WORLD HEALTH ORGANIZATION INTERNATIONAL AGENCY FOR RESEARCH ON CANCER

IARC MONOGRAPHS ON THE EVALUATION OF CARCINOGENIC RISKS TO HUMANS

VOLUME 77 SOME INDUSTRIAL CHEMICALS

2000 I A R C L Y O N FRANCE

WORLD HEALTH ORGANIZATION INTERNATIONAL AGENCY FOR RESEARCH ON CANCER

IARC MONOGRAPHS
ON THE

EVALUATION OF CARCINOGENIC RISKS TO HUMANS

Some Industrial Chemicals
VOLUME 77

This publication represents the views and expert opinions of an IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, which met in Lyon, 15–22 February 2000 2000

IARC MONOGRAPHS
In 1969, the International Agency for Research on Cancer (IARC) initiated a programme on the evaluation of the carcinogenic risk of chemicals to humans involving the production of critically evaluated monographs on individual chemicals. The programme was subsequently expanded to include evaluations of carcinogenic risks associated with exposures to complex mixtures, life-style factors and biological and physical agents, as well as those in specific occupations. The objective of the programme is to elaborate and publish in the form of monographs critical reviews of data on carcinogenicity for agents to which humans are known to be exposed and on specific exposure situations; to evaluate these data in terms of human risk with the help of international working groups of experts in chemical carcinogenesis and related fields; and to indicate where additional research efforts are needed. The lists of IARC evaluations are regularly updated and are available on Internet: http://www.iarc.fr/, under Publications. This project was supported by Cooperative Agreement 5 UO1 CA33193 awarded by the United States National Cancer Institute, Department of Health and Human Services. Additional support has been provided since 1986 by the European Commission, since 1993 by the United States National Institute of Environmental Health Sciences and since 1995 by the United States Environmental Protection Agency through Cooperative Agreement Assistance CR 824264.
©International Agency for Research on Cancer, 2000

Distributed by IARCPress (Fax: +33 4 72 73 83 02; E-mail: press@iarc.fr) and by the World Health Organization Distribution and Sales, CH-1211 Geneva 27 (Fax: +41 22 791 4857; E-mail: publications@)who.int) 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. Application for rights of reproduction or translation, in part or in toto, should be made to the International Agency for Research on Cancer. IARC Library Cataloguing in Publication Data
Some industrial chemicals / IARC Working Group on the Evaluation of Carcinogenic Risks to Humans (2000 : Lyon, France) (IARC monographs on the evaluation of carcinogenic risks to humans ; 77) 1. Carcinogens – congresses 2. Occupational Exposure – congresses I. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans II. Series ISBN 92 832 1277 0 ISSN 1017-1606 (NLM Classification: W1)

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CONTENTS
NOTE TO THE READER............................................................................................1 LIST OF PARTICIPANTS............................................................................................3 PREAMBLE ................................................................................................................9 Background..............................................................................................................9 Objective and Scope ................................................................................................9 Selection of Topics for Monographs ....................................................................10 Data for Monographs ............................................................................................11 The Working Group ..............................................................................................11 Working Procedures ..............................................................................................11 Exposure Data........................................................................................................12 Studies of Cancer in Humans ................................................................................14 Studies of Cancer in Experimental Animals..........................................................17 Other Data Relevant to an Evaluation of Carcinogenicity and its Mechanisms ..........................................................................................20 Summary of Data Reported ..................................................................................22 Evaluation ..............................................................................................................23 References..............................................................................................................27 GENERAL REMARKS..............................................................................................33 THE MONOGRAPHS................................................................................................39 Di(2-ethylhexyl) phthalate ....................................................................................41 Di(2-ethylhexyl) adipate ......................................................................................149 Cinnamyl anthranilate..........................................................................................177 Coumarin ............................................................................................................193 Ethylbenzene........................................................................................................227 ortho-Toluidine ....................................................................................................267 4-Chloro-ortho-toluidine ....................................................................................323 5-Chloro-ortho-toluidine ....................................................................................341 Diethanolamine....................................................................................................349 Triethanolamine ..................................................................................................381 N-Nitrosodiethanolamine ....................................................................................403 2,3-Dibromopropan-1-ol......................................................................................439 2,2-Bis(bromomethyl)propane-1,3-diol ..............................................................455 –iii–

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Glycidol ..............................................................................................................469 Nitromethane ......................................................................................................487 Pyridine................................................................................................................503 SUMMARY OF FINAL EVALUATIONS ..............................................................529 CUMULATIVE INDEX TO THE MONOGRAPHS SERIES..................................531

NOTE TO THE READER

The term ‘carcinogenic risk’ in the IARC Monographs series is taken to mean the probability that exposure to an agent will lead to cancer in humans. Inclusion of an agent in the Monographs does not imply that it is a carcinogen, only that the published data have been examined. Equally, the fact that an agent has not yet been evaluated in a monograph does not mean that it is not carcinogenic. The evaluations of carcinogenic risk are made by international working groups of independent scientists and are qualitative in nature. No recommendation is given for regulation or legislation. Anyone who is aware of published data that may alter the evaluation of the carcinogenic risk of an agent to humans is encouraged to make this information available to the Unit of Carcinogen Identification and Evaluation, International Agency for Research on Cancer, 150 cours Albert Thomas, 69372 Lyon Cedex 08, France, in order that the agent may be considered for re-evaluation by a future Working Group. Although every effort is made to prepare the monographs as accurately as possible, mistakes may occur. Readers are requested to communicate any errors to the Unit of Carcinogen Identification and Evaluation, so that corrections can be reported in future volumes.

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IARC WORKING GROUP ON THE EVALUATION OF CARCINOGENIC RISKS TO HUMANS: SOME INDUSTRIAL CHEMICALS Lyon, 15–22 February 2000 LIST OF PARTICIPANTS

Members S.J. Borghoff, CIIT, PO Box 12137, Research Triangle Park, NC 27709, United States J. Caldwell, Division of Biomedical Sciences, Imperial College School of Medicine, Sir Alexander Fleming Building, South Kensington, London SW7 2AZ, United Kingdom R.C. Cattley, Amgen, MS-15-2-B, One Amgen Center Drive, Thousand Oaks, CA 91320, United States S. Cordier, Unit of Epidemiological and Statistical Research on the Environment and Health, INSERM–U 170, 16 avenue Paul Vaillant Couturier, 94807 Villejuif Cedex, France N. Danford, Microptic Cytogenetic Services, 2 Langland Close, Mumbles, Swansea SA3 4LY, United Kingdom P.A. Demers, School of Occupational and Environmental Hygiene, University of British Columbia, 2206 East Mall, 3rd floor, Vancouver BC V6T 1Z3, Canada T.A. Dragani, Istituto Nazionale per lo Studio e la Cura dei Tumori, via G. Venezian 1, 20133 Milan, Italy J.K. Dunnick, National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709, United States E. Dybing, National Institute of Public Health, Department of Environmental Medicine, Postboks 4404 Torshov, 0403 Oslo, Norway (Chairman) E. Elovaara, Finnish Institute of Occupational Health, Topeliuksenkatu 41 a A, 00250 Helsinki, Finland L.R. Ferguson, Auckland Cancer Society, Research Centre, Private Bag 92019, Auckland 1000, New Zealand L. Fishbein, 4320 Ashford Lane, Fairfax, VA 22032, United States M. Gérin, University of Montréal, Occupational and Environmental Health, Faculty of Medicine, CP 6128–Centre-Ville, Montreal, Quebec H3C 3J7, Canada (Vice-Chairman) G.C. Hard, American Health Foundation, 1 Dana Road, Valhalla, NY 10595, United States –3–

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IARC MONOGRAPHS VOLUME 77

U. Hass, Institute of Food Safety and Toxicology, Mørkhøj Bygade 19, 2860 Søborg, Denmark K.S. Hougaard, Danish National Institute of Occupational Health, Department of Chemical Working Environment, Lersø Parkalle 105, 2100 Copenhagen Ø, Denmark T. Kauppinen, Finnish Institute of Occupational Health, Topeliuksenkatu 41 a A, 00250 Helsinki, Finland H. Kromhout, Environmental and Occupational Health Group, Wageningen University, PO Box 238, 6700 AAE Wageningen, The Netherlands L. Levy, MRC Institute for Environment and Health, University of Leicester, 94 Regent Road, Leicester LE1 7DD, United Kingdom M.E. McManus, Faculty of Biological and Chemical Sciences, University of Queensland, Brisbane Qld 4072, Australia F.E. Mirer, UAW Health and Safety Department, 8000 East Jefferson Avenue, Detroit, MI 48214, United States G.J. Mulder, Sylvius Laboratories, Wassenaarseweg 72, PO Box 9503, 2300 RA Leiden, The Netherlands S. Olin, International Life Sciences Institute, 1126 Sixteenth Street NW, Washington DC 20036, United States E. Parry, School of Biological Sciences, University of Wales, Singleton Park, Swansea SA2 8PP, United Kingdom D.H. Phillips, Section of Molecular Carcinogenesis, Haddow Laboratories, Institute of Cancer Research, 15 Cotswold Road, Sutton Surrey SM2 5NG, United Kingdom A. Pintér, Fodor József National Public Health Centre, National Institute of Environmental Health, Gyáli ut 2-6, 1097 Budapest, Hungary D. Schrenk, Universität Kaiserslautern, Lebensmittelchemie und Umwelttoxikologie, Erwin-Schrödinger-Strasse, Postfach 3049, 67653 Kaiserslautern, Germany P. Stewart, Division of Cancer Epidemiology and Genetics, National Cancer Institute, EPS 8102, 6120 Executive Blvd, Mail Stop 7240, Bethesda, MD 20892-7240, United States E. Ward, Industrywide Studies Branch, Division of Surveillance, Hazard Evaluations and Field Studies, National Institute for Occupational Safety and Health, R.A. Taft Laboratories, 4676 Columbia Parkway, Cincinnati, OH 45226, United States Representatives/Observers Representative of the National Cancer Institute D.G. Longfellow, Chemical and Physical Carcinogenesis Branch, Division of Cancer Biology, National Cancer Institute, 6006 Executive Blvd, Suite 220, MSC 7055, Rockville, MD 20892-7055, United States American Industrial Health Council R.M. David, Health and Environment Laboratories, Eastman Kodak Company, Rochester, NY 14652-6272, United States

PARTICIPANTS

5

L. Lehman-McKeeman, Human and Environmental Safety Division, Miami Valley Laboratories, Procter and Gamble Co., PO Box 538707, Cincinnati, OH 45253, United States European Centre for Ecotoxicology and Toxicology of Chemicals G. Gans, BASF Aktiengesellschaft, Z 470, D-67056 Ludwigshafen, Germany Technical Resources International Incorporated T. Junghans, Technical Resources Inc., 6500 Rock Spring Drive, Suite 650, Bethesda, MD 20817, United States IARC Secretariat R.A. Baan, Unit of Carcinogen Identification and Evaluation I. Burstyn, Environmental Cancer Epidemiology J. Cheney (Editor) M. Friesen, Unit of Nutrition and Cancer Y. Grosse, Unit of Carcinogen Identification and Evaluation J. Korte, Environmental Cancer Epidemiology V. Krutovskikh, Unit of Gene–Environment Interactions C. Malaveille, Unit of Endogenous Cancer Risk Factors A. t’ Mannetje, Environmental Cancer Epidemiology D. McGregor, Unit of Carcinogen Identification and Evaluation (Responsible Officer)1 C. Partensky, Unit of Carcinogen Identification and Evaluation J. Rice, Unit of Carcinogen Identification and Evaluation (Head of Programme) J. Wilbourn, Unit of Carcinogen Identification and Evaluation Technical assistance S. Egraz M. Lézère A. Meneghel D. Mietton J. Mitchell E. Perez S. Reynaud

1

Present address: 102 rue Duguesclin, 69006 Lyon, France

PREAMBLE .

.

and to publish in the form of monographs. quantitative evaluations of epidemiological data may be made in the Monographs. The term ‘carcinogen’ is used in these monographs to denote an exposure that is capable of increasing the incidence of malignant neoplasms. in some occupations and as a result of human habits) and of exposures to other agents. 1983. 1991a. the induction of benign neoplasms may in some circumstances (see p. The criteria established in 1971 to evaluate carcinogenic risk to humans were adopted by the working groups whose deliberations resulted in the first 16 volumes of the IARC Monographs series. 1988. such as radiation and viruses.. –9– 2. the title of the series was modified from IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans to IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. The Monographs programme has since been expanded to include consideration of exposures to complex mixtures of chemicals (which occur. with the help of international working groups of experts. Vainio et al. The terms ‘neoplasm’ and ‘tumour’ are used interchangeably. With Supplement 6 (IARC. Quantitative extrapolation from experimental data to the human situation is not undertaken. 1977. in order to reflect the widened scope of the programme. Those criteria were subsequently updated by further adhoc working groups (IARC. for example. 1987a). OBJECTIVE AND SCOPE The objective of the programme is to prepare. but without extrapolation beyond the range of the data available. critical reviews and evaluations of evidence on the carcinogenicity of a wide range of human exposures. the International Agency for Research on Cancer (IARC) initiated a programme to evaluate the carcinogenic risk of chemicals to humans and to produce monographs on individual chemicals. The second step is quantitative risk estimation. Detailed. 1978. The Monographs may also indicate where additional research efforts are needed. 1. 1982. which involves examination of all relevant information in order to assess the strength of the available evidence that certain exposures could alter the incidence of cancer in humans. . 1992). The Monographs represent the first step in carcinogenic risk assessment.IARC MONOGRAPHS PROGRAMME ON THE EVALUATION OF CARCINOGENIC RISKS TO HUMANS PREAMBLE BACKGROUND In 1969. 1979. 19) contribute to the judgement that the exposure is carcinogenic. 1987b.

for distribution to governments.. 1993 and 1998 gave recommendations as to which agents should be evaluated in the IARC Monographs series (IARC. . see also pp. 25–27). The scientific literature is surveyed for published data relevant to an assessment of carcinogenicity. Chemical analogues and compounds with biological or physical characteristics similar to those of suspected carcinogens may also be considered. 1976–1996) often indicate exposures that may be scheduled for future meetings. even in the absence of data on a possible carcinogenic effect in humans or experimental animals. and (b) there is some evidence or suspicion of carcinogenicity. 1998a. 3. which are the responsibility of individual governments and/or other international organizations. however. Ad-hoc working groups convened by IARC in 1984. Vainio et al. 1993. 1991. 1989. The term ‘agent’ is used to include individual chemical compounds.10 IARC MONOGRAPHS VOLUME 77 Some epidemiological and experimental studies indicate that different agents may act at different stages in the carcinogenic process. Other components of regulatory decisions vary from one situation to another and from country to country. from their inception. The evaluations of IARC working groups are scientific. 1991a. The aim of the Monographs has been. Therefore. The Monographs are also available from IARCPress in Lyon and via the Distribution and Sales Service of the World Health Organization in Geneva. Information on mechanisms may. and several mechanisms may be involved. Exposures to mixtures of agents may occur in occupational exposures and as a result of personal and cultural habits (like smoking and dietary practices). to evaluate evidence of carcinogenicity at any stage in the carcinogenesis process. The Monographs may assist national and international authorities in making risk assessments and in formulating decisions concerning any necessary preventive measures. physical agents (such as radiation) and biological factors (such as viruses). A survey of users in 1988 indicated that the Monographs are consulted by various agencies in 57 countries. independently of the underlying mechanisms. 1989. qualitative judgements about the evidence for or against carcinogenicity provided by the available data. regulatory bodies and interested scientists. be used in making the overall evaluation (IARC. 1984.b). These evaluations represent only one part of the body of information on which regulatory measures may be based. The IARC Monographs are recognized as an authoritative source of information on the carcinogenicity of a wide range of human exposures. 1991b. groups of related chemical compounds. no recommendation is given with regard to regulation or legislation. responding to different socioeconomic and national priorities. The IARC information bulletins on agents being tested for carcinogenicity (IARC. SELECTION OF TOPICS FOR MONOGRAPHS Topics are selected on the basis of two main criteria: (a) there is evidence of human exposure. About 3000 copies of each volume are printed. 1992. 1973–1996) and directories of on-going research in cancer epidemiology (IARC.

Only those data considered by the Working Group to be relevant to making the evaluation are included. only reports that have been published or accepted for publication in the openly available scientific literature are reviewed by the working groups. government or industry. The tasks of the group are: (i) to ascertain that all appropriate data have been collected. 4. on production and use and on occurrence. on analysis. In the sections on chemical and physical properties. with their addresses. government agency reports that have undergone peer review and are widely available are considered. (iii) to prepare accurate summaries of the data to enable the reader to follow the reasoning of the Working Group. WORKING PROCEDURES Approximately one year in advance of a meeting of a working group. including data storage and retrieval systems such as MEDLINE and TOXLINE. Working Group participants who contributed to the considerations and evaluations within a particular volume are listed. Each participant who is a member of a working group serves as an individual scientist and not as a representative of any organization. 6. on analysis. if their inclusion is considered pertinent to making a final evaluation (see pp. at the beginning of each publication. For chemicals and some complex mixtures. and (vi) to make an overall evaluation of the carcinogenicity of the exposure to humans. (v) to evaluate data relevant to the understanding of mechanism of action. Exceptions may be made on an ad-hoc basis to include unpublished reports that are in their final form and publicly available. DATA FOR MONOGRAPHS The Monographs do not necessarily cite all the literature concerning the subject of an evaluation. nominees of national and international agencies and industrial associations may be invited as observers.PREAMBLE 11 As significant new data on subjects on which monographs have already been prepared become available. With regard to biological and epidemiological data. and revised monographs are published. Subsequently. (iv) to evaluate the results of epidemiological and experimental studies on cancer. the major collection of data and the preparation of first drafts of the sections on chemical and physical properties. 5. . (ii) to select the data relevant for the evaluation on the basis of scientific merit. In certain instances. THE WORKING GROUP Reviews and evaluations are formulated by a working group of experts. unpublished sources of information may be used. In addition. 25–27). re-evaluations are made at subsequent meetings. the topics of the monographs are announced and participants are selected by IARC staff in consultation with other experts. relevant biological and epidemiological data are collected by the Carcinogen Identification and Evaluation Unit of IARC from recognized sources of information on carcinogenesis.

description of an industry or habit. epidemiology of infection and clinical disease other than cancer. edited and prepared for publication. the material obtained is sent to meeting participants. such as: historical perspectives. on production and use and on occurrence. the sources of exposure. the latest Chemical Abstracts Primary Name and the IUPAC Systematic Name are recorded. The first drafts are compiled by IARC staff and sent before the meeting to all participants of the Working Group for review. a comment is given in square brackets. In monographs on. for example. the people most likely to be exposed and the factors that contribute to the exposure are included at the beginning of each monograph. by direct contact with industries. other sections may be included. EXPOSURE DATA Sections that indicate the extent of past and present human exposure. Monographs on biological agents have sections on structure and biology. Information on uses may be obtained from published sources but is often complemented by direct contact with manufacturers. to prepare sections for the first drafts of monographs. The Working Group may conduct additional analyses of the published data and use them in their assessment of the evidence. groups of chemicals or complex mixtures include sections on chemical and physical data. the master copy of each monograph is verified by consulting the original literature. When an important aspect of a study. The available studies are summarized by the Working Group. physical agents. For chemical exposures. occupational exposures and cultural habits. should be brought to the attention of the reader.12 IARC MONOGRAPHS VOLUME 77 on production and use and on occurrence are carried out under a separate contract funded by the United States National Cancer Institute. Separate production data on some agents may not be available because their publication could disclose confidential information. 7. Efforts are made to supplement this information with data from other national and international sources. on analysis. After the meeting. the results of such supplementary analyses are given in square brackets. Most monographs on individual chemicals. units are converted when necessary for easier comparison. In general. methods of detection. in some cases. For biological agents. but the list is not necessarily comprehensive. The aim is to publish monographs within six months of the Working Group meeting. The Working Group meets in Lyon for seven to eight days to discuss and finalize the texts of the monographs and to formulate the evaluations. Representatives from industrial associations may assist in the preparation of sections on production and use. directly impinging on its interpretation. or is used by IARC staff. . Six months before the meeting. numerical findings are indicated as they appear in the original report. with particular regard to the qualitative aspects discussed below. the Chemical Abstracts Services Registry Number. chemistry of the complex mixture or taxonomy. other synonyms are given. Information on production and trade is obtained from governmental and trade publications and.

mention of their therapeutic uses does not necessarily represent current practice. soil. When available. for agents which do not occur naturally. mode of replication. methods of synthesis used in past and present commercial production and different methods of production which may give rise to different impurities are described. however. 1978–93). Methods for monitoring human exposure are also given. It should not. persistence and latency and host response are given. industries. Some of the trade names given may be those of mixtures in which the agent being evaluated is only one of the ingredients. a historical description is also given. nor does it imply judgement as to their therapeutic efficacy. target cells. occupations or processes. data relevant to identification. Some identified uses may not be current or major applications. which usually include Europe. The dates of first synthesis and of first commercial production of a chemical or mixture are provided. relevant specifications and available information on composition and impurities. Japan and the United States of America. The dates of first reported occurrence of an exposure are also provided. be inferred that those areas or nations are necessarily the sole or major sources or users of the agent. Information on the occurrence of an agent or mixture in the environment is obtained from data derived from the monitoring and surveillance of levels in occupational environments. that describe validated methods for analysing a wide variety of chemicals and mixtures. . In addition. when applicable. with emphasis on those widely used for regulatory purposes.PREAMBLE 13 taxonomy and structure are described. air. and the degree of variability is given. industries and occupations. foods and animal and human tissues. international trade and uses are obtained for representative regions. physical properties and levels of occupational exposure with time and place. For biological agents. and the coverage is not necessarily comprehensive. methods of detection and exposure assessment are described. Information on chemical and physical properties and. the epidemiology of infection is described. occurrence and biological activity are included. this information may allow a reasonable estimate to be made of the date before which no human exposure to the agent could have occurred. data on the generation. in particular. including their sensitivity. For biological agents. life cycle. information is given about all agents present. In the case of drugs. when available. Data on production. The IARC published a series of volumes. For processes. No critical evaluation or recommendation of any of the methods is meant or implied. A description of technical products of chemicals includes trade names. specificity and reproducibility. Environmental Carcinogens: Methods of Analysis and Exposure Measurement (IARC. water. For biological agents. persistence and bioaccumulation of the agent are also included. The purpose of the section on analysis or detection is to give the reader an overview of current methods. In the case of mixtures. noting variations in chemical composition.

14 IARC MONOGRAPHS VOLUME 77 Statements concerning regulations and guidelines (e. occupational exposure limits) are included for some countries as indications of potential exposures. They may. case–control studies and correlation (or ecological) studies. including vaccines and therapy. particularly when the information is considered to be a useful supplement to that in other reports or when they provide the only data available. Because individual exposure is not documented. Epidemiological studies of benign neoplasms. however. except in rare instances. the units of investigation are usually whole populations (e. that the concurrence of two events—that is. Their 8. but they may not reflect the most recent situation. to form the sole basis for inferring a causal relationship. In correlation studies. are described. legislation and control. Case series and case reports of cancer in humans may also be reviewed. presumed preneoplastic lesions and other end-points thought to be relevant to cancer are also reviewed by working groups. Rarely. strengthen inferences drawn from studies of cancer itself. a particular exposure and occurrence of a cancer—has happened rather more frequently than would be expected by chance. Those that are judged to be inadequate or irrelevant to the evaluation are generally omitted. and cancer frequency is related to a summary measure of the exposure of the population to the agent. in some instances. a causal relationship is less easy to infer from correlation studies than from cohort and case–control studies. Case reports usually lack complete ascertainment of cases in any population. relevant case reports or correlation studies may add materially to the judgement that a causal relationship is present. When taken together with case–control and cohort studies. They may be mentioned briefly. Case reports generally arise from a suspicion. since such limits are continuously reviewed and modified.g. For biological agents. STUDIES OF CANCER IN HUMANS (a) Types of studies considered Three types of epidemiological studies of cancer contribute to the assessment of carcinogenicity in humans—cohort studies. mixture or exposure circumstance under study. (b) Quality of studies considered The Monographs are not intended to summarize all published studies. based on clinical experience. however. Cohort and case–control studies relate the exposures under study to the occurrence of cancer in individuals and provide an estimate of relative risk (ratio of incidence or mortality in those exposed to incidence or mortality in those not exposed) as the main measure of association.g. . results from randomized trials may be available. The uncertainties surrounding interpretation of case reports and correlation studies make them inadequate.. The absence of information on regulatory status for a country should not be taken to imply that that country does not have regulations with regard to the exposure. definition or enumeration of the population at risk and estimation of the expected number of cases in the absence of exposure. pesticide registrations. maximal levels permitted in foods. in particular geographical areas or at particular times).

to reveal the possibility of reporting bias. In a cohort study. the statistical methods used to obtain estimates of relative risk. data on all cancer sites and all causes of death should have been given. the authors should have taken account in the study design and analysis of other variables that can influence the risk of disease and may have been related to the exposure of interest. 1987). Lack of clarity of any of these aspects in the reporting of a study can decrease its credibility and the weight given to it in the final evaluation of the exposure. or in the analysis. By ‘confounding’ is meant a situation in which the relationship with disease is made to appear stronger or weaker than it truly is as a result of an association between the apparent causal factor and another factor that is associated with either an increase or decrease in the incidence of the disease.PREAMBLE 15 inclusion does not imply acceptance of the adequacy of the study design or of the analysis and interpretation of the results. In a case–control study. the authors should have reported the basic data on which the conclusions are founded. The methods used should preferably have been the generally accepted techniques that have been refined since the mid-1970s. even if sophisticated statistical analyses were employed. It is necessary to take into account the possible roles of bias. . disease (or diseases) and exposure should have been well defined by the authors. Finally. Potential confounding by such variables should have been dealt with either in the design of the study. At the very least. absolute rates of cancer. In cohort studies. confounding and chance in the interpretation of epidemiological studies. mixture or exposure circumstance. In evaluating the extent to which these factors have been minimized in an individual study. and limitations are clearly outlined in square brackets at the end of the study description. 1980) and for cohort studies (Breslow & Day. by statistical adjustment. Further tabulations by time since exposure began and other temporal factors are also important. such as by matching. cohort and correlation studies. the effects of investigated factors other than the exposure of interest should have been reported. Firstly. By ‘bias’ is meant the operation of factors in study design or execution that lead erroneously to a stronger or weaker association than in fact exists between disease and an agent. and exposure should have been assessed in a way that was not related to disease status. Thirdly. they should have given the numbers of exposed and unexposed cases and controls in a case–control study and the numbers of cases observed and expected in a cohort study. confidence intervals and significance tests. These methods have been reviewed for case–control studies (Breslow & Day. working groups consider a number of aspects of design and analysis as described in the report of the study. Secondly. the study population. Internal comparisons of disease frequency among individuals at different levels of exposure should also have been made in the study. Cases of disease in the study population should have been identified in a way that was independent of the exposure of interest. comparisons with local rates of disease may be more appropriate than those with national rates. Most of these considerations apply equally to case–control. and to adjust for confounding should have been clearly stated by the authors.

If there are inconsistent results among investigations. the specificity of an association (an increased occurrence of cancer at one anatomical site or of one morphological type) adds plausibility to a causal relationship. cumulative exposure and time since exposure ceased. such as proto-oncogene mutation. although at best they allow only indirect inferences about the mechanism of action. The analysis of temporal relationships can be useful in formulating models of carcinogenesis. these data are not combined with those from later studies in any subsequent reassessment of the strength of the evidence. In particular. are reviewed and summarized when available. 1991a. Demonstration of a decline in risk after cessation of or reduction in exposure in individuals or in whole populations also supports a causal interpretation of the findings. when these are incorporated into epidemiological studies focused on cancer incidence or mortality. Associations that are replicated in several studies of the same design or using different epidemiological approaches or under different circumstances of exposure are more likely to represent a causal relationship than isolated observations from single studies.16 IARC MONOGRAPHS VOLUME 77 (c) Inferences about mechanism of action Detailed analyses of both relative and absolute risks in relation to temporal variables. A strong association (a large relative risk) is more likely to indicate causality than a weak association. this is considered to be a strong indication of causality. a judgement is made concerning the strength of evidence that the agent. Although rarely available. although absence of a graded response is not necessarily evidence against a causal relationship. mixture or exposure circumstance in question is carcinogenic for humans. and results of studies judged to be of high quality are given more weight than those of studies judged to be methodologically less sound.. If the risk of the disease in question increases with the amount of exposure. results from randomized trials showing different rates among exposed and unexposed individuals provide particularly strong evidence for causality. Such measurements may allow inferences to be made about putative mechanisms of action (IARC. duration of exposure. particularly when excess cancer occurrence is limited to one morphological type within the same organ. Although a carcinogen may act upon more than one target. When suspicion of carcinogenicity arises largely from a single study. such as age at first exposure. Vainio et al. time since first exposure. as well as markers of early steps in the carcinogenic process. although it is recognized that relative risks of small magnitude do not imply lack of causality and may be important if the disease is common. 1992). . such analyses may suggest whether a carcinogen acts early or late in the process of carcinogenesis. Special attention is given to measurements of biological markers of carcinogen exposure or action. such as DNA or protein adducts. the Working Group considers several criteria for causality. possible reasons are sought (such as differences in amount of exposure). In making its judgement. (d) Criteria for causality After the individual epidemiological studies of cancer have been summarized and the quality assessed.

it is biologically plausible and prudent to regard agents and mixtures for which there is sufficient evidence (see p. no individual study nor the pooled results of all the studies should show any consistent tendency for the relative risk of cancer to increase with increasing level of exposure. Such a judgement requires first of all that the studies giving rise to it meet. combined oral contraceptives. Animal strain. carcinogenicity in experimental animals was established or highly suspected before epidemiological studies confirmed their carcinogenicity in humans (Vainio et al. myleran. latent periods substantially shorter than 30 years cannot provide evidence for lack of carcinogenicity. It is important to note that evidence of lack of carcinogenicity obtained in this way from several epidemiological studies can apply only to the type(s) of cancer studied and to dose levels and intervals between first exposure and observation of disease that are the same as or less than those observed in all the studies. nevertheless.. 8methoxypsoralen plus ultraviolet A radiation. bischloromethyl ether and chloromethyl methyl ether (technical grade). when considered together. chlorambucil. age at start of treatment and survival are reported. to a sufficient degree. Moreover. due to sufficient population size. 4-aminobiphenyl. in the aggregate. numbers per group. 27) should also be taken into consideration. the period from first exposure to the development of clinical cancer is seldom less than 20 years. Although this association cannot establish that all agents and mixtures that cause cancer in experimental animals also cause cancer in humans. diethylstilboestrol. all studies that are judged to be methodologically sound should be consistent with a relative risk of unity for any observed level of exposure and. The possibility that a given agent may cause cancer through a speciesspecific mechanism which does not operate in humans (see p. . 24) of carcinogenicity in experimental animals as if they presented a carcinogenic risk to humans. The nature and extent of impurities or contaminants present in the chemical or mixture being evaluated are given when available. 2-naphthylamine. sex. STUDIES OF CANCER IN EXPERIMENTAL ANIMALS All known human carcinogens that have been studied adequately in experimental animals have produced positive results in one or more animal species (Wilbourn et al. chlornaphazine. in the absence of adequate data on humans. should provide a pooled estimate of relative risk which is at or near unity and has a narrow confidence interval. 9. in some cases. the possibility that bias. confounding or misclassification of exposure or outcome could explain the observed results should be considered and excluded with reasonable certainty. 1989). 1986. solar radiation.. estrogen replacement therapy/steroidal estrogens. 1995). thiotepa and vinyl chloride). mustard gas. they show evidence of lack of carcinogenicity. the standards of design and analysis described above.. betel quid with tobacco. azathioprine. Specifically. coal-tars. nonsteroidal estrogens. Experience with human cancer indicates that. melphalan. Tomatis et al. ciclosporin. coal-tar pitches.PREAMBLE 17 When several epidemiological studies show little or no indication of an association between an exposure and cancer. In addition. the judgement may be made that. cyclophosphamide. For several agents (aflatoxins.

(iii) whether the doses and duration of treatment were appropriate and whether the survival of treated animals was similar to that of controls. sex. consideration is given to the possibility of changes in the physicochemical properties of the test substance during collection. in the case of mixtures. for example. (iii) the spectrum of neoplastic response. (v) whether animals of each sex were used.. Those studies in experimental animals that are inadequate (e. and (viii) whether the data were adequately reported. and (iv) the possible role of modifying factors. For experimental studies of mixtures. (ii) the consistency of the results. The relevance of results obtained. from preneoplastic lesions and benign tumours to malignant neoplasms. and experiments on the carcinogenicity of known metabolites and derivatives. (vi) whether animals were allocated randomly to groups.18 IARC MONOGRAPHS VOLUME 77 Other types of studies summarized include: experiments in which the agent or mixture was administered in conjunction with known carcinogens or factors that modify carcinogenic effects. which may involve consideration of: (i) physical and chemical characteristics. (iii) the results of tests for genetic and related effects. 11). with animal viruses analogous to the virus being evaluated in the monograph must also be considered. An assessment is made as to the relevance to human exposure of samples tested in experimental animals. including studies on DNA adduct formation. (ii) constituent substances that indicate the presence of a class of substances. Chemical and toxicological interactions of the components of mixtures may result in nonlinear dose–response relationships. extraction. the Monographs are not intended to summarize all published studies. including (i) the experimental conditions under which the test was performed. studies in which the end-point was not cancer but a defined precancerous lesion. Considerations of importance to the Working Group in the interpretation and evaluation of a particular study include: (i) how clearly the agent was defined and. across species and target organ(s). as . strain. see below) or are judged irrelevant to the evaluation are generally omitted. for example. concentration and delivery. 1986). (vii) whether the duration of observation was adequate. (ii) whether the dose was adequately monitored. species. duration of follow-up. age. Guidelines for conducting adequate long-term carcinogenicity experiments have been outlined (e. recent data on the incidence of specific tumours in historical controls. poor survival. As mentioned earlier (p. (iv) whether there were adequate numbers of animals per group. storage.. Montesano et al. including route and schedule of exposure. proto-oncogene mutation and expression and suppressor gene inactivation.g. too few animals. They may provide biological and mechanistic information relevant to the understanding of the process of carcinogenesis in humans and may strengthen the plausibility of a conclusion that the biological agent under evaluation is carcinogenic in humans. If available. how adequately the sample characterization was reported. too short a duration.g. (a) Qualitative aspects An assessment of carcinogenicity involves several considerations of qualitative importance. particularly in inhalation experiments.

