur jaune pâle, qui contient jusqu'à 1,5% de matière volatile. Le produit de basse qualité peut contenir jusqu'à 10% de matière volatile. Le TBPP (degré de pureté > à 97%) a un point d'ébullition égal à 390°C, un point de fusion de 5,5°C et une tension de vapeur de 1,9 × 10-4 mmHg à 25°C. Il est faiblement soluble dans l'eau (8 mg/litre). Lorsqu'on le chauffe jusqu'à décomposition, c'est-à-dire au-dessus de 260-300°C, le TBPP libère des composés contenant du brome et du phosphore. Son coefficient de partage entre le n-octanol et l'eau (log Pow) est égal à 3,02. Il existe des méthodes d'analyse permettant le dosage du TBPP et de ses métabolites dans des échantillons biologiques ou d'autres matrices. 1.1.3 Transport, distribution et transformation dans l'environnement Le peu de données dont on dispose incitent à penser que le TBPP est relativement persistant dans l'environnement. Il ne semble pas que l'oxydation et la photodécomposition jouent un rôle important dans sa destinée. Cependant, il peut y avoir une hydrolyse impliquant les atomes de brome du groupement propyle, en particulier en milieu basique. Il peut s'évaporer de l'eau mais on ne dispose pas véritablement de données sur ce point. Bien qu'on ait signalé la possibilité d'une biodécomposition du TBPP (demi-vie 19,7 h.) dans les boues activées, on ne pense pas que cela constitue un processus important dans les sols et les eaux naturels. Dans la boue stérilisée, il n'y a pratiquement pas de décomposition. On a constaté que l'un des principaux produits de décomposition du TBPP était le phosphate de bis-2,3-dibromo-propyle. Le TBPP étant pratiquement insoluble dans l'eau, il est possible que l'adsorption sur les matières particulaires et sur les sédiments joue un rôle important. La valeur estimative du log de Koc (3,29) indique qu'il y a une forte adsorption au sol. Sur la base de cette valeur de Koc et de la faible solubilité dans l'eau du TBPP, on pense que ce composé n'est que lentement lessivé vers les eaux souterraines. Le TBPP va avoir tendance à s'accumuler dans les décharges publiques et autres lieux de ce genre, avec pour conséquence la possibilité d'une bioaccumulation. D'ailleurs, une étude portant sur un cyprin, Pimephales promelas, a permis de mettre en évidence un facteur de bioconcentration de 2,7, ce qui est faible, alors que le coefficient de partage n-octanol/eau (log de Pow) est de 3,02. En raison de sa faible tension de vapeur, on peut penser que le TBPP sera essentiellement sorbé sur les particules en suspension dans l'air. La décomposition thermique en milieu oxydant du TBPP à la température de 370°C entraîne la formation de bromure d'hydrogène et de composés bromés en C3 tels que des bromopropènes, des dibromopropènes ainsi que des di-et-tribromopropanes. 1.1.4 Concentrations dans l'environnement et exposition humaine On ne dispose que de données limitées sur les concentrations dans l'environnement et l'exposition humaine. Lors d'études effectuées au Japon en 1975, on a analysé 20 échantillons d'eau, de sol et de poissons sans y trouver de TBPP. En revanche, on a mis en évidence, sans le doser, du TBPP dans des particules en suspension dans l'air prélevé aux alentours d'une installation industrielle. Ce sont les enfants portant des vêtements de nuit traités par du TBPP qui, lorsque ce produit était autorisé, constituaient le groupe de population le plus particulièrement exposé à ce retardateur de flamme. On estime qu'à l'époque, la dose absorbée à travers la peau par ces enfants aux Etats-Unis d'Amérique était de l'ordre de 9 µg/kg de poids corporel et par jour. La Consumer Product Safety Commission des Etats-Unis a calculé que, sur une période de six ans, un enfant portant de tels vêtements pouvait absorber une dose totale d'au moins 2 à 77 mg de TBPP/kg de poids corporel. 1.1.5 Cinétique et métabolisme chez les animaux de laboratoire et l'homme Le TBPP est rapidement absorbé au niveau des voies digestives et à un rythme plus modéré par la voie percutanée chez les rats et les lapins. Chez le rat, le TBPP ou ses métabolites sont éliminés dans les 5 jours. L'élimination se produit à hauteur d'environ 50% dans les urines, 10% dans les matières fécales, 10-20% de son carbone étant rejetés sous forme de CO2 dans l'air expiré. Un jour après avoir administré par voie orale du TBPP radiomarqué à des rats, la radioactivité s'est retrouvée dans les limites de 0,2-6,6% au niveau du sang, du foie, des reins, des muscles, des tissus adipeux et de la peau. Le temps de demi-élimination de la radioactivité de tous ces organes était d'environ 2-4 jours. Au bout de 8 h., il ne restait encore, en concentrations notables, que du phosphate de bis-2,3-dibromopropyle dans la plupart des tissus. Après administration orale de TBPP à des rats, on a mis en évidence six métabolites dans les urines et la bile. Le principal métabolite urinaire, fécal, biliaire et tissulaire était le phosphate de bis-2,3-dibromopropyle. Un autre métabolite, le 2,3-dibromo- propanol, a été également mis en évidence chez des rats et des enfants qui portaient des vêtements traités par du TBPP. Les microsomes hépatiques métabolisent le TBPP en présence de NADPH et d'oxygène. Les principaux métabolites obtenus sont le phosphate de bis-2,3-dibromopropyle et le 2,3-dibromopropanol. On a montré qu'il se formait du phosphate de bis-2,3-dibromopropyle par oxydation au niveau du C3 et peut-être