Otherwise. it should nevertheless be suspected of being a carcinogen and requires further investigation. 1986). Both DNA damage and increased cell division are important aspects of carcinogenesis. as could saturation of processes such as DNA repair (Hoel et al. 1980. . the Working Group usually compares the proportions of animals developing each tumour type in each of the groups. it may be valid to combine them in assessing tumour incidence (Huff et al. If an agent or mixture induces only benign neoplasms that appear to be end-points that do not readily progress to malignancy. (c) Statistical analysis of long-term experiments in animals Factors considered by the Working Group include the adequacy of the information given for each treatment group: (i) the number of animals studied and the number examined histologically. 1983. activation. Since many chemicals require metabolic activation before being converted into their reactive intermediates. Gart et al. strain and age of the animal. Saturation of steps such as absorption. inactivation and elimination may produce nonlinearity in the dose–response relationship.PREAMBLE 19 well as in concurrent controls. The statistical methods used should be clearly stated and should be the generally accepted techniques refined for this purpose (Peto et al. the dose of the carcinogen and the route and length of exposure. when tumours are visible or when they may be considered ‘fatal’ because mortality rapidly follows tumour development.. depending on the particular agent under study and the target organ. (ii) the number of animals with a given tumour type and (iii) length of survival.. both metabolic and pharmacokinetic aspects are important in determining the dose–response pattern. These adjustments can include: comparisons of the proportions of tumour-bearing animals among the effective number of animals (alive at the time the first tumour is discovered). and the Mantel-Haenszel test or logistic regression. 1986). When there is no difference in survival between control and treatment groups... and cell proliferation is a strong determinant of dose–response relationships for some carcinogens (Cohen & Ellwein. life-table methods. 1989). The occurrence of lesions presumed to be preneoplastic may in certain instances aid in assessing the biological plausibility of any neoplastic response observed. Evidence of an increased incidence of neoplasms with increased level of exposure strengthens the inference of a causal association between the exposure and the development of neoplasms. consideration is given as to whether or not appropriate adjustments have been made for differences in survival. The form of the dose–response relationship can vary widely. Gart et al. When benign tumours occur together with and originate from the same cell type in an organ or tissue as malignant tumours in a particular study and appear to represent a stage in the progression to malignancy.. in the case where most differences in survival occur before tumours appear. sex. should be taken into account in the evaluation of tumour response. (b) Quantitative aspects The probability that tumours will occur may depend on the species. 1990).

micronucleus formation. the other data considered to be relevant are divided into those on absorption. gene mutation. which may include DNA damage. tobacco smoking). 25–27). are mentioned. classifying tumours as fatal or incidental may be difficult. . Tests of genetic and related effects are described in view of the relevance of gene mutation and chromosomal damage to carcinogenesis (Vainio et al. teratogenicity. The concentrations employed are given. The nature of the information selected for the summary depends on the agent being considered. aneuploidy and cell transformation. Data are given on acute and chronic toxic effects (other than cancer). immunotoxicity and endocrine effects. such comments are similar to those described for animal carcinogenicity tests on p.20 IARC MONOGRAPHS VOLUME 77 when occult tumours do not affect the animals’ risk of dying but are ‘incidental’ findings at autopsy.g. sister chromatid exchange. The available data are interpreted critically by phylogenetic group according to the end-points detected. In practice. For chemicals and complex mixtures of chemicals such as those in some occupational situations or involving cultural habits (e. metabolism and excretion. although they have not been fully evaluated. McGregor et al. commented upon. protein binding. 1992. and genetic and related effects. chromosomal aberrations. OTHER DATA RELEVANT TO AN EVALUATION OF CARCINOGENICITY AND ITS MECHANISMS In coming to an overall evaluation of carcinogenicity in humans (see pp. with regard to complex mixtures. Comparative information on the relationship between exposure and the dose that reaches the target site may be of particular importance for extrapolation between species. The Genetic and Related Effects data presented in the Monographs are also available in the form of Graphic Activity Profiles (GAP) prepared in collaboration with the United States Environmental Protection Agency (EPA) (see also 10. Concise information is given on absorption. the Working Group also considers related data. 1986).. toxic effects. data and references. 1999). Effects on reproduction. and mention is made of whether use of an exogenous metabolic system in vitro affected the test result. 18. distribution. Kinetic factors that may affect the dose–response relationship. detoxification and DNA repair processes. such as organ toxicity. The adequacy of the reporting of sample characterization is considered and. reproductive and developmental effects. increased cell proliferation. metabolic activation. where necessary. such as saturation of uptake. fetotoxicity and embryotoxicity are also summarized briefly. and comparisons of data on humans and on animals are made when possible... These data are given as listings of test systems. Several survival-adjusted methods have been developed that do not require this distinction (Gart et al. Studies that indicate the metabolic fate of the agent in humans and in experimental animals are summarized briefly. The presence and toxicological significance of cellular receptors is described. distribution (including placental transfer) and excretion in both humans and experimental animals.

cellular toxicity with regenerative proliferation. In-vitro tests for tumour-promoting activity and for cell transformation may be sensitive to changes that are not necessarily the result of genetic alterations but that may have specific relevance to the process of carcinogenesis. 1986)... Factors that may lead to misleading results in short-term tests have been discussed in detail elsewhere (Montesano et al. and any genetic alterations seen in human tumours) and other observations. Results from such tests may also give information about the types of genetic effect produced and about the involvement of metabolic activation.epa. immune response and the presence of tumour markers.PREAMBLE 21 Waters et al. The EPA/IARC GAP software and database may be downloaded free of charge from www. plants.g. lower eukaryotes. A critical appraisal of these tests has been published (Montesano et al. Structure–activity relationships that may be relevant to an evaluation of the carcinogenicity of an agent are also described. negative results in short-term tests with genetic end-points cannot be considered to provide evidence to rule out carcinogenicity of agents or mixtures that act through other mechanisms (e. Positive results in tests using prokaryotes. Negative results in tests for mutagenicity in selected tissues from animals treated in vivo provide less weight.. which might include cellular and tissue responses to infection. peroxisome proliferation) (Vainio et al. while others are to a greater or lesser degree associated with genetic effects (e. The demonstration that an agent or mixture can induce gene and chromosomal mutations in whole mammals indicates that it may have carcinogenic activity.g. For biological agents—viruses. The adequacy of epidemiological studies of reproductive outcome and genetic and related effects in humans is evaluated by the same criteria as are applied to epidemiological studies of cancer. When available. although this activity may not be detectably expressed in any or all species.. insects and cultured mammalian cells suggest that genetic and related effects could occur in mammals. 1992).g. Relative potency in tests for mutagenicity and related effects is not a reliable indicator of carcinogenic potency. 1986). receptor-mediated effects.gov/gapdb. molecular biology (integration and expression of viruses. partly because they do not exclude the possibility of an effect in tissues other than those examined. Some end-points described are clearly genetic in nature (e. bacteria and parasites—other data relevant to carcinogenicity include descriptions of the pathology of infection. Genetic or other activity manifest in experimental mammals and humans is regarded as being of greater relevance than that in other organisms. Moreover. gene mutations and chromosomal aberrations).. . unscheduled DNA synthesis). data relevant to mechanisms of carcinogenesis that do not involve structural changes at the level of the gene are also described. 1987) using software for personal computers that are Microsoft Windows® compatible.

are described. cultured mammalian cells and nonmammalian systems. These may include toxicological. For each animal species and route of administration. such as that on cytotoxicity and regeneration. (a) Exposure Human exposure to chemicals and complex mixtures is summarized on the basis of elements such as production. Toxicological information. (c) Carcinogenicity in experimental animals Data relevant to an evaluation of carcinogenicity in animals are summarized. 11 are considered for evaluating carcinogenicity. When available. the available data on end-points or other phenomena relevant to mechanisms of carcinogenesis from studies in humans. kinetic and metabolic considerations and evidence of DNA binding. and particularly animals that have developed cancer. The results of tests for genetic and related effects are summarized for whole mammals. experimental animals and tissue and cell test systems are summarized within one or more of the following descriptive dimensions: 11. case reports and correlation studies are also summarized. Only reports. . comparisons of such data for humans and for animals. receptor binding and hormonal and immunological effects. use. If the agent or mixture produced tumours after prenatal exposure or in singledose experiments. (b) Carcinogenicity in humans Results of epidemiological studies that are considered to be pertinent to an assessment of human carcinogenicity are summarized. Dose– response and other quantitative data may be given when available. For the agent. it is stated whether an increased incidence of neoplasms or preneoplastic lesions was observed. persistence of DNA lesions or genetic damage in exposed humans. that meet the criteria outlined on p. this is also indicated. When relevant. other than in abstract form. and data on kinetics and metabolism in experimental animals are given when considered relevant to the possible mechanism of the carcinogenic action of the agent.22 IARC MONOGRAPHS VOLUME 77 SUMMARY OF DATA REPORTED In this section. Negative findings are also summarized. (d) Other data relevant to an evaluation of carcinogenicity and its mechanisms Data on biological effects in humans that are of particular relevance are summarized. occurrence in the environment and determinations in human tissues and body fluids. the relevant epidemiological and experimental data are summarized. Structure–activity relationships are mentioned when relevant. Inadequate studies are generally not summarized: such studies are usually identified by a square-bracketed comment in the preceding text. Exposure to biological agents is described in terms of transmission and prevalence of infection. Quantitative data are given when available. mixture or exposure circumstance being evaluated. and the tumour sites are indicated.

process. chemically or biologically. (i) Carcinogenicity in humans The applicability of an evaluation of the carcinogenicity of a mixture. described below. survival of premalignant or malignant cells (immortalization. It is recognized that the criteria for these evaluations. is limited to the materials tested. cannot encompass all of the factors that may be relevant to an evaluation of carcinogenicity. as inferred from epidemiological studies. the Working Group may assign the agent. they may be grouped together for the purpose of a single evaluation of degree of evidence. occupation or industry on the basis of evidence from epidemiological studies depends on the (a) 12. and an agent may fall within more than one of them. alterations to metabolic activation/inactivation/DNA repair (iii) Evidence of relevant effects on cell behaviour (morphological or behavioural changes at the cellular or tissue level): for example. preneoplasia and hyperplasia. mutagenicity (effect on specific genes).PREAMBLE 23 (i) Evidence of genotoxicity (structural changes at the level of the gene): for example. chromosomal mutation/aneuploidy (ii) Evidence of effects on the expression of relevant genes (functional changes at the intracellular level): for example. alterations to the structure or quantity of the product of a proto-oncogene or tumour-suppressor gene. . immunosuppression). induction of mitogenesis. compensatory cell proliferation. EVALUATION Evaluations of the strength of the evidence for carcinogenicity arising from human and experimental animal data are made. early/late stage. Degrees of evidence for carcinogenicity in humans and in experimental animals and supporting evidence These categories refer only to the strength of the evidence that an exposure is carcinogenic and not to the extent of its carcinogenic activity (potency) nor to the mechanisms involved. adduct formation. structure–activity considerations. the action of an agent on the expression of relevant genes could be summarized under both the first and second dimensions. When the agents evaluated are considered by the Working Group to be sufficiently closely related. effects on metastatic potential (iv) Evidence from dose and time relationships of carcinogenic effects and interactions between agents: for example. In considering all of the relevant scientific data. whether for a single agent or a mixture. Thus. using standard terms. for example. An evaluation of degree of evidence. toxicokinetics These dimensions are not mutually exclusive. as defined physically. A classification may change as new information becomes available. as defined in animal carcinogenicity experiments. initiation/promotion/progression/malignant conversion. mixture or exposure circumstance to a higher or lower category than a strict interpretation of these criteria would indicate. even if it were known with reasonable certainty that those effects resulted from genotoxicity.

mixture or exposure circumstance and any studied cancer at any observed level of exposure. The evidence relevant to carcinogenicity from studies in humans is classified into one of the following categories: Sufficient evidence of carcinogenicity: The Working Group considers that a causal relationship has been established between exposure to the agent. or no data on cancer in humans are available. bias or confounding could not be ruled out with reasonable confidence. the possibility of a very small risk at the levels of exposure studied can never be excluded. a positive relationship has been observed between the exposure and cancer in studies in which chance. process or activity which is considered most likely to be responsible for any excess risk. A conclusion of ‘evidence suggesting lack of carcinogenicity’ is inevitably limited to the cancer sites. In addition. consistency or statistical power to permit a conclusion regarding the presence or absence of a causal association between exposure and cancer. conditions and levels of exposure and length of observation covered by the available studies. (ii) Carcinogenicity in experimental animals The evidence relevant to carcinogenicity in experimental animals is classified into one of the following categories: Sufficient evidence of carcinogenicity: The Working Group considers that a causal relationship has been established between the agent or mixture and an increased incidence of malignant neoplasms or of an appropriate combination of benign and malignant neoplasms in (a) two or more species of animals or (b) in two or more independent studies in one species carried out at different times or in different laboratories or under different protocols. That is. type of tumour or age at onset. Exceptionally. processes. the above categories may be used to classify the degree of evidence related to carcinogenicity in specific organs or tissues. mixture or exposure circumstance and cancer for which a causal interpretation is considered by the Working Group to be credible. mixture or exposure circumstance and human cancer. The Working Group seeks to identify the specific exposure. Evidence suggesting lack of carcinogenicity: There are several adequate studies covering the full range of levels of exposure that human beings are known to encounter.24 IARC MONOGRAPHS VOLUME 77 variability over time and place of the mixtures. bias and confounding could be ruled out with reasonable confidence. . site. Limited evidence of carcinogenicity: A positive association has been observed between exposure to the agent. Inadequate evidence of carcinogenicity: The available studies are of insufficient quality. In some instances. occupations and industries. a single study in one species might be considered to provide sufficient evidence of carcinogenicity when malignant neoplasms occur to an unusual degree with regard to incidence. The evaluation is focused as narrowly as the available data on exposure and other aspects permit. but chance. which are mutually consistent in not showing a positive association between exposure to the agent.

using terms such as weak. the body of evidence is considered as a whole. Then. physicochemical parameters and analogous biological agents. Inadequate evidence of carcinogenicity: The studies cannot be interpreted as showing either the presence or absence of a carcinogenic effect because of major qualitative or quantitative limitations. structure– activity relationships. or no data on cancer in experimental animals are available. This may include data on preneoplastic lesions. metabolism and pharmacokinetics. A conclusion of evidence suggesting lack of carcinogenicity is inevitably limited to the species. The data may be considered to be especially relevant if they show that the agent in question has caused changes in exposed humans that are on the causal pathway to carcinogenesis. or (b) there are unresolved questions regarding the adequacy of the design. . The strength of the evidence that any carcinogenic effect observed is due to a particular mechanism is assessed. because it is at least conceivable that certain compounds may be kept from human use solely on the basis of evidence of their toxicity and/or carcinogenicity in experimental systems. or (c) the agent or mixture increases the incidence only of benign neoplasms or lesions of uncertain neoplastic potential. however. in order to reach an overall evaluation of the carcinogenicity to humans of an agent. Such data may. the Working Group assesses if that particular mechanism is likely to be operative in humans. For complex exposures. The strongest indications that a particular mechanism operates in humans come from data on humans or biological specimens obtained from exposed humans. (b) Other data relevant to the evaluation of carcinogenicity and its mechanisms Other evidence judged to be relevant to an evaluation of carcinogenicity and of sufficient importance to affect the overall evaluation is then described. tumour pathology. The Working Group also determines the extent to which the materials tested in experimental systems are related to those to which humans are exposed. including occupational and industrial exposures. the chemical composition and the potential contribution of carcinogens known to be present are considered by the Working Group in its overall evaluation of human carcinogenicity. mixture or circumstance of exposure. (a) the evidence of carcinogenicity is restricted to a single experiment. never become available. genetic and related effects. Data relevant to mechanisms of the carcinogenic action are also evaluated. within the limits of the tests used. conduct or interpretation of the study. moderate or strong. Evidence suggesting lack of carcinogenicity: Adequate studies involving at least two species are available which show that. the agent or mixture is not carcinogenic.PREAMBLE 25 Limited evidence of carcinogenicity: The data suggest a carcinogenic effect but are limited for making a definitive evaluation because. e. (c) Overall evaluation Finally. tumour sites and levels of exposure studied. or of certain neoplasms which may occur spontaneously in high incidences in certain strains.g.

The categorization of an agent. Group 2 This category includes agents. there are no human data but for which there is evidence of carcinogenicity in experimental animals. an agent (mixture) may be classified in this category when there is inadequate evidence of carcinogenicity in humans. the degree of evidence of carcinogenicity in humans is almost sufficient. mixture or exposure circumstance may be classified in this category solely on the basis of limited evidence of carcinogenicity in humans. an agent. mixture or exposure circumstance is a matter of scientific judgement. In addition. The agent. reflecting the strength of the evidence derived from studies in humans and in experimental animals and from other relevant data. The exposure circumstance entails exposures that are carcinogenic to humans. at one extreme. sufficient evidence of carcinogenicity in experimental animals and strong evidence that the carcinogenesis is mediated by a mechanism that also operates in humans. .26 IARC MONOGRAPHS VOLUME 77 An evaluation may be made for a group of chemical compounds that have been evaluated by the Working Group. This category is used when there is limited evidence of carcinogenicity in humans and sufficient evidence of carcinogenicity in experimental animals. when supporting data indicate that other. Group 2A—The agent (mixture) is probably carcinogenic to humans. Agents. an agent (mixture) may be placed in this category when evidence of carcinogenicity in humans is less than sufficient but there is sufficient evidence of carcinogenicity in experimental animals and strong evidence in exposed humans that the agent (mixture) acts through a relevant mechanism of carcinogenicity. Exceptionally. an additional evaluation may be made for this broader group of compounds if the strength of the evidence warrants it. at the other extreme. Exceptionally. related compounds for which there is no direct evidence of capacity to induce cancer in humans or in animals may also be carcinogenic. and the designated group is given. as well as those for which. a statement describing the rationale for this conclusion is added to the evaluation narrative. In some cases. This category is used when there is sufficient evidence of carcinogenicity in humans. mixtures and exposure circumstances are assigned to either group 2A (probably carcinogenic to humans) or group 2B (possibly carcinogenic to humans) on the basis of epidemiological and experimental evidence of carcinogenicity and other relevant data. Group 1 —The agent (mixture) is carcinogenic to humans. mixtures and exposure circumstances for which. The exposure circumstance entails exposures that are probably carcinogenic to humans. mixture or exposure circumstance is described according to the wording of one of the following categories.

agents or mixtures for which there is inadequate evidence of carcinogenicity in humans but evidence suggesting lack of carcinogenicity in experimental animals.J. 3. 79).. It may also be used when there is inadequate evidence of carcinogenicity in humans but there is sufficient evidence of carcinogenicity in experimental animals. L. an agent.PREAMBLE 27 Group 2B—The agent (mixture) is possibly carcinogenic to humans. (1987) Statistical Methods in Cancer Research. consistently and strongly supported by a broad range of other relevant data. 82).N. The Analysis of Case–Control Studies (IARC Scientific Publications No. This category is used most commonly for agents. mixtures and exposure circumstances for which the evidence of carcinogenicity is inadequate in humans and inadequate or limited in experimental animals. 2.E. The exposure circumstance entails exposures that are possibly carcinogenic to humans. N. Group 4—The agent (mixture) is probably not carcinogenic to humans. Agents. & Wahrendorf. (1990) Cell proliferation in carcinogenesis. Lyon. mixtures and exposure circumstances that do not fall into any other group are also placed in this category. The Design and Analysis of Cohort Studies (IARC Scientific Publications No. Exceptionally. (1980) Statistical Methods in Cancer Research. IARCPress .E. 1007–1011 Gart. This category is used for agents or mixtures for which there is evidence suggesting lack of carcinogenicity in humans and in experimental animals. This category is used for agents.M. D. N. In some instances. & Day. Lyon. In some instances. 249. may be classified in this group. J. (1986) Statistical Methods in Cancer Research. Lee.E. P.E.. 1. IARCPress Breslow. Group 3—The agent (mixture or exposure circumstance) is not classifiable as to its carcinogenicity to humans. N. mixture or exposure circumstance for which there is inadequate evidence of carcinogenicity in humans but limited evidence of carcinogenicity in experimental animals together with supporting evidence from other relevant data may be placed in this group..E. Vol. & Ellwein. Krewski. N. agents (mixtures) for which the evidence of carcinogenicity is inadequate in humans but sufficient in experimental animals may be placed in this category when there is strong evidence that the mechanism of carcinogenicity in experimental animals does not operate in humans. Vol. The Design and Analysis of Long-term Animal Experiments (IARC Scientific Publications No. IARCPress Cohen. Tarone.B. Science. S. J. mixtures and exposure circumstances for which there is limited evidence of carcinogenicity in humans and less than sufficient evidence of carcinogenicity in experimental animals. 32). Vol. R. Lyon. & Day. References Breslow.

Wahrendorf Directory of On-going Research in Cancer Epidemiology 1992 (IARC Scientific Publications No. Coleman & J. 1032–1037 Huff.S. 28). Wagner Directory of On-going Research in Cancer Epidemiology 1983 (IARC Scientific Publications No. 117). Wagner Directory of On-going Research in Cancer Epidemiology 1980 (IARC Scientific Publications No. Muir & G.S.S. Wagner Directory of On-going Research in Cancer Epidemiology 1982 (IARC Scientific Publications No.L. Coleman & J.S. Muir & G. 38). Wagner Directory of On-going Research in Cancer Epidemiology 1984 (IARC Scientific Publications No. Edited by D. Muir & G.E. 86). Wahrendorf Directory of On-going Research in Cancer Epidemiology 1989/90 (IARC Scientific Publications No. Wagner Directory of On-going Research in Cancer Epidemiology 1987 (IARC Scientific Publications No. Kaplan. Edited by C. Coleman & J. 17). Wagner Directory of On-going Research in Cancer Epidemiology 1981 (IARC Scientific Publications No. Edited by C.G.. Muir & G.S. Edited by C. Muir & G. Démaret . Edited by C. Wagner Directory of On-going Research in Cancer Epidemiology 1978 (IARC Scientific Publications No.K. Science. & Haseman. Lyon. Wahrendorf Directory of On-going Research in Cancer Epidemiology 1988 (IARC Scientific Publications No.S. Muir & G. Edited by M. Muir & G.110). 50). (1983) Implication of nonlinear kinetics on risk estimation in carcinogenesis. Muir & G. Wagner Directory of On-going Research in Cancer Epidemiology 1979 (IARC Scientific Publications No. Edited by C.M. IARCPress Directory of On-going Research in Cancer Epidemiology 1976. Cancer Metastasis Rev.L. Edited by C. Lyon. D.S. 80). Edited by M. 1– 21 IARC (1973–1996) Information Bulletin on the Survey of Chemicals Being Tested for Carcinogenicity/Directory of Agents Being Tested for Carcinogenicity. J. Edited by C. 26). Edited by C. N. 93). Edited by M.S.. Edited by M.S. 62). Eustis. 8. Wagner Directory of On-going Research in Cancer Epidemiology 1977 (IARC Scientific Publications No. Edited by C. J. Numbers 1–17. Parkin & J..S. Wahrendorf & E.S. (1989) Occurrence and relevance of chemically induced benign neoplasms in long-term carcinogenicity studies.W. 69). Wagner Directory of On-going Research in Cancer Epidemiology 1985 (IARC Scientific Publications No. Coleman. IARCPress IARC (1976–1996). 219. 101). Muir & G. S. J. Muir & G. 46). Edited by C. Muir & G. Edited by C. Wahrendorf Directory of On-going Research in Cancer Epidemiology 1991 (IARC Scientific Publications No. & Anderson.28 IARC MONOGRAPHS VOLUME 77 Hoel. 35). Wagner Directory of On-going Research in Cancer Epidemiology 1986 (IARC Scientific Publications No. M.

Pb. van de Wiel. M.PREAMBLE 29 Directory of On-going Research in Cancer Epidemiology 1994 (IARC Scientific Publications No.K. Preamble (IARC intern. G. Some Aromatic Amines and Azo Dyes in the General and Industrial Environment (IARC Scientific Publications No. Dodet & I. Schuller & L. 8. Dodet & D. Castegnaro. O’Neill.K. Edited by I. Thain (1978) Vol. Rep. Dodet & I. 6. Sankaranarayanan. K. H. Sankaranarayanan. Castegnaro. H. 5.E. 18).R. Castegnaro. 22). M. O’Neill. O’Neill (1985) Vol. Chemicals. O’Neill (1988) Vol. tech. Preussmann. No.A. Zn (IARC Scientific Publications No. 3. J. Bartsch (1981) Vol. Cr. Bartsch (1983) Vol. N-Nitroso Compounds (IARC Scientific Publications No. Rappe. Cd. Bogovski. 7. Rep. Polychlorinated Dioxins and Dibenzofurans (IARC Scientific Publications No. Eisenbrand. 1. 78/003) IARC (1978–1993) Environmental Carcinogens. P. P. Wasserman (1978) Vol. Squirrell & W.D. 77/002) IARC (1978) Chemicals with Sufficient Evidence of Carcinogenicity in Experimental Animals— IARC Monographs Volumes 1–17 (IARC intern. Se.K. O’Neill. Scott. Edited by L. Stoloff.K.K. O’Neill (1991) Vol. Edited by L. Edited by R. Démaret IARC (1977) IARC Monographs Programme on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Spiegelhalder & H.K. 108). I. Preussmann. Rep. I. 85). Lyon. Lyon. Démaret Directory of On-going Research in Cancer Epidemiology 1996 (IARC Scientific Publications No. E. Fishbein & I. Castegnaro. Analysis of Polycyclic Aromatic Hydrocarbons in Environmental Samples (IARC Scientific Publications No. 40). Walker & A.A. Edited by D. IARCPress . 12. P. Fishbein (1986) Vol. Ni. Some Volatile Halogenated Hydrocarbons (IARC Scientific Publications No. Edited by R. Fishbein & I. Benzene and Alkylated Benzenes (IARC Scientific Publications No. M. 11. Edited by M. Water and Foodstuffs (IARC Scientific Publications No. Walker (1979) Vol. Methods of Analysis and Exposure Measurement. Volumes 1 to 29). 137).K.C. Supplement 4. 109). No. tech. IARCPress Vol. Edited by L. B. Industrial Processes and Industries Associated with Cancer in Humans (IARC Monographs. B. No. Edited by C. O’Neill (1993) IARC (1979) Criteria to Select Chemicals for IARC Monographs (IARC intern. 4. Methods for the Measurement of Vinyl Chloride in Poly(vinyl chloride). 2. Air. 10. O’Neill & H. Edited by R. Kunte & E. Analysis of Volatile Nitrosamines in Food (IARC Scientific Publications No. Fishbein. Seifert. Wahrendorf & E. Edited by B. Passive Smoking (IARC Scientific Publications No. Wahrendorf & E. 9. Some Mycotoxins (IARC Scientific Publications No. Hoffmann (1987) Vol. B. J. 130). B. Edited by L. Edited by I. Bartsch (1983) Vol. Indoor Air (IARC Scientific Publications No.K. Be. I. Some Metals: As.K. 45). H. 29). 71). 81). 79/003) IARC (1982) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. 68). tech. O’Neill & H. Buser. 44). Brunnemann. Edited by R.M.

. Pike. Supplement 2. & Shuker.. Jpn. pp. Gray... Lyon. J. tech. Rep.30 IARC MONOGRAPHS VOLUME 77 IARC (1983) Approaches to Classifying Chemical Carcinogens According to Mechanism of Action (IARC intern.. 98/004) McGregor. S.. No. J. H. S. IARCPress.. No. Vainio.B.M. (1989) Human carcinogens so far identified. tech. J. Complex Mixtures. 93/005) IARC (1998a) Report of an ad-hoc IARC Monographs Advisory Group on Physical Agents (IARC Internal Report No.. Day. S. IARCPress Montesano. D. 91/002) IARC (1991b) Report of an ad-hoc IARC Monographs Advisory Group on Viruses and Other Biological Agents Such as Parasites (IARC intern.. & Venitt. No. Lee. R. IARCPress IARC (1987b) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. In: IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. tech. Rep. Parish. Peto.G. R. Lyon. Richards. IARCPress IARC (1988) Report of an IARC Working Group to Review the Approaches and Processes Used to Evaluate the Carcinogenicity of Mixtures and Groups of Chemicals (IARC intern. Physical and Biological Agents and Exposure Circumstances to be Evaluated in Future IARC Monographs. 795–807 . tech. tech.. P.. tech. Rep. Overall Evaluations of Carcinogenicity: An Updating of IARC Monographs Volumes 1 to 42. Rep. No. 84/002) IARC (1987a) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. 89/004) IARC (1991a) A Consensus Report of an IARC Monographs Working Group on the Use of Mechanisms of Carcinogenesis in Risk Identification (IARC intern. M. eds (1999) The Use of Short and Medium-term Tests for Carcinogens and Data on Genetic Effects in Carcinogenic Hazard Evaluation (IARC Scientific Publications No. L. A..C. No. Lyon. Aitio. eds (1986) Long-term and Short-term Assays for Carcinogenesis—A Critical Appraisal (IARC Scientific Publications No. L. J. Lyon. 83/001) IARC (1984) Chemicals and Exposures to Complex Mixtures Recommended for Evaluation in IARC Monographs and Chemicals and Complex Mixtures Recommended for Long-term Carcinogenicity Testing (IARC intern. Groups of Chemicals. N. Report of an ad-hoc Working Group (IARC intern.. 98/002) IARC (1998b) Report of an ad-hoc IARC Monographs Advisory Group on Priorities for Future Evaluations (IARC Internal Report No. 146). Rice. Supplement 7. & Yamasaki. Rep. H. 83). 91/001) IARC (1993) Chemicals. Mixtures and Exposure Circumstances to be Evaluated in Future IARC Monographs. No. R. Bartsch. sensitive significance tests for carcinogenic effects in long-term animal experiments. Rep. Wilbourn. (1980) Guidelines for simple. J.N. J. Rep. Supplement 6.. 80. Wilbourn.. Lyon. H.E. Genetic and Related Effects: An Updating of Selected IARC Monographs from Volumes 1 to 42. IARCPress Peto.. & Wahrendorf. Report of an ad hoc Working Group (IARC intern. 88/002) IARC (1989) Chemicals. No. Cancer Res. Groups of Chemicals. Long-term and Short-term Screening Assays for Carcinogens: A Critical Appraisal. 311–426 Tomatis.

H. Heseltine.. 82. I.. Activity profiles for genetic and related tests. P.D. IARCPress Vainio.. L. Cancer.B.. L.. Lyon..D.N. 687–696 Wilbourn. & McMichael. In: IARC Monographs on the Evaluation of Carcinogenic Risks to Humans.J. 7. 116). Bull. A.F. J. Lohman. 339–348 (in French) Waters. Stack. Brady. H. Haroun.. eds (1992) Mechanisms of Carcinogenesis in Risk Identification (IARC Scientific Publications No. Gaudin. Heseltine. E.. 6. (1987) Appendix 1. Wilbourn.. pp..L. Carcinogenesis. C. N. A. Magee.H.. M. E.. Suppl. J. P. J. Sasco. (1995) Identification of human carcinogenic risk in IARC Monographs. Kaldor. H.J. Genetic and Related Effects: An Updating of Selected IARC Monographs from Volumes 1 to 42. 1853–1863 . (1986) Response of experimental animals to human carcinogens: an analysis based upon the IARC Monographs Programme.PREAMBLE 31 Vainio. Haroun. H.. IARCPress.M. A.. D. Partensky. & Vainio. & Eragne. & Vainio. Lyon.. C. McGregor. Partensky. H..

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no data. probably carcinogenic to humans. NOES was a nationwide observational survey conducted in a sample of 4490 establishments from 1981 to 1983. S. For these seven substances. new data have become available. 2B. The target population was defined as employees working in establishments or job sites in the United States of America employing eight or more workers in a defined list of Standard Industrial Classifications. Table 1. 2A. 7 (1987) 4-Chloro-ortho-toluidine Cinnamyl anthranilate Coumarin Di(2-ethylhexyl) adipate Di(2-ethylhexyl) phthalate N-Nitrosodiethanolamine ortho-Toluidine L ND ND ND ND ND I S L L L S S S L. Information on the extent of occupational exposures to many of these compounds in the United States was available from the National Occupational Exposure Survey (NOES) conducted by the United States National Institute for Occupational Safety and Health (NIOSH). possibly carcinogenic to humans. Previous evaluations of compounds covered in this volume Human Animal Overall evaluation 2A 3 3 3 2B 2B 2B Monograph volume (year) 48 (1990) Suppl. 3. ND. sufficient evidence. 7 (1987) Suppl. 7 (1987) Suppl.GENERAL REMARKS ON THE SUBSTANCES CONSIDERED Introduction This seventy-seventh volume of the IARC Monographs reviews sixteen industrial organic chemicals. Since these previous evaluations. –33– . 7 (1987) Suppl. I. 7 (1987) Suppl. the most recent previous evaluations are summarized below. limited evidence. 7 (1987) Suppl. and these have been incorporated into the monographs and taken into consideration in the present evaluations. inadequate evidence. seven of which have previously been evaluated one or more times. cannot be classified as to its carcinogenicity to humans Exposures This volume includes evaluations of the carcinogenicity of several chemical intermediates or additives to which a large number of workers are exposed in various industries.

i. disposal and movement to other off-site locations). The NOES had little or no coverage of agriculture. Data reported from selected categories of the TRI database are reported in these monographs. often varying mixtures of potential carcinogens other than the compound of interest. it must have presented a potential exposure for at least 30 min per week (on an annual average) or be used at least once per week for 90% of the weeks of the work year (NOES. it is often difficult to ascribe causality to a single agent. in some cases. as the salts dissociate readily under physiological conditions. private and business service and hospital industries. allow cancer excesses to be attributed to specific agents when there is mixed exposure. treatment. manufacturing. physical or biological agent or tradename product had to be observed in sufficient proximity to an employee such that one or more physical phases of that agent or product were likely to come into contact with or enter the body of the employee. Aromatic amines such as those evaluated in this volume are sometimes used commercially and/or in laboratory studies as their strong acid (e. underground injection and releases to land. energy recovery. when epidemiological studies of populations with mixed and complex exposures find positive results. NOES addressed recordable potential exposure that had to meet two criteria: (1) a chemical. Although epidemiological studies of some of the industries where exposure to chemicals considered in this volume occurs have been conducted.. mining. Due to changes in reporting requirements. wholesale/retail trade. or government operations. No exposure measurements were made. which is updated every year. These categories include air emissions (fugitive or non-point emissions plus stack or point emissions).e. The carcinogenicity of an amine and that of its strong acid salts are expected to be qualitatively similar. soil. transportation. Without such data. water. 1999). The presence of chemicals as intermediates.g. Categories typically not included in the monographs are classified as transfers (recycling. exposure to many of these chemicals has rarely been specifically assessed for epidemiological purposes. The Toxics Release Inventory (TRI) is a compilation of data reported by chemical manufacturers and processors to the United States Environmental Protection Agency under a regulation promulgated in 1986. and (2) the duration of the potential exposure had to meet the minimum duration guidelines.34 IARC MONOGRAPHS VOLUME 77 Generally. surface water discharges. apparent trends in reported releases do not always indicate an actual increase or decrease in quantities released. underground injection. these classifications emphasized coverage of construction.. TRI. it is difficult to evaluate exposure–response relationships which might. amounts transferred off-site for waste treatment. and source reduction and recycling data. contains data on the reported annual releases of toxic chemicals from industrial facilities in the United States to the environment—air. and for many compounds it may never . additives or contaminants in the workplace implies co-exposure to complex. finance/real estate. Quantitative estimates of historical exposure are often not available and therefore it is difficult to identify highly exposed subgroups or to estimate individual exposures. POTWs [Publicly Owned Treatment Works]. hydrochloride) salts. Thus.

N-Nitrosodiethanolamine has been detected in some bulk samples of soluble oils (National Institute for Occupational Safety and Health.and diethanolamine may be present in concentrations of 2–3% and triethanolamine may be present in concentrations of up to 25% of undiluted fluids.b. In the past. Emulsifiers are petroleum sulfonates and other detergents. phosphates and antimicrobial agents. some of which are carcinogenic (National Toxicology Program. This category has not been examined separately in epidemiological studies. In these cases. sulfur compounds and chlorinated compounds such as chlorinated paraffins. soluble oils. Semi-synthetic fluids contain smaller amounts of oil than soluble oils (3–30%). additional evidence from biomarkers of exposure or effect such as haemoglobin or DNA adducts in exposed humans to demonstrate and quantify genotoxic effects or other relevant mechanisms may allow a more definitive classification of potential carcinogenicity to humans. By the 1950s. nitrites were occasionally added to soluble oils. 1998). Soluble oils may frequently contain ethanolamines and antimicrobial agents of various types. 1984). Straight oils may contain elemental sulfur. Untreated and mildly treated mineral oils containing polycyclic aromatic hydrocarbons that were used in the past are recognized as human carcinogens (IARC. organic esters. but rather are primarily water with additives including buffers such as ethanolamines. Before the 1940s. Metalworking fluids can be divided into four broad categories: straight oils. ‘Soluble oils’ are emulsions of highly refined petrochemicals (30–85%) typically diluted 1:5 to 1:40 with water for use. Mono. semi-synthetic and synthetic fluids.. Straight oils are generally mineral oils (60–100%) and do not contain ethanolamines as additives. Oils were not always highly refined before the 1970s. One study (Sullivan et al. Synthetic fluids contain no oil. IARC. The semi-synthetic and synthetic fluids became more common in the 1960s and 1970s.GENERAL REMARKS 35 be possible to establish sufficient evidence of carcinogenicity in humans using traditional epidemiological data. metalworking fluids were predominantly straight oils. Refined straight oils continue to be used in lower-production operations and those requiring lubrication. They are typically diluted 1:10 to 1:40 for use. organic polyglycols. Diethanolamine may be converted to N-nitrosodiethanolamine in the presence of nitrosating agents. Metalworking fluids and ethanolamines Epidemiological studies relevant to the evaluation of di. . They are typically diluted 1:10 to 1:40 for use. 1986a. 1998) grouped semisynthetic oil exposure with soluble oil exposures. 1990). water-soluble fluids were being increasingly used in high-production operations. Such data were not available to the Working Group for most of the agents evaluated in this volume.and triethanolamine and N-nitrosodiethanolamine involve occupational cohorts exposed to metalworking fluids. along with the same mixture of additives mentioned below for synthetic fluids.

some synthetic and soluble fluids may contain chlorinated paraffins.AC mice carry the v-Ha-ras gene under a zeta-globin promoter and are specifically designed to detect chemical carcinogens and tumour promoters in skin carcinogenesis experiments. The transgenic and/or knock-out models. in particular. The role of peroxisome proliferation in evaluating carcinogenicity in humans has been discussed (IARC. microbial contaminants and metal and metal alloy contaminants (National Institute for Occupational Safety and Health.. (b) whether peroxisome proliferation (increases in peroxisome volume density or fatty acid β-oxidation activity) and hepatocellular proliferation have been demonstrated under the conditions of the bioassay. When. Two primary responses have been proposed to account for liver carcinogenesis in rats and mice by peroxisome proliferators. However. carcinogenicity experiments have been performed using genetically modified mice designed to be particularly susceptible to the induction of tumours at certain organ sites through specific mechanisms. to test genetic targets of carcinogenicity. The use of such animals for evaluation of the carcinogenicity of chemicals has been discussed (McGregor et al. 1995a)-releasing biocides. the relationship between peroxisome proliferation and liver tumours in rats or mice has been established. formaldehyde (see IARC. 1998). this should be considered relevant information in the evaluation of the possible risks for cancer in humans. The transgenic Tg. because of the limited available database on the responses of any particular genetically modified mice to chemical carcinogens. Genetically modified mice which lack one copy of an essential tumoursuppressor gene such as p53 model the situation in which a functioning copy of the suppressor gene has been lost in somatic cells of a normal individual. for any chemical.36 IARC MONOGRAPHS VOLUME 77 In addition to the chemicals evaluated in this volume. These are (i) induction of peroxisome . 1995b). and have been proposed to act by a mechanism involving peroxisomal proliferation in hepatocytes in those species. taking into account the following: (a) whether information is available to exclude mechanisms of carcinogenesis other than those related to peroxisome proliferation. triethanolamine and pyridine). may also be useful not only for carcinogen identification but also for studies aimed at identifying the mode of action of chemicals and. (c) whether such effects have been looked for and have not been found in adequately designed and conducted investigations of human groups and systems. Peroxisome proliferation Three of the compounds evaluated in this volume (di(2-ethylhexyl) phthalate. di(2ethylhexyl) adipate and cinnamyl anthranilate) are carcinogenic to the liver in mice and/or rats. Genetically modified animal models For three chemicals reviewed in this volume (diethanolamine. 1999). therefore. bioassays using these animals must be interpreted with caution.

43% Chlorine) in F344/N Rats and B6C3F1 Mice (NTP TR 305).G. No. B. Elliott. NC National Toxicology Program (1986b) Toxicology and Carcinogenesis Studies of Chlorinated Paraffins (C12. Part 2: Carbon Blacks. J.R. Tugwood. S.. Pharmacol. A. 217–362 IARC (1995b) Peroxisome Proliferation and its Role in Carcinogenesis (IARC Technical Report No. B. Lyon. 24). Cattley et al. Robinson. Lyon. 483–507 McGregor. Vol. 1998).. J. Lyon.S... OH National Toxicology Program (1986a) Toxicology and Carcinogenesis Studies of Chlorinated Paraffins (C23. 33.E. Cattley et al.A. & Venitt. Roberts. D. 146). Toxicol..G. 1993. 82–83. C. D. & Purchase.G. Research Triangle Park. 1996. Details are presented in the monograph on di(2ethylhexyl) phthalate.and Medium-term Tests for Carcinogens and Data on Genetic Effects in Carcinogenic Hazard Evaluation (IARC Scientific Publications No.M...F. C. IARCPress.GENERAL REMARKS 37 proliferation and (ii) increased hepatocellular proliferation (Lake. Lake. Polynuclear Aromatic Hydrocarbons.. J. Odum. T.M. 1994. I. (1998) Do peroxisome proliferating compounds pose a hepatocarcinogenic hazard to humans? Regul.D. Elcombe. (1994) Mechanistically-based human hazard assessment of peroxisome proliferator-induced hepatocarcinogenesis.. S1–S117 Cattley.. Lake. Ann. Kettle. Lyon. pp. and Exposures in the Textile Manufacturing Industry. Marsman. B. IARCPress Lake. exp.. (1995b) Mechanisms of hepatocarcinogenicity of peroxisome-proliferating drugs and chemicals. Cincinnati. Rice. IARCPress. R. S. Elcombe. NC . J. Wood Dust and Formaldehyde. B.. Brady. 1998). 62. Fenner. 47–60 IARC (1984) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Mineral Oils and Some Nitroarenes. together with the ability of peroxisome proliferators to inhibit apoptosis in rat and mouse hepatocytes and their properties as liver tumour promoters in these rodents. eds (1999) The Use of Short. Research Triangle Park. Schwetz. & Wahli. Toxicol. Rev. account for peroxisome proliferator-induced liver tumour formation in these species (Popp & Cattley. 673–681 Lake. D. Pastoor. Hum. W.. 55–72 IARC (1995a) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans.A. IARCPress National Institute for Occupational Safety and Health (1998) Criteria for a Recommended Standard: Occupational Exposure to Metalworking Fluids (DHHS (NIOSH) Publ.P.. J. 48.. Ashby et al. Vol. Toxicol. Such responses. 13 (Suppl 2). IARCPress IARC (1990) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans... Some Flame Retardants and Textile Chemicals... C.. Vol. Lett.. 1995a.. Popp... J. Tugwood. References Ashby. pp.B. 60% Chlorine) in F344/N Rats and B6C3F1 Mice (NTP TR 308). (1995a) Peroxisome proliferation: current mechanisms relating to nongenotoxic carcinogenesis. J. Pharmacol. B. J. Ishmael.b.. Toxicol. 98102). 35. DeLuca.. Lyon. 27.C. 1995a.

. D. Am. 653–665 Roberts.38 IARC MONOGRAPHS VOLUME 77 NOES (1999) National Occupational Exposure Survey 1981–83. (1998) Mortality studies of metalworking fluid exposure in the automobile industry: VI. Hallock. M. R. S. Sci. & Lake.Y. A case–control study of esophageal cancer. Hammond. 36–48 .. Smith.R. Eisen. Acad. (1996) Non-genotoxic hepatocarcinogenesis: suppression of apoptosis by peroxisome proliferators. Peroxisomes: Biology and Importance in Toxicology and Medicine. R.A. ind.. & Cattley..A. National Institute for Occupational Safety and Health Popp... P. R. Woskie. T. N. G. Med. 588–611 Sullivan.A. S.F. 804. Taylor and Francis. P. OH. Centers for Disease Control.C. Department of Health and Human Services.H.R.K..J.. eds. E. Cincinnati.. Unpublished data as of July 1999. Ann. Wegman.A. 34. J. D.. London. & Monson. (1993) Peroxisome proliferators as initiators and promoters of rodent hepatocarcinogenesis.E. pp.. B. Kriebel. J. In: Gibson. Tolbert. Public Health Service.

THE MONOGRAPHS .

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No. 205180-59-2 Chem. 1. Abstr.2-benzenedicarboxylate. phthalic acid.2-Benzenedicarboxylic acid. 1987).1 1.1. dioctyl phthalate. ethylhexyl phthalate. Nos: 8033-53-2.2 Structural and molecular formulae and relative molecular mass O C C O C2H5 O CH2 CH CH2 O CH2 CH CH2 C2H5 CH2 CH2 CH2 CH2 CH3 CH3 C24H38O4 –41– Relative molecular mass: 390. DEHP. Since that time. phthalic acid di(2-ethylhexyl) ester. bis(2-ethylhexyl) orthophthalate. 1. 1982) and March 1987 (IARC. new data have become available. and these have been incorporated into the monograph and taken into consideration in the present evaluation.1. in October 1981 (IARC. bis(2-ethylhexyl) ester IUPAC Systematic Names: Bis(2-ethylhexyl) phthalate. 40120-69-2. di-sec octyl phthalate.DI(2-ETHYLHEXYL) PHTHALATE This substance was considered by previous Working Groups. 2-ethylhexyl phthalate. bis(2-ethylhexyl) ester Synonyms: Bis(2-ethylhexyl) 1. phthalic acid dioctyl ester 1. 126639-29-0.56 . Reg. Name: 1. 137718-37-7. 50885-87-5. Serv.1 Exposure Data Chemical and physical data Nomenclature Chem. Abstr. octyl phthalate.: 117-81-7 Deleted CAS Reg.

Verschueren. 13. Kodaflex DOP.. Diacizer DOP. Octoil. 1 2 Lower values have been proposed. American Conference of Governmental Industrial Hygienists. ultraviolet [22080]. 1992.3 Chemical and physical properties of the pure substance (a) Description: Light-coloured liquid with a slight odour (Agency for Toxic Substances and Disease Registry. Witcizer 312 (National Toxicology Program. Vinycizer. 1999) (e) Spectroscopy data: Infrared (prism [28].01% (Aristech Chemical Corp. Sicol 150. WHO. 1991. Reomol D 79P. 1985. 1998. 1995) (i) Conversion factor2: mg/m3 = 16. and acidity (as acetic acid or phthalic acid). Lide. Truflex DOP. 1993. C-13 [5201]) and mass [NIST. 1996) (f) Solubility: Slightly soluble in water (0. slightly soluble in carbon tetrachloride (Environmental Protection Agency. 1996) (b) Boiling-point: 384 °C (Lide. Plasthall DOP. 1992. 0. Palatinol AH.981 g/cm3 at 25 °C (Lide. cork. glass wool.5 Analysis Detection and quantification of very low levels of di(2-ethylhexyl) phthalate are seriously limited by the presence of this compound as a contaminant in almost all laboratory equipment and reagents. DOP. 1993).. relative vapour density (air = 1).1%.4 Technical products and impurities Di(2-ethylhexyl) phthalate is available in a variety of technical grades (including a special grade for capacitor applications and a low residuals grade). Lide & Milne. Ergoplast. Flexol DOP.0–99.42 IARC MONOGRAPHS VOLUME 77 1. aluminium foil. Monocizer DOP.285 mg/L at 24 °C)1.0 × ppm 1. WHO. assuming a temperature of 25 °C and a pressure of 101 kPa . grating [18401]). Pittsburgh PX 138. glassware. 1992). 0. solvents and Teflon® sheets have all been found to be contaminated (Agency for Toxic Substances and Disease Registry. Eastman DOP Plasticizer. Trade names for di(2-ethylhexyl) phthalate include: Bisoflex. 1999) (d) Density: 0. Eviplast. Platinol AH. Hatco-DOP.1. based on models (Staples et al. 7.45) × ppm. 1997).1. 43511] spectral data have been reported (Sadtler Research Laboratories. Typical specifications are: minimal ester content. Verschueren.45 (Hansch et al. 1996) (h) Octanol/water partition coefficient (P): log P.. 1999) (g) Volatility: Vapour pressure. 1. 1999). Plastics. 8. 160 Pa at 200 °C.6 × 10–4 Pa at 25 °C. Staflex DOP. Fleximel. maximal moisture content.007–0. Etalon. Compound 889. 1999) (c) Melting-point: –55 °C (Lide.1.4 (Howard et al. Good-rite GP 264. rubber.6%. Calculated from: mg/m3 = (relative molecular mass/24. nuclear magnetic resonance (proton [9392]. 1980. Vestinol AH.. 99.

Romania. Information available in 1999 indicated that di(2-ethylhexyl) phthalate was produced by 30 companies in China. and one company each in Albania. 1.DI(2-ETHYLHEXYL) PHTHALATE 43 Determination of di(2-ethylhexyl) phthalate in air. four companies each in Argentina. 1998). footwear. Sweden. Kazakhstan. Colombia.. five companies each in Germany and the Russian Federation. South Africa.3 Use Di(2-ethylhexyl) phthalate is widely used as a plasticizer in flexible vinyl products. from approximately 114 000 tonnes in 1982 to over 130 000 tonnes in 1986 (Environmental Protection Agency. Turkey and Venezuela. 1993). two companies each in Belgium. It was first produced in commercial quantities in Japan around 1933 and in the United States in 1939 (IARC. Malaysia and Poland. shower curtains. Pakistan.2 Production The worldwide production of di(2-ethylhexyl) phthalate has been increasing during recent decades and in the late 1980s amounted to approximately 1 million tonnes per year (WHO. the United Kingdom and Viet Nam (Chemical Information Services. 12 companies in Japan. Ecuador. Indonesia. a high-performance liquid chromatography method for food has also been developed. Finland. Di(2-ethylhexyl) phthalate is produced commercially by the reaction of excess 2-ethylhexanol with phthalic anhydride in the presence of an acid catalyst such as sulfuric acid or para-toluenesulfonic acid. Di(2-ethylhexyl) phthalate is usually extracted from environmental samples with an organic solvent such as hexane. rainwear. 1996). flooring. production in Taiwan in 1995 was 207 000 tonnes. Selected methods for the analysis of di(2-ethylhexyl) phthalate in various matrices are presented in Table 1. In 1994. seven companies in Taiwan. dichloromethane or acetonitrile. Spain. food packaging materials and children’s . 1. three companies each in Canada. Ukraine. soil/sediments and food is usually accomplished by gas chromatographic analysis. production in Japan in 1995 was 298 000 tonnes. Production of di(2-ethylhexyl) phthalate in the United States increased during the 1980s. Iran. Brazil. Chile. production of di(2ethylhexyl) phthalate in the United States was 117 500 tonnes. water. upholstery. A purge-and-trap method has been developed for separation of di(2-ethylhexyl) phthalate from the fat in foods (Agency for Toxic Substances and Disease Registry. down from 241 000 tonnes in 1994 (Anon. 1982). the Philippines and the United States. 1999). eight companies in Mexico. wire and cable. Thailand. Korea (Republic of). Air samples are drawn through a solid sorbent material and desorbed with carbon disulfide. the Czech Republic. 1992). Peru. chloroform. Italy. Plastics may contain from 1 to 40% di(2-ethylhexyl) phthalate by weight and are used in consumer products such as imitation leather. 15 companies in India. France. tablecloths.

elute with acetonitrile. Di(2-ethylhexyl) phthalate is also used as a hydraulic fluid and as a dielectric fluid (a non-conductor of electric current) in electrical capacitors (Agency for Toxic Substances and Disease Registry. Poly(vinyl chloride) (PVC) containing di(2-ethylhexyl) phthalate is also used for tubing and containers for blood products and transfusions. dry. MS. 1998). flame ionization detection. leachate. concentrate Groundwater. clean-up GC/MS 10 μg/L GC/ECD 0. liquid detergents. desorb with carbon disulfide Extract in liquid–solid extractor. industrial and lubricating oils and defoaming agents during paper and paperboard manufacture (Environmental Protection Agency. decorative inks. exchange to hexane and concentrate Extract with dichloromethane. extract with dichloromethane. concentrate GC/PID 2. Other uses are in rubbing alcohol. 1989).44 IARC MONOGRAPHS VOLUME 77 Table 1.2] Environmental Protection Agency (1995b) [Method 506] Environmental Protection Agency (1999a) [Method 606] Environmental Protection Agency (1999b) [Method 625] Environmental Protection Agency (1999c) [Method 1625] Environmental Protection Agency (1996) [Method 8061A] Drinking-water. elute with dichloromethane. dry. exchange to hexane. isolate. munitions. PID. mass spectrometry. electron capture detection. photoionization detection toys. FID. Selected methods for the analysis of di(2-ethylhexyl) phthalate Sample matrix Sample preparation Assay procedurea GC/FID Limit of detection 10 μg/sample Reference Air Collect on cellulose ester membrane filter. gas chromatography. dry over sodium sulfate. concentrate GC/ECD 2.27 μg/L (aqueous) a Abbreviations: GC. and source water GC/MS 0. sediment Aqueous sample: extract with dichloromethane.0 μg/L GC/MS 2. soil. dry. sludge. ECD.5 μg/L Add isotope-labelled analogue. municipal and industrial Extract with dichloromethane.25 μg/L Wastewater. concentrate by evaporation Eller (1994) [Method 5020] Environmental Protection Agency (1995a) [Method 525. Solid sample: extract with dichloromethane/acetone (1:1) or hexane/acetone (1:1).5 μg/L Drinking-water Extract in liquid–liquid extractor. It is also used as an acaricide in .

eastern Asia. due to the chronic nature of the treatment. 1983).1 million tonnes. 765 (Towae et al.4. While occupational inhalation is a significant potential route of exposure... of which di(2-ethylhexyl) phthalate accounted for approximately 2.. 1. 245. because of the widespread use of di(2-ethylhexyl) phthalate in plastic containers and its ability to leach out of PVC. and others. calendering and coating operations). humans are exposed to this substance on a regular basis. umbilical catheterization and short-term cardiopulmonary by-pass can also result in high exposures (Huber et al. World consumption of phthalates in the early 1990s was estimated to be 3. 465. 490.25 million tonnes. 1972). di(2-ethylhexyl) phthalate aerosols are used to test the efficiency of high-efficiency particulate air filters during their manufacture (Roberts. Occupational exposure to di(2-ethylhexyl) phthalate occurs by inhalation. Patients undergoing haemodialysis are considered to have the highest exposure. in the testing of air filtration systems and as a component of cosmetic products (National Toxicology Program. 1992).1 Natural occurrence Di(2-ethylhexyl) phthalate is not known to occur naturally. 1. an inert ingredient in pesticides. Japan.4 Occurrence Concern regarding exposure to di(2-ethylhexyl) phthalate rose to prominence when Jaeger and Rubin (1970) reported its presence in human blood that had been stored in PVC bags. 1999). The extensive manufacture of di(2-ethylhexyl) phthalate-containing plastics has resulted in its becoming a ubiquitous environmental contaminant (Huber et al. essentially in the form of an aerosol (mist) because of the very low vapour pressure of the substance (Fishbein. extracorporeal membrane oxygenation. 1. a detector for leaks in respirators. Karle et al. . 1996. medical procedures such as haemodialysis. 1996). The same authors later reported the presence of di(2-ethylhexyl) phthalate in tissue samples of the lung. Further. 1991). blood transfusion.. Occupational exposure to di(2-ethylhexyl) phthalate may occur during its manufacture and its use mostly as a plasticizer of PVC (compounding. approximately 341 000 workers in the United States were potentially exposed to di(2ethylhexyl) phthalate.DI(2-ETHYLHEXYL) PHTHALATE 45 orchards. The estimated total consumption of di(2-ethylhexyl) phthalate by geographical region was (thousand tonnes): western Europe. 155.2 Occupational exposure According to the 1981–83 US National Occupational Exposure Survey.4. liver and spleen from patients who had received blood transfusions before death (Jaeger & Rubin. 1992). North America. Printing and painting occupations account also for a large number of workers potentially exposed (NOES. Indeed. 1997).

Environment Canada. 1985. Kelly et al.. 1. Giam et al.. 1985. WHO. other phthalates and hydrogen chloride. 1997). Meek et al. 1992. 1997) and the Netherlands (Wams. Human daily intakes of di(2-ethylhexyl) phthalate from various exposure pathways have been estimated (Table 3) (Meek & Chan. 27 tonnes of di(2-ethylhexyl) phthalate were released to air in 1995. 1992.. 1994. according to the Canadian National Pollutant Release Inventory (Environment Canada.. (1996) observed that concentrations in air reported in older studies were well above (up to 60 mg/m3) those determined later. 1985. its metabolites and total phthalates have been shown in a few studies to be higher in di(2-ethylhexyl) phthalate-exposed workers than in non-exposed workers and in post-shift samples than in pre-shift samples. WHO. Vainiotalo & Pfäffli. reported concentrations of total phthalates. 1993). Kelly et al.4. Canada (Meek & Chan.. In Canada. Dirven et al. . 1999d).. Nielsen et al. 1994.. use and disposal and its ubiquitous occurrence and stability in the environment. Wams. 1997). 1985. Huber et al. 1993. however. 1994.3 Environmental occurrence The environmental fate of phthalate esters has been reviewed (Staples et al. generally at very low levels.. 1987) because of the very large quantities that have been emitted during its production. 1982. water.46 IARC MONOGRAPHS VOLUME 77 Few data are available on levels of occupational exposure to di(2-ethylhexyl) phthalate (Table 2). Urinary levels of di(2-ethylhexyl) phthalate. paper and textile mills and refuse incinerators.. in food samples and in human and animal tissues (Peterson & Freeman. air emissions of di(2-ethylhexyl) phthalate from 298 industrial facilities in the United States amounted to 107 tonnes in 1997. depending on the type of industry (Liss et al. Agency for Toxic Substances and Disease Registry. Patients receiving blood products or undergoing treatments requiring extracorporeal blood circulation may be exposed by leaching of di(2-ethylhexyl) phthalate from PVC bags and tubing (Wams. Nielsen et al. in air. (a) Air According to the Toxics Release Inventory (Environmental Protection Agency. sediment soil and biota (with the highest levels found in industrial areas). phthalic anhydride. No standard method has been proposed for biological monitoring of exposure to di(2ethylhexyl) phthalate (Liss et al. 1987. these older studies. It is known to be widely distributed. 1984. e. Other exposures may occur concurrently with exposure to di(2-ethylhexyl) phthalate. Agency for Toxic Substances and Disease Registry. 1984. 1987. 1993a). 1994). 1994. The principal route by which it enters the environment is via transport in air or via leaching from plastics and plasticizer plants or other sources such as sewage treatment plants.. 1990). 1994).g. Di(2-ethylhexyl) phthalate is considered a priority and/or hazardous pollutant in the United States (Environmental Protection Agency. 1996). Huber et al. Sharman et al... precipitation..

technicians and maintenance workers Poly(vinyl chloride) processing industry Thick film department: calender operators/machine attendants Poly(vinyl chloride) processing plants Extrusion Extrusion Calendering Hot embossing Welding Injection moulding Compounding Thermoforming High-frequency welding Poly(vinyl chloride) processing plants Boot factory Mixing process Extruder process Cable factory Mixing process Extruder process USA ND–4.5–3 h 4 5 7 5 4 2 5 2 Personal—2-h 16 Range Personal— whole-shift 50b Nielsen et al.02 Netherlands 0.05 0. of samples Reference DI(2-ETHYLHEXYL) PHTHALATE Dirven et al.02 0.27 ± 0.26 0.02 Personal—2-h 16 11 8 13 0.05 ± 0.01 ± 0.02 ± 0.02 < 0. (1985) Vainiotalo & Pfäffli (1990) Liss & Hartle (1983) Sampling No.05 0.3 0.28 0.1–0.01 ± 0.12 0.02 0.8 Area—1.3 0.5 ± 0.11a Sweden 0. (1993a) 47 . Workplace air levels of di(2-ethylhexyl) phthalate Production Country Air concentration (mg/m3) Mean Di(2-ethylhexyl) phthalate manufacturing plant Chemical operators.4c Finland 0.05–0.18 0.2 ± 0.1–1.03 ± 0.Table 2.24 0.01–0.81 0.5 0.2 0.01–1.

29 ND. not detected Only six measurements were above the detection limit c Presented as total phthalates. but where di(2-ethylhexyl) phthalate was the main plasticizer b .06–0.01–0.48 Table 2 (contd) Production Country Air concentration (mg/m3) Mean Various plants Di(2-ethylhexyl) phthalate manufacture Two aerosol filter testing facilities Poly(vinyl chloride) sheet processing plant a IARC MONOGRAPHS VOLUME 77 Sampling No.14 0. of samples Reference Range Personal—4–5 h Roberts (1983) 8 4 3 USA ND 0.

drink 1.03–0. Erie and Ontario was estimated to amount to 16.. Somewhat similar levels in air. respectively (Eisenreich et al.75 L water and ingest 35 mg soil b Assumed to weigh 13 kg.. The concentration was one to two orders of magnitude lower in maritime air than in continental atmospheres (Giam et al.014 14 000 12–19 yearsd 0. drink 0.064 8900–9100 0. 1980.. 1978.03–0.9 L water and ingest 35 mg soil d Assumed to weigh 57 kg.5 yearsa Ambient air: Great Lakes region Indoor air Drinking-water Food Soil Total estimated intake 0.3 860 130–380 7900 0.1 ng/m3 (Giam et al. The total deposition of di(2-ethylhexyl) phthalate into Lakes Superior. 3 990 60–180 18 000 0.3 950 20–70 7200 0. 1992: Agency for Toxic Substances and Disease Registry. the Gulf of Mexico and on Enewetak Atoll in the North Pacific and levels ranged from not detectable to 4. 2 ng/m3 ) of di(2ethylhexyl) phthalate have been found in the Great Lakes ecosystem (Canada and United States).5 L water and ingest 20 mg soil Di(2-ethylhexyl) phthalate concentrations of up to 790 ng/m3 have been found in urban and polluted air. . 1980). Concentrations of di(2-ethylhexyl) phthalate in the atmosphere of the northwestern Gulf of Mexico averaged 1. Huron. breathe 2 m3 air.4 1200 30–100 13 000 0.04 8200 20–70 yearse 0. wash-out by rain appears to be a significant removal process (Atlas & Giam. 1981). drink 0. 1992). Giam et al. breathe 23 m3 air.7 tonnes per year..3 850 20–60 4900 0. breathe 21 m3 air.03–0. 1981). 6 ng/L).04–0.5–4 yearsb 0. The concentration of di(2-ethylhexyl) phthalate in precipitation ranged from 4 to 10 ng/L (mean. 11. breathe 5 m3 air. Atmospheric fluxes to the Great Lakes are a combination of dry and wet removal processes. WHO. 1981. Atlas & Giam.3 L water and ingest 20 mg soil e Assumed to weigh 70 kg.DI(2-ETHYLHEXYL) PHTHALATE 49 Table 3. breathe 12 m3 air. drink 1.042 19 000 5–11 yearsc 0. between 0. Michigan.16 ng/m3 for ten samples.5 and 5 ng/m3 (mean. 5. but usually the levels in ambient air are well below 100 ng/m3 (WHO.0 and 3.03 5800 From Meek & Chan (1994) a Assumed to weigh 6 kg. with 57% of the compound measured in the vapour phase only. Di(2-ethylhexyl) phthalate released into air can be carried for long distances in the troposphere and it has been detected over the Atlantic and Pacific Oceans.8 L water and ingest 50 mg soil c Assumed to weigh 27 kg. drink 0. 12. 1993). 1978. 1984. Di(2-ethylhexyl) phthalate in air has been monitored in the North Atlantic.03–0. Estimated daily intake of di(2-ethylhexyl) phthalate by the population of Canada Substrate/medium Estimated intake for various age ranges (ng/kg bw per day) 0–0.

1993). 1987. There is a paucity of data concerning concentrations of di(2-ethylhexyl) phthalate concentrations in indoor air.5 to 31 ng/m3 (normalized) and from 34. Texas.0 ng/m3 (range. respectively (Bove et al. ranged from < 2 to 12 000 ng/L (average. 1990). respectively. The total annual fallout of di(2-ethylhexyl) phthalate in Sweden was estimated to be 130 tonnes (Thurén & Larsson. The median air concentration was 2. It was suggested that suspended particulate exposure to di(2-ethylhexyl) phthalate is one. The concentration of di(2-ethylhexyl) phthalate in air at Lyngby. 1983). respectively. The concentrations of di(2-ethylhexyl) phthalate ranged from 20.5 μg/m2 per month). The yearly average concentrations at three air sampling stations in New York City in 1978 ranged from 10 to 17 ng/m3. at the two monitoring stations (Guidotti et al. 1977).. in a number of Oslo dwellings. Belgium (Cautreels et al. (b) Water and sediments In general. 0. Di(2-ethylhexyl) phthalate has been shown to account for 69 and 52% of the total amount of phthalates adsorbed to sedimented dust and particulate matter. Agency for Toxic Substances and Disease Registry. 1997). 1985). 1999d).50 IARC MONOGRAPHS VOLUME 77 In Sweden. as reported in the Environmental Protection Agency Toxics Release Inventory (Environmental Protection Agency.. It was found at levels of 11–210 μg/100 mg [110– 2100 mg/kg] sedimented dust in 38 dwellings and at levels of 24–94 μg/100 mg [240–940 mg/kg] suspended particulate matter (mean ± SD.8 μg/m2 per month (range. Levels of 26–132 ng/m3 were measured in four samples from a residential area in Antwerp. concentrations of di(2-ethylhexyl) phthalate in fresh waters are in the range of < 0.5 ng/m3 (normalized).3–77 ng/m3) and the average fallout was 23. although occasionally much higher values have been observed (~ 100 μg/L) when water basins are surrounded by heavy concentrations of industry (WHO. although its volatilization from plastic products has been noted (Wams... 1998). Di(2-ethylhexyl) phthalate concentrations in water from Galveston Bay. 1981). Denmark was calculated to be 22 ng/m3 based on the analysis of snow samples (Løkke & Rasmussen.. 1993). During 1995. 1978). somewhat higher than those found earlier for the Mississippi Delta .. four sets of samples from two monitoring stations of the breathable fraction of atmospheric particulates including phthalates were measured in the air of Rieti urban area in Italy. 60 ± 30) in six dwellings. Di(2-ethylhexyl) phthalate has been detected in 24% of 901 surface water supplies at a median concentration of 10 μg/L and in 40% of 367 sediment samples at a median concentration of 1000 μg/kg in samples recorded in the STORET database in the United States (Staples et al. 600 ng/L) (Murray et al. 6.to three-fold higher than the estimated vapour phase exposure (Øie et al.0– 195.1–10 μg/L.8 to 503. air concentrations of di(2-ethylhexyl) phthalate were measured at 14 monitoring stations (53 samples). Surface water discharges of di(2-ethylhexyl) phthalate from 298 industrial facilities in 1994 in the United States amounted to 264 kg. 1992. Agency for Toxic Substances and Disease Registry.

. 1983. Ritsema et al. 1986)..98 μg/L.. Liverpool.6 μg/L was found in the River Rhine near Lobith and levels ranging from < 0. 1989. The Netherlands (Ritsema et al. average. In a 12-day survey. which serves as a sink (WHO. 1980. Di(2-ethylhexyl) phthalate has been reported in the leachate from municipal and industrial landfills at levels ranging from < 0. Concentrations ranged from 0. 1995). Giam & Atlas.DI(2-ETHYLHEXYL) PHTHALATE 51 (23–225 ng/L.3 ng/L were found in the IJsselmeer. 1984). and ranged from 190 to 700 μg/kg near industrial discharges in marine sediments around coastal Taiwan (Jeng.10 μg/L and 0. Rhode Island. 1989).. 1984).. Ray et al..01 to 150 μg/mL (Ghassemi et al. Since di(2-ethylhexyl) phthalate is lipophilic..125 to 0.3 μg/L between January 1992 and February 1993. 1979. Thurén. United States. 1992). it was shown that biodegradation by the surface microlayer biota accounted for at least 30% of the removal of di(2-ethylhexyl) phthalate (Davey et al. Preston & Al-Omeron. In a study of the degradation of di(2-ethylhexyl) phthalate . Levels of di(2-ethylhexyl) phthalate up to 720 ng/L were found in two sampling stations of the Mississippi River in the summer of 1984 (DeLeon et al. 1990). Water solubility is a major factor limiting the degradation of phthalate esters under methanogenic conditions.32–3. Levels of up to 1.39–1.693 μg/L (Preston & Al-Omran. Di(2-ethylhexyl) phthalate has been measured in rivers and lake sediments in Europe (Schwartz et al.9 μg/L di(2-ethylhexyl) phthalate were found in rivers of the greater Manchester area. 1986). 1995). average.1 to 0. 1986. 1986. Tan. It has also been detected in 13% of 86 samples of urban storm water runoff evaluated for the National Urban Runoff Program at concentrations ranging from 7 to 39 μg/L (Cole et al.. it tends to be absorbed onto sediment.029 to 70 mg/kg. 1995). concentrations of di(2-ethylhexyl) phthalate in sediments were above 1000 mg/kg (Thurén. Levels of dissolved di(2-ethylhexyl) phthalate in samples from the River Mersey estuary.. 1990) and at unspecified levels as contaminants of the Elbe River and its tributaries in Germany during the period 1992–94 (Franke et al. Hollyfield & Sharma. 1989). The highest value was in samples taken near an industrial effluent discharge (Thurén. di(2-ethylhexyl) phthalate at levels ranging from 0. 1978). 1986). United Kingdom. Levels of di(2-ethylhexyl) phthalate in water samples from 12 stations in the Klang River Basin in central Malaysia ranged from 3. ranged from 0. Near direct discharge points from industry in Sweden and Malaysia. United Kingdom (Fatoki & Vernon. 1989) and in river and bay sediments in the United States (Peterson & Freeman.2 to 0. 130 ng/L) (Giam et al. Levels of di(2-ethylhexyl) phthalate in two rivers in southern Sweden were 0.. In experimental studies of a marine environment of Narragansett Bay. 70 ng/L) and the Gulf of Mexico coast (6–316 ng/L. 1995).1 to 64. The highest levels of phthalates in the water and sediment samples were collected near industrial areas where direct discharge points were found (Tan. 1982.

1993). the culture readily degraded 2-ethylhexanol via 2-ethylhexanoic acid to methane. 1994. 33 tonnes of di(2-ethylhexyl) phthalate were released from Canadian facilities to land (Environment Canada. 1983). Agency for Toxic Substances and Disease Registry. Sharman et al.3 mg/day per individual.5 mg/kg dry matter (Wams. Petersen. 2-ethylhexanol and mono(2-ethylhexyl) phthalate in a methanogenic phthalic acid ester-degrading enrichment culture at 37 °C. 1992. The highest levels of di(2-ethylhexyl) phthalate have been measured in milk products. Giam & Wong. 1987). 1990. According to the Canadian National Pollutant Release Inventory. 1995). 1994). (d) Foods The most common route of human exposure to di(2-ethylhexyl) phthalate is through food contamination. Five soils and leachate-sprayed soils from the Susquehanna River basin in Pennsylvania and New York had levels of di(2-ethylhexyl) phthalate ranging from 0. Ejlertsson et al. meat and fish as well as in other products which have a high fat content. 1996). 1993. WHO.. butter. 1993. Residues of di(2ethylhexyl) phthalate in soil collected in the vicinity of a di(2-ethylhexyl) phthalate manufacturing plant amounted to up to 0. 1994). The average daily exposure from food in the United States has been estimated to be about 0. margarine. 1999d). Di(2-ethylhexyl) phthalate has been found at generally low levels in a broad variety of foods. Releases of di(2ethylhexyl) phthalate to land from 298 industrial facilities in the United States in 1997 amounted to 32 137 kg (Environmental Protection Agency. cereals. 1997). fish and other seafood (Cocchieri. 1986.2 mg/kg (Russell & McDuffie. manufacturing processes or from the animals which had produced the milk or meat (Giam & Wong. . 1997). Gilbert.52 IARC MONOGRAPHS VOLUME 77 and its intermediate hydrolysis products. The use of di(2-ethylhexyl) phthalate in food contact applications is reported to be decreasing (Page & Lacroix.. It can originate from PVC wrapping materials. 1992. cheese. Gilbert. meat. 1987. 1997). Contaminated soil in the Netherlands was found to contain up to 1. Bauer & Herrmann. Additionally. di(2-ethylhexyl) phthalate from various sources such as food wraps is released to municipal waste. mono(2-ethylhexyl) phthalate was degraded to stoichiometric amounts of methane with phthalic acid as a transient intermediate. Castle et al. (c) Soil The principal source of di(2-ethylhexyl) phthalate release to land is disposal of industrial and municipal waste to landfills (Agency for Toxic Substances and Disease Registry. 1978). with a maximum exposure of 2 mg/day (WHO. while di(2-ethylhexyl) phthalate remained unaffected throughout the 330-day experimental period (Ejlertsson & Svensson.5 mg/kg (Persson et al... Waste disposal of PVC products containing varying amounts of di(2-ethylhexyl) phthalate to landfills is another source (Swedish Environmental Protection Agency. 1987. including milk. Agency for Toxic Substances and Disease Registry. 1996. 1991.001 to 1.

When di(2-ethylhexyl) phthalate was not present in the wrapper.28 μg/kg. Concentrations of di(2-ethylhexyl) phthalate in 10 samples of retail cream and 10 samples of butter obtained in the United Kingdom ranged from 0.55 μg/g (Sharman et al. an average level in the butter of 9. whereas low-fat milk contained from < 0. the concentration of di(2-ethylhexyl) phthalate was below 0.05 μg/g [mg/L] in the central collecting tank.9 mg/kg. pooled milk samples from doorstep delivery in different regions of the country contained < 0. In the United Kingdom.01 to 0. Thirteen retail milk and cream products from Spain had levels of di(2-ethylhexyl) phthalate ranging from < 0.01–0. An investigation of residues of di(2-ethylhexyl) phthalate in retail whole milk samples from 14 Danish dairies about six months after the use of di(2-ethylhexyl) phthalate-plasticized milk tubing was banned in Denmark in August 1989 revealed a mean concentration lower than 50 μg/L (Petersen.9 to 11.. Samples of Norwegian milk obtained at various stages during collection. respectively. On processing the di(2-ethylhexyl) phthalate was concentrated in the cream at levels up to 1. Analysis of the wrappers showed little correlation between the levels of di(2-ethylhexyl) phthalate in the total wrapper and the corresponding food.93 μg/g. transportation and packaging operations showed no apparent trends in phthalate contamination. Retail samples of Canadian butter and margarine wrapped in aluminium foil–paper laminate were found to contain di(2-ethylhexyl) phthalate at levels up to 11.9 mg/kg and six samples of margarine (454 g each) had levels ranging from 0.005 μg/g [mg/L].09 μg/g [mg/L] di(2ethylhexyl) phthalate.03 μg/g [mg/L] and rose to 0. a background level of di(2-ethylhexyl) phthalate from about 3 to 7 mg/kg was found in butter while.4 μg/g.2 to 2.5 to 7.DI(2-ETHYLHEXYL) PHTHALATE 53 Di(2-ethylhexyl) phthalate was determined in milk. Ten samples of butter (454 g each) had levels of di(2-ethylhexyl) phthalate ranging from 2. 1992).8 to 11.01 to 0. Concentrations for each individual cow averaged 0. Di(2-ethylhexyl) phthalate was found in both the packaging and in a number of contacted foods sampled in a 1985–89 survey as part of the Canadian Health Protection Branch Total Diet Program.4 mg/kg of the phthalate was found (Page & Lacroix. In control milk samples obtained by hand-milking. Low levels (65 μg/kg [L] average in beverages and 29 μg/kg average in foods) associated with the use of di(2-ethylhexyl) phthalateplasticized cap or lid seals were found in a variety of glass-packaged foods. with di(2-ethylhexyl) phthalate present in the wrapper. It was . di(2-ethylhexyl) phthalate in milk tubing has been replaced by other types of plasticizers. cream.12 and 0. Spain and the United Kingdom.7 μg/g and 2. 1990). such as di(2-ethylhexyl) adipate (see monograph in this volume) and diisodecyl phthalate (Castle et al.3 mg/kg.07 μg/g [mg/L]. 1991).. In Norway and the United Kingdom. with total phthalate levels (expressed as di(2-ethylhexyl) phthalate equivalents) in the raw milk of between 0. 1994). butter and cheese samples from a variety of sources from Norway. Milk samples were collected from a dairy in Norway at various stages of the milking process to determine the extent of migration of di(2-ethylhexyl) phthalate from plasticized tubing used in commercial milking equipment.

Di(2-ethylhexyl) phthalate was found (mg/kg wet weight) in the following commercial fish (pooled samples of 10 individuals each): herring (fillets).7 μg/L (mean. 1998). 84 and 52% of Italian plastic packaged salted meat.50. It was also found in 13 poly(ethylene terephthalate) bottled drinking-water samples (sealed with caps with plastic internal gasket) at levels ranging from 2. Rock et al. Di(2-ethylhexyl) phthalate was found in nine varieties of factory-packaged non-frozen meats at levels that ranged from 0.21 and 2.3 and 3. the concentration of di(2-ethylhexyl) phthalate increases with storage time and it is converted by a plasma enzyme to a more toxic metabolite.5 mg/kg. 1972.. 1993). 1981.19. and redfish (fillets).010 (Musial et al. 71. The mean di(2-ethylhexyl) phthalate levels ranged between 0.4 to 17. on a butter–fat basis.... di(2-ethylhexyl) phthalate was also found in 12 glass bottled drinking-water samples (sealed with caps with plastic internal gasket) at levels ranging from 2. plaice (fillets).0 μg/L). with hardly any differences between the food items (Gruber et al. respectively.2 mg/kg in halibut and 2. 1995). 1981).71. Rock et al.1–3. < 0. 2.9 mg/kg. Cole et al. infusion–transfusion. poultry and fish ranged from 0..2 mg/kg] and. Low incidence and low levels of di(2-ethylhexyl) phthalate were found in total diet samples of fruits and vegetables (mostly not detected to 0. dialysis systems or feeding tubes and catheters in disposable medical devices). 4.. baby food and milk samples. platelet concentrates and plasma during storage. 5.1 mg/kg in pollack and in two smoked salmon samples were 0.8 μg/L (mean.38 mg/kg (Cocchieri.1 to 3. and in all the cheese and vegetable soups samples. mothers’ milk and cows’ milk were analysed for their content of phthalates found a relatively narrow range of 50–210 mg/kg di(2-ethylhexyl) phthalate. Levels in factory-packaged fish were 0.4 mg/kg.g. 6. Analysis of dairy food composite samples showed the presence of di(2-ethylhexyl) phthalate in all samples at 0. < 0. It is known to leach from PVC blood packs into whole blood.. In an investigation of 2-ethyl-1-hexanol as a contaminant in some samples of bottled Italian drinking-water.5 μg/L) (Vitali et al.010.07 mg/kg) (Page & Lacroix.6 mg/kg and.02 to 1. 1995). mackerel (fillets).54 IARC MONOGRAPHS VOLUME 77 found in 14 types of cheese at levels up to 5. 1978. in total diet cereal products. baby food. mono(2-ethylhexyl) phthalate (Rock et al.5 mg/kg [average. 1978). Di(2-ethylhexyl) phthalate has been detected in the blood and tissues of patients receiving blood transfusions and haemodialysis treatments (Jaeger & Rubin.. 6.7 mg/kg (Page & Lacroix. Di(2-ethylhexyl) phthalate was detected in 80. 10. ranged from 0.1 to 2. cod (liver). these levels averaged about 8 mg/kg di(2-ethylhexyl) phthalate. Sasakawa & Mitomi.7 to 31. (e) Exposure from medical devices Di(2-ethylhexyl) phthalate at concentrations up to 40% by weight is generally used as a plasticizer in PVC materials which have been widely used for a variety of medical purposes (e. jam. 1978. The levels in total diet samples of meat. . A German study in which 22 samples of baby milk. 1986).

6 mg/mL (Sjöberg et al. 1986). 1985b). in six patients receiving a third blood unit. Di(2-ethylhexyl) phthalate was detected in whole blood at levels ranging from 16. .8 and 19. Immediately after the transfusions.6 μg/mL in cryoprecipitate and 7. In 13 newborn infants receiving a second blood unit..4 to 15. the serum levels ranged from 24.and mono(2-ethylhexyl) phthalates were similar in PVC bags stored for four days or less (Rock et al. Both values were independent of the storage period (Sasakawa & Mitomi.9 to 93. respectively.1 to 21. the plasma levels of di(2-ethylhexyl) phthalate in six patients varied between 3. 1978).0 and 15.3 and 0. There were indications that about 30% of the infused di(2-ethylhexyl) phthalate originated from parts of the transfusion set other than the blood bag.5 ± 6.6 to 55. and 14-day storage levels of 432 ± 24.20 mg/kg bw. 1991).8 mg/L and. In an additional study to investigate further the disposition of di(2-ethylhexyl) phthalate and mono(2-ethylhexyl) phthalate during a single exchange transfusion in four newborn infants.4 and 11.. 1991. The half-life of this phase was approximately 10 h (Sjöberg et al. In one study of newborn infants who received exchange transfusion.1 mg/L (Plonait et al. Huber et al. the amounts of di(2-ethylhexyl) phthalate and mono(2-ethylhexyl) phthalate infused ranged from 0.7 ± 4.6 mg/L and subsequently declined rapidly (reflecting its distribution within the body).2 mg/L) after a single exchange transfusion. Serum levels of di(2-ethylhexyl) phthalate in 16 newborn infants undergoing exchange transfusion indicated an undetectable level (< 1 mg/L) before exchange but levels ranging from 6. The accumulation of di(2-ethylhexyl) phthalate in platelet-poor plasma stored for seven and 14 days in PVC bags sterilized by steam.05 to 0. while mono(2-ethylhexyl) phthalate in these samples ranged between 3. 362 ± 10 and 275 ± 15 mg/L..1 μg/mL [mg/L] and in packed cells at levels ranging from 32.9 mg/L.8 μg/mL in fresh frozen plasma. In newborn infants subjected to a single exchange transfusion. The concentrations in blood of both di.5 μg/mL [mg/L] in PVC blood bags stored at 5 °C.. Dine et al. 1993)..6 mg/L serum (average. 428 ± 22 and 356 ± 23 mg/L. 1996. The degree of exposure to di(2-ethylhexyl) phthalate was assessed in 11 patients undergoing haemodialysis for treatment of renal failure and showed that on average. 1996a)... while mono(2-ethylhexyl) phthalate levels in the corresponding samples ranged from 2. respectively (Dine et al. ethylene oxide or irradiation revealed seven-day storage levels of di(2-ethylhexyl) phthalate of 378 ± 19.8 to 3. concentrations of di(2-ethylhexyl) phthalate in plasma from the blood taken from the transfusion set varied between 36.8 to 46..4 ± 2. Christensson et al.. 1991.3 to 87. Mettang et al. Approximately 30% of the infused amount of di(2-ethylhexyl) phthalate was withdrawn during the course of each transfusion.1 mg/L. These levels increased with storage. respectively.1 mg/L. the plasma levels of di(2-ethylhexyl) phthalate levels ranged between 5.DI(2-ETHYLHEXYL) PHTHALATE 55 1986. 1985a). The average content was 6. 12. followed by a slower elimination phase.8 and 84. the serum levels of di(2-ethylhexyl) phthalate ranged from 12.

p = 0.017). 0.073–0.11 mg/L).1–0.129 mg/L.17) to 0. 0.05 mg/L) (Christensson et al.05 mg/mL (range.022 mg/L (range.05–0. Assuming a schedule of treatment three times per week.8–360 mg.097–0. with a range of 3. Remarkably high concentrations of phthalic acid (0. di(2-ethylhexyl) phthalate was not detected (< 0. 0. 1996a). 1991).058 mg/L) (p = 0. During the first 4 h of dwell time.075–0. < 0.4 mg/L) (Nässberger et al.659 mg/L.034 mg/L. range.016–0. 2-ethylhexanol and phthalic acid in seven elderly patients undergoing regular CAPD were compared with those in six aged-matched healthy controls during a 4-h dwell period.8 to 4.137–0.33 ± 0.58 mg/L) were similar to those of di(2-ethylhexyl) phthalate (1.038– 0. the average patient in the study would have received approximately 16 g di(2-ethylhexyl) phthalate over the course of a year. In three of the CAPD patients and in all of the predialysis patients. while the concentration of di(2-ethylhexyl) phthalate remained stable.56 IARC MONOGRAPHS VOLUME 77 an estimated 105 mg di(2-ethylhexyl) phthalate was extracted from the dialyser during a single 4-h dialysis session.177 mg/L (range.062) (Mettang et al.10 mg/L (range. with a range of 23.231 mg/L.6) (detection limit. and these concentrations tended to increase during dwell time but without statistical significance (0. 0.9 mg/L in four of seven continuous ambulatory peritoneal dialysis (CAPD) patients.064 mg/L) (p = 0. 0.466 mg/L) were found in the CAPD bags before use. respectively.017).2 mg/L serum in 17 haemodialysis patients after dialysis and 0. respectively) than in controls (median. Levels of di(2-ethylhexyl) phthalate ranging from < 1 to 4100 μg/mL [mg/L] in the condensate from water traps of six respirators have been reported.30–1.0195 mg/L. 1985).087 mg/L (range. Estimation of the inhalatory di(2-ethylhexyl) phthalate exposure to five artificially ventilated preterm infants over a 24-h period yielded values ranging between 1 μg/h and 4200 μg/h.005–0. During treatment with tubing containing di(2-ethylhexyl) phthalate.135 mg/L.006–0.167 mg/L.097 mg/L) to 0. Time-averaged circulating concentrations of mono(2-ethylhexyl) phthalate during the session (1. and 0. A comparative evaluation of haemodialysis tubing plasticized with di(2ethylhexyl) phthalate and with tri-2-ethylhexyl trimellitate was made in 11 patients (10 men. 0. respectively) in patients (median. 1987).012 mg/L. and 0.032–0. the concentrations of mono(2-ethylhexyl) phthalate and 2-ethylhexanol in dialysate consistently decreased from 0. respectively). Serum concentrations of di(2-ethylhexyl) phthalate and phthalic acid were significantly higher (p = 0. 0. in no case could the hydrolysis product mono(2-ethylhexyl) phthalate be detected (< 0. range. 0.1 mg/L).. 0. Di(2- .023–0.7–56 g (Pollack et al.027 and p = 0. range. range.239 mg/L) to 0. range.026. The concentration of mono(2-ethylhexyl) phthalate in the fluid of CAPD bags before use was four times higher than that of di(2-ethylhexyl) phthalate. one woman) with chronic renal failure on haemodialysis for a period of six months. The degree of exposure to and the fate of di(2-ethylhexyl) phthalate and derived mono(2-ethylhexyl) phthalate. Di(2-ethylhexyl) phthalate was found at concentrations ranging from 0. 0.91 ± 2... range.210 mg/L.70 mg/L (range. and from 0.079 mg/L. 0. 0. 0. the plasma level of di(2-ethylhexyl) phthalate rose from 0.025 mg/L.. 0.