également en position C2 du TBPP. Outre le phosphate bis-2,3-dibromopropyle, on retrouve de la 2-bromoacroléine, de l'acide 2-bromoacrylique ainsi que des composés propylliques hydroxylés et des métabolites conjugués avec du glutathion. Du S-(2,3-dihydroxypropyl)glutathion ayant été mis en évidence dans la bile de rats, on a avancé l'hypothèse que le TBPP et/ou le phosphate de bis-2,3-dibromopropyle subissent une conjugaison directe par le glutathion en présence de glutathion S-transférase avec formation, comme métabolites, d'ions épisulfonium. Par activation, le TBPP forme des produits qui se fixent par liaisons covalentes aux protéines et à l'ADN in vivo et in vitro. Après injection intrapéritonéale de TBPP tritié à des souris, à des hamsters et à des cobayes mâles, qui sont moins sensibles à la néphrotoxicité induite par cette substance que les rats, on a observé un degré analogue de liaison covalente aux protéines dans le foie et les reins. Chez le rat mâle, qui est beaucoup plus sensible à la néphrotoxicité induite par ce produit, on a constaté qu'il y avait beaucoup plus de composé radiomarqué fixé aux protéines rénales qu'aux protéines hépatiques. Mis en présence de microsomes hépatiques provenant de souris, de cobayes, de hamsters et de sujets humains, le TBPP est dans tous les cas métabolisé en intermédiaires génotoxiques. Toutefois, les métabolites réactifs du TBPP se forment beaucoup plus lentement en présence de microsomes hépatiques d'origine humaine qu'en présence de microsomes prélevés sur rongeurs. Après avoir administré à des rats une dose néphrotoxique de TBPP et de ses analogues radiomarqués, on a constaté que le degré de liaison covalente aux protéines décroissait dans l'ordre suivant: reins, foie et testicules. D'après les résultats d'études in vitro et in vivo au cours desquelles on a comparé les lésions produites au niveau de l'ADN rénal, on est incité à penser qu'il y a formation de phosphate de bis-2,3-dibromopropyle au niveau du foie par oxydation, catalysée par le P450, du TBPP en position C2 ou C3. Ce phosphate de bis-2,3-dibromopropyle est ensuite transporté vers les reins où il est métabolisé en intermédiaire réactif qui endommage l'ADN et se fixe aux protéines rénales. L'activation qui se produit au niveau du rein ne semble pas impliquer le P450 mais s'effectuer plutôt par l'intermédiaire d'un métabolisme dépendant du GSH. Des études in vitro avec du TBPP et certains de ses analogues radiomarqués ont montré que l'oxydation du TBPP comportait l'incorporation d'un atome d'oxygène provenant de l'eau. Cela implique que l'oxydation en position C2 du reste propyle donne naissance à une alpha-bromocétone réactive qui est capable d'alkyler directement les protéines ou de s'hydrolyser pour donner du phosphate de bis-2,3-dibromopropyle et une bromo-alpha-hydroxycétone réactive. 1.1.6 Effets sur les mammifères de laboratoire et les systèmes d'épreuve in vitro La toxicité du TBPP est faible, qu'il s'agisse de la toxicité aiguë par voie orale à court terme ou de la toxicité aiguë par voie percutanée. Pour le rat, la DL50 par voie orale est > 2 g/kg et pour le lapin, la DL50 par voie cutanée dépasse 8 g/kg de poids corporel. On a observé une atteinte rénale très étendue (nécrose cellulaire au niveau des tubules proximaux) chez des rats mâles à qui l'on avait injecté par voie intrapéritonéale une seule dose de 100 mg de TBPP par kg de poids corporel. Chez des rats soumis respectivement pendant quatre semaines ou 90 jours à des épreuves de toxicité orale au cours desquelles du TBPP leur avait été administré par gavage ou mêlé à leur nourriture, on a observé une augmentation de l'incidence et de la gravité des néphrites chroniques aux doses supérieures ou égales à 25 mg/kg de poids corporel. Chez des lapins, l'application cutanée quotidienne de TBPP à des doses supérieures ou égales à 2,2 g/kg de poids corporel pendant 4 semaines a entraîné une dégénérescence au niveau du foie et des reins. Tous les lapins sont morts dans les quatre semaines. En revanche, aucun animal n'est mort lors d'une autre étude avec des doses allant jusqu'à 250 mg/kg de poids corporel. Des lapins qui avaient subi chaque semaine pendant 90 jours une application cutanée de 2,27 g de TBPP/kg de poids corporel ont présenté des anomalies rénales, une atrophie testiculaire et une aspermatogénèse. Aux doses de 1,1 ou 0,22g de TBPP, on n'a observé aucune irritation cutanée ou oculaire chez les lapins et il n'y a pas eu non plus de sensibilisation cutanée chez des cobayes. Deux études de tératogénicité ont été effectuées sur des rats. L'une d'entre elles comportait des doses allant jusqu'à 125 mg/kg de poids corporel et n'a pas permis de mettre en évidence d'effet tératogène. Lors d'une autre étude où la dose administrée était de 200 mg/kg de poids corporel, on a observé une augmentation significative des variations affectant le squelette chez les foetus et, aux doses de 50 et 100 mg/kg de poids corporel, une diminution sensible de l'indice de viabilité. Les auteurs en ont conclu que l'effet observé était dû à l'action toxique du composé sur les femelles gestantes. Chez des rats auxquels on avait administré du TBPP, on a constaté des lésions étendues de l'ADN dans divers organes. In vitro, on a constaté que le TBPP produisait la rupture des brins d'ADN dans des cellules KB d'origine humaine. Le TBPP a également induit une synthèse non programmée de l'ADN dans des hépatocytes de foie de rat, ce phénomène n'étant toutefois pas constaté dans des cellules épithéliales de prépuce humain. Plusieurs études ont révélé que le TBPP provoquait des mutations, notamment par substitution des paires de base, chez des souches de Salmonella typhimurium, avec ou sans activation métabolique. L'étude des mutations géniques directes sur cellules de hamsters chinois V79 avec ou sans activation métabolique, a donné des résultats négatifs. Toutefois, en présence de microsomes hépatiques de rats préalablement traités par du phénobarbital, on a observé un effet positif. Un effet faiblement positif a également été observé avec des cellules lymphomateuses de souris (locus L5178YTK). Dans des cellules V79 de hamsters chinois, on a constaté que le TBPP augmentait le nombre d'échanges entre chromatides soeurs. En revanche il n'y avait pas d'augmentation du nombre des aberrations chromosomiques, ni dans les cellules de hamsters chinois, ni dans les cellules de moelle osseuse murine, ni dans les cellules lymphoïdes humaines en culture. Dans des cellules fibroblastiques humaines diploïdes (lignée HE 2144), on a observé des échanges entre chromatides soeurs mais pas d'aberrations chromosomiques, l'épreuve étant effectuée sans activation métabolique. Toutefois la recherche in vitro d'aberrations chromo-somiques dans des lignées cellulaires de hamsters chinois a donné un résultat positif. Un résultat positif a également été obtenu lors de la recherche de micronoyaux dans des cellules de moelle osseuse provenant de hamsters chinois. Une autre épreuve de ce genre, portant cette fois sur des souris, a permis d'observer un effet faiblement positif. Les études effectuées sur Drosophila melanogaster ont montré que le TBPP augmentait les mutations récessives létales liées au sexe dans les gamètes mâles ainsi que chez les mâles adultes et il y avait induction de translocations réciproques. Dans l'épreuve de l'oeil en mosaïque w/w+, le TBPP a suscité une réaction fortement positive. Un certain nombre d'études ont été menées pour tenter d'élucider les mécanismes qui sont à la base de la mutagénicité et/ou de la génotoxicité induites par le TBPP. Ainsi la mutagénicité du TBPP pour les bactéries s'effectue par l'intermédiaire du système des monooxygénases microsomiques. Par ailleurs, lors d'une réaction qui est sous la dépendance du NADPH et de l'oxygène, il y a activation du TBPP par le cytochrome P450. Des microsomes préparés à partir de foies d'animaux traités par du phénobarbital ou des PCBs entraînent un accroissement de la mutagénicité. Le phosphate de mono- et de bis-2,3-dibromopropyle sont moins mutagènes que le TBPP. Des études in vitro ont montré que l'oxydation de la molécule de TBPP au niveau du C3 donnait naissance à un puissant mutagène à action directe, la 2-bromoacroléine qui se lie également à l'ADN. On a mis en évidence des différences interspécifiques dans la bioactivation du TBPP en métabolites mutagènes pour la souche TA 100 de Salmonella typhimurium. A cet égard, les microsomes hépatiques de souris étaient plus efficaces que ceux de cobayes, de hamsters et de rats. Trois études de transformation cellulaire ont été menées à l'aide de cellules C3H/10T1/2. Dans l'une d'entre elles, on a obtenu un effet positif, mais les deux autres ont donné des résultats négatifs. Lors d'études à long terme, on a administré par voie orale du TBPP à des souris et à des rats et on en a appliqué sur la peau de souris femelles. Chez les souris, on a constaté, après administration par voie orale, qu'il se formait chez les deux sexes des tumeurs au niveau de la portion cardiaque de l'estomac et des poumons, ainsi que des tumeurs bénignes ou malignes au niveau du foie chez les femelles et au niveau des reins chez les mâles. Chez les rats, des tumeurs bénignes ou malignes se sont formées au niveau des reins chez les mâles, les tumeurs rénales étant bénignes chez les femelles. L'application de TBPP sur la peau de souris femelles a entraîné l'apparition de tumeurs de la peau, des poumons, de la portion cardiaque de l'estomac et de la cavité buccale. On peut conclure de ces études que le TBPP est doté de pouvoir cancérogène chez la souris et le rat. Après administration d'un métabolite du TBPP, le phosphate de bis-2,3-dibromopropyle, par voie orale à des rats, on a constaté l'apparition de tumeurs digestives chez les deux sexes. Il s'agissait de papillomes et d'adénocarcinomes de la langue, de l'oesophage et de la portion cardiaque de l'estomac, ainsi que d'adénocarcinomes de l'intestin avec en outre des adénomes et des carcinomes hépatocellulaires. On a également procédé à l'application cutanée d'un autre métabolite du TBPP, le 2,3-dibromo-1-propanol, à des souris et à des rats. Chez les rats mâles, on a constaté un accroissement de l'incidence des tumeurs malignes de la peau, du nez, de la muqueuse buccale, de l'oesophage, de la portion cardiaque de l'estomac, de l'intestin grêle et du gros intestin, de la glande de Zymbal, du foie, du rein, de la vaginale, et de la rate. Chez les rats femelles, on constatait une incidence accrue de tumeurs malignes affectant la peau, le nez, la muqueuse buccale, l'oesophage, la portion cardiaque de l'estomac, l'intestin grêle et le gros intestin, la glande de Zymbal, le foie, le rein, la glande clitoridienne, et les glandes mammaires. Chez les souris mâles, il y avait également une incidence plus élevée des tumeurs malignes au niveau de la peau, de la portion cardiaque de l'estomac, du foie et des poumons, tandis que chez les femelles l'accroissement des tumeurs malignes se manifestait au niveau de la peau et de la portion cardiaque de l'estomac. 