The serum levels of di(2-ethylhexyl) phthalate after 14 and 24 days of ECMO support were 26.. and levels of 3. The exposure to di(2-ethylhexyl) phthalate for a 4-kg infant on ECMO support for 3–10 days was estimated to be 42–140 mg/kg (Shneider et al. heart and testicular tissues.5 mg/L respectively.4 mg/kg di(2-ethylhexyl) phthalate were found in liver.5.1 mg/L per hour). The Food and Drug Administration (1999) permits the use of di(2-ethylhexyl) phthalate in the United States as a component of adhesives used in food packaging. 1998. Wilkinson & Lamb.. and the estimated exposure over 3–10 days was 10–35 mg/kg bw. The World Health Organization has established an international drinking-water guideline for di(2-ethylhexyl) phthalate of 8 μg/L (WHO. and trace quantities were found in the brain. The rate of di(2-ethylhexyl) phthalate extraction from the model PVC circuits was linear with time (rate.DI(2-ETHYLHEXYL) PHTHALATE 57 ethylhexyl) phthalate (0. 1. The Czech Republic has set a maximum limit for plastic materials for di(2-ethylhexyl) phthalate of 50 mg/g as a component of plastic products permitted for contact with food (UNEP. as a plasticizer in resinous and polymeric coatings used in food packaging. 1988) Serum samples and autopsy specimens were examined from two infants with congenital diaphragmatic hernia who had received life support with extracorporeal membrane oxygenation (ECMO).0 and 0.3 ± 5. 1999). Exposure of children to di(2-ethylhexyl) phthalate by migration from PVC toys and other articles into saliva has been reported. as a component of cellophane where total phthalates do . as a component of defoaming agents used in the manufacture of paper and paperboard used in food packaging. in particular di(isononyl) phthalate (Steiner et al. as a flow promoter in food contact surfaces not to exceed 3 wt% based on monomers. it has been replaced in most countries by other plasticizers..5 Regulations and guidelines Occupational exposure limits for di(2-ethylhexyl) phthalate are given in Table 4. The Environmental Protection Agency (1998) has set the maximum contaminant level (MCL) for di(2ethylhexyl) phthalate in drinking-water at 6 μg/L in the United States. 1997). For standard 3–10-day treatment courses. Since then.7 mg/L. 1989). 1.8 and 33. No leaching of di(2ethylhexyl) phthalate from heparin-coated PVC circuits was detected (Karle et al. 3. 1993).. Until the early 1980s.5 and 4. the mean peak plasma concentration of di(2-ethylhexyl) phthalate was 8.23 mg/kg wet weight) was found in the lung tissue of one infant who died of pneumothorax soon after birth following artificial ventilation (Roth et al. 1999). A more recent study of 18 infants on ECMO life support also reported leaching of di(2-ethylhexyl) phthalate from the PVC circuits at linear rates that were dependent on the surface area of the circuit. respectively. di(2-ethylhexyl) phthalate was the predominant plasticizer used in soft PVC children’s products.

Occupational exposure limits for di(2-ethylhexyl) phthalatea Countryb Year Concentration (mg/m3)b 5 10 5 10 5 10 5 10 5 10 5 (Ca) 5 (sk) 10 5 10 5 (sk) 10 5 10 5 5 5 1 5 1 5 10 3 5 5 5 10 Interpretationb Argentina Australia Belgium Canada Czech Republic Denmark Finland France Germany Hungary Ireland Japan Netherlands Philippines Poland Russian Federation Slovakia Sweden Switzerland United Kingdom 1991 1993 1993 1994 1993 1993 1998 1993 1999 1993 1997 1998 1993 1993 1998 1993 1993 1993 1993 1993 TWA STEL TWA STEL TWA STEL TWA STEL TWA STEL TWA TWA STEL TWA TWA TWA STEL TWA STEL TWA TWA TWA TWA STEL STEL TWA STEL TWA STEL TWA TWA STEL . Table 4. The European Pharmacopoeia identifies di(2-ethylhexyl) phthalate as a substance that may be used in the manufacture of PVC plasticized containers and tubing for human blood and blood components.58 IARC MONOGRAPHS VOLUME 77 not exceed 5%. at a level of not more than 40% in the plastic (Council of Europe. 1997). as a component of surface lubricants used in the manufacture of metallic articles that contact food and as a food-packaging plasticizer for foods of high water content.

Most subjects (135/221) were hired after 1965 and the process was completely enclosed in 1966. STEL. Ca. short-term exposure limit. restrictions on use of phthalates as plasticizers (softeners) in PVC toys and baby care items (Anon. sk. rattles and teethers shall not intentionally contain di(2-ethylhexyl) phthalate [ASTM F 963-96a] (American Society of Testing and Materials. C. Altogether. Singapore and Viet Nam In the United States. Several countries in Europe also have proposed. New Zealand. threshold limit value c The following countries follow the exposure limits suggested by the ACGIH: Bulgaria. A3. eight deaths . time-weighted average. but appeared to be complete for the remaining cohort. including di(2-ethylhexyl) phthalate. Jordan. The European Union has temporarily banned the use of six phthalates. there is a voluntary industry standard that states that pacifiers. animal carcinogen. or are considering. 2.DI(2-ETHYLHEXYL) PHTHALATE 59 Table 4 (contd) Countryb Year Concentration (mg/m3)b Interpretationb United States OSHA (PEL) NIOSH (REL) ACGIHc (TLV) a 1999 1997 1999 5 5 (Ca) 10 5 (A3) TWA TWA STEL TWA From Finnish Ministry of Social Affairs and Health (1998).. Information on vital status for foreigners [number not stated] was obtained for only 55% of them. Cohort study Occupational exposure Studies of Cancer in Humans The mortality of 221 workers in a di(2-ethylhexyl) phthalate production plant in Germany was followed between 1940 and 1976. suspected of being a carcinogen. the Rheinhessen-Pfalz land) and national rates. Deutsche Forschungsgemeinschaft (1999). UNEP (1999) b Abbreviations: TWA. skin designation. Occupational Safety and Health Standards (1999). REL. permissible exposure limit. 1999). National Library of Medicine (1998). Reference rates were obtained from local populations (the city of Ludwigshafen. Republic of Korea. potential occupational carcinogen. No information on level of exposure was provided. PEL. Colombia. in toys and other articles intended for children aged under three years of age and designed to be put in the mouth. TLV. recommended exposure limit. 1997).

were fed diets containing 3000 or 6000 mg/kg diet (ppm) di(2-ethylhexyl) phthalate (> 99% pure) for 103 weeks.9 expected using local rates [standardized mortality ratio.0 expected using national rates. 19/50. immunodeficiency) or incidental exposures from treatment (viruses. p = 0.60 IARC MONOGRAPHS VOLUME 77 occurred during the follow-up period versus 15. In male mice. Survival at the end of the study was more than 60% in males and more than 50% in females. 1991). 14/50. 3.50. six weeks of age.1. high-dose.13 expected) and one from bladder papilloma (0.3(e)).1 3. p = 0. 9/50. In conclusion. . The Cochran–Armitage test also indicated a significant trend (p = 0. low-dose. 0.] Dialysis patients Long-term dialysis patients are likely to experience elevated exposures to di(2ethylhexyl) phthalate. Fisher’s exact test). Fisher’s exact test). Cancer risk among dialysis patients has been specifically studied because of concern about medical conditions (for example.4.99] and 17. low-dose. In females. [The Working Group noted that the majority of the cohort members were employed after exposure levels had been considerably reduced. 29/50. 14/48.013. The incidence of hepatocellular adenomas and carcinomas combined was also increased in males (control.. One death from pancreatic cancer (0. significant increases in the incidence of hepatocellular carcinomas were seen (control. through frequent and protracted exposure to substances leached from surgical tubing during dialysis (see Section 1. However. There was a clear doserelated decrease in body weight gain in females.002. Due to the medical condition of this population.01 expected) occurred among workers with a long exposure time (≥ 20 years). low-dose.1 Studies of Cancer in Experimental Animals Oral administration Mouse Groups of 50 male and 50 female B6C3F1 mice. the Working Group was not aware of any study of dialysis patients for which study methods were suitable for the evaluation of carcinogenic risk associated with di(2ethylhexyl) phthalate. follow-up is usually very short and incompatible with induction of chemical carcinogenesis. p = 0.. 95% confidence interval. p = 0.006. significant increases in the incidence of hepatocellular carcinomas were observed (control. 1978). All surviving mice were killed at 104–105 weeks. drugs) (Inamoto et al.22–0.018). 0. 7/50. highdose. exposure to di(2ethylhexyl) phthalate resulting from treatment has not been studied as such in relation to cancer risk. and that the methods for this study were poorly described. No further report on a longer follow-up for this cohort was available to the Working Group (Thiess et al. 25/48. 3.022. 0/50. High-dose males had a slightly decreased body weight gain.

1982. National Toxicology Program.. 14/60 (100 ppm). High-dose male rats had significant increases (p = 0. More than 60% of the animals survived to the end of the study.05. 17/50. 7/65. about 80% of the untreated controls and treated animals in the other groups survived. 19/65. high-dose.7%) for 104 weeks.1982.001. six weeks of age. 37/70 (6000 ppm) and 15/55 (recovery group). There was a clear dose-related decrease in body weight gain in females.2 Rat Groups of 50 male and 50 female Fischer 344 rats. 18/50. Two subgroups of 15 additional mice in the 0 and 6000 ppm groups were designated for measurement of cell proliferation.DI(2-ETHYLHEXYL) PHTHALATE 61 high-dose. All surviving rats were killed at 104–105 weeks. Hepatocellular tumour incidences in males were: 8/70 (control). 1999) 3. low-dose.] The incidence of hepatocellular carcinomas alone or neoplastic nodules alone was not significantly increased. 6/49.1.05. There was a dose-related decrease in body weight gain in both sexes but no effect on survival. Fisher’s exact test) (David et al. p < 0. All but the 100 ppm group differed significantly (p < 0. the incidence of hepatocellular carcinomas was increased in high-dose rats (8/50. Fisher’s exact test) and of hepatocellular adenoma and carcinoma combined (control. Groups of 65 male and 65 female B6C3F1 mice. Other groups of 50 males and 50 females served as controls. Another group of 65 male and 65 female mice received the highest concentration for 78 weeks and then control diet for a further 26 weeks (recovery group). Fisher’s exact test) compared with controls (0/50) and that of neoplastic nodules was also . In female rats.001. The survival of male mice treated with 6000 ppm was significantly lower than that of the controls. 12/49). 6000-ppm and recovery groups differing significantly from the concurrent control group (p < 0.003. 21/65 (500 ppm). Fisher’s exact test) from the control group. All surviving mice were killed at 105 weeks for histopathological examination. were fed diets containing 0. p < 0. 3/50. 1500 or 6000 ppm di(2-ethylhexyl) phthalate (purity.. 44/70 and 30/55. 12/50. 4/60. five to six weeks of age. 27/65 (1500 ppm). The respective incidences in females were 3/70. The Cochran–Armitage test also indicated a significant trend (p = 0.007).. 1985). 100. were fed diets containing 6000 or 12 000 mg/kg diet (ppm) di(2-ethylhexyl) phthalate (> 99% pure) for 103 weeks. These additional animals were included in the final analysis. high-dose. 99. Kluwe et al. trend and Fisher’s exact tests) (Kluwe et al. low-dose. with the 1500-ppm. biochemical analysis for peroxisome proliferation and histopathological evaluation and were killed at week 79. 500. [The Working Group noted that the term neoplastic nodule is now generally assumed to represent hepatocellular adenomas. 1983. more than 60% of the animals survived until the end of the study. In females.01. Fisher’s exact test) in the combined incidence of hepatocellular carcinomas and neoplastic nodules (control. p = 0. Ten of these animals per group and sex were killed at week 105 for biochemical analyses of peroxisome proliferation. 1/50.

1/18 low-dose. 1/55. .62 IARC MONOGRAPHS VOLUME 77 increased in high-dose females (5/50. 99.03. Kluwe et al. 34/80 (12 500 ppm. the Fisher’s exact test was used. Ten of these animals per group and sex were killed at week 105 for biochemical analyses of peroxisome proliferation. p < 0.7%) for up to 104 weeks. eight weeks of age. Di(2-ethylhexyl) phthalate did not induce foci of altered hepatocytes as judged by basophilia. 1982.05). Neoplastic nodules or hepatocellular carcinomas were seen in 0/18 control. 1990). six weeks of age. Several studies using smaller numbers of animals have also been reported. 22/80 (p < 0. were fed diets containing 0.05). 1987). 2500and 12 500-ppm groups were designated for measurement of cell proliferation.. were fed a diet containing 2% di(2-ethylhexyl) phthalate (purity.. National Toxicology Program.05) and 10/55 (p < 0. p < 0. Groups of 20 female Fischer 344 rats.2% di(2-ethylhexyl) phthalate [purity not specified] for two years. Neoplastic nodules and/or hepatocellular carcinomas were observed in 1/10 controls and 11/14 treated rats [no statistical analysis given] (Rao et al. 1982. 1987). 4/50 (p < 0. The incidence of hepatocellular carcinomas and neoplastic nodules combined was also increased in low-dose (6/49. six weeks of age. Another group of 65 male and 65 female rats received the highest concentration for 78 weeks and then control diet for a further 26 weeks (recovery group)..10 or 1. p < 0. 1983. 2500 or 12 500 ppm di(2-ethylhexyl) phthalate (purity. All surviving rats were killed at 105 weeks for histopathological examination. Both the neoplastic nodules and hepatocellular carcinomas were negative for γ-glutamyltranspeptidase (γ-GT) (Rao et al.. 1999).05) (David et al. 1985). 11/65 (2500 ppm. although they do not report a comparison of their findings with conventional liver trimming and sectioning techniques]. and these animals were included in the final tumour analyses. About 65% of males and females survived until the end of the study. Hepatocellular tumour incidences in males were: 5/80 (control). 4/55 (500 ppm). Groups of 65 male and 65 female Fischer 344 rats.001) females compared with controls (0/50) (Kluwe et al. These studies are reviewed below. 98%) for 95 weeks. ATPase-deficiency or glucose-6-phosphatase-deficiency (Cattley et al.028) compared with controls (0/50). some of which were not designed for carcinogenicity testing. p < 0. Fifteen additional rats in the 0-. p < 0.05). biochemical analyses for peroxisome proliferation and histopathological evaluation and were killed at 79 weeks. Groups of 10 male Fischer 344 rats. 500.05) and 18/55 (recovery group. The respective incidences in females were 0/80. 0. For group comparison.. 100.012) and high-dose (13/50. 0. 3/65. [The authors suggest that their finding of a high incidence of liver tumours was due to their gross slicing technique. Groups of 10–14 male Fischer 344 rats were fed a diet containing 0 (control) or 2% di(2-ethylhexyl) phthalate (98% pure) for 108 weeks.005). 5/50 (100 ppm). All liver lobes were sliced at 1–2-mm intervals and the number and size of grossly visible lesions recorded. p = 0. were fed a diet containing 0 (control).01). 1/19 mid-dose and 6/20 high-dose rats (p < 0.. Neoplastic nodules and/or hepatocellular carcinomas were found in 0/18 controls and 6/10 rats fed di(2ethylhexyl) phthalate (p < 0.

3 Intraperitoneal administration Hamster: Groups of 50 male and 50 female Syrian golden hamsters. were administered 0 (control) or 3 g/kg bw di(2-ethylhexyl) phthalate (> 99% pure) by intraperitoneal injection once per week. six weeks of age. but not in the low-dose animals... 1994).. Median survival in controls was 709 days in males and 507 days in females and that in treated animals was 703 days in males and 522 days in females. no liver tumours had developed in 10 di(2ethylhexyl) phthalate-treated rats or in 10 controls.2 Inhalation exposure Hamster: Groups of 65–80 male or female Syrian golden hamsters. 12 weeks of age. 48. No difference in survival was seen between controls and the di(2-ethylhexyl) phthalate-treated groups. There were no differences in survival between groups (range of median survival being 629–686 days for males and 465–495 days for females). 72 and 96.and high-dose groups. Hamsters were maintained for their natural lifespan. once per two weeks or once per month (total doses.DI(2-ETHYLHEXYL) PHTHALATE 63 A total of 520 male Sprague-Dawley rats (180 g) were fed diets containing 0. 1988). 1988). At 52 weeks.02.2 or 2% (200. A group of 18 untreated animals served as controls. while at 78 weeks.] .. hepatocellular carcinomas or neoplastic nodules were found in 3/7 di(2-ethylhexyl) phthalate-treated rats and 0/8 controls (Hayashi et al. 17 male Fischer 344 rats were fed a diet containing 2% (20 000 ppm) di(2ethylhexyl) phthalate [purity unspecified] for up to 78 weeks. were exposed by whole-body inhalation to 15 μg/m3 di(2-ethylhexyl) phthalate vapour (> 99% pure) for 24 h per day on five days per week until natural death. 24–54 g/kg bw). 0. dose-dependent decrease in body weights in the mid. For biochemical assays and histopathological examinations. 1991) [The Working Group noted the inadequate reporting. [The Working Group noted the low concentration and that it was selected to simulate occupational exposure. 0.] 3. > 99%) for 102 weeks [group sizes not specified]. 7–18 rats were killed at weeks 24. 3. Tumour incidence was not increased in di(2-ethylhexyl) phthalate-treated hamsters (Schmezer et al.] As part of a larger experiment for studying the characteristics of hepatocarcinogenesis. The number of animals at terminal sacrifice was not given. 2000 or 20 000 ppm) di(2-ethylhexyl) phthalate (purity. There was a significant. [The Working Group noted limitations in the dosing schedule. No hyperplastic nodules or hepatocellular carcinomas were reported in either the control or treated animals (Ganning et al. Data on survival were not reported. The incidence of tumours was not increased by treatment (Schmezer et al. Total exposure over the lifetime was 7–10 mg/kg bw.

seven. Two weeks later. 99%) for up to 18 months.6%) compared with those in mice receiving NDEA alone (foci. 6000 or 12 000 ppm di(2-ethylhexyl) phthalate for up to six months.4 mm3 for the control. At 18 months. Groups of 10–20 mice were killed at two.. 1983). but there was an increase in the volume of the foci (0. 1984).4.64 IARC MONOGRAPHS VOLUME 77 3.6%). carcinomas were found in 3/10 mice treated with NDEA alone and in 10/10 and 18/20 mice treated with NDEA + di(2-ethylhexyl) phthalate at the low and mid doses. 6. All doses of di(2-ethylhexyl) phthalate increased the numbers of all lesions at the time periods studied compared with mice receiving NDEA alone. In mice treated with di(2ethylhexyl) phthalate alone. All mice given 12 000 ppm di(2-ethylhexyl) phthalate with NDEA initiation died by nine months and 11/20 had liver carcinomas. 3000. the mice were fed diets containing 0. 1986). Few hepatocellular foci were seen at two. The differential effects of short.6. four weeks of age. were fed diets containing 0. 28. four. Male B6C3F1 mice.1 Mouse Liver: Male B6C3F1 mice. 84 or 168 days and were killed at 168 days. 3. 50. the mice were fed diets containing 3000 ppm di(2-ethylhexyl) phthalate for periods of one. There was also an increase in lesion number and size (Ward et al. 6000. while numerous foci and neoplasms were seen in mice given di(2-ethylhexyl) phthalate after NDEA.4 Administration with known carcinogens and modifying agents There are numerous reports of studies of the initiating or promoting activities of di(2-ethylhexyl) phthalate given in combination with known carcinogens or promoting agents. the number of foci per unit volume of liver was similar in mice at all doses of di(2-ethylhexyl) phthalate. adenomas and carcinomas were determined. The numbers of mice with hepatocellular foci. Selected studies (complex protocols involving multiple promoting agents and special procedures were excluded) are summarized below and in Tables 5 and 6.6. . 9. 1.4. there was an increase in incidences of hepatocellular foci (45. 3000-. four or six months in mice treated with NDEA alone or di(2-ethylhexyl) phthalate alone.or long-term exposure to di(2-ethylhexyl) phthalate were studied in male B6C3F1 mice. 2/30 had liver carcinomas (Ward et al. two weeks later. By the end of the study. No tumours were found at six months in mice receiving NDEA alone.8 and 46.. four and six months after NDEA treatment. At five weeks of age. 67%) and adenomas (20.. respectively) (Ward et al. received a single intraperitoneal injection of 80 mg/kg bw N-nitrosodiethylamine (NDEA) in tricaprylin. Groups of 10 mice were killed at two. 20%. Mice were given an intraperitoneal injection of 80 mg/kg bw NDEA at four weeks of age. 17. When di(2-ethylhexyl) phthalate was fed after NDEA treatment for 28 or more days. received a single intraperitoneal injection of 80 mg/kg bw NDEA in tricaprylin and. adenomas. 6000 or 12 000 ppm di(2-ethylhexyl) phthalate (purity. 3000. respectively. 0. four weeks of age.and 12 000-ppm groups. six or 18 months.

i.p. i. i. i. i.g. Oral 2 weeks 2 weeks 2 weeks 1 week 1 week 1 week 2 weeks 2 weeks 2 weeks 2 weeks 3 weeks 2 weeks 4 weeks 2 weeks 1 week 1 week 1 week 1 week 4 weeks 3000 mg/kg diet/6 months 6000 mg/kg diet/6 months 12 000 mg/kg diet/6 months 3000 mg/kg diet/28 days 3000 mg/kg diet/84 days 3000 mg/kg diet/168 days 3000 mg/kg diet/18 months 6000 mg/kg diet/18 months 12 000 mg/kg diet/18 months 12 000 mg/kg diet/24 weeks 12 000 mg/kg diet/6 months 12 000 mg/kg diet/14 weeks 12 000 mg/kg diet/31 weeks 3000 mg/kg diet/6 weeks 10 mg/kg 3 × weekly/ 11 weeks 100 mg/kg 3 × weekly/ 11 weeks 200 mg/kg 3 × weekly/ 11 weeks 500 mg/kg 3 × weekly/ 11 weeks 12 000 ppm /24 weeks Oral Oral Oral Oral Oral Oral Oral Oral Oral Oral Oral Oral Oral Oral i. i. Oral + + + + + + + + + + – – – – – – + + – Ward et al. i. i.p. i. (1986) Ward et al. i. (1983) Ward et al. i. Oral i.g.g. (1983) Ward et al.Table 5.p. i.p. (1983) Ward et al.p.p.p. (1986) Ward et al.p.p.p. i. (1990) DI(2-ETHYLHEXYL) PHTHALATE 65 . (1986) Weghorst et al.p.g. (1986) Williams et al. (1984) Ward et al.g.p.g. i. (1985) Ward et al. i. i. (1984) Ward et al. (1988) Oesterle & Deml.g.g.p. i.F) Fischer 344 rats (M) Fischer 344 rats (M) Fischer 344 rats (M) Fischer 344 rats (M) Sprague-Dawley rats (F) Sprague-Dawley rats (F) Sprague-Dawley rats (F) Sprague-Dawley rats (F) Fischer 344 rats (M) 80 mg/kg bw NDEA 80 mg/kg bw NDEA 80 mg/kg bw NDEA 80 mg/kg bw NDEA 80 mg/kg bw NDEA 80 mg/kg bw NDEA 80 mg/kg bw NDEA 80 mg/kg bw NDEA 80 mg/kg bw NDEA 5 mg/kg bw NDEA 150 mg/kg bw NDEA 282 mg/kg bw NDEA 200 mg/kg diet AAF 7 weeks PH/200 mg/kg NDEA 8 mg/kg NDEA 8 mg/kg NDEA 8 mg/kg NDEA 8 mg/kg NDEA 200 ppm AAF 7 weeks i. Selected promotion studies on di(2-ethylhexyl) phthalate (DEHP) with known carcinogens and modifying factors Tumour type Species/strain (sex) Known carcinogen (initiator) Route of administration Interval between initiator and promoter Dose and duration of DEHP Route of administration Promoting activity for DEHP Reference Liver B6C3F1 mice (M) B6C3F1 mice (M) B6C3F1 mice (M) B6C3F1 mice (M) B6C3F1 mice (M) B6C3F1 mice (M) B6C3F1 mice (M) B6C3F1 mice (M) B6C3F1 mice (M) C3H/HeNCr mice (M. (1988) Oesterle & Deml (1988) Oesterle & Deml (1988) Oesterle & Deml (1988) Maruyama et al. (1987) Ito et al. (1993/94) Popp et al. (1984) Ward et al. i.

N-nitrosodiethylamine. 2-acetylaminofluorene. BBN.. 30 000 ppm uracil weeks 8–11 Oral 0 weeks 3000 ppm/16 weeks Oral – Hagiwara et al. i.66 Table 5 (contd) IARC MONOGRAPHS VOLUME 77 Tumour type Species/strain (sex) Known carcinogen (initiator) Route of administration Interval between initiator and promoter Dose and duration of DEHP Route of administration Promoting activity for DEHP Reference Kidney Fischer 344 rats (M) Urinary bladder Fischer 344 rats (M) 5000 ppm BBN weeks 1–4. intragastric. M. N-ethyl-N-hydroxyethylnitrosamine. partial hepatectomy . PH. (1988) AAF. female.g. N-butyl-N-(4-hydroxybutyl)nitrosamine.. i. (1990) 500 mg/kg diet EHEN/2 weeks Oral 0 weeks 12 000 ppm/24 weeks Oral + Kurokawa et al. F. intraperitoneal injection.p. male. NDEA. EHEN.

Initiation studies on di(2-ethylhexyl) phthalate (DEHP) with promoting agents DI(2-ETHYLHEXYL) PHTHALATE Species/strain DEHP initiation Route of administration Interval between initiation and promotion 2 weeks 2 weeks 2 weeks Dose and duration of promoter Route of administration Initiating activity of DEHP Reference Mouse B6C3F1 (M) Fischer 344 rats (F) Fischer 344 rats (F) 25 or 50 g/kg bw 10 g/kg bw DEHP 6. 24 h after PH 12 000 mg/kg diet DEHP 12 weeks Gavage Oral Oral 500 mg/L phenobarbital 6 or 18 months 200 ppm AAF 2 weeks 1. (1986) Garvey et al. 39 weeks Drinkingwater Diet Gavage Diet – – Ward et al. 2-acetylaminofluorene. AAF.Table 6. carbon tetrachloride 67 . (1987) – Garvey et al.5 mL/kg CCl4 once 500 mg/kg diet phenobarbital. 12. (1987) PH. CCl4. partial hepatectomy.

3. 1986). The numbers of adenomas per liver were also increased (NDEA-treated males. Ward et al. NDEA + di(2-ethylhexyl) phthalate-treated females. Thus the study showed no evidence of initiating activity of di(2-ethylhexyl) phthalate (Ward et al. NDEA + di(2-ethylhexyl) phthalate-treated females. NDEA-treated females. were given a single intragastric dose of 25 or 50 g/kg bw di(2-ethylhexyl) phthalate (99% pure).2). 0. Skin: Groups of 25 female SENCAR mice. six to eight weeks of age. One week later. NDEA + di(2-ethylhexyl) phthalate-treated males. 366. Two weeks later. 15 days of age. enhanced only slightly the numbers of papillomas (0. four weeks of age.3 mm3) (Weghorst et al. At weaning (four weeks of age). Groups of 10 male and five female C3H/HeNCr mice. 7. 169). 1993/94).4.. NDEA-treated females. Di(2ethylhexyl) phthalate in combination with NDEA increased the average numbers of foci per liver (NDEA-treated males. 1986). 2). To test di(2-ethylhexyl) phthalate as a second-stage promoter.44 versus 2. mice received applications of 100 mg per animal di(2ethylhexyl) phthalate (99% pure) twice weekly for 28 weeks. received a single application of 20 μg 7. groups of 7–20 male B6C3F1 mice. 1/10 mice given 25 g/kg di(2-ethylhexyl) phthalate.. mice received TPA for two weeks followed by di(2-ethylhexyl) phthalate for 26 weeks. when tested as a complete promoter (28 weeks of exposure).. received either a single intraperitoneal injection of 5 mg/kg bw NDEA or saline.8. 2/15 mice given 50 g/kg bw di(2-ethylhexyl) phthalate + phenobarbital. Di(2-ethylhexyl) phthalate. 2/20 mice given 25 g/kg di(2-ethylhexyl) phthalate + phenobarbital.2 mL acetone on the skin of the back. 176. hepatocellular carcinomas were found in 0/7 mice given 50 g/kg di(2-ethylhexyl) phthalate.2 Rat Liver: Groups of 10 female Fischer 344 rats. In male mice. 47. TPA and di(2-ethylhexyl) phthalate controls were included.12-dimethylbenz[a]anthracene (DMBA) in 0. 15.4 mm3 versus 1. 1985. 12-O-Tetradecanoylphorbol 13-acetate (TPA) control groups received 2 μg TPA. All mice were killed at 28 weeks of age and the number and size of hepatic foci were measured. treatment with NDEA + di(2-ethylhexyl) phthalate yielded larger adenomas than those seen in mice treated with NDEA alone (2. received a single intraperitoneal injection of 150 mg/kg bw NDEA followed three weeks later by . At 18 months. The authors concluded that di(2-ethylhexyl) phthalate was a second-stage promoter (Diwan et al.88 per mouse versus DMBA alone 0 per mouse) but significantly (p < 0. mice were divided into two groups and fed diets containing either 0 or 12 000 ppm di(2-ethylhexyl) phthalate [purity unspecified] for 24 weeks..01) enhanced papillomas when given for 26 weeks after two weeks of TPA first-stage promotion (6. 3/17 mice given phenobarbital alone and 0/10 untreated mice [statistical analysis not given]. NDEA + di(2-ethylhexyl) phthalate-treated males. Groups of 10–17 controls were used. Appropriate acetone.68 IARC MONOGRAPHS VOLUME 77 In a study to test di(2-ethylhexyl) phthalate for initiating activity. phenobarbital was given as a promoting agent at a concentration of 500 mg/L in the drinking-water for six or 18 months. seven weeks of age.

All rats were killed at week 8.2% di(2-ethylhexyl) phthalate (99. No neoplasms or nodules were identified. 1988).6 for NDEA alone) (Ito et al. In foci that were induced by AAF. Di(2-ethylhexyl) phthalate (99. the volume percentage was not . Di(2-ethylhexyl) phthalate-treated rats had no increase in foci staining positively for glutathione S-transferase placental form (8. The incidence of foci with expression of γ-GT was not affected by di(2-ethylhexyl) phthalate treatment (Oesterle & Deml. Lower doses were ineffective. Male Fischer 344 rats were fed 200 ppm 2-acetylaminofluorene (AAF) for seven weeks to induce hepatocellular altered foci. glucose-6-phosphatase. and produced no significant enhancement of the occurrence of AAFinduced liver neoplasms (3/6 compared with 3/12) (Williams et al. as detected histochemically. adenosine triphosphatase or fibronectin. Although the numbers of haematoxylin/eosin-stained foci were increased in di(2-ethylhexyl) phthalate-treated rats. they were fed a diet containing 3000 ppm di(2-ethylhexyl) phthalate [purity unspecified] for six weeks. and were then fed diets containing either 0 or 12 000 ppm di(2-ethylhexyl) phthalate (purity. and were subsequently fed 0 or 12 000 ppm di(2-ethylhexyl) phthalate (98% pure) in the diet. Male Fischer 344 rats were fed diets containing 200 ppm AAF for seven weeks to induce hepatocellular altered foci. The initiating activity of di(2-ethylhexyl) phthalate was examined after single and sub-chronic dosing.2% in the diet followed by various known promotion regimens. 1988).5% pure) was administered as a single oral dose (10 g/kg bw) or by 12 weeks of feeding at a concentration of 1. such as phenobarbital treatment or partial hepatectomy.. 98%) for 24 weeks. There was no increase in number or mean volume of foci in liver sections examined using multiple histological markers and no tumours were identified. Two weeks later.5% pure) for three or six months. di(2-ethylhexyl) phthalate reduced the activity of γ-GT. At week 3.. but showed no promoting effect on iron-excluding altered hepatic foci induced by AAF. Di(2-ethylhexyl) phthalate did not increase the number of foci or the mean volume of the foci. they were subjected to a partial hepatectomy. as identified by five different histological markers (Popp et al.. No evidence of induction of hepatocellular altered foci or hepatic neoplasms was found when di(2-ethylhexyl) phthalate was given alone for 24 weeks.DI(2-ETHYLHEXYL) PHTHALATE 69 a diet containing 1. Di(2-ethylhexyl) phthalate exerted weak promoting activity in weanling female Sprague-Dawley rats after doses of 200 or 500 mg/kg bw.. Di(2-ethylhexyl) phthalate fed for 24 weeks increased basophilic foci. 1987). but did not increase the number. Groups of 18–20 male Fischer rats (weighing 160 g) were given a single intraperitoneal injection of 200 mg/kg bw NDEA. indicating that di(2-ethylhexyl) phthalate had no initiating activity (Garvey et al. 1987). mean volume or volume percentage of foci detected by deficiencies in iron storage.1985). given three times per week by gavage for 11 consecutive weeks after initiation with a single oral dose of 8 mg/kg bw NDEA.5 per cm2 versus 11. The incidence of ATPase-deficient foci was enhanced about two-fold compared with rats treated with NDEA alone.

67 mg/kg bw once [exact week of dosing for both chemicals not given].. Rats were killed at 27 weeks. six weeks old. p < 0. 50. 3. 0. liver carcinomas occurred in 0/50 control. 1990). seven weeks of age. 1996). The modifying potential of di(2-ethylhexyl) phthalate on second-stage N-butyl-N(4-hydroxybutyl)nitrosamine (BBN)-initiated urinary bladder carcinogenesis was investigated in male Fischer 344 rats.. Hamsters were maintained for their natural lifespan.2% in the diet for 24 weeks. . 3.5 3. 1/50 low-dose.0%.. p < 0.01) (Kurokawa et al. Uracil was administered during weeks 9–11 at a dietary level of 3.05 Fisher’s exact test) high-dose mice (Astill et al. were given 2-ethylhexanol by gavage five times weekly at doses of 0. 200 and 250 mg/kg bw for 18 months.3. Body weight gain was reduced by 24–26% in the high-dose group and mortality was dose-related. Surviving animals were killed at the end of week 20 of the experiment. Di(2-ethylhexyl) phthalate increased the numbers of rats with renal (tubular) cell tumours (EHEN + di(2-ethylhexyl) phthalate 65% versus 20% for EHEN alone..01) and the mean number of tumours per kidney (EHEN + di(2-ethylhexyl) phthalate 1. 0. Di(2-ethylhexyl) phthalate did not promote hyperplastic lesions (papillary or nodular) of the urinary bladder or papillomas induced by BBN (Hagiwara et al.2.2% in the diet) for experimental weeks 5–8 and weeks 12–20. Urinary system: Groups of 20 male Fischer 344 rats were given 0.3 Hamster Groups of 50 male and 50 female Syrian golden hamsters.1 versus EHEN alone 0. using a uracil-accelerated transitional-cell proliferation model. Six-week-old animals received 0. 3/50 mid-dose and 5/50 (p < 0.6 or 1. N-Nitrosodimethylamine (NDMA) was given orally at 1. were given intraperitoneal injections of 3 g/kg bw di(2-ethylhexyl) phthalate (> 99% pure) either once. Di(2-ethylhexyl) phthalate did not affect tumour yield (liver tumours: 16/50 and 9/50 in di(2-ethylhexyl) phthalate + NDMA and NDMA males.05% N-ethyl-Nhydroxyethylnitrosamine (EHEN) for two weeks in the diet followed by di(2-ethylhexyl) phthalate [purity unspecified] at a concentration of 0 or 1. or once per week for two or four weeks.5. 1988). 1988). 1990).05% BBN in their drinking-water for four weeks followed by di(2-ethylhexyl) phthalate [purity unspecified] (0.70 IARC MONOGRAPHS VOLUME 77 increased and no difference in the numbers of iron storage foci was seen (Maruyama et al. In females..4. and 6/50 and 6/50 in di(2-ethylhexyl) phthalate + NDMA and NDMA females) [statistical analysis not given] (Schmezer et al.1 Carcinogenicity of the metabolite 2-ethylhexanol Mouse Groups of 50 male and 50 female B6C3F1 mice. Survival was reduced among hamsters receiving NDMA.

150 or 500 mg/kg bw for 104 weeks. Figure 1 summarizes the metabolic pathways responsible for the metabolism of di(2-ethylhexyl) phthalate in humans and in animals. Huber et al. 1991. 1988. who excreted between 60 and 90% of the infused dose in the urine collected for 24 h after transfusion. five times weekly. at doses of 0.DI(2-ETHYLHEXYL) PHTHALATE 71 3. The quantitative urinary metabolic profile of di(2-ethylhexyl) phthalate reported by Schmid and Schlatter (1985) is quite similar to that found by Albro et al. These authors reported that 11–15% of the dose was excreted in the urine and a urinary elimination half-life of about 12 h can be estimated from the data. metabolism and excretion Humans 4.5% of the dose was excreted in the urine within 24 h. (1945) administered single oral doses of 5 and 10 g di(2-ethylhexyl) phthalate to two human subjects and reported that approximately 4. The major phase I metabolites of di(2-ethylhexyl) phthalate in human urine are mono(2- . with 11 and 33 % of the dose recovered each day in the urine. (1993a). but at the much lower dose of 30 mg per person. 50. metabolism and excretion of di(2-ethylhexyl) phthalate in humans (Lawrence & Tuell. 1984. the two volunteers also received 10 mg di(2-ethylhexyl) phthalate daily for four days. oral and intravenous routes. 1989. Schmid and Schlatter (1985) also administered di(2-ethylhexyl) phthalate orally to two human subjects. A dose-related depression of body weight gain in male and female rats and increased mortality in high-dose female rats were observed. Thomas & Thomas. Shaffer et al.5. Other Data Relevant to an Evaluation of Carcinogenicity and its Mechanisms Absorption.1. who determined the urinary metabolites of di(2-ethylhexyl) phthalate in leukaemia patients who received platelet transfusions from bags containing di(2-ethylhexyl) phthalate. using the widely accepted metabolite nomenclature of Albro and Lavenhar (1989). distribution. In the same study. 4. 1996). 1999). In contrast.. Albro & Lavenhar. inhalation. Doull et al. who studied the excretion of di(2-ethylhexyl) phthalate metabolites in five workers occupationally exposed to di(2-ethylhexyl) phthalate. six weeks of age. were given 2-ethylhexanol by gavage.1 4. Kamrin & Mayor. In a very early study. there being no evidence of accumulation. There was no increase in the incidence of tumours in any treated group (Astill et al.2 Rat Groups of 50 male and 50 female Fischer 344 rats. (1982).1 Human exposure to di(2-ethylhexyl) phthalate can occur via the dermal. The high level of exposure has prompted many studies on the absorption. distribution.. These data are also in good agreement with the results of Dirven et al.. 1979. 1996. Rubin and Schiffer (1976) reported data from two patients receiving platelet transfusions from bags containing di(2-ethylhexyl) phthalate. Burg.