1.1.7 Effets sur l'homme On ne dispose que de données limitées concernant les effets du TBPP sur l'homme. Quelques études ont été consacrées à la recherche chez l'homme d'un effet sensibilisateur que le TBPP pourrait avoir sur la peau. Les résultats obtenus montrent que ce produit n'a qu'un faible pouvoir sensibilisateur et aucune irritation cutanée n'a été observée. Toutefois les personnes qui avaient présenté une réaction positive au TBPP pur ont également réagi lorsqu'on les a mises en contact avec des tissus qui en contenaient. 1.1.8 Effets sur les autres êtres vivants au laboratoire et dans leur milieu naturel On ne possède que très peu de données concernant les effets du TBPP sur les autres êtres vivants. Par exemple, des poissons rouges (Caraccius auratus) qui avaient été exposés à du TBPP à raison de 1 mg/litre sont morts tous les 6 en l'espace de 5 jours. La CE50 relative à l'inhibition de la croissance de la semence d'avoine se situait à 1000 mg/kg de terre. Cette concentration a provoqué l'inhibition totale de la croissance des semences de navet ( Brassica rapa sp.). 1.2 Conclusions Le TBPP a été utilisé naguère comme retardateur de flamme pour en imprégner les tissus, en particulier destinés à la confection de vêtements de nuit pour enfants, mais on est guère renseigné sur ses autres applications. C'est essentiellement par contact avec des tissus traités par ce composé que la population générale a pu être contaminée. On n'a guère de renseignements non plus sur l'exposition des ouvriers employés à la production commerciale du TBPP ainsi qu'à son utilisation pour la fabrication de divers produits, ni d'ailleurs sur les dangers qu'il représente. En raison de la rareté des données, il n'est pas possible de parvenir à des conclusions définitives quant aux niveaux d'exposition ou aux dangers que le TBPP fait courir aux êtres vivants dans leur milieu naturel, l'homme mis à part. Les études sur l'animal ont montré que le TBPP pouvait être absorbé au niveau des voies digestives et, dans une moindre proportion, par la voie percutanée. Il peut également être résorbé par cette dernière voie chez l'homme. Chez le rat, il se révèle être très largement métabolisé dans le foie en phosphate de bis-2,3- dibromopropyle, qui constitue le principal métabolite mis en évidence dans les urines, et, dans une moindre proportion, en 2,3- dibromopropanol. En outre, on a retrouvé de petites quantités d'autres métabolites bromés du TBPP. La présence de 2,3-dibromopropanol a été également observée chez des personnes qui portaient des tissus traités par le TBPP. La principale voie d'élimination est la voie urinaire, le composé étant excrété en très faible proportion sous sa forme initiale. Quant à la principale voie métabolique, elle semble faire intervenir le cytochrome P450 et les glutathion- S-transférases. D'après les données dont on dispose, on peut conclure que le TBPP ne présente qu'une faible toxicité aiguë chez l'animal de laboratoire. Des études au cours desquelles on a administré de manière répétée de fortes doses de TBPP, on permis de mettre en évidence des lésions rénales et hépatiques chez le rat ainsi qu'une atteinte testiculaire chez le lapin. Le composé a également un indéniable effet génotoxique dans plusieurs systèmes d'épreuve, tant in vitro qu' in vivo. Des effets cancérogènes ont également été relevés chez le rat et la souris. Deux de ses métabolites, le phosphate de bis-2,3- dibromopropyle et le 2,3-dibromopropanol, produisent également des effets cancérogènes chez l'animal de laboratoire. Il n'est pas irritant chez l'animal et son pouvoir sensibilisateur chez l'homme est faible. En 1977, la Consumer Product Safety Commission des Etats-Unis d'Amérique (Commission pour protection du consommateur) a interdit l'utilisation de vêtements d'enfants traités par le phosphate de tris-2,3-dibromoproyl, par crainte que ce composé ne soit cancérogène pour l'homme et en raison du risque non négligeable encouru par les personnes portant des vêtements confectionnés à l'aide de tissus imprégnés. Depuis lors, l'utilisation de ce composé comme retardateur de flamme dans les produits destinés à la consommation courante est très sévèrement réglementée dans un certain nombre d'autres pays et son utilisation dans les textiles est interdite. 1.3 Recommandations En raison de ses effets toxiques, le TBPP ne doit plus être utilisé dans le commerce. Au cas où, pour certains usages, il n'existerait pas de substituts moins dangereux au TBPP, il faudrait entreprendre des études pour s'assurer de l'absence d'exposition et de danger pour l'homme et l'environnement. 2. Bis-2,3-dibromopropyle La base de données relatives au phosphate de bis-2,3-dibromopropyle et à ses sels est insuffisante pour en permettre l'évaluation ou en justifier l'usage commercial. D'après les données disponibles on peut penser que ce composé pourrait être mutagène et cancérogène. Il ne sera pas possible d'évaluer ce produit tant qu'on ne disposera pas de données complémentaires sur ses propriétés physiques et chimiques, sa production et son usage, son transport, sa distribution, sa transformation et sa concentration dans l'environnement ainsi que l'exposition humaine auxquels il donne lieu, sa cinétique et son métabolisme chez l'animal et l'homme, ses effets sur les animaux de laboratoire, l'homme et les systèmes d'épreuve in vitro ainsi que son action sur les autres êtres vivants au laboratoire et dans leur milieu naturel. Il est également nécessaire d'obtenir davantage de données concernant son pouvoir mutagène sur au moins deux points d'aboutissement. RESUMEN Y EVALUACION; CONCLUSIONES Y RECOMENDACIONES 1. El fosfato de tris(2,3-dibromopropilo) 1.1 Resumen y evaluación 1.1.1 Producción y utilización El fosfato de tris(2,3-dibromopropilo) (FTBP) se produjo por primera vez hacia 1950. Se sabe que en 1959 hubo producción con fines comerciales. En 1975 la producción de FTBP en los Estados Unidos de América se estimó entre 4100 y 5400 toneladas. Que se sepa, el FTBP no se produce ni utiliza corrientemente en la actualidad a nivel mundial como retardador de ignición en productos textiles, pero puede utilizarse en polímeros empleados para otros fines. El FTBP era un importante retardador de ignición de la celulosa y de tejidos de triacetato y de poliéster, especialmente en ropa de dormir para niños, pero este empleo se ha prohibido en varios países de Europa, los Estados Unidos de América (1977) y el Japón (1978). El FTBP se utiliza tanto dentro del tejido como sobre el mismo. Si se encuentra dentro del tejido, no puede extraerse con disolventes y, por consiguiente, probablemente no esté disponible para su absorción cutánea. Sin embargo, si se halla en la superficie de la fibra, puede extraerse durante el lavado o por acción del ácido acético, de otros disolventes y de la saliva y está disponible para la absorción cutánea. En este caso habrá pérdida sustancial de FTBP superficial del tejido durante la utilización y/o el lavado de los productos acabados, y se contaminará el medio ambiente. Además, se sabe que hay emisión de FTBP al medio ambiente en las operaciones de acabado de textiles y en la evacuación final de desechos sólidos que contienen FTBP. 1.1.2 Propiedades físicas y químicas Puede obtenerse FTBP de dos calidades por lo menos. El producto de alto grado de pureza es un líquido transparente, amarillo pálido y viscoso, que tiene hasta un 1,5% de componentes volátiles. La calidad de baja pureza puede contener hasta un 10% de componentes volátiles. El punto de ebullición del FTBP (pureza > 97%) es de 390°C, su punto de fusión de 5,5°C y su presión de vapor de 1,9 × 10-4 mmHg a 25°C. La solubilidad del FTBP en el agua es baja (8 mg/litro). Cuando se calienta hasta su descomposición, a una temperatura superior a 260-300°C el FTBP emite compuestos que contienen bromo y fósforo. El coeficiente de reparto n-octanol/agua (log Pow) es de 3,02. Se dispone de métodos analíticos para determinar la presencia de FTBP y sus metabolitos en muestras biológicas y otras matrices. 1.1.3 Transporte, distribución y transformación en el medio ambiente La limitada información disponible sugiere que el FTBP es relativamente persistente en el medio ambiente. La oxidación y la fotodegradación probablemente no tengan un efecto significativo en su destino. Sin embargo, puede haber hidrólisis de los átomos de bromo del grupo propílico, especialmente en condiciones básicas. Puede producirse volatilización a partir del agua, pero no se dispone de datos efectivos. Aunque se han notificado casos de biodegradación del FTBP (semivida 19,7 h) en aguas residuales activadas, no se considera que ésta constituya un proceso importante en suelos y aguas naturales. En fangos esterilizados casi no se produce descomposición. Se encontró fosfato de bis(2,3-dibromopropilo) como principal producto de la descomposición. Como el FTBP es prácticamente insoluble en agua, la adsorción en partículas en sedimento puede ser un proceso importante. Un log Koc estimado (3,29) sugiere una fuerte adsorción en el suelo. Sobre la base de este valor de Koc y de la baja solubilidad medida en agua, sólo se prevé una lixiviación lenta del FTBP a las aguas subterráneas. El FTBP tenderá a acumularse en basureros y otros vertederos de desechos, lo que tal vez dé lugar a la acumulación biológica. Un estudio sobre bioacumulación en Pimephales promelas mostró un factor de bioconcentración de 2,7, que es bajo, mientras que el coeficiente de reparto n-octanol/agua (Log Pow) es de 3,02. Debido a su baja presión de vapor, se prevé que el FTBP sea principalmente objeto de sorción en las partículas en suspensión en el aire. La degradación oxidativa térmica del FTBP a 370 oC mostró que se forman bromuro de hidrógeno y compuestos C3-bromados, tales como bromopropenos, dibromopropenos, y dibromopropanos y tribromopropanos. 