IX. phthalic acid. I. mono(2-(2-hydroxyethyl)hexyl) ester. This chart is based on the work of Huber et al. mono(4-carboxy2-ethylhexyl) ester. V. mono(2-ethyl-6-hydroxyhexyl) ester . X. phthalic acid. phthalic acid. Major pathways involved in the metabolism of di(2-ethylhexyl) phthalate Di(2-ethylhexyl) phthalate HO CH2 CH CH2 CH3 O C OH CH2 CH2 CH2 CH3 HOOC CH2 CH CH2 CH3 CH2 CH2 CH2 CH3 2-Ethylhexanol 2-Ethylhexanoic acid Mono(2-ethylhexyl) phthalate C O CH2 CH O CH2 CH2 CH2 CH2 CH3 IARC MONOGRAPHS VOLUME 77 ω 1-Oxidation of hexyl chain CH3 ω Oxidation of hexyl chain ω Oxidation of ethyl chain R CH CH2 CH2 CH2 CH3 CH OH CH3 R CH CH2 CH2 CH2 CH3 R CH CH2 CH2 CH2 CH2OH IX CH2 CH2OH VII CH2 CH3 X (ω 1)-Oxidation ω-Oxidation ω-Oxidation R CH CH2 CH2 CH2 CH3 C CH3 O R CH CH2 CH2 CH2 CH3 R CH CH2 CH2 CH2 COOH VI CH2 COOH IV CH2 CH3 V β-Oxidation α-Oxidation O C OH R CH CH2 CH2 COOH CH2 CH3 R CH CH2 CH2 CH2 CH3 R= C O CH2 O II COOH I The numbering of the metabolites is based on the nomenclature of Albro and Lavenhar (1989). mono(2-ethyl-5-oxohexyl) ester. phthalic acid. phthalic acid. mono(5-carboxy-2-ethylpentyl) ester. phthalic acid. VI. mono(2-carboxyhexyl) ester. mono(2-ethyl-5-hydroxyhexyl) ester.72 Figure 1. phthalic acid. VII. phthalic acid. II. IV. mono(2-(carboxymethyl)hexyl) ester. (1996) and Astill (1989).

2 mg/kg bw di(2-ethylhexyl) phthalate.d) observed in rats (Plonait et al..7 times higher than the concentration of metabolite V.. 323 μg/L at 30 min. The same authors found a marked interindividual variation in the conjugation of mono(2-ethylhexyl) phthalate. while only 32–45% of metabolite V was present in the conjugated form. Metabolites VI and IX were reported by Dirven et al. Schmid & Schlatter. Similar levels of exposure (1. and concluded that metabolism via (ω–1)-hydroxylation is favoured over ω-hydroxylation in humans. in newborn infants was similar (Sjöberg et al. In two of the five subjects studied. 1985a). from which a transfusion of 2. 1982. and compounds V. with a half-life of 28 min. The terminal half-life of the active metabolite of di(2-ethylhexyl) phthalate. (1977) and Sjöberg et al. In adult haemodialysis patients. Sjöberg et al. (1993b) to be almost completely conjugated. (1978) determined the disappearance of di(2-ethylhexyl) phthalate in seven adult patients following dialysis. (1982) and Schmidt and Schlatter (1985) reported that up to 80% of the urinary metabolites of di(2-ethylhexyl) phthalate in humans were excreted as glucuronide conjugates. Albro et al.DI(2-ETHYLHEXYL) PHTHALATE 73 ethylhexyl) phthalate..2 mg/kg bw) have been reported in newborn infants during single exchange transfusions (Sjöberg et al. (1985b) and Plonait et al. mono(2-ethylhexyl) phthalate. 1972. 1993). whereas in urine samples from the other three subjects. Dirven et al. The plasma levels ranged from 0. Both Albro et al. VI and IX (Figure 1. Rubin and Schiffer (1976) reported peak blood plasma levels of di(2-ethylhexyl) phthalate in adult patients transfused with platelets ranging from 3 to 8 mg/L and described the kinetics of plasma disappearance as mono-exponential. (1993) also described a rapid decline in serum di(2-ethylhexyl) phthalate levels in term newborns similar to that seen in adults. This is a low level compared with di(2-ethylhexyl) phthalate levels in whole blood stored in blood bags for up to 21 days (Jaeger & Rubin. only 20–38% of mono(2ethylhexyl) phthalate was not conjugated.. with the half-life of the terminal phase being approximately 10 h. (1985b) reported that plasma levels of di(2-ethylhexyl) phthalate declined biexponentially after transfusions in newborn infants. Peck et al. The mean serum levels were 606 μg/L immediately after dialysis. However. 77–100% of mono(2-ethylhexyl) phthalate was in the free form. Conjugates of di(2ethylhexyl) phthalate metabolites with sulfate. as Stern et al.6 mg/kg bw di(2-ethylhexyl) phthalate. 167 μg/L at 1 h and 145 μg/L at 3 h after completion of dialysis. taurine and glycine have not been found in any species studied to date. (1993b) found that the urinary concentration of metabolites VI and IX was 1. 1985. 1978. (1985c.. Rock et al. They concluded that most of the di(2-ethylhexyl) phthalate present in serum at the completion of dialysis is likely to disappear within 5–7 h.. 1985b). di(2-ethylhexyl) phthalate can persist in serum for a much longer time and it is postulated that either there are different kinetics resulting from immature metabolism or there is a biphasic disappearance pattern.3–2.7 to 4.5 L of blood to a 70-kg person would give an exposure of 1. Dirven et al. in preterm infants. circulating levels of mono(2-ethylhexyl) phthalate are similar to . Sjöberg et al. 1993b). Lewis et al. 1979)..3 to 1.

7 μg/mL mono(2-ethylhexyl) phthalate were reached . They reported that the median values of three major di(2-ethylhexyl) phthalate metabolites studied (VI. Albro & Lavenhar.. 1978). For example. Dirven et al. blood and other tissues (Albro. 1984.2 Experimental systems The absorption and disposition of di(2-ethylhexyl) phthalate has been extensively investigated in laboratory animals (Thomas & Thomas.. The first step in the metabolism of di(2-ethylhexyl) phthalate in all species is the hydrolysis of one of the two ethylhexyl side-chains to yield mono(2-ethylhexyl) phthalate and 2-ethylhexanol. 1987..3-fold) in urine samples collected at the end of the workday compared with urine samples collected at the start of the workday. Teirlynck and Belpaire (1985) studied the disposition of both di(2-ethylhexyl) phthalate and mono(2-ethylhexyl) phthalate in immature male Wistar rats and reported that. It is generally accepted that in all mammalian species the majority of di(2-ethylhexyl) phthalate reaching the intestine is absorbed as hydrolysis products rather than as the intact diester (Albro & Lavenhar. Hence.2 ± 8. although the airborne exposure levels were within a similar range. The level of pancreatic lipase in the intestine of laboratory animals has been shown to exhibit marked species and strain differences (Albro & Thomas.8 ± 1. a meaningful interpretation of these data is impeded by the lack of understanding of the toxicokinetics of di(2-ethylhexyl) phthalate metabolites in humans. Huber et al.. patients undergoing transfusion may be exposed to mono(2-ethylhexyl) phthalate at doses of up to one tenth those of di(2-ethylhexyl) phthalate (Rock et al. 1993a. After oral administration of di(2-ethylhexyl) phthalate. plasma concentrations of 8. after a single oral dose of di(2-ethylhexyl) phthalate (2.. Lower levels of di(2-ethylhexyl) phthalate-hydrolysing enzyme activity are expressed in the liver. as dermal absorption through preparations of human skin in vitro is low (Scott et al.7 μg/mL di(2ethylhexyl) phthalate and 63. 1989. 4. However. Huber et al.. this pathway is primarily catalysed by pancreatic lipase. 1996). 1993a). (1993a) evaluated the inhalation exposure to di(2-ethylhexyl) phthalate in a group of nine volunteers in a boot factory in an attempt to evaluate the absorption and disposition of the plasticizer. 1986.8 g/kg).2–2. 1985).1. Further. Furthermore. see Figure 1) were significantly increased (1. 1999). 1996. Astill... di(2-ethylhexyl) phthalate can be hydrolysed by plasma proteins in blood products to mono(2-ethylhexyl) phthalate. Doull et al. IX and V. 1989.. 1996). showing that this metabolite is formed after systemic intake in humans (Pollack et al. Occupational exposure to di(2-ethylhexyl) phthalate based on personal air sampling data in the range 9–1266 μg/m3 has been reported (Dirven et al. a similar comparison in six cable factory workers detected no statistically significant increase in post-shift urinary di(2-ethylhexyl) phthalate metabolite concentrations compared with pre-shift values.74 IARC MONOGRAPHS VOLUME 77 those of di(2-ethylhexyl) phthalate. Absorption of di(2-ethylhexyl) phthalate via the lungs is considered to be the major route of occupational exposure. 1973). 1989). As pointed out by the authors. Huber et al. Dirven et al.

According to this study. rats and mice. respectively. the excretion of di(2ethylhexyl) phthalate in rats varied depending on whether it was administered by gavage or in the diet.2 h. di(2-ethylhexyl) phthalate was rapidly and extensively metabolized. respectively. Di(2-ethylhexyl) phthalate is excreted largely in the urine by both African green monkeys and humans (> 60% in 24 h) as glucuronide conjugates (~80%) of the oxidation products of mono(2-ethylhexyl) phthalate. only 16% of the urinary metabolites of di(2-ethylhexyl) phthalate in cynomolgus monkeys were in the form of hydrolysable products (Astill. The efficiency of di(2-ethylhexyl) phthalate elimination from the gastrointestinal tract differs between species. ranging from 28% in monkeys. In rats. 1982). with the faecal and urinary routes accounting for 25. after a single 100 mg/kg bw gavage dose of di(2-ethylhexyl) [carbonyl-14C]phthalate.9 mg/kg bw oral dose of [14C]di(2-ethylhexyl) phthalate. . In dogs. The opposite was the case in pigs.. In male Wistar rats given a single 2. the figures showed that the faecal route of excretion dominated (75%). respectively. faecal elimination accounted for approximately 50% of the dose in cynomolgus monkeys. Rhodes et al. (1980) fed 50 mg/kg bw di(2-ethylhexyl) phthalate per day to male Sprague-Dawley rats. 33% in rats to 37% in mice over approximately the first 96 h. within four days. The predominant hydrolysable urinary metabolites were metabolite IX (26.6 mg/kg bw [14C]di(2-ethylhexyl) phthalate.1%) and mono(2-ethylhexyl) phthalate (19. Urinary excretion levels were also similar across the three species. These authors reported that 45% and 7% of di(2-ethylhexyl) phthalate were absorbed by marmosets at doses of 100 mg/kg bw and 2000 mg/kg bw. 14% of the dose was excreted in the bile. In contrast to the extent of conjugation of urinary metabolites in African green monkeys. Astill (1989) observed that. In African green monkeys. 1989). 37.7% of the radioactivity. male pure-bred beagles and male immature swine of the Hormel strain for 21–28 days and then administered a single dose of 14C-labelled di(2-ethylhexyl) phthalate at 50 mg/kg bw on the last day. Daniel and Bratt (1974) found 42% and 57% of the dose in the urine and faeces within seven days. (1986) found that the absorption of di(2-ethylhexyl) phthalate was considerably lower in marmosets than in rats. Di(2-ethylhexyl) phthalate excretion in urine and faeces and the subsequent metabolic patterns of di(2-ethylhexyl) phthalate were stated to be quantitatively similar in cynomolgus monkeys. in rats fed 2. with the urinary route accounting for 20. Ikeda et al.DI(2-ETHYLHEXYL) PHTHALATE 75 after 3 h.5 and 53. The plasma concentration of mono(2-ethylhexyl) phthalate declined with a half-life of 5.7% and 79. whereas the corresponding value for di(2-ethylhexyl) phthalate could not be estimated due to very low levels of the parent diester. However. Peck and Albro (1982) also showed that di(2-ethylhexyl) phthalate was rapidly and extensively metabolized in both African green monkeys and humans. 1981. male Fischer 344 rats and male B6C3F1 mice. In the same study.4%. with urinary excretion being 80% in the form of glucuronide conjugates (Albro et al.6%) and faecal excretion in monkeys accounted for approximately 8% of the administered di(2-ethylhexyl) phthalate 48 h after infusion.2% of the administered radioactivity were excreted in the urine and faeces.

1989). further metabolism (shortening of the carbon chains) of the dicarboxylic acid products of ω-oxidation by α.or β-oxidation. The liver. 1989. Albro & Lavenhar. mono(2-ethylhexyl) phthalate. 1996. Chu et al. (1978) found comparable levels in the liver. (1978) studied the metabolism and distribution of mono(2-ethylhexyl) phthalate in rats after oral dosing and found that the intestine contained the highest tissues levels after 24 h. Doull et al. Astill. They also reported that 80% of the 14C-dose of mono(2-ethylhexyl) phthalate was eliminated 24 h after oral administration. heart. Further. As mentioned above. the bulk of a di(2-ethylhexyl) phthalate dose is absorbed as the mono-ester. our understanding of the distribution of intact di(2-ethylhexyl) phthalate is limited. with other organs containing approximately 10–25% of the level of the liver. Huber et al.76 IARC MONOGRAPHS VOLUME 77 In making species comparisons. in plasma containing ethanol or in the plasticizer leached from the plastic into the plasma was 263. 1989. 181 and 31 min. 1984). 1982). cited in Thomas & Thomas. Twenty minutes after the administration of an intravenous dose of [14C]mono(2-ethylhexyl) phthalate. respectively (Miripol et al. (1999) as follows (see Figure 1): 1.. lung and muscle each contained approximately half the level in the intestine. hydroxylation of the terminal carbon atoms (ω-oxidation) of both the hexyl and ethyl side-chains and the penultimate carbon (ω–1-oxidation) of the hexyl chain. Because distribution studies have monitored total radioactivity. and following absorption this metabolite is subjected to extensive oxidative metabolism mediated by cytochrome P450 enzymes (Albro & Lavenhar. and 3. Albro and Lavenhar (1989) reported that intact di(2-ethylhexyl) phthalate was not absorbed until the dose reached 500 mg/kg bw in CD-1 mice and 450 mg/kg bw in Fischer 344 rats. no threshold was seen in B6C3F1 mice: the amount of di(2-ethylhexyl) phthalate absorbed was directly proportional to the dose down to a dose of 20 mg/kg bw. conversion of these hydroxyl groups to either a carboxylic acid (ω-oxidation) or a ketone (ω–1-oxidation).. 83. 1989. the disposition halflife in the rat of [14C]di(2-ethylhexyl) phthalate solubilized in aqueous polysorbate emulsion. The metabolism of mono(2-ethylhexyl) phthalate has been summarized by Doull et al. 2.. more of the parent diester reaches the circulation at high doses (Albro et al. Astill (1989) reported that prolonged feeding or the use of high doses of up to 1000 mg/kg bw did not influence the absorption of di(2-ethylhexyl) phthalate from the gastrointestinal tract of male Fischer 344 rats. careful interpretation of data is required. in plasma containing polysorbate emulsion. 1975. In contrast. kidney and bladder. For example. 1999). Astill. 1984. 1999).. Chu et al. Species differences in the metabolism of di(2-ethylhexyl) phthalate have been reported and attempts have been made to explain the susceptibility of animals to di(2ethylhexyl) phthalate-induced hepatic peroxisome proliferation based on their metabolic profiles (Doull et al. 72% in the urine and 8% in the faeces. as the physical state of the di(2-ethylhexyl) phthalate used can affect the outcome (Thomas & Thomas.. While most of a dose of di(2-ethylhexyl) phthalate is absorbed as mono(2-ethylhexyl) phthalate. .

1% and 14%. Scott et al.and five-day-old) in vitro are capable of metabolizing mono(2-ethylhexyl) phthalate.. resulting from two oxidative steps. Elcombe et al.. 1986.. 18. 1987.5% in hamsters (Albro et al. this metabolite could account for as much as 30% of urinary metabolites (Albro et al. 1993b). 1981. In rats and mice. 1999). Mono(2-ethylhexyl) phthalate undergoes extensive metabolism in rats. Dermal absorption of di(2-ethylhexyl) phthalate is slow in rats (Melnick et al.. hamsters and humans. while mice and hamsters excrete 1.. two other studies have reported that this metabolite comprises approximately 20–30% of urinary metabolites in humans (Schmid & Schlatter.9% in African green monkeys. Astill (1989) reported that 5. 1985.4% and 0. Fetal exposure to [14C]di(2-ethylhexyl) phthalate or its metabolites occurs following intraperitoneal administration to pregnant rats (Singh et al.. (1993b) involved occupational exposure of humans via inhalation. The study by Dirven et al.. 8.3% in humans to 4. Elcombe & Mitchell.. 1982). 1985. with approximately 75% of all hydrolysable urinary metabolites consisting of dicarboxylic acids. Keith et al. 1982). In rats given high oral doses of di(2-ethylhexyl) phthalate (2000 mg/kg bw) . metabolite IX accounted for 13.. 1981.. 1988)..3% of a 100-mg/kg bw single gavage dose of di(2-ethylhexyl) phthalate was excreted in the urine in the first 24 h as metabolite V by cynomolgus monkeys. 1996. the possibility that the route of administration affected the metabolic profile cannot be ruled out. Dirven et al.. 1989.3% of hydrolysable urinary metabolites. Liver microsomes from fetuses (21 days of gestation) and neonates (one. Chu et al.. These are the keto and hydroxyl metabolites of mono(2-ethylhexyl) phthalate that are produced via ω–1-oxidation..6% of hydrolysable metabolites of di(2-ethylhexyl) phthalate was excreted as mono(2-ethylhexyl) phthalate and this metabolite ranged from 71. 1982) were collected following intravenous administration of di(2-ethylhexyl) phthalate. 1975). 1996.DI(2-ETHYLHEXYL) PHTHALATE 77 Mono(2-ethylhexyl) phthalate and metabolites VI and IX are considered to be primarily responsible for the peroxisome-proliferating activity of di(2-ethylhexyl) phthalate (Mitchell et al. whereas in African green monkeys. Therefore. Doull et al. respectively (Albro et al. 1987. which accounts for 51. rats and mice. 28. Astill (1989) reported that similar amounts of metabolite IX were excreted in the urine of cynomolgus monkeys. 1992.3% of the urinary metabolites of di(2-ethylhexyl) phthalate in rats (Albro et al. Cornu et al. 1982). 1998)..2% in guinea-pigs. 1981). 1992. 1981. liver microsomes from mature animals metabolize mono(2-ethylhexyl) phthalate at a rate similar to those from five-day-old rats. mono(2-ethylhexyl) phthalate was present only in trace amounts in rat urine. It should be emphasized that the above data for African green monkeys and humans in the studies by Albro and coworkers (Albro et al.. respectively. demonstrating rapid postnatal development of mono(2-ethylhexyl) phthalate (ω–1)-hydroxylase activity in rats (Sjöberg et al. Albro & Lavenhar. rats and mice..7%.3% and 12. In mice. Further. In another comparative study. African green monkeys. guinea-pigs and humans excrete approximately 5% of a dose of di(2-ethylhexyl) phthalate in the urine as metabolite V. mainly metabolited V. Deisinger et al. In contrast. 18. Interestingly. whereas all the remaining studies reported findings following a single oral dose. In contrast.

respectively).. however... 4. 1992).9 h compared with 3. but only slightly or non-sensitizing to human skin (Shaffer et al.. While occupational exposure via inhalation is considered to be a major route of human exposure to di(2-ethylhexyl) phthalate.78 IARC MONOGRAPHS VOLUME 77 during lactation. 1986).2. The level of mono(2-ethylhexyl) phthalate in the fetus was approximately 1% that of di(2-ethylhexyl) phthalate (Tomita et al.3.2). 40 or 60 days old. 25 μg/mL 6 h after dosing) were transported into the milk (Dostal et al.as in 60-day-old rats. There are few data on effects of occupational exposure specifically to di(2-ethylhexyl) phthalate (WHO. respectively.1 h and 2.. 1987a).g. levels of di(2-ethylhexyl) phthalate in the fetuses were 522 μg/g and 426 μg/g. It was concluded that the toxicokinetic differences may in part explain the age-dependent effect of di(2-ethylhexyl) phthalate on the testes (see Section 4. The dams were killed at 24-h intervals starting on days 8 and 11 until day 20 of gestation.1 Toxic effects Humans Dermally applied di(2-ethylhexyl) phthalate was considered to be moderately irritating. The concentration was less than that in maternal blood and less than 1% of the administered dose. Radiolabelled di(2-ethylhexyl) phthalate (10 mL/kg) was administered by oral gavage to ddY-SLC mice on gestation day 8. Placental transfer of di(2-ethylhexyl) phthalate has been observed following intraperitoneal administration of di(2-ethylhexyl) [carbonyl-14C]phthalate on gestational day 5 or 10 in rats (Singh et al. Three and 12 h after exposure. The AUC (area under the plasma curve) for 0–30 h was significantly higher (about two-fold) in the 25-day-old rats than in the older rats. 1952).2 4. amniotic fluid and placenta at all time points. (1985d) also studied the kinetics of orally administered di(2-ethylhexyl) phthalate and its excretion in groups of 10 rats 25. but these . Two adults given single oral doses of either 5 or 10 g di(2-ethylhexyl) phthalate exhibited no untoward effects apart from mild gastric disturbances and moderate diarrhoea at the higher dose (Shaffer et al. In a study involving workers at a Swedish PVC-processing factory. the half-life was..g. 216 μg/mL 6 h after dosing) and smaller. not significantly higher in the 25-day-old rats (3. Sjöberg et al. there is no information available on the toxicokinetics or metabolic disposition of this compound in animals exposed via this route. peripheral nervous system symptoms and signs were investigated in 54 workers exposed to di(2-ethylhexyl) phthalate and other phthalate diesters. Radioactivity was detected in fetal tissues. large amounts of di(2-ethylhexyl) phthalate (e. Some workers showed various peripheral nervous system symptoms and signs.. 1975). 1945). The amount of di(2-ethylhexyl) phthalate-derived products excreted in the urine was twice as high in 25. Mallette & von Haam. yet significant amounts of mono(2-ethylhexyl) phthalate (e. The radioactivity peaked at 48 h and declined rapidly thereafter. 1945.8 h at 40 and 60 days.

Lawrence et al. 1992). 1982). ECMO is considered to be the clinical intervention that results in the highest intravenous dose of di(2-ethylhexyl) phthalate (Karle et al. 1987) is not adequate for evaluation. Various clinical parameters of liver. 1966. IARC.DI(2-ETHYLHEXYL) PHTHALATE 79 were not related to the level of exposure to phthalate diesters. Others have suggested that more cautious evaluation..3 to 33.. 4.5 mg/kg bw given up to six times by intraperitoneal injection on alternate days) in male Wistar rats (approximately 150 g bw). Furthermore.. One case of occupational exposure to di(2-ethylhexyl) phthalate was associated with asthma in a worker at a PVC-processing plant (WHO. Based on subjective ultrastructural evaluation of one subject. pulmonary and cardiac dysfunction were found to be unaffected in treated infants. There was no relationship between severity of pruritus and post-dialysis serum concentrations of di(2-ethylhexyl) phthalate. 1984.. Mettang et al.] The toxicity of di(2-ethylhexyl) phthalate was evaluated in 28 term infants with respiratory failure. 1975). (1998) evaluated the systemic toxicity of di(2-ethylhexyl) phthalate (0–7.2 Experimental systems In-vivo studies of general toxic effects Acute oral LD50 values for di(2-ethylhexyl) phthalate ranging from 26. Nair et al. Calley et al. 1945. would be needed to determine conclusively whether peroxisome proliferation due to di(2-ethylhexyl) phthalate occurs in dialysis patients (Huber et al.9 g/kg bw in rats. (1984. 18 of whom received extracorporeal membrane oxygenation (ECMO) and were compared with 10 untreated infants.2. 1996). Animals were evaluated by organ . [The Working Group noted that biopsy study of Ganning et al. 8 μg/mL). 1985). 1987). However. mice. no effect was seen after one month of dialysis. None of the workers reported symptoms indicating work-related obstructive lung disease and conventional lung function tests showed no association with exposure to phthalate diesters (Nielsen et al. LD50 values after intraperitoneal administration were 30.7 g/kg bw in rats and 14. guinea-pigs and rabbits have been reported (Shaffer et al.. phthalic acid or 2-ethylhexanol. Di(2-ethylhexyl) phthalate is leached in significant amounts from the PVC tubing used in transfusion and dialysis. (1996b) investigated the relationship between di(2-ethylhexyl) phthalate exposure and uraemic pruritus in dialysis patients. even though the rate of administration ranged up to 2 mg/kg bw di(2ethylhexyl) phthalate over 3–10 days (mean peak plasma concentration. 1997). including objective measurements.. Dialysis patients were studied for evidence of liver peroxisome proliferation in biopsy samples (Ganning et al. 1945. 1975. Lawrence et al.8 g/kg bw in mice (Shaffer et al... in a liver biopsy from another subject after 12 months of dialysis. an increased number of peroxisomes was reported to be present. increased numbers of biopsy intervals. serum concentrations of di(2-ethylhexyl) phthalate and these related compounds were not significantly different between patients with or without uraemic pruritus.2 and 37. and appropriate controls. mono(2-ethylhexyl) phthalate..

resulting in mean di(2ethylhexyl) phthalate intakes of 143. 1000 or 2000 mg/kg bw) in male Sprague-Dawley rats. alanine aminotransferase. 4. respectively. 1996). 14. 797 or 1414 mg/kg bw per day in females. No evidence of toxicity was observed. Suckling rats (starting at 6–21 days of age) suffered severe growth retardation at doses of 1000 mg/kg bw and death at 2000 mg/kg bw.. 21. to 146 and 124% of control values in male and female rats. lactic dehydrogenase. 16. 100. 737 or 1440 mg/kg bw per day in males and 154. 1. and body weight gain and food consumption were not affected. brain and testis) and plasma clinical chemistry (γ-GT. Significant dose-dependent increases in relative liver weight to 116–204% of control values were observed in both males and females at all levels of di(2-ethylhexyl) phthalate treatment. associated with marked seminiferous tubule atrophy. 1997). 3.2. 38 and 375 mg/kg bw per day in males and 0.2. Significant increases in relative liver weight.4. beginning at 6. light microscopy (liver.0% di(2-ethylhexyl) phthalate for 17 weeks. Lethality was observed at doses of 1000 mg/kg bw starting at 14 days of age but not at six days or ≥ 16 days. postweanling male and female Sprague-Dawley (CD) rats were fed diets containing 0.0 and 2.. Apart from the liver.80 IARC MONOGRAPHS VOLUME 77 weight (testis and liver). 10. Dostal et al. age may be an important factor in sensitivity to the lethal effects of oral di(2-ethylhexyl) phthalate..0 and 2. respectively (Gray et al. while older rats showed only decreased weight gain at 2000 mg/kg bw. 500 or 5000 ppm di(2-ethylhexyl) phthalate for 13 weeks (Poon et al. A number of other studies have examined the toxic effects of di(2-ethylhexyl) phthalate in rodents and other species following oral administration.2% di(2-ethylhexyl) phthalate in the diet. In rats. (1987b) evaluated the effects of di(2-ethylhexyl) phthalate (five daily oral doses of 0. respectively. 50. 42 and 419 mg/kg bw per day in females. were observed only in animals given 5000 ppm di(2-ethylhexyl) phthalate. 1977). young male and female Sprague-Dawley rats (10 per sex per group) were fed diets containing 5.7. which is characterized by increases in the number of . Young. alkaline phosphatase). heart. Such effects in rodents include peroxisome proliferation.4. no relative organ weights were affected in male and female rats given 0. No clinical signs of toxicity were observed. In another study. Di(2-ethylhexyl) phthalate levels of 1. 42 or 86 days of age. Morphological examination revealed minimal to mild centrilobular hypertrophy in the liver and mild to moderate seminiferous tubule atrophy in the testis in male rats fed 5000 ppm di(2-ethylhexyl) phthalate and Sertoli cell vacuolation of minimal nature in male rats fed 500 ppm di(2-ethylhexyl) phthalate. Relative testis weight was reduced in male rats fed 5000 ppm di(2-ethylhexyl) phthalate.0% resulted in reduced testis weights in male rats. In-vivo studies of hepatic peroxisome proliferation in rats and mice Many studies have examined the hepatic biochemical and morphological effects of di(2-ethylhexyl) phthalate (reviewed in Huber et al. Mean di(2-ethylhexyl) phthalate intakes were 0.

Di(2-ethylhexyl) phthalate increased relative liver weights and markedly induced hepatic carnitine acetyltransferase activity in both species (up to 25-fold in rats and 10-fold in mice). In rats fed 12 500 ppm di(2ethylhexyl) phthalate. both mono(2-ethylhexyl) phthalate and 2-ethylhexanol increased relative liver weight and produced hepatic peroxisome proliferation. 1984). two or 13 weeks of administration. there was an increase in hepatocyte replicative DNA synthesis (measured after continuous bromodeoxyuridine administration (osmotic pump) for three days before sampling) after one week (but not after two or 13 weeks) and an increase in hepatic peroxisomal β-oxidation activity after one.5–4% di(2-ethylhexyl) phthalate in the diet for one or four weeks and male Swiss Webster mice (20–30 g) were fed 2 or 4% di(2-ethylhexyl) phthalate in the diet for one or four weeks (Reddy et al. there was no increase in hepatocyte replicative DNA synthesis (measured after continuous bromodeoxyuridine three days before sampling) after one.. there was no statistically significant increase in hepatic peroxisomal βoxidation activity after one. In mice fed 1000 ppm di(2-ethylhexyl) phthalate. Male and female Fischer 344 rats and B6C3F1 mice were fed di(2-ethylhexyl) phthalate for up to 13 weeks (David et al. the induction of peroxisomal and microsomal fatty acidoxidizing enzymes and hepatocellular hyperplasia. 1975). While phthalic acid had no effect. Male Fischer 344 rats (body weight. 2-ethylhexanol and phthalic acid at doses equimolar to 2000 mg/kg bw per day di(2-ethylhexyl) phthalate for seven days. Treatment caused increases in relative liver weight and in microsomal cytochrome P450 content. two and 13 weeks’ administration. two or 13 weeks’ administration (bromodeoxyuridine labelling was not evaluated at this lower dietary concentration of di(2-ethylhexyl) phthalate). In addition.. This study also demonstrated that di(2-ethylhexyl) phthalate was a hypolipidaemic agent.. 1999). Rats were also treated with mono(2-ethylhexyl) phthalate.DI(2-ETHYLHEXYL) PHTHALATE 81 hepatocellular peroxisomes. Young male Wistar rats were given 2000 mg/kg bw di(2-ethylhexyl) phthalate per day by gavage for periods of 3–21 days (Lake et al. Ultrastructural examination revealed marked peroxisome proliferation and a dilation of the smooth and rough endoplasmic reticulum. di(2ethylhexyl) phthalate treatment induced cyanide-insensitive palmitoyl-coenzyme A (CoA) oxidation activity and microsomal lauric acid 12-hydroxylase activity in the . 1976). as serum triglyceride levels were reduced to one seventh of control values in rats and one third of control values in mice. Treatment of five-week-old male Sprague-Dawley rats with 1000 mg/kg bw di(2ethylhexyl) phthalate per day by gavage for 14 days caused increased relative liver weight and hepatic peroxisome proliferation (Lake et al. Some increase in hepatic catalase activity (approximately two-fold) was observed and subjective (non-morphometric) ultrastructural examination revealed marked peroxisome proliferation. but there was an increase in hepatic peroxisomal β-oxidation activity after one. two and 13 weeks’ administration. In mice fed 10 000 and 17 500 ppm di(2-ethylhexyl) phthalate. 100–150 g) were fed 0..

1998). Such . although earlier onset and greater severity were seen in the SV129 strain control mice than in the knock-out mice. Isseman et al. Studies of peroxisome proliferator-activated receptor α (PPARα) It has been established that the peroxisome proliferation and hepatocellular proliferation effects of di(2-ethylhexyl) phthalate and other peroxisome proliferators in rodent liver are mediated through activation of the peroxisome proliferatoractivated receptor α (PPARα). Elcombe & Mitchell. This nuclear receptor is a member of the steroid hormone receptor superfamily. In this study.. (1986) fed diets containing 0. In non-hepatocytic systems in vitro.01–2. Reddy et al. 1996). an excellent correlation between the enzymatic marker of the peroxisomal fatty acid β-oxidation cycle and changes in peroxisome morphometry was observed. 1995. while for 2-ethylhexanol the ‘proximate’ peroxisome proliferator was 2-ethylhexanoic acid (Mitchell et al. Di(2-ethylhexyl) phthalate-induced hepatic peroxisome proliferation in rodents is not due to the parent diester per se.25–2. the two ‘proximate’ peroxisome proliferators were found to be the (ω–1)-hydroxy (IX) and keto (VI) metabolites. 1992... induction of mRNA expression for peroxisomal acyl CoA oxidase and microsomal CYP4A) that were observed in SV129 strain control mice (Ward et al. the latter reflects induction of cytochrome P450 isoforms in the CYP4A subfamily.. While the former is considered to be a specific marker of the peroxisomal fatty acid β-oxidation cycle. Its role in mediating the hepatic effects of peroxisome proliferators was demonstrated with knock-out mice that do not express the PPARα receptor (Lee et al. This relatively high intake of di(2-ethylhexyl) phthalate induced histological evidence of testicular and renal toxicity in both strains of mice. Di(2-ethylhexyl) phthalate at dietary levels of 0. addition of mono(2-ethylhexyl) phthalate results in activation of PPARα (Issemann & Green.5% to young male and female Fischer 344 rats for 21 days produced dose-related increases in relative liver weight and in cyanide-insensitive palmitoyl-CoA oxidation and lauric acid 12-hydroxylase activities (Barber et al..2% in the diet for 24 weeks showed none of the hepatic effects (liver weight increase. 1993). cyanide-insensitive palmitoylCoA oxidation activity and peroxisome volume density were observed. 1986. Induction of these two enzymatic markers has also been observed in other studies.0% di(2-ethylhexyl) phthalate to young male Fischer 344 rats for 30 days. Peters et al. 1993). but rather to its metabolites.. 1990. Cornu et al. Dose-related increases in relative liver weight.. 1985. These results establish an absolute requirement for PPARα in the manifestation of hepatic effects of di(2-ethylhexyl) phthalate. demonstrating that peroxisomal cyanide-insensitive palmitoyl-CoA oxidation is a good marker for peroxisome proliferation in rodent liver.82 IARC MONOGRAPHS VOLUME 77 liver. With mono(2-ethylhexyl) phthalate. CYP4A induction is generally associated with peroxisome proliferation (Huber et al. 1997).. 1987). and indicate a contributory role of the receptor in mediating extrahepatic toxicity. Knock-out mice fed di(2-ethylhexyl) phthalate at 1. Lewis & Lake.

. caused transactivation of a responsive reporter gene construct. In another study. male Sprague-Dawley rats were fed a diet containing 2% di(2ethylhexyl) phthalate for two years (Lake et al. Male Fischer 344 rats (seven to nine weeks old) were fed di(2-ethylhexyl) phthalate in the diet for various periods up to 365 days (Marsman et al.... 1989). mono(2-ethylhexyl) phthalate and 2-ethylhexanoic acid. Two studies have demonstrated that oral administration of di(2-ethylhexyl) phthalate to mice results in increased replicative DNA synthesis in hepatocytes.. Levels of conjugated dienes were increased in liver homogenates and morphological examination of liver sections revealed increased lipofuscin deposition in non-nodular/tumorous areas of the liver. . rates of replicative DNA synthesis were similar to those in control animals. Other hepatic studies in vivo Other hepatic effects of di(2-ethylhexyl) phthalate in rodent liver include effects on replicative DNA synthesis and oxidative stress. whereas at later time points. Di(2-ethylhexyl) phthalate was found to increase levels of lipofuscin.4-fold increase in replicative DNA synthesis (James et al. Over the 365-day treatment period. Di(2-ethylhexyl) phthalate produced a burst of replicative DNA synthesis during treatment days 1–8.2% di(2-ethylhexyl) phthalate in the diet for periods of 1–12 months. Conway et al. In the first study. 1998). In COS-1 cells transfected to express high levels of mouse or human PPARα. as demonstrated by cyanide-insensitive palmitoyl-CoA oxidation activity and peroxisome morphometry. Treatment with di(2-ethylhexyl) phthalate resulted in sustained stimulation of cyanide-insensitive palmitoyl-CoA activity and produced up to a twofold increase in levels of 8-hydroxydeoxyguanosine in hepatic DNA. 1989). but not di(2ethylhexyl) phthalate. Replicative DNA synthesis was studied employing subcutaneously implanted osmotic pumps to continuously infuse [3H]thymidine over seven-day periods (Marsman et al. this level being maintained throughout the rest of the treatment period (Conway et al.DI(2-ETHYLHEXYL) PHTHALATE 83 systems were also used to study the ability of di(2-ethylhexyl) phthalate and its metabolites to activate nuclear receptors PPARα from mouse and human (Maloney & Waxman. (1990a. feeding a diet containing 6000 ppm di(2-ethylhexyl) phthalate to male B6C3F1 mice for seven days resulted in a sevenfold increase in replicative DNA synthesis in hepatocytes.. di(2-ethylhexyl) phthalate produced a sustained stimulation of peroxisome proliferation. Liver sections were processed for autoradiography and subsequent determination of the hepatocyte labelling index. 1988). while no increase was observed when the animals were fed for 14 or 28 days (Smith-Oliver & Butterworth. 1987).b) investigated the relationship between hepatic peroxisome proliferation and levels of 8-hydroxydeoxyguanosine in hepatic DNA. 1987). In the second study. 1988. Takagi et al. 1999). Male Fischer 344 rats (six weeks old) were fed 1. as a marker of oxidative stress. administration of oral di(2-ethylhexyl) phthalate (1150 mg/kg bw per day) to male B6C3F1 mice for two days resulted in a 2. three-fold after 39 days of treatment.

84 IARC MONOGRAPHS VOLUME 77 Other hepatic effects have also been attributed to di(2-ethylhexyl) phthalate. Interspecies comparisons in vivo Many studies have demonstrated marked species differences in hepatic peroxisome proliferation. carnitine acetyltransferase: up to 1000% increase in rats versus 180% increase in hamsters). male mice (20–30 g) and male guinea-pigs (320–340 g) were fed 2% di(2ethylhexyl) phthalate in the diet for two weeks (Osumi & Hashimoto. 1999). In another study. hepatic β-oxidation activity and hepatocyte DNA replication. Male and female marmosets (24 months old) were also given 1000 mg/kg bw per day di(2-ethylhexyl) phthalate by daily intraperitoneal . In the same study.. and decreases in hepatocyte apoptosis were observed in the rats but not the guinea-pigs. In rats. 1986).. cyanide-insensitive palmitoyl-CoA oxidation: up to 1400% increase in rats versus 200% increase in hamsters. Increases of about 30% in hepatic phosphatidylcholine and phosphatidylethanolamine were observed in male Wistar rats fed 2% di(2-ethylhexyl) phthalate in the diet for seven days (Mizuguchi et al. male Fischer 344 rats and male Dunkin-Hartley guinea-pigs were given 950 mg/kg bw per day by gavage for four days (Hasmall et al. Significant increases in liver weight. this species was clearly less responsive to di(2-ethylhexyl) phthalate-induced hepatic peroxisome proliferation than rats (liver weight: up to 176% increase in rats versus 122% increase in hamsters. 1984). While some effect was also observed in Syrian hamsters. 2000). carnitine acetyltransferase: 2100% increase in rats versus 162% increase in hamsters). Male Wistar rats (250–350 g). rats and Syrian hamsters were also treated with 500 mg/kg bw mono(2-ethylhexyl) phthalate per day. Male and female Wistarderived rats (six to eight weeks old) and male and female marmosets (12–18 months old) were given 2000 mg/kg bw per day di(2-ethylhexyl) phthalate by gavage for 14 days (Rhodes et al. but none in guineapigs.. As with di(2-ethylhexyl) phthalate. male Sprague-Dawley rats (five weeks old) and male Syrian hamsters (five weeks old) were given di(2-ethylhexyl) phthalate at doses of 25–1000 mg/kg bw per day by gavage for 14 days (Lake et al. cyanideinsensitive palmitoyl-CoA oxidation: 1380% increase in rats versus 171% increase in hamsters.. with a more marked effect in male than in female Sprague-Dawley rats. A number of studies have compared the ability of di(2-ethylhexyl) phthalate to induce hepatic peroxisome proliferation in rats and primates. treatment with di(2-ethylhexyl) phthalate produced dose-related increases in relative liver weight and hepatic cyanide-insensitive palmitoyl-CoA oxidation and carnitine acetyltransferase activities. 1978). In another study. mono(2-ethylhexyl) phthalate produced a greater increase in relative liver weight and a greater stimulation of enzyme activities in rats than in Syrian hamsters (liver weight: 65% increase in rats versus 10% increase in hamsters. male and female Sprague-Dawley rats (140–170 g). Marked induction of enzyme activity was also observed in the mice. Hepatic cyanide-insensitive palmitoyl-CoA oxidation activity was significantly increased in both rat strains.

treatment with di(2-ethylhexyl) phthalate increased relative liver weight and produced hepatic peroxisome proliferation. In rats.. No such effects were observed in marmosets given di(2-ethylhexyl) phthalate by either oral or intraperitoneal administration.DI(2-ETHYLHEXYL) PHTHALATE 85 injection for 14 days. Male Fischer 344 rats were fed diets containing 100–25 000 ppm di(2-ethylhexyl) phthalate for 21 days. 1995). di(2-ethylhexyl) phthalate did not produce morphological changes in the livers of male and female marmosets or in the testes of the males. the results are considered not to have demonstrated peroxisome proliferation. Furthermore. A preliminary dose-setting study indicated that 2500 mg/kg bw di(2-ethylhexyl) phthalate per day was close to the maximum tolerated dose in this species. these results indicate that di(2-ethylhexyl) phthalate treatment did not produce testicular damage in adult marmosets. 1995). Treatment with di(2-ethylhexyl) phthalate did not affect relative liver weight or hepatic cyanideinsensitive palmitoyl-CoA oxidation and carnitine acetyltransferase activities. Groups of four male and four female marmosets (final body weight. Thus. In rats. although small increases in mean peroxisomal volume were noted in male marmosets given 500 or 2500 mg/kg bw di(2-ethylhexyl) phthalate per day. 1987). In addition. Ultrastructural examination did not reveal significant changes in peroxisome numbers per hepatocyte or in peroxisome volume density. Many of the known characteristics of peroxisome proliferation in vivo. carnitine acetyltransferase and lauric acid 12-hydroxylase in male cynomolgus monkeys. 1998). . di(2-ethylhexyl) phthalate treatment produced a dose-related increase in relative liver weight and in enzymatic markers and ultrastructural evidence (subjective evaluation) of hepatic peroxisome proliferation. in addition to demonstrating a lack of hepatic peroxisome proliferation. differential induction of peroxisomal enzyme activities and stimulation of replicative DNA synthesis. as demonstrated by ultrastructural examination and increased cyanide-insensitive palmitoyl-CoA oxidation and lauric acid hydroxylation. around 360 g) were given 100. have been demonstrated in cultured rat and mouse hepatocytes (IARC. while male cynomolgus monkeys were given 100 or 500 mg/kg bw di(2-ethylhexyl) phthalate per day by gavage for 25 days (Short et al. no treatment-related changes were observed by light or electron microscopic examination of liver sections.. Significant suppression of body weight gain was observed in male marmosets given 2500 mg/kg di(2-ethylhexyl) phthalate per day. Treatment with di(2-ethylhexyl) phthalate did not affect relative liver weight or activities of cyanide-insensitive palmitoyl-CoA oxidation. 500 or 2500 mg/kg bw di(2-ethylhexyl) phthalate per day by gavage for 13 weeks (Kurata et al. As peroxisomal volume density is considered the most accurate morphometric measurement of peroxisome proliferation by ultrastructural evaluation (IARC. Interspecies comparisons with hepatocytes in vitro Peroxisome proliferation may also be demonstrated in vitro in cultured rat and mouse hepatocytes. including increased number and size of peroxisomes.