1.1.4 Niveles ambientales y exposición humana Los datos sobre los niveles ambientales y la exposición humana son limitados. Estudios realizados en el Japón en 1975 mostraron que 20 muestras de agua, suelo y peces no contenían FTBP. Se identificó la presencia de FTBP en partículas en suspensión en el aire en los alrededores de una planta industrial, pero no se cuantificaron. Los niños que llevaban ropa de dormir tratada con FTBP fueron el grupo de la población general particularmente expuesto a este retardador de ignición. La absorción estimada a través de la piel de los niños que llevaban ropa de dormir tratada con FTBP en los Estados Unidos de América se calculó en 9 µg/kg de peso corporal por día. La Comisión de Seguridad de los Productos de Consumo de los Estados Unidos de América calculó que, en un periodo de seis años, un niño que lleve ropa tratada con FTBP podría absorber en total 2-77 mg de FTBP/kg de peso corporal o más. 1.1.5 Cinética y metabolismo en animales de laboratorio y en seres humanos El FTBP se absorbe rápidamente a través del tracto gastro- intestinal y a una velocidad moderada a través de la piel en la rata y el conejo. En la rata, el FTBP o sus metabolitos se eliminan en cinco días. Aproximadamente el 50% se elimina por la orina, el 10% por las heces y el 10-20% se exhala en forma de CO2. Un día después de la administración oral de FTBP marcado a ratas, se encontró radiactividad en la sangre, el hígado, los riñones, los músculos, la grasa y la piel con valores comprendidos entre el 0,2% y el 6,6%. El periodo de semieliminación de la radiactividad de dichos órganos fue de aproximadamente 2-4 días. Después de ocho horas, solamente el fosfato de bis(2,3-dibromopropilo) seguía presente en concentraciones sustanciales en la mayor parte de los tejidos. Después de la administración oral de FTBP a ratas, se identificaron seis metabolitos en la orina y en la bilis. El principal metabolito en la orina, las heces, la bilis y los tejidos fue el fosfato de bis(2,3-dibromopropilo). También se identificó el metabolito 2,3-dibromopropanol en ratas y en niños que llevaban ropa tratada con FTBP. Los microsomas del hígado metabolizan el FTBP en presencia de NADPH y oxígeno. Los principales metabolitos son el fosfato de bis(2,3-dibromopropilo) y el 2,3-dibromopropanol. Se ha mostrado que el fosfato de bis(2,3-dibromopropilo) se forma por oxidación en la posición C3 y posiblemente también en la posición C2 del FTBP. Además del fosfato de bis(2,3-dibromopropilo), se han encontrado 2-bromoacroleína, ácido 2-bromoacrílico, y compuestos propil- hidroxilados y metabolitos conjugados con glutatión. Se identificó la presencia de S-(2,3-dihidroxipropil)glutatión en la bilis de ratas y se sugirió que el FTBP y/o el fosfato de bis(2,3- dibromopropilo) son conjugados directamente con el glutatión por la glutatión S-transferasa, formándose iones episulfonio como metabolitos. Se ha mostrado que el FTBP se activa para formar productos que se enlazan de forma covalente con las proteínas y el ADN in vivo e in vitro. Después de inyecciones intraperitoneales de FTBP tritiado, el ratón, el hámster y el cobayo machos, que son menos sensibles a la nefrotoxicidad inducida por el FTBP que la rata, mostraron niveles semejantes de enlace covalente con las proteínas en el hígado y los riñones. En la rata macho, muy susceptible a la nefrotoxicidad inducida por el FTBP, los átomos marcados se habían fijado a las proteínas de riñon en cantidad mucho mayor que las proteínas del hígado. Los microsomas del hígado de ratón, cobayo, hámster y humano metabolizaron todos ellos el FTBP formando productos intermedios genotóxicos. Sin embargo, la tasa de formación de metabolitos reactivos del FTBP por acción de los microsomas del hígado humano fue menor a la de los microsomas del hígado de roedores. El enlace del FTBP marcado y análogos en ratas a las que se había administrado una dosis nefrotóxica mostró que el número de enlaces covalentes a las proteínas era máximo en los riñones; les seguían el hígado y los testículos. Los resultados de estudios comparativos in vitro e in vivo sobre lesiones del ADN renal parecen indicar que el fosfato de bis(2,3-dibromopropilo) se forma en el hígado por oxidación mediada por el P450 en las posiciones C2 o C3 del FTBP. El fosfato de bis(2,3 dibromopropilo) se transporta a los riñones, donde se metaboliza formando productos intermedios reactivos que lesionan el ADN y se enlazan con las proteínas del riñón. La activación en el riñón no parece realizarse con intervención del P450 sino por medio del metabolismo depen-diente del glutatión. Estudios in vitro con FTBP marcado y productos análogos mostraron que, en la oxidación, al FTBP se incorpora un átomo de oxígeno del agua. Ello significa que la oxidación en la posición C2 del grupo propílico produce una alpha- bromocetona reactiva que puede alkilizar la proteína directamente o hidrolizarla produciendo fosfato de bis(2,3-dibromopropilo) y una bromo-alpha-hidroxicetona reactiva. 1.1.6 Efectos en mamíferos de laboratorio y en sistemas de prueba in vitro La toxicidad oral aguda y de corto plazo y la toxicidad cutánea aguda del FTBP son bajas. La DL50 oral para la rata es > 2 g/kg y la DL50 dérmica para el conejo es > 8 g/kg de peso corporal en ambos casos. Se observaron lesiones renales extensas (necrosis de las células de los tubos proximales) en ratas macho después de una sola inyección intraperitoneal de 100 mg de FTBP/kg de peso corporal. Estudios de cuatro semanas y de 90 días sobre toxicidad oral del FTBP (administrado por sonda o en la alimentación) a ratas mostraron un aumento relacionado con la dosis en la incidencia de nefritis crónica y su gravedad a niveles de dosis de 25 mg/kg de peso corporal o más. En conejos, aplicaciones cutáneas cotidianas de 2,2 g de FTBP/kg de peso corporal o más durante cuatro semanas ocasionaron cambios degenerativos en el hígado y los riñones. Todos los conejos murieron en cuatro semanas. No se registraron muertes en otro estudio con niveles de dosis de hasta 250 mg/kg de peso corporal. En una prueba de 90 días en conejos, aplicaciones cutáneas semanales de 2,27 g/kg de peso corporal dieron lugar a cambios renales, atrofia testicular y aspermatogénesis. No se observó irritación cutánea ni ocular en conejos a niveles de dosis de 1,1 g ó 0,22 g de FTBP y tampoco se observó sensibilización cutánea en cobayos. Se realizaron dos estudios sobre teratogenicidad en ratas. En un estudio, a niveles de dosis de hasta 125 mg/kg de peso corporal no se observó teratogenicidad. En otro estudio, a un nivel de dosis de 200 mg/kg de peso corporal se observó un aumento significativo en las variaciones esqueléticas de los fetos, y con 50 y 100 mg/kg de peso corporal se obtuvo un índice de viabilidad significativamente más bajo. Los autores llegaron a la conclusión de que el efecto observado se debía a toxicidad materna. Se encontraron lesiones extensas del ADN en diversos órganos de ratas a las que se había administrado FTBP. In vitro, se ha mostrado que el FTBP induce ruptura de las hebras de ADN en las células KB humanas. Indujo síntesis imprevista del ADN en hepatocitos de ratas, pero no en células epiteliales del prepucio en el hombre. El FTBP resultó mutagénico en varios estudios realizados en Salmonella typhimurium, especialmente en cepas sensibles a la sustitución de pares de bases, con y sin activación metabólica. Las valoraciones de mutación génica anterógrada efectuadas en células V79 de hámster de China, con y sin activación metabólica, dieron resultados negativos. Sin embargo, se obtuvo un efecto positivo en presencia de microsomas del hígado de ratas tratadas previamente con fenobarbital. Se obtuvo un efecto positivo débil con células de linfoma de ratón (locus L5178YTK). El FTBP aumentó el número de intercambios de cromátides hermanas en células V79 de hámster de China, pero no indujo aberraciones cromosómicas en células de hámster de China, células de médula ósea de ratón y células linfoides humanas de cultivo. Se encontraron intercambios de cromátides hermanas, pero no aberraciones cromosómicas, en fibroblastos humanos diploides (línea HE 2144) sin activación metabólica. Sin embargo, en una prueba in vitro sobre aberración cromosómica con la línea celular de hámster de China, el FTBP dio resultados positivos. Se obtuvo un resultado positivo con FTBP en una prueba de formación de micronúcleos en células de médula ósea de hámster de China. Otro estudio en ratones sobre formación de micronúcleos mostró un efecto positivo débil. Estudios con Drosophila melanogaster mostraron que el FTBP hacía aumentar el número de efectos recesivos letales ligados al sexo en células germinales masculinas y en machos adultos y se inducían traslocaciones recíprocas. El FTBP mostró una respuesta fuertemente positiva en la valoración de mosaicismo ocular w/w+. Se han realizado varios estudios para dilucidar los mecanismos de la mutagenicidad y/o la genotoxicidad inducidas por el FTBP. La mutagenicidad bacteriana ocasionada por el FTBP está mediada por el sistema de la monooxigenasa microsómica. El citocromo P450 activa el FTBP en una reacción que depende del NADPH y del oxígeno. Los microsomas preparados a partir del hígado de animales tratados con fenobarbital o con bifenilos policlorados acusaron un aumento de la mutagenicidad. Los fosfatos de mono(2,3-dibromopropilo) y bis(2,3-dibromopropilo) son menos mutagénicos que el FTBP. Estudios in vitro han mostrado que la oxidación en la posición C3 de la molécula de FTBP produce el potente mutágeno 2-bromoacroleína de acción directa, que también se enlaza con el ADN. Se han notificado diferencias entre especies en la bioactivación del FTBP con transformación en metabolitos mutagénicos para cepas TA 100 de Salmonella typhimurium. Los microsomas del hígado de ratones fueron más eficaces que los de cobayos, hámsters y ratas. Se realizaron tres estudios sobre transformación celular en los que se utilizaron células C3H/10T1/2. En un estudio se observó un efecto positivo, pero en los otros dos los resultados fueron negativos. Se probó el FTBP en ratones y ratas por administración oral y en ratones hembra por aplicación cutánea en estudios a largo plazo. En los ratones, el FTBP administrado por vía oral produjo tumores de preestómago y pulmón en los animales de ambos sexos, tumores hepáticos benignos y malignos en las hembras y tumores renales benignos y malignos en los machos. En ratas, el FTBP produjo tumores renales benignos y malignos en los machos y tumores renales benignos en las hembras. Después de la aplicación cutánea a ratones hembra, el FTBP produjo tumores en la piel, el pulmón, el preestómago y la cavidad bucal. De esos estudios puede concluirse que el FTBP tiene un potencial carcinogénico en ratones y ratas. El fosfato de bis(2,3-dibromilpropilo), un metabolito del FTBP administrado por vía oral a ratas produjo tumores del sistema digestivo en ambos sexos. Entre los tumores encontrados había papilomas y adenocarcinomas de lengua, esófago y preestómago, adenocarcinomas de intestino, y adenomas y carcinomas hepato-celulares. Otro metabolito del FTBP, el 2,3-dibromo-1-propanol, se ensayó en ratas y ratones por aplicación cutánea. En ratas macho se observó mayor incidencia de neoplasias de piel, nariz, mucosa bucal, esófago, preestómago, intestino delgado y grueso, glándula de Zymbal, hígado, riñón, túnica vaginal y bazo. En ratas hembra se registró mayor incidencia de neoplasias de piel, nariz, mucosa bucal, esófago, preestómago, intestino delgado y grueso, glándula de Zymbal, hígado, riñón, glándula clitorídea y mama. En ratones macho se observó un aumento de la incidencia de neoplasias de piel, preestómago, hígado y pulmón, y en ratones hembra, un aumento de la incidencia de neoplasias de piel y preestómago. 