Mono(2-ethylhexyl) phthalate was also shown to induce cyanide-insensitive palmitoyl-CoA oxidation and.While both caused concentration-dependent induction of cyanide-insensitive palmitoyl-CoA oxidation in rat hepatocytes.5 mM mono(2-ethylhexyl) phthalate for 72 h (Elcombe & Mitchell. Metabolite VI induced cyanide-insensitive palmitoyl-CoA oxidation and lauric acid hydroxylation in cultured . Doull et al. In another study with hepatocytes from Wistar-derived rats (180–220 g).. 1986).2 mM mono(2-ethylhexyl) phthalate or 1 mM 2-ethylhexanol for 48 h (Gray et al. male Alderley Park guinea-pigs (400–500 g). 1986).0 mM metabolite VI produced concentration-dependent increases in lauric acid hydroxylation.. Hepatocytes were isolated from Wistar-derived rats (180–220 g) and treated for 72 h with 0–0. 1999). Both di(2-ethylhexyl) phthalate metabolites increased carnitine acetyltransferase activity about nine-fold. male marmosets (350–500 g) and three human liver samples (renal transplant donors) were treated with 0–0.. Treatment with mono(2-ethylhexyl) phthalate and metabolites VI and IX (see Figure 1) resulted in a concentration-dependent induction of cyanide-insensitive palmitoyl-CoA oxidation. 1985). treatment with 0. 1995. In studies with hepatocytes from male Sprague-Dawley rats (180–220 g). Hepatocytes isolated from Wistar-derived rats (180–220 g).86 IARC MONOGRAPHS VOLUME 77 Hepatocytes isolated from male Wistar rats (180–250 g) were treated with 0. Hepatocytes were isolated from male Sprague–Dawley rats (180–220 g).5 mM mono(2-ethylhexyl) phthalate or metabolite IX for 72 h (Mitchell et al. Treatment with 20–200 μM mono(2-ethylhexyl) phthalate for 70 h caused strong induction of cyanide-insensitive palmitoyl-CoA oxidation activity in rat hepatocytes (up to 600% of control levels). male Syrian hamsters (70–80 g) and male Dunkin-Hartley guinea-pigs (400–450 g). While there was a concentration-dependent induction of cyanide-insensitive palmitoyl-CoA oxidation in rat hepatocytes. Species comparisons of hepatic peroxisomal proliferation have also included studies of human and non-human primate primary hepatocyte cultures. 1983). In addition.5 mM mono(2-ethylhexyl) phthalate and 0–1.0 mM 2ethylhexanol for 48 h resulted in induction of carnitine acetyltransferase activity about 15-fold and six-fold. while no marked effect was observed in Syrian hamster (up to 120% of control) or guinea-pig (down to 80% of control) hepatocytes (Lake et al. 1982). Hepatocytes were isolated from male Wistar-derived rats (180–220 g) and male Alderley Park guinea-pigs (400–500 g) and treated with 0–0. Treatment with metabolites I and V resulted in only small effects on the enzymatic markers of peroxisome proliferation. 1985). 1986). Primary hepatocyte cultures may also be employed to study species differences in hepatic peroxisome proliferation (IARC.. by ultrastructural examination.. no such effect was observed in guinea-pig hepatocytes. respectively (Gray et al. no induction was observed in guinea-pig or human hepatocytes and only small non-concentrationdependent effects were observed in marmoset hepatocytes. metabolite VI was shown by subjective ultrastructural examination to cause proliferation of peroxisomes (Elcombe & Mitchell. to increase numbers of peroxisomes. 0–0.5 mM mono(2ethylhexyl) phthalate and some mono(2-ethylhexyl) phthalate metabolites (Mitchell et al..2 mM mono(2-ethylhexyl) phthalate and 1.

Treatment with mono(2-ethylhexyl) phthalate induced β-oxidation activity. rabbit and cynomolgus monkey hepatocytes. Hepatocytes were isolated from male Fischer 344 rats and from three human liver (liver transplantation donors). The treatment of rat hepatocytes with 0–300 μM of mono(2-ethylhexyl) phthalate or metabolite VI resulted in a concentration-dependent induction of cyanide-insensitive palmitoyl-CoA oxidase and lauric acid 12-hydroxylase activities. Hepatocytes were isolated from male Wistar-derived rats.. male Dunkin-Hartley guinea-pigs (350 g). two dogs (age. but had no significant effect in either guinea-pig or human hepatocytes. sex not stated) (Hildebrand et al. Hepatocytes were isolated from male Fischer 344 rats and from two human liver samples (liver surgery patients).. Treatment with 200 μM mono(2-ethylhexyl) phthalate for either 48 or 72 h induced carnitine acetyltransferase activity in cultured rat but not human hepatocytes (Butterworth et al.DI(2-ETHYLHEXYL) PHTHALATE 87 rat hepatocytes. Treatment with up to 1100 μM metabolite VI for 72 h caused a concentration-dependent induction of cyanide-insensitive palmitoyl-CoA oxidation in rat hepatocytes. Hepatocytes were isolated from male Alderley Park (Wistar-derived) rats. Similarly.. male Alderley Park (Dunkin-Hartley) guinea-pigs and . Hepatocytes were isolated from male Wistar rats (200 g). lauric acid hydroxylation activity was not induced in marmoset or human hepatocytes treated with 0–2. male New Zealand rabbits (2500 g) and cynomolgus monkeys (two to three years old) and treated with mono(2-ethylhexyl) phthalate or with metabolites VI or V for 72 h (Dirven et al. rabbit or cynomolgus monkey hepatocyte cultures. In collagen sandwich cultures. 1989). whereas treament with 0–600 μM of either compound had no effect on lauric acid 12-hydroxylase activity in guinea-pig..0 mM metabolite VI and guinea-pig and human hepatocytes with 0–2.0 mM metabolite VI resulted in no induction of cyanide-insensitive palmitoyl-CoA oxidation activity. In contrast. metabolite V had very little effect. replicative DNA synthesis and inhibited apoptosis induced by transforming growth factor β (TGFβ) in cultured rat but not human hepatocytes (Hasmall et al. breed and sex not stated) and two human subjects (69–71 years of age. Small increases in cyanide-insensitive palmitoyl-CoA oxidase activity were observed at mono(2-ethylhexyl) phthalate and metabolite VI concentrations of 600 μM. male Alderley Park guinea-pigs and three human liver samples (liver transplant donors). Hepatocytes were isolated from male Wistar rats. but no such effect was observed in cultured human hepatocytes (Elcombe et al. 1996). the rat hepatocytes responded to di(2ethylhexyl) phthalate in the culture medium with slightly increased carnitine acetyltransferase activity.. In guinea-pig. 1993c). 1999). male Alderley Park (Swiss) mice. 1999). treatment of marmoset hepatocytes with 0–1. Ultrastructural examination revealed an increase in the numbers of peroxisomes in rat hepatocytes.0 mM metabolite VI. while dog and human hepatocytes did not respond. neither mono(2-ethylhexyl) phthalate nor metabolite VI had any effect on cyanide-insensitive palmitoyl-CoA oxidase activity at concentrations of up to 300 μM.

3 4. 1992). Increased peroxisomal ω-oxidation (at 250–750 μM). An effect of di(2-ethylhexyl) phthalate on estrogen metabolism has been reported (Eagon et al. The testicular toxicity of di(2-ethylhexyl) phthalate is described in Section 4.. 1995). and inhibition of apoptosis (at 250–1000 μM) were observed in rat hepatocytes. Other effects The effect of di(2-ethylhexyl) phthalate in diet (2% for 21 days) on lipoprotein metabolism in male Wistar rats was evaluated (Mocchiutti & Bernal.88 IARC MONOGRAPHS VOLUME 77 male captive bred (ICI.3. 1997). Peroxisome proliferators have also been shown to induce replicative DNA synthesis in cultured rodent hepatocytes (IARC. 1994).. as evaluated by functional observational battery and motor activity testing (Moser et al. Alderley Park) marmosets. 1995).. estrogen 2-hydroxylase.2% di(2-ethylhexyl) phthalate for four.. . several peroxisome proliferators have failed to induce replicative DNA synthesis in human hepatocyte cultures (Doull et al. 1999). Hepatocytes were isolated from male Wistar-derived rats and from three human liver samples (liver transplantation donors) and treated with 2ethylhexanoic acid and some other peroxisome proliferators for 72 h (Elcombe et al. no effect was observed in human hepatocytes.. Hepatocytes were isolated from male Fischer 344 rats and three humans and treated in culture with 250–2000 μM mono(2-ethylhexyl) phthalate (Hasmall et al. replicative DNA synthesis (at 500–1000 μM). 1999).3. While 2-ethylhexanoic acid induced replicative DNA synthesis in cultured rat hepatocytes. 1996).1 Reproductive and developmental effects Humans No data were available to the Working Group. eight or 16 weeks had significantly increased serum estradiol levels. and a male-specific estrogen-sequestering protein. Male Fischer 344 rats fed diets containing 1. The observed reduction in plasma triglyceride levels was associated with (and attributed to) increased activity of extrahepatic lipoprotein lipase. None of these parameters was affected by mono(2-ethylhexyl) phthalate in human hepatocytes. Female Fischer 344 rats treated with a single oral dose (up to 5000 mg/kg bw) or with repeated doses (up to 1500 mg/kg bw per day for 14 days) of di(2-ethylhexyl) phthalate showed no neurobehavioural effects. Treatment with either 2-ethylhexanol or 2-ethylhexanoic acid for 72 h produced a concentration-dependent induction of cyanide-insensitive palmitoyl-CoA oxidation in rat and mouse but not guineapig or marmoset hepatocytes (Cornu et al. This was explained by the observation that these rats showed significant loss of hepatic activity of a major male estrogen-metabolizing enzyme.. In contrast. 4.

external. 0. Groups of 22–25 Fischer 344 rats were fed diets containing 0.3.. growth and malformations. It was noted that di(2-ethylhexyl) phthalate impairs the fertility in both sexes of adult rats at doses above 100 mg/kg bw per day. 10 and 15.0.2 μm) of 0.. decreases in sperm production and a depletion of testicular zinc.. 1988).2 Experimental systems The teratogenicity and reproductive toxicity of di(2-ethylhexyl) phthalate have been reviewed (Huber et al. Fetal body weight was reduced in both treated groups. but was not consistent with the species sensitivity to peroxisome proliferation (e. 1. haemangiomas of the legs) but not skeletal abnormalities was also evident in the high-dose group (Singh et al. The only dose-related effect noted was an increase in the extent of retarded development (renal pelvic dilatations) in the highest-exposure group. 1972). visceral and . Di(2-ethylhexyl) phthalate was associated with a reduction in relative testis weight. 0.0% di(2-ethylhexyl) phthalate (> 99% pure) on days 0–20 of gestation (Tyl et al. 1. viability.5 or 2. and that several studies indicate that di(2-ethylhexyl) phthalate is embryotoxic and teratogenic in rodents. No dose-related effects were noted. Both the embryotoxic and testicular effects in adults were considered to be observed at doses above those at which peroxisome proliferation was recorded.. Groups of 25 Wistar rats were exposed by head–nose inhalation to di(2-ethylhexyl) phthalate (> 99% pure) at aerosol concentrations (mass median aerodynamic diameter (MADD) 50% of < 1.. Effects on testicular development in rats following exposure to di(2-ethylhexyl) phthalate prenatally and during suckling or during adolescence at dose levels below those associated with peroxisome proliferation have been reported (Poon et al. 1988).g. Fetuses were examined on gestation day 20 for growth. 1996).2. 1997.05 and 0. (a) Developmental toxicity studies (i) Rats Sprague-Dawley rats were administered intraperitoneal injections of 5 or 10 mL/kg bw [4930 or 9860 mg/kg] di(2-ethylhexyl) phthalate on gestation days 5.0 mg/L (the maximum concentration that could be achieved)] for 6 h per day on gestation days 6–15.g.1.5..3 mg/L [a range-finding study noted a dose-related trend for hepatic peroxisome proliferation at levels of 0. The remaining five females per group were allowed to litter and their offspring were examined for postnatal growth and viability. Twenty females were killed on gestation day 20 and their fetuses examined for viability. resorptions increased from about 8% in controls and the low-dose group to 27% in the high-dose group and an increase in the incidence of abnormalities (e. 1998). guinea-pigs were more sensitive to the testicular effects than were Syrian hamsters). Arcadi et al.5 and 1. 0. The testicular response appeared to be species-specific.. The metabolite mono(2-ethylhexyl) phthalate was judged to be more potent in causing both teratogenicity and reproductive toxicity.. but no mechanism for either response was identified. The only maternal effect seen was a reduction in body weight on post-partum day 21 in the high-dose group (Merkle et al. 0.DI(2-ETHYLHEXYL) PHTHALATE 89 4.

In another study. 1995). Maternal body weight gain effects and reduced fetal weights were evident at levels of 1. 200 and 1000 mg/kg bw per day by oral . The effects on testicular development in the offspring of rats exposed to di(2ethylhexyl) phthalate in utero were studied by Tandon et al. microphthalmia. The only sign of reproductive toxicity was an 8% decrease in pup birth weight (National Toxicology Program. 0. There were no effects upon maternal body weight or clinical findings and the only developmental effects were a delay in parturition and micro/anophthalmia at 750 and 1125 mg/kg bw. 750 or 1125 mg/kg bw di(2-ethylhexyl) phthalate per day by gavage on gestation days 6–15. No significant increase in the incidence of variations or malformations was observed. 1997a).g.0% and above. suggesting alterations in Sertoli cell function and germ cell maturation. No treated pups survived to postnatal day 6 and malformations (e. sorbitol dehydrogenase. Exposure to either level resulted in decreased maternal weight gain on gestation days 6–8 and a significant increase in females with fully resorbed litters. Testicular weight was significantly reduced in the offspring only at 31 days of age. Alterations in the activity of testicular γ-GT. In a screening assay for developmental toxicity. (1991). viability and malformations. 61 and 91 days of age.. 1997). Di(2-ethylhexyl) phthalate was one of several phthalate esters evaluated for prenatal toxicity in Wistar rats (Hellwig et al. In a follow-up study. Groups of six Wistar rats were exposed to 0 or 1000 mg/kg di(2-ethylhexyl) phthalate by gavage for the entire period of gestation and spermatogenesis was assessed in the offspring at 31. groups of 16–21 Fischer 344 rats were exposed to di(2-ethylhexyl) phthalate (98% pure) by oral gavage at doses of 0. Epididymal sperm counts were reduced by about 22% at 91 days of age. it was reported that the high-dose level reduced the body weight of the parental generation by 25%. These effects were in contrast to the earlier study.25. Fetal viability was reduced at the highest exposure level.5 or 1% di(2-ethylhexyl) phthalate in the diet during gestation and offspring were evaluated for reproductive function and fertility. Fischer 344 rats were exposed to 0. so a comparison study was made using dose levels of 1125 and 1500 mg/kg bw administered on gestational days 6–15 or 6–19. 500. while reductions in food consumption were seen at the mid.. Maternal food intake was decreased and water consumption and liver weights were increased at all dose levels. In the summary of the study. but the authors stated that in the 6–15-day groups. increased prenatal mortality. missing innominate artery and cleft palate) were present in a few pups that were found dead. 1995). Groups of 10 females received di(2-ethylhexyl) phthalate at doses of 0.90 IARC MONOGRAPHS VOLUME 77 skeletal malformations and variations. there were significant maternal weight loss. 1125 or 1500 mg/kg bw per day on gestation days 6–19 (Narotsky & Kavlock. 40. 0. groups of 10–12 Fischer 344 rats were exposed to 0.and high-dose level. delay in parturition and eye malformations (Narotsky et al. Detailed results were not presented. lactate dehydrogenase and β-glucuronidase were noted.. especially at the youngest age. 333. The litters were followed postnatally for growth.

which was assumed to be due to increased water intake. the exposure was roughly calculated to correspond to 3. Low-dose rats exhibited only a few elongated spermatids in tubules showing a pervious lumen. 42. The increase in liver weight was not dose-related. predominantly of the tail. Pregnancy rate. 56 days). 28. Four adult male rats exposed to the same levels via the drinkingwater for 42 days did not show any significant change in either kidney. At the end of the observation period. there were pronounced postimplantation loss. 35.. absence of elongated spermatids and spermatozoa and tubular lumen filled with cellular deposits. Also in the highdose group. The alterations were less pronounced at week 8. Groups of 12 female Long-Evans rats were exposed daily to drinking water containing 32. A reduction in kidney weight (absolute and relative) was observed at both dose levels.5 or 325 μL/L di(2-ethylhexyl) phthalate from day 1 of pregnancy to day 21 after the delivery. eight pups per group were killed. the histological findings included a generalized disorganization of the tubular epithelium. vertebral column and sternum. The perinatal exposure caused severe histological damage to the testes. The highest dose level decreased food consumption and body weight gains. Female pups exposed perinatally to 325 μL/L di(2-ethylhexyl) phthalate showed a significantly increased time necessary to perform the beam walking test. A dose-dependent reduction in testicular weight (absolute and relative) was observed and did not appear to return to normal with growth. Perinatal exposure produced no significant changes in body weight gain in the pups. Histopathology of the kidneys. 1998). and only minor histological damage of the testes. Female pups were used for behavioural assessment 30 days after birth in the ‘beam walking’ test.5 and 30–35 mg/kg di(2-ethylhexyl) phthalate per day during pregnancy. urinary tract. At 21 and 28 days of age. there were still severe histopathological changes in the testes of pups. body weight gain and gross appearance in the dams were not affected by the treatment. designed to assess the locomotor activity by employing a learned avoidance test.0–3. brain. reduced numbers of live fetuses and an increase in malformations. during suckling this value increased by at least 30%. indicating a behavioural effect expressed as reduced locomotor activity. There are reports indicating major organ toxicity in the offspring including irreversible testicular effects following exposure to di(2-ethylhexyl) phthalate prenatally and during suckling (Arcadi et al. there was a gross disorganization of the seminiferous tubular structure. and increased relative liver and kidney weights at term. with spermatogonia detached from the basal membrane.DI(2-ETHYLHEXYL) PHTHALATE 91 gavage on gestation days 6–15. liver and testes was also studied. Based on estimated water intake. gonads. . dilation of renal tubuli and light fibrosis) between weeks 0 and 4 of age. accompanied by histopathological findings (shrinkage of renal glomeruli with signs of glomerulonephritis. In high-dose animals. Pup body weight gain and kidney. liver or testis relative weights. At different times after delivery (21. two dams in this group showed vaginal haemorrhage during treatment. at 56 days. detachment of the spermatogonial cells from basal membrane and absence of spermatocytes in both exposure groups. liver and testis weights were measured.

All four biochemical parameters showed significant decreases in the di(2-ethylhexyl) phthalate-treated pups relative to control. In the livers of the pups (5). a concentration of 25. were significantly reduced. Food consumption was reduced in the dams dosed on days 14–18. (1985) exposed groups of five dams (albino rats) to 0 or 2000 mg/kg bw di(2-ethylhexyl) phthalate. mammary gland weights. 6–10 or 14–18 of lactation (Dostal et al. and protein were increased relative to control rats. Two pups from each litter were killed 3–4 h after the third dose. plasma contained virtually no di(2-ethylhexyl) phthalate (< 0. and concentration of cytochrome P450. In treated rats.to eight-fold in treated dams at all three stages of lactation. whereas milk lactose was significantly decreased. Litters of seven pups were dosed with di(2-ethylhexyl) phthalate through mothers’ milk throughout the lactation period (from parturition to day 21). Pooled liver homogenates were prepared for an assay of the activities of arylhydrocarbon hydroxylase. Relative liver weights were increased in the lactating dams at all three stages of lactation but not in the suckling pups. aniline hydroxylase and ethylmorphine Ndemethylase. 1987a). Pup body weights were recorded at five-day intervals and on day 21 when the pups were killed. resulting in a high milk:plasma ratio for di(2-ethylhexyl) phthalate and a low milk:plasma ratio for . Twofold increases in these enzyme activities were also observed in pups suckling the treated dams. and total milk solids.7 ± 0. The body weight of the di(2ethylhexyl) phthalate-treated pups was lower than that of the control group throughout the whole period. Plasma cholesterol and triglyceride concentrations were decreased by 30–50%. the dams were killed and milk and mammary glands were collected.. lipid.3 μg/g di(2-ethylhexyl) phthalate was found.5 μg/mL) but substantial amounts of mono(2-ethylhexyl) phthalate (76 μg/mL). The body weights of lactating rats and of their suckling pups were significantly reduced at all treatment intervals. The hepatic peroxisomal enzyme activities (cyanide-insensitive palmitoyl-CoA oxidase and carnitine acetyltransferase) were increased five.92 IARC MONOGRAPHS VOLUME 77 (ii) Postnatal studies in rats Parmar et al. Two hours after dosing on day 17. 10 pups per litter were removed from the dams to allow milk to accumulate. Six hours after the last dose. In contrast. both absolute and relative. Female Sprague-Dawley rats were given five oral doses of 2000 mg/kg bw per day di(2-ethylhexyl) phthalate (> 99% pure) in corn oil by gavage on days 2–6. Increased activities of cyanide-insensitive palmitoyl-CoA oxidase and carnitine acetyltransferase in dams and pups were observed. relative liver weight was similar in the two groups. Hypolipidaemia was observed in treated lactating rats at all three stages of lactation. The rats were killed 24 h after the last dose. The transfer of di(2-ethylhexyl) phthalate into the milk of lactating rats was shown in groups of female Sprague-Dawley rats given three oral doses of 2000 mg/kg bw per day di(2-ethylhexyl) phthalate in corn oil by gavage on days 15–17 of lactation. Absolute liver weight was significantly decreased in the di(2ethylhexyl) phthalate-treated pups. Milk collected 6 h after the third dose contained 216 μg/mL di(2-ethylhexyl) phthalate and 25 μg/mL mono(2-ethylhexyl) phthalate.

In a neonatal rat model used to assess di(2-ethylhexyl) phthalate toxicity following intravenous administration. beginning at the age of six (one week old). 14–16 (two weeks old). epididymis. Absolute and relative liver weights were significantly increased at 100 mg/kg bw per day in all age groups (except for one-week-old rats) and in all age groups at higher dose levels. and a significant decrease in the activity of acid phosphatase and sorbitol dehydrogenase at 31 and 61 days of age compared with controls. virtually all pups in the three youngest age groups died. in suckling rats only small decreases (not significant) occurred. After two doses of 2000 mg/kg bw per day. whereas significant decreases in plasma triglycerides were observed in weanling and adult rats.8 mg/kg bw di(2-ethylhexyl) .and two-week-old) than in weanling (threeweek-old) and adult controls. The authors concluded that exposure to di(2-ethylhexyl) phthalate during early life through mother’s milk causes biochemical alterations which may affect the functional development of the testis. twoand three-week-old rats. 61 or 91 days when testes. In di(2-ethylhexyl) phthalate-treated rats. groups of 12 neonates. 21 (three weeks old).and 12-week-old rats showed significantly decreased body weight but no fatalities. 1987a). were injected intravenously with 30. The offspring of di(2ethylhexyl) phthalate-treated mothers showed a significant increase in the activity of testicular γ-GT. The pups were killed at the age of 31. 100. 91. In suckling rats. The activities of these enzymes were similar in control rats of all ages. lactate dehydrogenase and β-glucuronidase. Absolute kidney weight was reduced in some cases. Five daily doses of 1000 mg/kg bw per day caused significant decreases in body weight gain in one-. (1990) performed a study on testis development in male rats after exposure during the suckling period. plasma cholesterol concentrations were significantly reduced in weanling and adult rats given doses of 1000 mg/kg bw per day or more. Groups of 10 male Sprague-Dawley rats were given 0. Tandon et al.7 or 164. two to four days of age. Plasma cholesterol concentrations were higher in suckling control rats (one. 42 (six weeks old) or 86 (12 weeks old) days (Dostal et al.. The activities of cyanide-insensitive palmitoyl-CoA oxidase and carnitine acetyltransferase were increased in a dose-dependent manner in all age groups. plasma cholesterol levels were increased at 1000 mg/kg bw per day. The control group was given the vehicle. whereas relative kidney weight was increased at doses of 1000 mg/kg bw per day or more in rats three or more weeks old. whereas six. 10. No effect on these testicular enzymes was seen in 91day-old rats.DI(2-ETHYLHEXYL) PHTHALATE 93 mono(2-ethylhexyl) phthalate. Plasma triglyceride levels in the control group were similar at all ages. prostate and seminal vesicles were examined. 1987b).8. 1000 or 2000 mg/kg bw per day di(2-ethylhexyl) phthalate (> 99% pure) in corn oil by gavage for five days. Di(2-ethylhexyl) phthalate and mono(2-ethylhexyl) phthalate were not detected in the plasma of the pups (Dostal et al. Groups of four female rats were given vehicle (groundnut oil) or 2000 mg/kg bw per day di(2-ethylhexyl) phthalate orally for 21 days from parturition.. Morphological examination revealed increased peroxisome proliferation in neonatal as well as in adult rats.

(iii) Mice In a study designed to identify the gestational days when mice are particularly sensitive to di(2-ethylhexyl) phthalate. Dose levels of 0.. In the 0.0. myeloschisis and tail anomalies were observed.1% and above significantly reduced embryonic viability. stomach. 5.5. 7. corresponding to 1/30. The incidences of fetal deaths were 8% and 5%. exencephaly. 0. Doses of 5 or 10 mL/kg bw given on day 7 led to 100% fatality of the fetuses.g. malformations such as exencephaly. 1.5 mL/kg bw on day 7 or 8 of gestation. 1980). 2. Body weight gains and average weight gain per day were significantly and dose-dependently decreased from days 4 to 21 of the treatment period..5.5 mL/kg bw on day 7.5 or 7. High rates of external (e. open eyelid. Maternal body weight at the end of pregnancy was reduced at dose levels of 0. The number of resorptions was increased largely depending on the dose and particularly on the day of dosing. 1/4. club foot and bent tail) and skeletal anomalies (e. groups of three to eight female ddY-Slc(SPF) mice. spleen. 8. respectively. lungs. Dose levels of 2.0% by weight throughout gestation and effects on the developing fetus were assessed (Shiota & Nishimura. The average body weights of the fetuses were decreased at all dose levels.2% di(2-ethylhexyl) phthalate group.2. duodenum and caecum) were prepared for histopathological evaluation. but particularly by 2. heart. Control neonates were injected with a solution of 4% BSA or saline.. 7.g. A high and doserelated increase was observed in animals dosed on days 7 and 8 of gestation at all dose levels tested.1. regardless of the day of maternal exposure. kidneys. Neonates were examined for signs of toxicity immediately after treatment and again 1–3 h later. 9 or 10 (Tomita et al. 10 or 30 mL/kg bw di(2-ethylhexyl) phthalate (> 99% pure. The numbers of live fetuses were greatly reduced by treatment on gestational days 7–9.5 mL/kg di(2-ethylhexyl) phthalate given to mice on day 7 or 8 of gestation induced a high incidence of gross and skeletal abnormalities including encephaly. Absolute and relative liver weights were significantly increased in a dose-related manner. 1/6. eyes. when 10 mL/kg bw di(2-ethylhexyl) phthalate was administered on day 9 or 10 of gestation. No conclusive histopathological alterations were detected in the tissues.94 IARC MONOGRAPHS VOLUME 77 phthalate in 4% bovine serum albumin (BSA) solution for 18 consecutive days (Greener et al. 7.. 8.2% and higher. 1982a). 0. liver. Animals were killed 24 h after the last treatment (postnatal days 20–23) and a complete necropsy was performed and selected tissues (brain. 1/3 or 1/1 of the acute oral LD50) by gavage on day 6. 0. injection site.4 or 1. or were untreated. were given 0. Di(2-ethylhexyl) phthalate was administered in the diet to groups of 7–24 ICR mice at levels of 0. . There was no information on maternal toxicity. 1/12. 1982). seven to eight weeks of age. abnormal vertebrae and ribs) were noted in groups receiving 2. open eyelid and club foot. 0.05–30 mL/kg di(2ethylhexyl) phthalate on gestational day 6.5 or 7. Groups of 6–19 ddY-Slc × CBA mice were administered 0.05. 1987). Doseand stage-dependent effects on fetal development were observed.. 9 or 10 of gestation (Yagi et al.

05% level.025 or 0. laevocardia. Fetal body weights were reduced by about 23% and relative liver weights increased by about 22% in rats exposed by oral gavage to 1000 mg/kg bw di(2-ethylhexyl) phthalate on gestational days 6–15. CD-1 mice were exposed via the diet to 0. 0. 1989).5 or 25 mmol/kg). beginning at the 0. 1000. and the offspring were evaluated for acquisition of developmental landmarks. short and kinky tails. This dose also reduced maternal weight gain during pregnancy and decreased the activity of several enzymes (dehydrogenase.10% and above reduced fetal viability and. 500.10%. although no malformations were observed (Srivastava et al. central nervous system. One of 11 females died in the highest-dose group. Control females were untreated [no maternal effects were reported]. with the order of potency suggesting that 2-ethylhexanoic acid may be the proximate teratogen [malformation data not shown]. 0. 2-ethylhexanol (6. 1000 or 2000 mg/kg bw or by intraperitoneal injection (3–9 per group) at doses of 0. The summary report of the study indicates that no effects were observed on reproductive toxicity in either generation (National Toxicology Program. 1988). All three chemicals induced malformations (hydronephrosis. malate dehydrogenase. Fetal body weight was reduced in females at 0. Using a similar experimental design.05. misplaced digits and bowed radius). whereas this effect was not observed until the 0. adenosine triphosphatase and cytochrome c oxidase) in fetal livers. Concentrations of 0. 1997b).. 0. No effects on the fetuses were observed following intraperitoneal exposure.5 mmol/kg) or 2-ethylhexanoic acid (6. 250.15% di(2-ethylhexyl) phthalate (> 99% pure) on days 0–17 of gestation (Tyl et al. 4000 or 8000 mg/kg bw on days 7–9 of gestation (Shiota & Mima. di(2-ethylhexyl) phthalate exposure caused resorptions.10 or 0. When given by the oral route.25 or 12.. septal defects.DI(2-ETHYLHEXYL) PHTHALATE 95 Groups of 9–11 Slc-ICR mice were exposed to di(2-ethylhexyl) phthalate by oral gavage at concentrations of 0. increased the incidence of malformations (affecting the eye. Maternal body weight gain was reduced and liver weights were increased at 0. Controls received the olive oil vehicle. anencephaly. ectrodactyly..10% and above. axial skeleton and tail). viability.25 or 12. heart.05% di(2-ethylhexyl) phthalate in the diet on days 0–17 of gestation. external. visceral and skeletal malformations and variations. 1985).025. growth and morphology on day 20 of gestation (Ritter et al. (b) Mechanistically oriented developmental toxicity studies Groups of 7–10 Wistar rats were dosed by gavage with undiluted di(2-ethylhexyl) phthalate (12. 2000.5 mmol/kg) on day 12 of gestation and fetuses were examined for viability. Groups of 24–30 CD-1 mice were fed diets containing 0. these authors reported that mono(2ethylhexyl) phthalate was not toxic to development in the mouse. reduced fetal weight and an increased incidence of malformations (exencephaly. tail anomalies) at doses of 1000 mg/kg and above. spontaneous locomotor activity and fertility. 1987). Fetuses were examined on gestational day 17 for growth. 0.01. In another study. 500.15% level in male offspring. although two of three females exposed to the high dose died. . 0. open eyelids.

0% (0. 737 or 1440 mg/kg bw per day in males) for two.. (c) Reproductive toxicity studies (i) Rats Sprague-Dawley rats received daily intraperitoneal injections of 50 or 100 mg/kg bw di(2-ethylhexyl) phthalate on alternate days for 20 days. six or 17 weeks. incidence of malformations) was seen in mice of both genotypes. In eight-dayold pups. 1999).2-bis(para-chlorophenyl)ethylene)) (Gray et al.1-dichloro-2. Testicular lesions of this nature have not been observed with known anti-androgens. were administered di(2-ethylhexyl) phthalate by gavage at 0 or 1000 mg/kg on days 8–9 of gestation. 2. The reproductive effects of 10 known or suspected anti-androgens on sexual differentiation were investigated in male rats. growth retardation. 143. di(2-ethylhexyl) phthalate induced lower body weight. including atrophy and agenesis. suggesting that the developmental effects are not PPARα-mediated. Offspring were evaluated on gestational days 10 and 18.96 IARC MONOGRAPHS VOLUME 77 Di(2-ethylhexyl) phthalate (0.and high-dose groups were lower than ..0. Groups of 10–13 pregnant female mice. In the liver. Groups of 10 Long-Evans hooded rats were administered 0 or 750 mg/kg di(2-ethylhexyl) phthalate by gavage from day 14 of pregnancy to day 3 of lactation. In the offspring. The absolute and relative testicular weights of rats in the mid. which indicates that di(2ethylhexyl) phthalate affects the developing male reproductive system by a mechanism that is distinct from that of previously described reproductive toxicants. cyanide-insensitive palmitoyl-CoA oxidase activity showed similar increases in pups and adult females. 1994). several males from different litters displayed haemorrhagic testes that were visible by gross examination of the inguinal region..5. Similar developmental toxicicity (resorptions.e. 1. procymidone. Pup growth was impaired at the two lowest doses.2.b). vinclozolin. retained nipples and high levels of testicular and epididymal abnormalities. or 2. Groups of five or 15 male and female Sprague-Dawley rats were exposed to di(2ethylhexyl) phthalate in the diet at concentrations of 0. Additional analysis showed that di(2ethylhexyl) phthalate induced maternal hepatic CYP4A1 mRNA in the wild-type females only. The profile of the effects induced by di(2-ethylhexyl) phthalate was different from that induced by some known androgen-receptor antagonists (i.5 or 5 g/kg per day) was administered to Fischer 344 rats by oral gavage from birth through lactation day 21 and the activity of several peroxisomal enzymes was determined in the livers. 1982). and both genotypes showed induced MT-1 and zinc levels in the livers and reduced serum and fetal concentrations after exposure to di(2-ethylhexyl) phthalate (Peters et al.. decreased anogenital distance. 0. either homozygous wild type (+/+) or PPARα null (–/–).p′-DDE (1. No pups survived exposure to doses of 2. kidneys and brains of the females and their offspring (Cimini et al. Prostatic and testicular zinc levels were reduced by this treatment (Curto & Thomas. 0. 1.5 g/kg per day. 1997a. p.

Testicular morphology was characterized by a marked shrinkage of the seminiferous tubules.2 or 2% (0. Testicular effects were also investigated after oral administration of 2000 mg/kg bw di(2-ethylhexyl) phthalate for seven consecutive days to 13-week-old male Wistar rats (Saxena et al. 1985). Degeneration was observed in about 40% of the seminiferous tubules.02. These changes were observed after exposure for two weeks (Gray et al. The size of the litters at birth was significantly reduced at the highest dose (Agarwal et al. Histopathologically. Oral administration of di(2-ethylhexyl) phthalate at levels of 0. di(2-ethylhexyl) phthalate inhibited spermatogenesis and general seminiferous tubular atrophy at the end of the study was reported. 250.DI(2-ETHYLHEXYL) PHTHALATE 97 those of control rats. the germinal epithelium consisting of Sertoli cells. 1250.8%. .. 1986). the majority of tubules showed a lack of spermatogenesis. In a 102-week study. 5000 or 20 000 ppm di(2-ethylhexyl) phthalate (> 99% pure) for 60 days resulted in dosedependent reductions in body weight and testis. 0. 1985). or 700 mg/kg bw per day) (Ganning et al. there were reductions in the sperm density in the epididymus and percentage of motile sperm. 1986). After a recovery period of 45 days after termination of di(2ethylhexyl) phthalate administration. while abnormal sperm shapes. testicular zinc and serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were elevated.. Loss of succinic dehydrogenase. but some tubules had intact epithelium.. 10 treated animals were killed and compared with 10 rats fed control diet for an additional 45 days without further administration of di(2ethylhexyl) phthalate. the testes showed severe atrophy of the seminiferous epithelium and loss of spermatogonia at the highest dose. adult male Sprague-Dawley rats were exposed to di(2-ethylhexyl) phthalate in the diet at dose levels of 0. 1991). γ-GT and lactate dehydrogenase and a decrease in the activity of acid phosphatase in the testes. 7. The percentage of spermatogenic tubules in a representative cross-section was 0 (total atrophy) or 12. In all dose groups. 320. 1977). NADH-diaphorase and acid phosphatase activity and increases in adenosine triphosphatase. very few spermatogonia and several multinucleated cells. One day after the last administration. At this dose level. epididymis and prostate weights beginning at 5000 ppm. Dietary exposure of groups of 24 adult male Fischer 344 rats to 0. 0.. The authors interpreted these findings as indicating germ-cell depletion and deterioration of the germinal epithelium in the testes (Parmar et al.. glucose-6-phosphate dehydrogenase and alkaline phosphatase activity were observed in treated rats. The reversibility of testicular effects was studied after oral administration of 2000 mg/kg bw per day di(2-ethylhexyl) phthalate for 14 days (Oishi. 500 or 1000 mg/kg bw to adult albino rats for 15 days resulted in a significant decrease in sperm count in the epididymus and increased activity of β-glucuronidase. The interstitial tissue and Leydig cells appeared normal. 70. [The Working Group noted that the study was designed to study the effects of phthalates on the liver and there was little information on testicular effects]. Histological examination revealed severe seminiferous tubular atrophy and cessation of spermatogenesis related to the dietary level of di(2-ethylhexyl) phthalate.