1.1.7 Efectos en el ser humano Se dispone de datos limitados sobre los efectos del FTBP en el ser humano. Se ha ensayado el FTBP para determinar su potencial de sensibilización cutánea en unos pocos estudios en seres humanos. Los resultados de éstos indican que el FTBP tiene un bajo potencial de sensibilización y no ha habido irritación cutánea. Sin embargo, las personas que mostraron una respuesta positiva de sensibilización al FTBP puro también reaccionaron cuando se expusieron a tejidos tratados con FTBP. 1.1.8 Efectos en otros organismos en laboratorio y en el medio natural Hay muy pocos datos sobre los efectos del FTBP en otros organismos. Seis carpas doradas (Carassius auratus) expuestas a 1 mg de FTBP/litro murieron todas a los cinco días. La CE50 de inhibición del crecimiento en semillas de avena fue de 1000 mg/kg de suelo. Esta concentración causó una inhibición del 100% del crecimiento en semillas de nabo ( Brassica rapa sp.). 1.2 Conclusiones El FTBP se ha utilizado como retardador de ignición en tejidos, en particular en ropa de dormir para niños, pero hay información insuficiente sobre su utilización para otros fines. La exposición de la población general se ha efectuado principalmente por contacto con tejidos tratados con FTBP. Hay poca información sobre la exposición de los trabajadores y los riesgos que para éstos entrañan la producción comercial de FTBP y su utilización en diversos productos. Debido a la escasez de datos, no pueden sacarse conclusiones firmes respecto de los niveles de exposición y los riesgos del FTBP para organismos en el medio ambiente distintos del ser humano. Estudios en animales han mostrado que el FTBP puede absorberse a través del tracto gastrointestinal y, en menor medida, de la piel. El FTBP también puede absorberse a través de la piel en el ser humano. En la rata, el FTBP parece metabolizarse extensamente en el hígado convirtiéndose en fosfato de bis(2,3-dibromopropilo), que es el principal metabolito detectado en la orina, y, en menor medida, en 2,3-dibromopropanol. Además, se han encontrado pequeñas cantidades de otros metabolitos bromados del FTBP. También se ha detectado 2,3-dibromopropanol en seres humanos que llevaron tejidos tratados con FTBP. La principal vía de eliminación es la orina y una cantidad muy pequeña se excreta en la forma del compuesto originario. La principal vía metabólica parece ser la del metabolismo de las S-transferasas del citocromo P450 y del glutatión. Sobre la base de los datos disponibles se puede concluir que el FTBP tiene una baja toxicidad aguda para animales de experimentación. Estudios sobre la administración repetida de dosis relativamente elevadas de FTBP han revelado lesiones renales y hepáticas en ratas y también toxicidad testicular en conejos. El FTBP ha producido un claro efecto genotóxico en varios sistemas de prueba, tanto in vitro como in vivo. Se observaron efectos carcinogénicos en ratas y ratones. Se ha observado que los metabolitos fosfato de bis(2,3-dibromopropilo) y 2,3-dibromopropanol también tienen efectos carcinogénicos en animales de experimentación. No se registraron efectos irritativos en animales y se observó un bajo potencial de sensibilización en seres humanos. En 1977, la Comisión de Seguridad de los Productos de Consumo de los Estados Unidos de América prohibió las prendas de vestir para niños tratadas con fosfato de tris(2,3-dibromo-propilo), debido a la preocupación de que esta sustancia química pudiera ser carcinogénica para el ser humano y a la posibilidad de una exposición humana significativa por contacto con los tejidos tratados. Desde entonces, la utilización de esta sustancia como retardador de ignición en productos de consumo se ha restringido rigurosamente en varios otros países y se ha prohibido en los productos textiles. 1.3 Recomendaciones Debido a sus efectos tóxicos, el FTBP no se debería utilizar ya comercialmente. Si se identifican aplicaciones para las cuales no hay alternativas menos peligrosas que el FTBP, deberían realizarse estudios para demostrar la ausencia de exposición humana y ambiental y de riesgos para el ser humano y para el medio ambiente. 2. El fosfato de bis(2,3-dibromopropilo) La base de datos sobre el fosfato de bis(2,3-dibromopropilo) y sus sales es insuficiente para hacer una evaluación y para respaldar su utilización comercial. Sobre la base de los datos disponibles, hay algunos indicios de que esta sustancia puede ser mutagénica y carcinogénica. Esta sustancia no puede evaluarse a menos que llegue a disponerse de datos adicionales sobre sus propiedades físicas y químicas; su producción y utilización; su transporte, distribución y transformación en el medio ambiente; los niveles ambientales y la exposición humana; su cinética y metabolismo en animales y en seres humanos; sus efectos en mamíferos de laboratorio, en seres humanos y en sistemas de prueba in vitro; y sus efectos en otros organismos en el laboratorio y en el medio natural. También se necesitan más datos sobre mutagenicidad en relación con dos variables de evaluación por lo menos.