Relative testis weight and epididymal sperm counts were reduced at the highest dose. In a 90-day study.. six or 12 weeks of age. At 5000 ppm. Decreased testicular zinc levels did not appear to be related to the effect on spermatogenesis. Di(2-ethylhexyl) phthalateexposed females had prolonged estrous cycles. Offspring from this treatment showed normal fertility at six weeks of age. the absolute and relative testis weights were significantly reduced.0 mg/L di(2-ethylhexyl) phthalate for 6 h per day on five days per week for 28 days and had normal fertility when mated with untreated females two and six weeks later (Klimisch et al. aged one. the treated females had reduced preovulatory granulosa cell estrogen production. 1992). 37. .7. two-. received five daily doses ranging from 10 to 2000 mg/kg bw per day di(2-ethylhexyl) phthalate by oral gavage and testicular effects were examined 24 h after the last dose (Dostal et al. 5.05 or 1. multifocal or complete atrophy of the seminiferous tubules with complete loss of spermatogenesis and cytoplasmic vacuolation of the Sertoli cells lining the tubules in 9/10 rats (Poon et al. but there was no evidence of testicular histopathological effects. Adult male albino rats received 0... 1994)..and six-week-old rats. In the 500-ppm dose group. a high incidence of minimal to mild Sertoli cell vacuolation was observed in 7/10 rats. 1997). No other effects were noted at this dose level. The activity of testicular lactate dehydrogenase was increased at this dose. Effects on female reproductive function were evaluated in groups of six to nine Sprague-Dawley rats exposed by gavage to 2 g/kg di(2-ethylhexyl) phthalate (purity.01. 50. 0. 0. Losses of spermatocytes were evident in rats exposed to 1000 or 2000 mg/kg bw per day at ages of two weeks or older. At the end of the exposure. were exposed by inhalation to 0. rats had significantly increased absolute and relative liver weights and relative kidney weight. groups of 10 young male Sprague-Dawley rats (105–130 g at initiation of dosing) were given 0. with secondary increases in FSH and insufficient LH surge for ovulation. three. 1988).6 or 375. relative liver weights were increased in the high-dose males. nine weeks of age. Feed consumption and body weight gain were not affected.98 IARC MONOGRAPHS VOLUME 77 Male Sprague-Dawley rats. as was the activity of several enzymes relevant to spermatogenesis (aldose reductase and sorbitol dehydrogenase). 0. No clinical signs of toxicity were observed. although there was histological evidence of decreased maturation of spermatids at doses as low as 500 mg/kg bw per day. two. At 5000 ppm. 1992). 3. Sertoli cell number was transiently reduced in rats exposed to 1000 mg/kg bw per day at one week of age. three.4. 500 or 1000 mg/kg di(2-ethylhexyl) phthalate by gavage for 15 days and were killed 24 h later (Siddiqui & Srivastava. 500 or 5000 ppm (0. > 99%) by gavage daily for 1–12 days (Davis et al.2 mg/kg bw) di(2-ethylhexyl) phthalate per day in the diet for 13 weeks. bilateral. Relative testicular weight was reduced at doses of 1000 mg/kg bw per day in one-. Endocrinologically. suppressed or delayed ovulations and smaller preovulatory follicles. Groups of 10 male Wistar rats. Microscopic examination revealed mild to moderate. while 2000 mg/kg was required for this effect when the animals were 12 weeks of age.

1982). In males. Prostatic and testicular zinc levels were not affected by treatment with di(2-ethylhexyl) phthalate (Curto & Thomas.01. development and reproductive performance of the F1 generation.05% di(2-ethylhexyl) phthalate (> 99% pure) in the diet (NTIS.DI(2-ETHYLHEXYL) PHTHALATE 99 (ii) Mice Adult Swiss Webster mice received daily intraperitoneal injections of 50 or 100 mg/kg bw di(2-ethylhexyl) phthalate for five days or alternate daily intraperitoneal injections of 50 or 100 mg/kg bw di(2-ethylhexyl) phthalate for 20 days. During the neonatal period. Both male and female mice were exposed during a seven-day premating period and were then randomly grouped as mating pairs. The study was carried out to examine the effect of prenatally administered di(2-ethylhexyl) phthalate on the growth. viability. 200 or 600 mg/kg bw per day. A two-generation study in CD-1 mice was performed by feeding 0. the percentage of prenatal mortality was increased at the high dose from 9% to 26. number of pups per litter. 0. No other effects of di(2-ethylhexyl) phthalate upon growth. A cross-over mating trial conducted with F0 mice showed a decrease in fertility for both treated males and treated females. The doses were equivalent to about 19.1 and 0.3% (equivalent to 20. epididymis. eye opening.3% di(2-ethylhexyl) phthalate caused significantly reduced weights of the testes. testis descent. or vaginal opening or spontaneous locomotor activity) were observed on postnatal days 14. All high-dose males but one showed some degree of bilateral atrophy of the seminiferous tubules. age of acquisition for developmental landmarks (incisor eruption. wire grasping. 48 or 95 mg/kg bw per day. 0. Exposure to 0.3% di(2-ethylhexyl) phthalate also caused an increased incidence of abnormal sperm forms. 0. the percentage of viable pups was significantly decreased at the dose of 0. Reproductive function was evaluated by measuring the number of litters per breeding pair.1 or 0. Di(2-ethylhexyl) phthalate did not significantly decrease body-weight gain in the high-dose group. respectively) and a control group of 40 male and 40 female mice received basal diet (Lamb et al. For F1 litters. Sperm analysis showed a significant decrease in the percentage of motile sperm and a significantly decreased sperm concentration in cauda epididymis.01. Treatment continued for the 98-day cohabitation period and for 21 days thereafter.. The F1 generation was mated within dose groups at sexual maturity and F2 offspring were evaluated for viability and growth at postnatal day 4. .4%. 21 or 50.05% di(2-ethylhexyl) phthalate. Dietary levels of 0.025 or 0. 1987). prostate and seminal vesicles. No pups were born when treated females were mated with control males. In a fertility assessment by continuous breeding. proportion of pups born alive and mean pup weight. Only four litters out of 20 were born to treated males mated with control females and the proportion of pups born alive was decreased. groups of 20 male and 20 female CD-1 mice were fed di(2-ethylhexyl) phthalate (> 99% pure) at dietary levels of 0.3% di(2-ethylhexyl) phthalate produced dose-dependent and significant decreases in fertility and in the number and proportion of pups born alive. 1988).

seminal vesicles or prostate (Kurata et al. in germ-cell apoptosis. The fertility (evaluated by the ability of the motile spermatozoa to fertilize normal cycling females) was reduced from 90 to 75%. Fifteen mice were dosed orally with 1000 mg/kg bw di(2-ethylhexyl) phthalate [purity not specified] in 0. metabolism and distribution rather than to changes in tissue sensitivity (Heindel & Powell. Expression of Fas in the germ cell is related to apoptosis. The authors suggested that these events may be linked via signal transduction events (Richburg & Boekelheide. Histopathological changes were observed in the testes from 10 mL/kg and in the ovaries from 1 mL/kg. 1989). and of the Fas ligand (FasL). This response was accompanied at first by a decrease. (iii) Marmosets In a 13-week study. those showing the staining reaction indicative of apoptosis) were present in same regions as the heightened Fas expression.e. Younger rats tend to be more sensitive to the testicular effects of phthalates. 5 and 10 and were evaluated on day 21 (Agarwal et al. (3) altered metabolic function and (4) altered FSH reactivity.100 IARC MONOGRAPHS VOLUME 77 Groups of male and female ICR mice received doses of di(2-ethylhexyl) phthalate ranging from 1 to 100 mL/kg bw by subcutaneous injections on experimental days 1. 1998).. 1993). and theories have been proposed related to (1) reduced testicular zinc levels. 100. At 15 mL/kg. (d) Mechanistic-based reproductive toxicity studies The Sertoli cell appears to be the primary site of phthalate toxicity in the testes. and terminal deoxynucleotide transferase (TdT)-mediated dUTP-biotin nick-end labelling (TUNEL)-positive cells (i.. Administration of 2 g/kg mono(2-ethylhexyl) phthalate by oral gavage to 20-day-old Fischer 344 rats resulted in a collapse of Sertoli cell vimentin filaments within 3 h of exposure. 1996). it has been demonstrated that expression of Fas. Body-weight gain was significantly depressed at 2500 mg/kg bw. (2) altered hormonal status. This suggests that damage to Sertoli cells increases the . a transmembrane receptor protein. Thirty mice of an inbred colony were used to study the effect of di(2-ethylhexyl) phthalate on reproductive function (Jain & Joshi. Subsequently. Doses of 10 mL/kg or greater reduced the fertility of both males and females. respectively. 1992). relative testis weights were reduced. No significant changes were observed in testis weights or histopathology of the testis.. is up-regulated in the Fischer 344 rat testes following exposure to 2 g/kg mono(2-ethylhexyl) phthalate. 500 or 2500 mg/kg bw di(2-ethylhexyl) phthalate. and then several hours later by an increase. while changes in ovarian and testicular ATPase and lysosomal enzymes occurred at 10 mL/kg. epididymis. 1991). groups of four mature male marmosets were given daily doses of 0. although this difference appears to be related to changes in absorption. which occur in the germ and Sertoli cells. None of these alone appears to account for the observed testicular effects (reviewed in Boekelheide.1 mL olive oil for one week. Sperm density and sperm motility were also significantly reduced.

The other half served as a control group. 1997). and of increased activity of β-glucuronidase and γ-GT. The estrogenic activities of phthalates were investigated in competitive ligandbinding assays. The involvement of testosterone in the testicular atrophy caused by di(2-ethylhexyl) phthalate was examined by co-administration of testosterone (1 mg/kg bw) subcutaneously with 2000 mg/kg bw di(2-ethylhexyl) phthalate [purity not specified] in groundnut oil to adult male Wistar rats for 15 days (Parmar et al.and 10-week-old males. Co-administration of testosterone seemed to normalize the sperm count and the activity of testicular enzymes. yeast and mammalian gene expression assays and in a uterotrophic assay. In four-week-old rats fed a diet containing 2. One group received a synthetic diet containing 20% casein and the other a diet containing 8% starch.DI(2-ETHYLHEXYL) PHTHALATE 101 output of FasL. lactate dehydrogenase and β-glucuronidase and decreased the activity of sorbitol dehydrogenase and acid phosphatase. but recovery was slower when treatment continued throughout puberty.. the testicular weight was not affected. Four-... The group on the low-protein diet had a more severe response to di(2-ethylhexyl) phthalate in terms of sperm count. A marked decrease in testicular weight (% of control) was seen in four-weekold rats. The role of testosterone in the testicular toxicity of di(2-ethylhexyl) phthalate has not been fully elucidated..0% di(2ethylhexyl) phthalate. support estrogen-inducible growth in yeast cells or demonstrate any estrogenic activity in mammalian assays in vivo (Zacharewski et al. Administration of di(2-ethylhexyl) phthalate reduced the sperm count and also significantly increased the activity of γ-GT. The weights of the seminal vesicles and ventral prostate were reduced in the four. 5–50% of the tubules were atrophic. Di(2-ethylhexyl) phthalate did not compete for estrogen receptors. testosterone propionate (200 μg/kg per day in corn oil) or FSH was given subcutaneously. di(2-ethylhexyl) phthalate produced uniform seminiferous tubular atrophy. Simultaneous administration of testosterone or FSH with di(2-ethylhexyl) phthalate did not affect the development of testicular atrophy. In four-week-old rats. In 10week old rats. Gray and Butterworth (1980) found an age-dependent induction of testicular atrophy in rats. with younger rats being more sensitive than older ones. A study of the influence of a low-protein diet on the testicular toxicity of di(2ethylhexyl) phthalate was performed in adult male Wistar rats (12 weeks old) (Tandon et al. 1998). half of the rats in each group received 1000 mg/kg bw di(2-ethylhexyl) phthalate [purity not stated] in 0. After 15 days of consumption. 1992).2 mL groundnut oil orally for 15 days. Several reports refer to increased or decreased testosterone levels in plasma and testicular tissue.and 15-week-old Wistar rats were administered 2800 mg/kg bw di(2-ethylhexyl) phthalate [purity not specified] dissolved in corn oil by gavage for 10 days. leading to increased germ-cell loss in an effort to maintain testicular homeostasis (Lee et al. 1987). 10. induce luciferase activity in transfected MCF-7 cells or stably transfected HeLa cells. the testicular effects were reversible within 12 weeks when treatment was stopped before puberty. comprising loss of spermatids and spermatocytes. In some experiments. .

1985d). There was also a marked reduction in the number of spermatogonia. Groups of eight male Sprague-Dawley rats (four. VI. Di(2-ethylhexyl) phthalate had no effect in 15-week-old rats. Body weight gain was retarded in all treated groups. there were a marked reduction in the number of germ cells. To determine which compound or compounds were responsible for the testicular damage after oral administration of di(2-ethylhexyl) phthalate. a high occurrence of degenerating cells and a reduction in the tubular diameter.102 IARC MONOGRAPHS VOLUME 77 but prevented the reduction of accessory gland weights. the majority of tubules were totally devoid of germ cells. The authors concluded that the difference in response to di(2-ethylhexyl) phthalate of male rats of different ages may be due to higher absorption of the metabolite mono(2-ethylhexyl) phthalate in younger animals. Counting of degenerated cells per tubular cross-section was carried out. The interstitial tissue appeared to be unaffected in all dosed animals. (1986a) administered di(2-ethylhexyl) phthalate and five of its major metabolites (mono(2ethylhexyl) phthalate. In the 25-day-old rats. The number of degenerated spermatocytes and spermatids was increased in animals receiving mono(2-ethylhexyl) phthalate. Testicular weight was markedly reduced and severe testicular damage occurred in the 25. 10 or 15 weeks of age) were used to study the age-dependence of effects on male reproductive organs (Gray & Gangolli. Histologically. The liver weight was increased in all three age groups.. no such effects were seen in animals given the mono(2-ethylhexyl) phthalate-derived metabolites. Groups of eight male Sprague-Dawley rats (25. Administration to four-week-old rats produced a marked reduction in absolute weights of the testes. In another study. (1986b) examined the age-dependent testicular toxicity of di(2-ethylhexyl) phthalate (1000 and 1700 mg/kg bw in the diet for 14 days) in rats at 25. Sjöberg et al. Sjöberg et al. Groups of six male Sprague-Dawley rats (35 days old at the start of the experiment) were given 2. 40 and 60 days of age.and 40-day-old rats given doses of 1700 mg/kg bw. The rats were given 2800 mg/kg bw di(2-ethylhexyl) phthalate orally for 10 days. No changes were found in the 60-day-old rats. Only a few seminiferous tubules (1–10%) were affected in the 1000 mg/kg bw group at any age of exposure. In the youngest age group. No effects were observed in 15-week-old rats. or IX) of mono(2-ethylhexyl) phthalate) for five days. the absolute testicular weight was decreased and histopathological examination showed severe testicular damage. but the seminal vesicle and prostate weights were significantly reduced. There was only a slight reduction in testis weight in 10-week-old rats.7 mmol/kg bw di(2-ethylhexyl) phthalate (1055 mg/kg bw) or one of the metabolites. the testes of the four-week-old rats showed severe atrophy affecting . No testicular damage was observed following oral doses of di(2-ethylhexyl) phthalate or 2-ethylhexanol. 1986). 40 or 60 days old) were dosed with 0 or 1000 mg/kg bw di(2-ethylhexyl) phthalate in corn oil by gavage for 14 days (Sjöberg et al. 2-ethylhexanol and three identified metabolites (V. seminal vesicles and prostate. whereas the older animals were unaffected. In some animals that were allowed to survive for 100 days. The authors suggested that this indicated that the damage was not completely reversible.

sorbitol dehydrogenase. 4. TA98. 50. 4..1 Humans No data were available to the Working Group. TA1538. TA102. 250 or 500 mg/kg bw di(2-ethylhexyl) phthalate in groundnut oil were given for 30 consecutive days to groups of six male rats. lactate dehydrogenase and γ-GT occurred thus at much lower levels of di(2-ethylhexyl) phthalate and before the histopathological changes. In the same study. β-glucuronidase and acid phosphatase were studied in 25-day-old male Wistar rats (Parmar et al. which were populated only by Sertoli cells. βGlucuronidase activity was elevated at 250 or 500 mg/kg bw.4. The Leydig cells and the fibroblasts appeared to be normal. To elucidate further the mechanisms responsible for the enhanced sensitivity of the testes of developing animals to di(2-ethylhexyl) phthalate. TA1537. TA1535.4. The data from this and other evaluations of the genetic and related effects of di(2-ethylhexyl) phthalate have been reviewed (Huber et al.4 Genetic and related effects Di(2-ethylhexyl) phthalate was one of the compounds used in the IPCS evaluation of short-term tests for carcinogenicity (IPCS. 4.DI(2-ETHYLHEXYL) PHTHALATE 103 virtually all the tubules. A single dose of 1000 mg/kg bw mono(2ethylhexyl) phthalate reduced fluid and protein production to around 50% of the concurrent control group and to 25% after three repeated doses. In the 10-week-old rats. 100. while acid phosphatase decreased at the same dose levels. γ-GT. 1985). 1995).. Doses of 0. No histological abnormalities were seen in testes from the 15-week-old rats.2 Experimental systems (see Table 7 for references) Di(2-ethylhexyl) phthalate was not mutagenic to Salmonella typhimurium strains TA100. the effects of mono(2-ethylhexyl) phthalate on Sertoli cell function in immature rats were studied by measuring the secretion of seminiferous tubule fluid and androgen-binding protein. these histological changes were evident in 5–50% of the tubules. the activities of the testicular enzymes associated with spermatogenesis including lactate dehydrogenase. a dose-dependent and significant increase in the activities of lactate dehydrogenase and γ-GT was noted. 1996). while activity of sorbitol dehydrogenase decreased. There was an exposure-related and significant decrease of absolute and relative testicular weight at all dose levels. The administration also resulted in marked destructive changes in the advanced germ cell layers and marked degrees of vacuolar degeneration in the testes at 250 and 500 mg/kg bw. spermatogonia and occasional primary spermatocytes. The significant alterations in the activities of sorbitol dehydrogenase. From 50 mg/kg bw. TA97 or the TA7000 series in the . the remainder appearing to be essentially normal.

TA97. TA98. TA98. reverse mutation Salmonella typhimurium TA100. TA7001. TA7003. TA97. TA1537. TA102. (1985) Inge-Vechtomov et al. (1983) Robertson et al. reverse mutation Salmonella typhimurium TA100. TA1535. TA1537. reverse mutation Escherichia coli WP2 uvrA. (1998) Yoshikawa et al. TA7005. (1985) Gee et al. TA97. Genetic and related effects of di(2-ethylhexyl) phthalate Test system Result a Without exogenous metabolic system Bacillus subtilis rec. TA1535. TA98. TA98. TA7006. TA98. gene conversion Saccharomyces cerevisiae. TA1537. (1985) Rexroat & Probst (1985) Zeiger & Haworth (1985) Zeiger et al. TA7004. TA102. TA1538. reverse mutation Salmonella typhimurium TA1537. reverse mutation Salmonella typhimurium TA100. reverse mutation Salmonella typhimurium TA100. gene conversion – – – – – – – – – – – – – (+) – – With exogenous metabolic system NT – – – – – – – – – – – – (+) – – 500 μg/disc 500 9860 4000 2000 10 000 5000 10 000 5000 10 000 10 000 1000 2000 5000 2000 1000 Tomita et al. reverse mutation Salmonella typhimurium TA100. (1982b) Liber (1985) Kirby et al. TA1535. (1983) Arni (1985) Brooks et al. TA98. (1985) Doseb (LED or HID) Reference IARC MONOGRAPHS VOLUME 77 . TA98.104 Table 7. TA98. TA102. forward mutation Salmonella typhimurium TA100. reverse mutation Salmonella typhimurium TA100. (1985) Nohmi et al. differential toxicity Salmonella typhimurium. reverse mutation Salmonella typhimurium TA100. (1983) Yoshikawa et al. TA7002. (1983) Baker & Bonin (1985) Matsushima et al. gene conversion Saccharomyces cerevisiae. TA98. TA97. TA1538. reverse mutation Saccharomyces cerevisiae. TA98. reverse mutation Salmonella typhimurium TA100. TA1535.

mutation Aspergillus nidulans non-disjunction Aspergillus nidulans. (1985) Fujikawa et al. (1985) Carere et al. (1985) Inge-Vechtomov et al. (1985) Carls & Schiestl (1994) Würgler et al. (1985) Arni (1985) Inge-Vechtomov et al. (1985) Carere et al. somatic mutation Drosophila melanogaster. forward mutation Saccharomyces cerevisiae. crossing-over/recombination Drosophila melanogaster. forward mutation Saccharomyces cerevisiae DEL assay. reverse mutation Saccharomyces pombe. mitotic crossing-over Saccharomyces cerevisiae. somatic mutation + + – – – – – – – – – + – ? – – (+) (+) – With exogenous metabolic system + + – – – NT NT NT – – – + – – – 1500 5000 5000 5000 1000 9900 9900 9900 1000 5000 1000 1500 5000 5900 200 000 39 000 μg/g food 6930 μg/cm2 food surface 780 μg/g food 39 000 μg/g food Mehta & von Borstel (1985) Parry & Eckardt (1985) Parry & Eckardt (1985) Arni (1985) Inge-Vechtomov et al. somatic mutation Drosophila melanogaster. reverse mutation Saccharomyces cerevisiae. reverse mutation Saccharomyces cerevisiae. (1985) Carere et al. (1985) Mehta & von Borstel (1985) Parry & Eckardt (1985) Loprieno et al. (1985) Doseb (LED or HID) Reference DI(2-ETHYLHEXYL) PHTHALATE 105 . homozygosis Aspergillus nidulans haploid.Table 7 (contd) Test system Result a Without exogenous metabolic system Saccharomyces cerevisiae. reverse mutation Saccharomyces cerevisiae. gene conversion Saccharomyces cerevisiae D6. gene conversion Saccharomyces cerevisiae. homozygosis Saccharomyces cerevisiae. aneuploidy Saccharomyces cerevisiae. (1985) Vogel (1985) Würgler et al. and ICR recombination Drosophila melanogaster.

(1983) Amacher & Turner (1985) Myhr et al. Tk locus in vitro Gene mutation. B6C3F1 mouse primary hepatocytes in vitro Gene mutation. Tk locus in vitro Gene mutation. 18 600 μg/g food 7540 μg/g food 7540 μg/g food 7540 μg/g food 3900 39 000 9750 3900 3900 3900 10 000 1000 390 980 2500 4900 7. Unscheduled DNA synthesis. DNA double strand breakage in vivo Drosophila melanogaster. (1984) Kornbrust et al. wing spot test. rat primary hepatocytes Unscheduled DNA synthesis. (1989) Kawai (1998) Kawai (1998) Kawai (1998) Bradley (1985) Douglas et al. sex-linked recessive lethal mutation Drosophila melanogaster. Unscheduled DNA synthesis. Chinese hamster ovary cells in vitro DNA single-strand breaks. (1988) Butterworth et al. Tk locus in vitro Gene mutation. (1985) Zimmering et al. (1985) Doseb (LED or HID) Reference IARC MONOGRAPHS VOLUME 77 NT – NT NT NT NT NT NT NT – – – (+) . (1985) Schmezer et al. mouse lymphoma L5178Y cells. mutation in vivo DNA single-strand breaks. (1985) Oberly et al. rat primary hepatocytes Unscheduled DNA synthesis. Tk locus in vitro – – – – – – – – – – – – – – (+) ? – (+) With exogenous metabolic system 20 inj. Unscheduled DNA synthesis. (1985) Astill et al. rat primary hepatocytes UIA. mouse lymphoma L5178Y cells. rat primary hepatocytes URP. rat primary hepatocytes URP. (1986) Smith-Oliver & Butterworth (1987) Kirby et al. (1984) Probst & Hill (1985) Williams et al. rat hepatocytes in vitro DNA strand breaks.5 Yoon et al. sex-linked recessive lethal mutation Drosophila melanogaster. rat or Syrian hamster hepatocytes in vitro Unscheduled DNA synthesis. mouse lymphoma L5178Y cells.106 Table 7 (contd) Test system Result a Without exogenous metabolic system Drosophila melanogaster. DNA repair test in vivo Drosophila melanogaster. mouse lymphoma L5178Y cells.

in vitro Chromosomal aberrations. (1985) Matthews et al. mouse lymphoma L5178Y cells. Tk locus in vitro Gene mutation. Chinese hamster ovary cells in vitro Sister chromatid exchange. (1993) Abe & Sasaki (1977) Ishidate & Odashima (1977) Phillips et al. (1985) Priston & Dean (1985) Douglas et al. mouse lymphoma L5178Y cells. Chinese hamster ovary cells in vitro Sister chromatid exchange. Chinese hamster ovary cells in vitro Chromosomal aberrations. (1985) Doseb (LED or HID) Reference DI(2-ETHYLHEXYL) PHTHALATE Astill et al. ouabain resistance in vitro Sister chromatid exchange. Tk locus in vitro Gene mutation. (1995) Fritzenschaf et al. (1985) Gulati et al. (1986) Garner & Campbell (1985) Styles et al. (1985) Müller-Tegethoff et al. BALB/c-3T3 mouse cells.Table 7 (contd) Test system Result a Without exogenous metabolic system Gene mutation. ouabain resistance in vitro Gene mutation. mouse lymphoma L5178Y cells. Chinese hamster Don cells in vitro Sister chromatid exchange. rat liver RL4 cells in vitro Micronucleus formation. (1985) Abe & Sasaki (1977) Douglas et al. Chinese hamster ovary cells in vitro Chromosomal aberrations. (1982) Danford (1985) Gulati et al. (1985) Ishidate & Sofuni (1985) 107 . Chinese hamster liver cells in vitro Chromosomal aberrations. Chinese hamster lung cells in vitro – – – – NT – – (+) – – – + – – – – – – With exogenous metabolic system – – – – – NT – – NT – NT NT NT NT NT NT – – 9800 250 200 9800 1960 3900 3900 5000 1000 3900 3900 NG 3900 160 781 50 5000 4130 Styles et al. Syrian hamster embryo cells. Chinese hamster Don cells in vitro Chromosomal aberrations. ouabain resistance in vitro Gene mutation. mouse lymphoma L5178Y cells. Chinese hamster lung cells in vitro Chromosomal aberrations. rat hepatocytes in vitro Micronucleus formation. Chinese hamster ovary cells in vitro Micronucleus formation.

BALB/3T3 mouse cells Cell transformation. RLV/Fischer rat Cell transformation. human lymphocytes in vitro Sister chromatid exchange. human hepatocytes in vitro Gene mutation. BALB/3T3 mouse cells Cell transformation. (1986) Lawrence & McGregor (1985) Sanchez et al. Syrian hamster embryo cells.9 1 4 10 30 1. (1993) Danford (1985) Parry (1985) Priston & Dean (1985) Matthews et al. Syrian hamster embryo cellsd Cell transformation. Syrian hamster embryo cells. rat liver RL4 cells in vitro Cell transformation. (1974) Doseb (LED or HID) Reference IARC MONOGRAPHS VOLUME 77 . Chinese hamster liver cells in vitro Mitotic aberrations. SA7/Syrian hamster embryo cells Cell transformation. Syrian hamster embryo cells. Chinese hamster primary liver cells in vitro Aneuploidy.2 39 1000 500 39 3900 1000 1000 39 75 Priston & Dean (1985) Tsutsui et al. rat liver RL4 cells in vitro Chromosomal aberrations. clonal assay Ornithine decarboxylase superinduction. Syrian hamster embryo cells in vitro Aneuploidy. clonal assay Cell transformation. clonal assay Cell transformation. human lymphocytes. (1985) Astill et al. clonal assay Cell transformation.108 Table 7 (contd) Test system Result a Without exogenous metabolic system Chromosomal aberrations. human lymphocytes (metabolic activation by co-cultured with rat liver cells) in vitro Chromosomal aberrations. (1984) Crespi et al. (1998) Butterworth et al. (1987) Barrett & Lamb (1985) Sanner & Rivedal (1985) Mikalsen et al. clonal assay Cell transformation. C3H10T½ mouse cells Cell transformation. Syrian hamster embryo cells. (1990) Mikalsen & Sanner (1993) Tsutsui et al. C3H10T½ mouse cells Cell transformation. Syrian hamster embryo cells. (1985) Obe et al. human lymphocytes in vitro – – (+) (+) – – – (+) – + + + + (+) – + + + – – – – – With exogenous metabolic system NT + NT NT NT – – (+) NT NT NT NT NT + NT NT NT NT NT – – (+) NT 1000 39 50 50 1000 25 000 20 40 3. Syrian hamster embryo cells Unscheduled DNA synthesis. (1989) Turner et al. (1985) Lindahl-Kiessling et al. TK and HPRT loci in vitro Sister chromatid exchange. (1998) Suk & Humphreys (1985) Hatch & Anderson (1985) Dhalluin et al. (1993) Dhalluin et al.

microbial mutagenicity DNA strand breaks. (1984) Doseb (LED or HID) Reference DI(2-ETHYLHEXYL) PHTHALATE Unscheduled DNA synthesis. Fischer 344 rat hepatocytes in vivo Unscheduled DNA synthesis. (1999) DiVincenzo et al. (1988) Smith-Oliver & Butterworth (1987) 109 . (1985) Elliott & Elcombe (1987) Takagi et al. (1990b) Tamura et al. Fischer 344 rat liver in vivo Unscheduled DNA synthesis. 30 days + 500 × 1 po 5000 × 1 po 12 000 mg/kg diet 28 d 6000 mg/kg diet 28 d Stenchever et al. (1990a) Takagi et al. Sprague-Dawley rat hepatocytes in vivo Unscheduled DNA synthesis. 150 × 14 po or 12 000 ppm diet. (1984) Cattley et al. (1976) Anderson et al. Fischer 344 rat liver in vivo DNA oxidative damage. (1991) Cattley & Glover (1993) Butterworth et al. Sprague Dawley rat urine. Fischer 344 rat liver in vivo DNA single-strand breaks.Table 7 (contd) Test system Result a Without exogenous metabolic system Chromosomal aberrations. 1–2 w 20 000 mg/kg diet 78w 12 000 mg/kg diet 22w 500 × 1 po. B6C3F1 mouse hepatocytes in vivo – – – Kornbrust et al. Wistar rat liver in vivo DNA oxidative damage. human lymphocytes in vitro Chromosomal aberrations. Fischer 344 rat liver in vivo DNA oxidative damage. human fetal lung cells in vitro Comet assay on human blood in vitro Body fluids. human lymphocytes in vitro Aneuploidy. (1976) Tsuchiya & Hattori (1976) Stenchever et al. Fischer 344 rat hepatocytes in vivo – – – + – – +c +c – – – With exogenous metabolic system NT NT NT – – 60 160 6 156 2000 × 15 po 2000 × 28 po 12 000 mg/kg diet 1 y 12 000 mg/kg diet.

(1986) Douglas et al. (1985) Albro et al. (1983) Kornbrust et al. (1985) Hasmall & Roberts (1997) Tomita et al. B6C3F1 mouse erythrocytes in vivo Chromosomal aberrations. (1984) Doseb (LED or HID) Reference IARC MONOGRAPHS VOLUME 77 NT . (1983) Tomita et al. Fischer 344 rat hepatocytes in vivo Cell transformation.110 Table 7 (contd) Test system Result a Without exogenous metabolic system Gene mutation. (1974) Autian (1982) Agarwal et al. Chinese hamster V79 cells in vitro Inhibition of gap-junctional intercellular communication. (1986) Putman et al. ICR Swiss mice in vivo Aneuploidy. (1999) Malcolm et al. ICR Swiss mice in vivo Dominant lethal test.1 Gunz et al. (1982b) Gupta et al. cynomolgus monkey liver cells in vivo Inhibition of gap-junctional intercellular communication. Syrian hamster embryos in vivo/in vitro Binding (covalent) to Fischer 344 rat hepatocyte DNA in vitro Binding (covalent) to Fischer 344 rat liver DNA in vivo Binding (covalent) to Fischer 344 rat liver DNA in vivo Binding (covalent) to Fischer 344 rat liver DNA in vivo Binding (covalent) to Fischer 344 rat liver DNA in vivo Inhibition of gap-junctional intercellular communication. (1982) Däniken et al. mice in vivo [strain not specified] Dominant lethal test. (1985) Lutz (1986) Pugh et al. (1984) Gupta et al. lacI transgenic C57BL/6 mouse liver in vivo Micronucleus formation. (1982b) Singh et al. Chinese hamster V79 cells in vitro – – – – + + + + – + – – – – – – + – NT NT With exogenous metabolic system 6000 mg/kg diet 120 d 5000 × l po 6000 × 5 ip 4900 × 5 po 7500 × 1 po 12 780 × 1 ip 980 × 3 sc 980 × 3 sc 12 000 mg/kg diet 7d 7500 po 390 10 000 mg/kg diet 11 d 10 000 mg/kg diet 4w 2000 × 3 po 500 × 1 po 500 × 14 po 3 0. Fischer 344 rat bone marrow in vivo Chromosomal aberrations. mice in vivo Micronucleus formation. (1993) Astill et al. Syrian hamster embryos in vivo Dominant lethal test.

Syrian hamster embryo cells in vitro Inhibition of gap-junctional intercellular communication. (1993) Cruciani et al. Chinese hamster V79 cells in vitro Inhibition of gap-junctional intercellular communication.2 μL/plate 1000 μg/plate 139 Tomita et al. TA98. (1991) Smith-Oliver & Butterworth (1987) + + + + + With exogenous metabolic system NT NT NT NT NT 5 10 30 78 10 6000 × 5 ip 5200 × 5 ip Elmore et al. Chinese hamster V79 cells and Syrian hamster embryo cells in vitro Sperm morphology. reverse mutation Unscheduled DNA synthesis. (1982b) Kirby et al. TA 98.Table 7 (contd) Test system Result a Without exogenous metabolic system Inhibition of gap-junctional intercellular communication. (1986) Douglas et al. reverse mutation Salmonella typhimurium TA100. differential toxicity Salmonella typhimurium TA100. TA 97. Chinese hamster V79 cells in vitro Inhibition of gap-junctional intercellular communication. B6C3F1 mouse primary hepatocytes in vitro + – – – – NT – – – NT 400 μg/disc 1250 μg/plate 0. (1983) Dirven et al. TA1537. Chinese hamster V79 cells in vitro Inhibition of gap-junctional intercellular communication. reverse mutation Salmonella typhimurium TA100. (1985) Malcolm & Mills (1989) Mikalsen & Sanner (1993) Vang et al. Sprague Dawley rats in vivo Metabolites Mono(2-ethylhexyl) phthalate (MEHP) Bacillus subtilis rec. (1997) Doseb (LED or HID) Reference DI(2-ETHYLHEXYL) PHTHALATE – – Douglas et al. TA1535. (1986) 111 . (1982b) Tomita et al. TA1538. B6C3F1 mice in vivo Sperm morphology. TA102.

TA98. reverse mutation Mono(2-ethyl 5-oxohexyl) phthalate (VI) Salmonella typhimurium TA100. (1991) – – 1000 μg/plate Dirven et al. (1991) – – 1000 μg/plate Dirven et al.8 56 23 417 28 Kirby et al. clonal assay Cell transformation. TA97. Tk locus in vitro Sister chromatid exchange. (1997) Doseb (LED or HID) Reference IARC MONOGRAPHS VOLUME 77 – – – NT NT – 139 500 po × 14 1000 μg/plate Butterworth et al.3 μL/mL 25 2. Syrian hamster embryo cells. (1993) Tsutsui et al. (1982b) Tsutsui et al. TA102. Chinese hamster V79 cells in vitro Chromosomal aberrations in Syrian hamster embryo cells in vitro Cellular transformation in Syrian hamster embryo cells in vitro Cell transformation. (1984) Elliot & Elcombe (1987) Dirven et al. reverse mutation Mono(5-carboxyl 2-ethylpentyl) phthalate (V) Salmonella typhimurium TA100. human primary hepatocytes in vitro DNA strand breaks. (1990) Sanchez et al. C3H10T½ mouse cells Inhibition of gap-junctional intercellular communication. (1983) Tomita et al. TA98. Wistar rat liver in vivo Mono(2-ethyl 5-hydroxyhexyl) phthalate (IX) Salmonella typhimurium TA100. Chinese hamster V79 cells and Syrian hamster embryo cells in vitro Unscheduled DNA synthesis. mouse lymphoma L5178Y cells. TA97.112 Table 7 (contd) Test system Result a Without exogenous metabolic system Gene mutation. (1987) Cruciani et al. TA102. (1993) Mikalsen et al. TA97. reverse mutation – + – – + – + With exogenous metabolic system – NT + (+) NT NT NT 0. (1991) . TA98. TA102.

w. positive. reverse mutation Gene mutation. oral. TA98.3 μL/mL 500 Tomita et al. ip. year. (1982b) Kirby et al. inconclusive LED. week. (1983) Tomita et al. TA1535. y. (+).16 μM 12-O-tetradecanoylphorbol 13-acetate b 113 . intraperitoneal. lowest effective dose. po. ?. mouse lymphoma L5178 cells. in-vitro tests. differential toxicity a Doseb (LED or HID) With exogenous metabolic system Reference DI(2-ETHYLHEXYL) PHTHALATE – – – – NT – – NT 500 1 μL/plate 0. (1982b) +. subcutaneous c No oxidative damage in kidney DNA d Positive if followed by 5 h 0. in-vivo tests. day.Table 7 (contd) Test system Result a Without exogenous metabolic system 2-Ethylhexanol Bacillus subtilis rec. mg/kg bw/day. μg/mL. NT. d. weakly positive. sc. differential toxicity Salmonella typhimurium TA100. highest ineffective dose. TA1537. HID. negative. TA1538. –. (1983) Kirby et al. not tested. Tk locus in vitro Phthalic acid Bacillus subtilis rec.

Only three of these studies for chromosomal aberrations included an exogenous metabolic activation system. In a single study. di(2-ethylhexyl) phthalate induced a small increase in sister chromatid exchange frequencies in Chinese hamster ovary cells cultured without but not with exogenous metabolic activation. using Syrian hamster embryo cells. one. although neither compound was active when administered alone. Unscheduled DNA synthesis was not induced in rat or mouse primary hepatocytes exposed to di(2-ethylhexyl) phthalate. neither was sexlinked recessive lethal mutation induced in two studies in D. It induced aneuploidy in a single study. cerevisiae in the presence or absence of exogenous metabolic activation. DNA single-strand breaks were not induced by di(2-ethylhexyl) phthalate in primary cultures of rat or Syrian hamster hepatocytes or in Chinese hamster ovary (CHO) cells. Ouabain-resistant mutants were not induced in mouse lymphoma or BALB/c3T3 cells. In a single study. It did not induce gene mutations in Saccharomyces cerevisiae strain D7 in four of five studies. It was also not mutagenic to Escherichia coli WP2 uvrA in the presence or absence of exogenous metabolic activation. cerevisiae. mitotic recombination was not induced in S. Di(2-ethylhexyl) phthalate induced cell transformation in the Syrian hamster embryo clonal assay. mitotic recombination was not induced by di(2-ethylhexyl) phthalate. melanogaster treated with di(2-ethylhexyl) phthalate in the feed or by injection. found an increase in aberration frequency. Of these. In one of two studies. Neither mutation nor genetic crossing-over was induced in Aspergillus nidulans cultured with di(2-ethylhexyl) phthalate in the absence of exogenous metabolic activation in one study. Only one of six studies reported induction of gene mutations at the Tk locus in mouse lymphoma L5178Y cells exposed to di(2-ethylhexyl) phthalate in the absence of an exogenous metabolic activation system. melanogaster. When administered to D. Weak effects were detected for the induction of aneuploidy and mitotic division aberrations in Chinese hamster lung cells. whereas the induction of micronuclei by di(2-ethylhexyl) phthalate in Syrian hamster embryo cells has been reported. di(2-ethylhexyl) phthalate and N-nitrosodiethylamine induced DNA double-strand breakage and DNA repair. Mitotic gene conversion was induced in two out of five studies with S.114 IARC MONOGRAPHS VOLUME 77 presence or absence of exogenous metabolic activation. the virally enhanced SA7/Syrian hamster embryo assay and . Chromosomal aberrations were not induced by di(2-ethylhexyl) phthalate in any of eight studies in various types of cultured cells in the absence of metabolic activation. Di(2-ethylhexyl) phthalate did not induce micronuclei in Chinese hamster ovary cells or in cultured rat hepatocytes. A small increase in somatic mutation frequency was reported in the eye-colour spot test with Drosophila melanogaster exposed to di(2-ethylhexyl) phthalate in the feed but no effect was observed in two independent wing-spot tests. In other studies conducted only without metabolic activation. it caused no increase in sister chromatid exchanges in either Chinese hamster Don cells or rat liver RL4 cells.

Di(2-ethylhexyl)phthalate did not increase the mutation frequency in the lacI gene of liver DNA isolated from female lacI transgenic mice given di(2-ethylhexyl) phthalate in the feed for 120 days. Di(2-ethylhexyl) phthalate did not induce DNA strand breaks in rat liver or unscheduled DNA synthesis in rat or mouse liver following single or multiple oral treatments. In single studies with human lymphocytes in vitro. or aneuploidy in human fetal lung cells cultured without metabolic activation. Di(2-ethylhexyl) phthalate did not increase the frequency of chromosomal aberrations in human lymphocytes in the absence of exogenous metabolic activation in vitro. six or nine months. A significant increase in 8-OH-dG was measured in liver DNA at the one. Urine samples from rats treated by gavage with 15 daily doses of di(2-ethylhexyl) phthalate were not mutagenic to S. typhimurium strains TA100. Di(2-ethylhexyl) phthalate treatment of Syrian hamster embryo cells in a two-stage exposure with 12-O-tetradecanoylphorbol 13-acetate resulted in superinduction of ornithine decarboxylase. Micronuclei were not induced in mice fed with di(2-ethylhexyl) phthalate. an early event in morphological transformation. mouse JB6 epidermal cells were transformed by di(2-ethylhexyl) phthalate without activation and in one of two studies a weak response was reported in the C3H10T½ cell transformation assay with di(2-ethylhexyl) phthalate in either the absence or presence of exogenous metabolic activation. Di(2-ethylhexyl) phthalate inhibited gap-junctional intercellular communication in Chinese hamster V79 cells in six of seven studies. One single study using human lymphocytes found that di(2-ethylhexyl) phthalate did not induce sister chromatid exchange. but not in one study of liver cells of cynomolgus monkeys in vivo. TA1537. BALB/c-3T3 cells were not transformed by di(2-ethylhexyl) phthalate with or without metabolic activation.and 12-month sampling times but not at three. the formation of 8-hydroxydeoxyguanosine (8-OHdG) was measured as an indicator of DNA oxidative damage in liver and kidney of rats exposed to di(2-ethylhexyl) phthalate in the diet for one to two weeks: 44–64% increased levels of 8-OH-dG were observed in liver but not kidney DNA. Similar results were reported in a follow-up study in which rats received di(2-ethylhexyl) phthalate in the diet for one year. no effect was seen after a one-stage treatment with di(2-ethylhexyl) phthalate alone. nor did it give rise to covalent DNA binding in rat hepatocytes in vitro or rat liver in vivo. A single study investigating the effect of di(2-ethylhexyl) phthalate on human blood cells by the Comet assay reported induction of DNA damage but none in the presence of exogenous metabolic activation. TA1538 or TA98. In one study. TA1535. Di(2-ethylhexyl)phthalate did not induce chromosomal aberrations in rat bone marrow sampled 6 h after the last of five daily treatments . di(2-ethylhexyl) phthalate did not induce unscheduled DNA synthesis or gene mutations at the TK or HPRT loci without exogenous metabolic activation. while another study found weak induction when the lymphocytes were co-cultured with rat liver cells.DI(2-ETHYLHEXYL) PHTHALATE 115 RLV/Fischer rat assay without the addition of an exogenous metabolic activation system. In a single study.

The peroxisome-proliferating effect of di(2-ethylhexyl) phthalate has been related most specifically in susceptible species to metabolites VI. this function was also inhibited in rat and mouse hepatocytes. Di(2-ethylhexyl) phthalate did not induce changes in sperm morphology in mice or rats.. As reported in an abstract (Baker et al. 1996). and neither this metabolite nor 2-ethylhexanol induced mutations in mouse lymphoma cells in vitro. mono(2-ethyl-5-hydroxyhexyl) phthalate (IX). mono(5-carboxy-2-ethylpentyl) phthalate (V) and 2-ethylhexanol. 1996). Negative results were also obtained in the Bacillus subtilis rec assay with mono(2ethylhexyl) phthalate. Gapjunctional intercellular communication was inhibited by mono(2-ethylhexyl) phthalate in Syrian hamster embryo cells and in Chinese hamster V79 cells. DNA strand breaks were not induced in rat liver in vivo. Unscheduled DNA synthesis was not induced in either mouse or human primary hepatocyte cultures with mono(2-ethylhexyl) phthalate. Ashby & Clapp. so that this reported mutagenic activity of di(2-ethylhexyl) phthalate is questionable. Di(2-ethylhexyl) phthalate was reported to induce dominant lethal mutations in mice in three studies. 4. Chromosomal aberrations and cell transformations were induced in Syrian golden hamster embryos exposed transplacentally to di(2-ethylhexyl) phthalate for 24 h after pregnant females were treated on day 11 of gestation. Metabolites Five metabolites of di(2-ethylhexyl) phthalate. It also induced transformation in Syrian hamster embryo cells. None induced mutations in S. mono(2-ethyl-5-oxohexyl) phthalate (VI). Mono(2-ethylhexyl) phthalate induced sister-chromatid exchange in Chinese hamster V79 cells and chromosomal aberrations in Syrian hamster embryo cells. however. 2-ethylhexanol and phthalic acid. In the single study with mono(2-ethylhexyl) phthalate. IX and mono(2-ethylhexyl) phthalate. . 1989. analysis of the disposition data does not provide an explanation for the observed species differences in di(2ethylhexyl) phthalate-induced hepatic peroxisome proliferation. Two re-evaluations (Adler & Ashby. mono(2-ethylhexyl) phthalate.5 Mechanistic considerations Significant species differences have been observed in the absorption and disposition of di(2-ethylhexyl) phthalate. typhimurium. were tested in genotoxicity assays. One study reported that di(2-ethylhexyl) phthalate was antimutagenic and co-recombinogenic in combination with N-ethyl-N-nitrosourea in the mouse spot test (Fahrig & Steinkamp-Zucht. but not in Syrian hamster or human hepatocytes. but not in mouse C3H10T½ cells.116 IARC MONOGRAPHS VOLUME 77 administered by gavage and it did not induce aneuploidy in hepatocytes of rats fed a diet containing di(2-ethylhexyl) phthalate for seven days. 1995) of these studies considered that cytotoxicity can interfere with the recognition of a dominant lethal effect.

. Cattley et al. No study has yet compared the responsiveness to di(2-ethylhexyl) phthalate of human versus rodent livers in vivo. Several lines of investigation have enhanced our understanding of the molecular basis of peroxisome proliferation. have demonstrated that administration of di(2-ethylhexyl) phthalate metabolites does not lead to peroxisome proliferation in vitro. A growing body of evidence concerning the molecular basis of peroxisome proliferation.b. In biopsies from humans receiving hypolipidaemic drugs.b. Marked species differences in hepatic peroxisome proliferation have been reported (Ashby et al. 1994. 1995. This can theoretically result in increased levels of mutation by increasing the frequency of replicative DNA synthesis as well as increasing the number of hepatocytes at risk. Lake.. Takagi et al. Cattley et al.. modulation of hepatocellular proliferation by peroxisome proliferators has been implicated in the mechanism of carcinogenesis. unlike those with hepatocytes from mice and rats. hepatocellular proliferation is probably involved in the promotion of growth of preneoplastic hepatocytes. including formation of active metabolites. there was no effect or changes were much smaller than those that would be produced in rodent hepatocytes at equivalent dose levels (Lake. 1995a. Evidence from humans. Taken together.. In rodent liver. Similarly. Furthermore. Chronic administration of peroxisome proliferators to rodents results in sustained oxidative stress due to overproduction of peroxisomal hydrogen peroxide.DI(2-ETHYLHEXYL) PHTHALATE 117 The weight of evidence for di(2-ethylhexyl) phthalate and for other rodent peroxisome proliferators in general demonstrates that they do not act as direct DNAdamaging agents. several studies using human hepatocytes. While peroxisome proliferation may be readily demonstrated in cultured rat and mouse hepatocytes.. IARC. There is clear evidence that di(2-ethylhexyl) phthalate causes acute and sustained hepatocellular proliferation under bioassay conditions which resulted in liver tumours in rats (Marsman et al. Lake et al. these disposition data do not provide an explanation for the species differences in response to di(2-ethylhexyl) phthalate.. primates and humans. This can theoretically generate reactive oxygen species which can damage DNA and other intracellular targets. 1988. The induction of peroxisomal fatty acid β-oxidation and increases in peroxisomal volume density by di(2-ethylhexyl) phthalate in vivo under bioassay conditions (Marsman et al.. rats and mice indicates that differences in disposition. increased replicative DNA synthesis and induction of peroxisomal and microsomal fatty . 1988). 1989. are of a quantitative. Supporting data on induction of oxidative stress (lipofuscin accumulation. However. 1987) supports this hypothesis. 1995a. such effects are not observed in hepatocytes from non-responsive species including guinea-pigs. with important implications for cancer risk and species differences. peroxisome proliferation. 8-OH-dG in DNA) in rat liver by di(2-ethylhexyl) phthalate are available (Conway et al. there are no data for mouse liver. not qualitative nature. 1998). 1990a).. increased organ weight. 1998). indicates why human livers and hepatocytes would be expected to be refractory to induction of peroxisome proliferation and carcinogenesis by di(2-ethylhexyl) phthalate. summarized below. however.

A study of PPARα knock-out mice treated with di(2-ethylhexyl) phthalate in vivo demonstrated that the increased liver weights and induction of peroxisomal and microsomal enzymes are absolutely dependent on PPARα (Ward et al. The species differences. WY-14. 1998). and other aspects of interaction with transcriptional regulatory proteins. 1998. Peters et al.. these results demonstrate that peroxisome proliferation.. Peroxisome proliferator response elements (PPREs) have been found in genes for both peroxisomal and microsomal fatty acid-oxidizing enzymes (Lake... Moreover. the presence or absence of active PPREs in the promoter region of specific genes. Palmer et al. some of these tumours were malignant. unlike wild-type mice. but did .118 IARC MONOGRAPHS VOLUME 77 acid-oxidizing enzymes require the expression of functional PPARα. Studies of PPARα activation in vitro have shown that di(2-ethylhexyl) phthalate metabolites are capable of activating PPARα. 0/9 and 0/9. Taken together.643 at 0. Marked species differences in the expression of PPARα mRNA have been demonstrated between rodent and human liver. 1998). in three of these mice.. 1998).643...643 did not produce liver tumours in PPARα-deficient mice (Peters et al. particularly with respect to humans compared to rats and mice. Tugwood et al.643 produced multiple hepatocellular tumours in 6/6 wild-type mice. the same treatment produced no tumours in PPARα-deficient mice (0/9). Using a sensitive and specific immuno/DNA binding assay. Most of the samples (13/20) contained no detectable PPARα activity. Ward et al. 1997a. Palmer et al. In this study. 1995b. The insensitivity of human liver to rodent peroxisome proliferators is associated with low levels of expression of PPARα in human liver. 1997a). with the latter expressing 1–10% of the levels found in mouse or rat liver (Palmer et al. These include the level of expression and functional capability of PPARα..1% in the diet or control diet was fed for 11 months to PPARα-deficient and wild-type mice and the livers were removed and examined. respectively). (1998) have shown that active PPARα protein is expressed at variable concentrations in human livers. and no preneoplastic lesions were found by microscopy. related hepatic effects and carcinogenesis induced by peroxisome proliferators are all absolutely dependent on functional PPARα. 1995. 1994. Cattley et al.. WY-14. Aoyama et al. Recent evidence confirms that species differences can involve more than one aspect of PPARα-mediated regulation of gene expression. the potent peroxisome proliferator Wy-14. 1996. 1998). The study compared 20 different human livers and found that those with the highest levels of PPARα protein expression contained less than 10% of the level in mice. three died and were lost to evaluation and one was euthanized and examined when moribund before 11 months. In contrast. can be potentially attributed to several aspects of PPARα-mediated regulation of gene expression.. This nuclear receptor is a member of the steroid hormone receptor superfamily and binds to DNA as a heterodimer with the retinoid X receptor (RXR). Of nine PPARα-deficient mice fed WY-14. Studies with PPARα knockout mice have demonstrated that all the above effects are lost in these mice (Lee et al. No gross tumours or microscopic evidence of preneoplasia were seen in either wild-type mice or PPARαdeficient mice on the control diet (incidence.

g. 1995) or lack of co-activating proteins such as steroid receptor co-activator-1 (SRC-1) and PPAR-binding protein (PBP) (Zhu et al. (1998). The truncated human PPARα mRNA was expressed in vitro. (1999).DI(2-ETHYLHEXYL) PHTHALATE 119 exhibit PPRE-binding activity that did not correspond to PPARα. The truncated human PPARα resulting from mRNA mis-splicing. 1997). Other human PPARs may compete with PPARα. TR) for dimerization with RXR (Juge-Aubry et al. differential species sensitivity to peroxisome proliferators could depend on genespecific factors.. while no truncated PPARα mRNA was found in livers of rats and mice. an additional element of transcriptional regulation. In addition to the role of receptor expression and other transcriptional regulators. at least in part. suggesting that the low level of endogenous PPARα is the major factor in the non-responsiveness of these cells. In most human samples studied. the low levels of PPARα protein detected in human liver were lower than those estimated from RNA analysis and this was explained by the finding that a significant fraction of PPARα mRNA is mis-spliced in human liver. It is possible that the low level of PPARα in human liver may explain.. 1999. Such data support a ‘threshold through competition’ concept... 1994. the apparent lack of responsiveness of human hepatocytes to peroxisome proliferators. these results demonstrate that a certain fraction of human PPARα is probably truncated and inactive and may interfere with the function of any full-length human PPARα present in human hepatocytes.. All six subjects aged 10 years or less lacked detectable PPARα activity. in part due to titration of coactivator CREB-binding protein. has been further characterized by Gervois et al. Taken together. which contains similar levels of PPARα and RXR to those found in intact human liver. it was found that PPREs are mainly bound by other competing proteins that may block peroxisome proliferator responsiveness. the promoter regions containing PPRE responsible for transcriptional activation of the rodent gene are not present in the promoter region of the human gene (Lambe et al. This may be due to competition for binding to PPREs by other transcription factors such as HNF4. Woodyatt et al. Peroxisome proliferator responsiveness is unlikely to be a linear function of PPARα expression levels... competition of PPARα with other hormone receptors (e. LXRα) leading to formation of non-functional heterodimers (Miyata et al. In the case of peroxisomal acyl-coenzyme A oxidase. identified by Palmer et al. It is possible that similar differences in promoter regions of . for example PPAR* (hNUC1) can repress the activation of hPPARα (Jow & Mukherjee. 1997). ARP-1 and TR (Palmer et al. binding of PPARα to other transcription factors (e. In support of this hypothesis. 1995). increasing PPARα levels by heterologous expression in the Huh cell line conferred responsiveness to peroxisome proliferators. (1998) have shown that transcriptional responses to peroxisome proliferators are not evident in the Huh cell line. a necessary step for gene activation and (b) interfere with gene activation by expressed full-length human PPARα. They found that the truncated PPARα mRNA accounted for 25–50% of total PPARα mRNA in 10 human liver samples.g. 1999).. Palmer et al. In addition. However. Miyamoto et al.. where it was shown to (a) fail to bind to PPRE. 1996).

. 1998). 1998). Significantly. may be explained by differences in the mechanism of action of PPARα in relation to these hypolipidaemic genes compared with those that regulate hypertrophic or hyperplastic responses: the former may have a lower threshold of activation and may require lower concentrations of receptors due to different binding affinities for different PPREs.. Cattley et al.. Guinea-pigs are also refractory to the hepatic effects of rodent peroxisome proliferators (reviewed in Doull et al. plasma lipoprotein levels are not changed by fibrate treatment. which is not associated with any hypertrophic or hyperplastic response. Dirven et al. 3. The existence of such a response. 1999). In summary: 1. In humans.. 1998. Di(2-ethylhexyl) phthalate produces liver tumours in rats and mice. In normal rabbits.. recent studies with rabbits suggest a further potential model for the refractory nature of human liver to peroxisome proliferation (Staels & Auwerx. In addition to results from guinea-pigs. This process is essential for liver hypertrophy and . Elcombe et al. including di(2-ethylhexyl) phthalate metabolites (Elcombe & Mitchell. The weight of evidence for di(2-ethylhexyl) phthalate and its metabolic products demonstrates that they do not act as direct DNA-damaging agents. 1998. fibrate treatment of transgenic apo A-I rabbits results in increased plasma HDL and human apo A-I concentrations due to induction of the human apoA-I transgene expression in the rabbit liver. Under conditions of the bioassays. 4. The hypolipidaemic response. However. This demonstrates that PPARαmediated effects of fibrates on human lipoprotein gene expression can occur in the absence of peroxisome proliferation. 1996) and. 1998). like humans... Significant responses (peroxisome proliferation. Bell et al. However. 2. fibrates increase plasma levels of high-density lipoprotein (HDL) via induction of human apo A-I gene expression. The insensitivity towards peroxisome proliferators exhibited by human hepatocytes is reflected in guinea-pigs. has been attributed to PPARα-mediated regulation of genes encoding lipoprotein lipase and various lipoproteins. Rodent peroxisome proliferators exercise their pleiotropic effects in liver due to activation of PPARα. 1993c.120 IARC MONOGRAPHS VOLUME 77 other genes could also help to explain differences between rodent and human responses to peroxisome proliferators. these species do exhibit hypolipidaemic responses when exposed to some rodent peroxisome proliferators (Lake. Differences in the activation properties for different PPRE-containing promoters have been demonstrated (Hsu et al. in spite of low levels of PPARα. this induction of lipoprotein gene expression occurs without affecting liver weight or peroxisomal acyl-coenzyme A oxidase activity. induction of fatty acid-oxidizing enzymes and stimulation of replicative DNA synthesis) believed to be associated with hepatocarcinogenesis in rodents are not observed in humans or guinea-pigs. di(2-ethylhexyl) phthalate induces peroxisome proliferation and cell replication in liver that are characteristic of a peroxisome proliferator in mice and rats. express similar low levels of PPARα (Bell et al. 1986. 1995a. The fibrate effect on human apo A-I is mediated by PPARα that interacts with a PPRE in its promoter. Tugwood et al. 1995)..

Hepatic peroxisome proliferation has not been adequately evaluated in studies of human livers following exposure to di(2-ethylhexyl) phthalate in vivo. 6. such as during dialysis and transfusion. . 5. However. at concentrations usually below 1 mg/m3. indicate that humans can reasonably be predicted to be refractory to induction of peroxisome proliferation and hepatocellular proliferation by di(2-ethylhexyl) phthalate.DI(2-ETHYLHEXYL) PHTHALATE 121 5. The evidence indicates that the mechanism of peroxisome proliferation induced by di(2-ethylhexyl) phthalate in rat hepatocytes does not operate in humans. where concentrations up to 10 mg/kg have been reported. 7. It is found in ambient air at levels usually below 100 ng/m3. Overall.2 Human carcinogenicity data One small study of workers in a di(2-ethylhexyl) phthalate production plant did not show any excess of cancer mortality. as well as other peroxisome proliferators. widely used as a plasticizer in flexible poly(vinyl chloride) products at concentrations of up to 40%. meat and fish and in other products with a high fat content. the effect of treatment of human and mouse hepatocytes with di(2ethylhexyl) phthalate metabolites which are active in rat hepatocytes. hyperplasia and eventual hepatocarcinogenesis in response to peroxisome proliferators. The absence of a significant response of human liver to induction of peroxisome proliferation and hepatocellular proliferation is explained by several aspects of PPARα-mediated regulation of gene expression. these findings indicate that the increased incidence of liver tumours in mice and rats treated with di(2-ethylhexyl) phthalate results from a mechanism that does not operate in humans. however. The highest levels of di(2-ethylhexyl) phthalate in foods are found in milk products. this study did not have adequate power to detect a potential excess risk. Di(2-ethylhexyl) phthalate is ubiquitous in the general environment as a result of its widespread use in poly(vinyl chloride) products. as well as in a number of other minor applications. Occupational exposure occurs mainly by inhalation as an aerosol during its manufacture and its use as a plasticizer in poly(vinyl chloride) product manufacturing plants. can result in large direct exposures. 5. The leaching of di(2-ethylhexyl) phthalate from flexible plastics used in medical devices.1 Summary of Data Reported and Evaluation Exposure data Di(2-ethylhexyl) phthalate is a liquid of low volatility. 5.

Taken together. A considerable amount of information on the hepatic effects of orally administered di(2-ethylhexyl) phthalate indicates that it causes hepatic peroxisome proliferation (ultrastructural effects and enzyme induction). the data indicate that di(2-ethylhexyl) phthalate does not cause observable toxicity following oral and intravenous exposure. administration of di(2-ethylhexyl) phthalate following administration with known carcinogens enhanced the incidences of hepatocellular preneoplastic foci. hepatomegaly and increased replicative DNA synthesis in rats and mice. marmosets and cynomolgus monkeys evaluated under the same or similar experimental conditions did not exhibit peroxisome proliferation responses. In all species studied. no promoting activity of di(2-ethylhexyl) phthalate was demonstrated.. the bulk of a di(2-ethylhexyl) phthalate dose was absorbed as the monoester. one showed enhancement of renal tubule tumours by di(2-ethylhexyl) phthalate.122 IARC MONOGRAPHS VOLUME 77 5. In a number of similar studies in rats and in one study in hamsters. 5. while species differences have been observed in the absorption and disposition of di(2-ethylhexyl) phthalate. Hepatocellular tumours were produced consistently in both species. adenomas and carcinomas.g. mono(2-ethylhexyl) phthalate. enzyme induction and hepatomegaly have been observed (ultrastructural effects and replicative DNA synthesis have not been evaluated). In two N-nitrosamine-initiation target organ models in rats. they do not provide an explanation for the species differences in hepatic peroxisome-proliferating activity. but do not contribute information relevant to the evaluation of human carcinogenicity. At a lower magnitude in Syrian hamsters. The peroxisome-proliferating effects of di(2-ethylhexyl) phthalate in susceptible species (e. However. the compound underwent rapid metabolism. This ester is also formed by esterases in the body following intravenous administration and is subject to extensive oxidative metabolism by the cytochrome P450 system. No initiating activity of di(2-ethylhexyl) phthalate was found in the liver of mice or rats. in general. Guinea-pigs. whereas the other showed no promotion of urinary bladder tumours. The literature on potential toxic effects of di(2-ethylhexyl) phthalate following human exposure is limited. Following oral administration. rats and mice) have primarily been related to mono(2-ethylhexyl) phthalate and two other specific metabolites. Studies of di(2-ethylhexyl) phthalate . with the urine and faeces being the major routes of excretion. In a number of initiation/promotion studies in strains of mice susceptible to liver carcinogenesis.3 Animal carcinogenicity data Di(2-ethylhexyl) phthalate was tested for carcinogenicity by oral administration in the diet in two experiments in mice and six experiments in rats.4 Other relevant data The absorption and disposition of di(2-ethylhexyl) phthalate has been investigated extensively in humans and laboratory animals.

Di(2-ethylhexyl) phthalate has been studied extensively for its genotoxic effects in a wide range of test systems. Low levels of mutation were induced in Drosophila melanogaster in somatic cells in some studies. human hepatocytes. Oral exposure of rats and mice to di(2-ethylhexyl) phthalate during organogenesis caused malformations and fetal death.643. Young animals were much more sensitive to gonadal effects than adults and in some cases. A study in knock-out mice suggested that the developmental effects are not PPARα-mediated. The Sertoli cells in the testes appear to be the main target of the testicular toxicity. In one study using small groups of adult marmosets. Oral exposure of adult rats and mice caused effects on fertility in males and females and serious effects on the testicles. based on lack of response to peroxisome proliferators in PPARα-deficient mice. No mutagenic activity was observed in bacteria. most notably. marmoset or. In fungi. mouse and. In cultured mammalian cells. Proposed mechanistic hypotheses relate to reduced testicular zinc levels. WY14. Irreversible testicular damage has been observed in male rat pups exposed prenatally and during suckling via maternal exposure to drinking water containing the compound. altered hormonal status. sister chromatid exchange or chromosomal . Syrian hamster hepatocyte cultures in vitro elicited markers of peroxisome proliferation. to a lesser extent. Oral administration of di(2-ethylhexyl) phthalate failed to elicit markers of peroxisome proliferation in PPARα-deficient mice. altered metabolic function and altered follicle-stimulating hormone reactivity. oral exposure did not cause testicular toxicity at doses higher than those producing testicular effects in adult rats. cynomolgus monkey. dog. to mediate these responses in mice. both in vitro and in vivo. all but two studies failed to show any evidence of recombinational events or mutation. Metabolites of di(2-ethylhexyl) phthalate caused activation of PPARαmediated gene expression in mammalian cell co-transfection assays. but no germ-cell mutations or DNA damage were induced in these insects. while the same or similar experimental conditions did not elicit markers of peroxisome proliferation in primary cultures of either guinea-pig. A single study in yeast for aneuploidy was positive.DI(2-ETHYLHEXYL) PHTHALATE 123 metabolites in primary rat. In one study with another peroxisome proliferator. the onset of occurrence of the testicular effects was earlier in young animals. Differences between responsive rodents and humans in various aspects of PPARα-mediated regulation of gene expression are consistent with the lack of activity of di(2-ethylhexyl) phthalate metabolites in hepatocyte cultures from 12 people studied to date. rabbit. mutation. No data on reproductive and developmental effects in humans were available. carcinogenesis was shown to be dependent on the same receptor. The majority of these studies did not reveal any activity. no primary DNA damage. PPARα. Hepatic peroxisome proliferation depends on a nuclear receptor. Dose-dependent testicular effects were seen in young rats exposed to di(2-ethylhexyl) phthalate in the diet. while the same treatment elicited this response in normal mice.

Overall evaluation Di(2-ethylhexyl) phthalate is not classifiable as to its carcinogenicity to humans (Group 3). Res. J. Gene mutations were not induced in the liver of dosed mice in a single study and there was no evidence for induction of chromosomal aberrations in mice or rats.. (1985) Antifertility and mutagenic effects in mice from parenteral administration of di-2-ethylhexyl phthalate (DEHP). natl Cancer Inst. 71–84 . 6. Dominant lethal effects were reported to be induced in male mice. Therefore. (1989) The present lack of evidence for unique rodent germ-cell mutagens.5 Evaluation There is inadequate evidence in humans for the carcinogenicity of di(2-ethylhexyl) phthalate. 5. Lawrence.124 IARC MONOGRAPHS VOLUME 77 aberrations were induced (except in a single study for DNA strand breakage). Mutat.. but re-evaluation of these data did not confirm this conclusion. in the embryos of dosed pregnant Syrian hamsters. environ. J. 58. and unscheduled DNA synthesis was not induced in the liver of either rats or mice. In vivo. Toxicol.-L. I. 212. J. There is sufficient evidence in experimental animals for the carcinogenicity of di(2-ethylhexyl) phthalate. J. M. 1635–1641 Adler. (1977) Chromosome aberrations and sister chromatid exchanges in Chinese hamster cells exposed to various chemicals. the mechanism by which di(2-ethylhexyl) phthalate increases the incidence of hepatocellular tumours in rats and mice is not relevant to humans. whereas transformation of cells was induced in a number of different systems. however. & Ashby. Health. D. 16. the Working Group took into consideration that (a) di(2-ethylhexyl) phthalate produces liver tumours in rats and mice by a non-DNA-reactive mechanism involving peroxisome proliferation.. S. & Sasaki. References Abe. (b) peroxisome proliferation and hepatocellular proliferation have been demonstrated under the conditions of the carcinogenicity studies of di(2-ethylhexyl) phthalate in rats and mice. & Autian.K. 55–66 Agarwal. neither covalent binding to DNA nor DNA strand breakage was induced in several studies on rat liver. W. In making its overall evaluation of the carcinogenicity to humans of di(2-ethylhexyl) phthalate.H. Aberrations were induced. and (c) peroxisome proliferation has not been documented in human hepatocyte cultures exposed to di(2-ethylhexyl) phthalate nor in the liver of exposed non-human primates.

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and these have been incorporated in the monograph and taken into consideration in the evaluation.58 –149– . new data have become available. Abstr. Serv. DOA. 63637-48-9.2 Structural and molecular formulae and relative molecular mass O C O CH2 (CH2)4 C O CH2 O CH C2H5 (CH2)3 CH3 C2H5 CH (CH2)3 CH3 C22H42O4 Relative molecular mass: 370. No. Abstr.1. octyl adipate 1. bis(2-ethylhexyl) ester IUPAC Systematic Names: Adipic acid bis(2-ethylhexyl) ester.: 103-23-1 Deleted CAS Reg.1 1. 70147-21-6 Chem. DEHA. Since that time.DI(2-ETHYLHEXYL) ADIPATE This substance was considered by previous working groups in October 1981 (IARC. dioctyl adipate. hexanedioic acid. 1.1. Reg. Nos: 39393-67-4. bis(2-ethylhexyl) adipate Synonyms: BEHA.1 Exposure Data Chemical and physical data Nomenclature Chem. 1987). 1. 1982) and March 1987 (IARC. dioctyl ester. Name: Hexanedioic acid.

Velsicol Chemical Corp.45) × ppm. 8. 1998. Lankroflex DOA. 1.. flash-point. Kodaflex DOA. 1996) Density: 0.3 (a) (b) (c) (d) (e) Chemical and physical properties of the pure substance Description: Light-coloured. 1995). oily liquid (Verschueren. assuming a temperature of 25 °C and a pressure of 101 kPa . 346 Pa at 200 °C (Lewis. Verschueren. 99–99. 1996) (g) Volatility: Vapour pressure. 1997. Aldrich Chemical Co.1 (Verschueren. Trade names for di(2-ethylhexyl) adipate include Adimoll DO. (C.. Flexol A 26. ADO (lubricating oil). 1995. Reomol DOA. 1993.1.. 1993) Melting-point: –67.8 °C (Lide & Milne. acidity. Sicol 250.1. 2000).16 × ppm Technical products and impurities 1. 1996) (i) Conversion factor1: mg/m3 = 15. Effomoll DOA.P.150 IARC MONOGRAPHS VOLUME 77 1. 1993) (h) Octanol/water partition coefficient (P): log P. Eastman Chemical Co. Monoplex DOA.10% max. Ergoplast AdDO.9%. Aldrich Chemical Co. Hatcol 2908. very soluble in acetone... Wickenol 158. Effomoll DA. Solutia. Adipol 2EH. Truflex DOA.25 meq/100 g max. 1996) Boiling-point: 417 °C (Lewis..5 Analysis Di(2-ethylhexyl) adipate can be extracted from a water sample by passing this water through a cartridge or disk containing a solid inorganic matrix coated with a chemically bonded C18 organic phase (liquid–solid extraction).4 Di(2-ethylhexyl) adipate is commercially available with the following specifications: purity. ADO. Hall Co.05–0. 0. undated. Diacizer DOA. 1996. moisture. Witamol 320. 1998) (f) Solubility: Very slightly soluble in water (< 200 mg/L at 20 °C) (Verschueren. Eastman DOA Plasticizer. 1996). 1998.1. 1 Calculated from: mg/m3 = (relative molecular mass/24. Arlamol DOA. diethyl ether and ethanol (Lide & Milne. National Institute for Standards and Technology. Organic material eluted from the liquid–solid extraction cartridge or disk with dichloromethane is analysed for di(2ethylhexyl) adipate by gas chromatography/mass spectrometry (Environmental Protection Agency. Plastomoll DOA. Plasthall DOA. Crodamol DOA. 1996) Spectroscopy data: Infrared (grating [8003]). nuclear magnetic resonance [943C] and mass spectral data have been reported (Lide & Milne.922 g/cm3 at 20 °C (Lide & Milne.. Vestinol OA. Sansocizer DOA. 196 °C (Lewis. Inc. 0. Bisoflex DOA. 1996).

France and Germany. five companies each in India and Taiwan. Peru. mainly as an aerosol. 1992. cellophane and hydroxyethyl cellulose films. Romania. No measurements of di(2-ethylhexyl) adipate exposure in manufacturing and processing industries are available. 1. Exposure may also occur during the manufacture of rubber products.3 Use Di(2-ethylhexyl) adipate is used primarily as a plasticizer in the flexible vinyl industry and is widely used in flexible poly(vinyl chloride) (PVC) food film (cling film). Russia. 1.DI(2-ETHYLHEXYL) ADIPATE 151 1. as many as 15 600 workers in the United States were potentially exposed to di(2-ethylhexyl) adipate (NOES. National Toxicology Program. Information available in 1999 indicated that di(2-ethylhexyl) adipate was manufactured by eleven companies in Japan.2 Occupational exposure According to the 1981–83 National Occupational Exposure Survey. eight companies in the United States. the Czech Republic. Italy and the United Kingdom and one company each in Belgium. 1996. the Republic of Korea. Colombia. lubricants and hydraulic fluids (Opresko. 1996. four companies each in Canada. polystyrene and dammar wax and in cosmetics (cellulose-based liquid lipsticks) (Cadogan & Howick.1 Occurrence Natural occurrence Di(2-ethylhexyl) adipate is not known to occur as a natural product. It is used as a solvent and as a component of aircraft lubricants. the Netherlands. 1999). It is commonly blended with di(2-ethylhexyl) phthalate (see monograph in this volume) and di(isooctyl) phthalate in PVC and other polymers. two companies each in Argentina. 1984). 1999).2 Production Di(2-ethylhexyl) adipate can be prepared by the reaction of adipic acid and 2-ethylhexanol in the presence of an esterification catalyst such as sulfuric acid or para-toluenesulfonic acid (National Library of Medicine. during its manufacture and its use. particularly as a plasticizer of PVC films and in other materials used in food packaging such as adhesives. Verschueren.4. nonferrous wire. 1999). China. three companies each in Brazil. Chile. Mexico and Spain. It is important in the processing of nitrocellulose and synthetic rubber. . South Africa. 1999). Australia. cellulose acetate butyrate. 1. Turkey and Venezuela (Chemical Information Services. Occupational exposure may occur through inhalation.4.4 1. cosmetics. in plasticizing polyvinyl butyral.

. IARC.. 1979.0 μg/L with an average of 0.46 μg/L (Felder et al. Di(2-ethylhexyl) adipate is relatively insoluble in water and is likely to partition to sediment and biota in the aquatic environment. 1982. 1998).08 mg/m3) near a cool rod machine (operating temperature of 190 °C) used to cut PVC film in a meat cutting and wrapping department of a grocery store (Daniels et al. It was detected in ‘finished’ drinking-water in New Orleans. at an average concentration of 0. (b) Water Di(2-ethylhexyl) adipate has been detected infrequently in fresh water. A survey of 23 natural surface water sites in 12 states showed that 7% of 82 samples contained di(2-ethylhexyl) adipate at levels ranging from 0.. as reported in the Toxics Release Inventory (Environmental Protection Agency. (a) Air According to the Toxics Release Inventory (Environmental Protection Agency.. WHO. 1996). In the United States. during PVC blending operations and cutting of PVC film. Louisiana. 1996). and from consumer use and disposal of finished products (IARC. Environmental Protection Agency.08–0. 1985). Cook (1980) estimated from test emission data that maximum di(2-ethylhexyl) adipate concentrations of 0.25 mg/m3 and 0. 1.10 μg/L but not in drinking-water in two smaller nearby cities (IARC. It has also been identified in Europe .2 mg/m3 in workroom air could be reached in hot wire operations. 1979). generally at < 1 μg/L (Sheldon & Hites. respectively (Van Houten et al. air emissions of di(2-ethylhexyl) adipate from 148 industrial facilities amounted to approximately 315 000 kg in 1994 in the United States. 1996).. 1996). Surface water discharges of di(2-ethylhexyl) adipate from 148 industrial facilities in the United States in 1994 amounted to 560 kg.152 IARC MONOGRAPHS VOLUME 77 Workers wrapping meat are potentially exposed to particulate di(2-ethylhexyl) adipate while cutting PVC films by drawing them across a heated cutter (hot wire or cool rod process) (Smith et al. 1974). 1982). 1986. 1986). Di(2-ethylhexyl) adipate was found at microgram-per-litre levels in two of five samples of finished water from a water-treatment plant in the United States (WHO.3 μg/L (Sheldon & Hites. the National Institute for Occupational Safety and Health reported non-detectable levels of di(2-ethylhexyl) adipate (less than 0. Felder et al.4. 1983). Di(2-ethylhexyl) adipate was detected in the Delaware River at levels of 0. 1982.25 to 1. Exposure concentrations of 0.14 mg/m3 just above the hot wire of a PVC film cutting machine have been reported in tests simulating normal operating conditions when the wire was operated at 182 °C and 104 °C.3 Environmental occurrence Di(2-ethylhexyl) adipate may be released into the environment during its manufacture and distribution.

cereals.0 mg/kg in fruits and vegetables. Page & Lacroix. poultry. Ranges of di(2-ethylhexyl) adipate levels were 1.. 1987. north of Philadelphia in 1977 and at levels of 90 and 10 μg/L at sampling sites at influent and effluent waste-treatment sites. (c) Soil Releases of di(2-ethylhexyl) adipate to land from 148 industrial facilities in the United States in 1994 amounted to 67 000 kg. (d) Biodegradation and bioconcentration Model experiments with acclimated activated sludge systems have shown essentially complete biodegradation of relatively high concentrations (~ 20 mg/L) of di(2ethylhexyl) adipate to carbon dioxide and water in 35 days (Saeger et al. . 1996). fruit. Felder et al. 1994. Startin et al. 1995. respectively (Sheldon & Hites. A bioconcentration study with bluegill showed that di(2-ethylhexyl) adipate is not an accumulative or persistent chemical in this species of fish (Felder et al. bread rolls and sandwiches).0–72. 1986). particularly to fatty foods such as cheese and meat. 1996). 1996) and in the Great Lakes of North America at levels of 0.. Castle et al. Startin et al. as reported in the Toxic Release Inventory (Environmental Protection Agency. WHO. A United Kingdom survey of di(2-ethylhexyl) adipate levels in 83 retail samples wrapped in plasticized PVC films was reported by Castle et al. 1987. fruits and vegetables (Castle et al. 1995.01–7. Di(2-ethylhexyl) adipate was found at levels of 2000 μg/L near a chemical plant source near the Delaware River..0–212 mg/kg in baked goods and sandwiches. 11.. 1982. baked goods and sandwiches.6 mg/kg in cooked chicken portions. Page & Lacroix.0 μg/L (Hrudey et al. meat. 27. vegetables and baked goods (cakes.DI(2-ETHYLHEXYL) ADIPATE 153 as a trace level contaminant of the River Rhine (WHO.0 mg/kg in cheese. 1986). cheese.. margarine. Petersen et al. Mercer et al.. (e) Food Food is the major source of exposure of the general population to di(2-ethylhexyl) adipate because of its migration...8–135.. 9. butter. The level of di(2-ethylhexyl) adipate in meat exposed to plasticized film was not reduced significantly by volatilization or chemical transformation on subsequent cooking by grilling or frying. cheese. from PVC films used for packaging that have been plasticized with di(2-ethylhexyl) adipate (IARC. 1987.. Foodstuffs analysed (from both retail and take-away outlets) included fresh meat and poultry. 1979).8 mg/kg in uncooked meat and poultry. 1987. ready-cooked poultry. 1976. 1976). Di(2-ethylhexyl) adipate has been found at generally low levels in a broad variety of foods including milk.. (1987). Gilbert et al.4–48. 1990. and < 2. 1995).

Australia. 181 and 109 mg/kg. whilst lower levels were found for wrapping of unfilled buttered sandwiches. fish. Levels of migration of di(2-ethylhexyl) adipate into purchased ready-cooked meats. cooked meats. respectively. microwave power setting. Selected foods (260 samples) packaged in materials with potential to contribute plasticizers to the food and available food composites (98 samples) obtained from the Canadian Health Protection Branch Total Diet Program were analysed for plasticizers including di(2ethylhexyl) adipate and di(2-ethylhexyl) phthalate. 77% of the films used for fatty foodstuffs sampled from importers. In a study in New South Wales. Di(2-ethylhexyl) adipate was found in food-contacting PVC film and as a migrant in store-wrapped meat. respectively. Five out of 42 samples (12%) of fresh meat packaged in PVC film gave positive results.154 IARC MONOGRAPHS VOLUME 77 The highest levels of migration of di(2-ethylhexyl) adipate from PVC films during home-use and microwave cooking in the United Kingdom were observed for cheese. wholesalers and retail shops . and beef (minced). the nature of the food simulant and the initial concentration of the plasticizer in the film. 75 and 29 mg/kg. ham. at levels ranging from 31 to 429 mg/kg. Badeka and Kontominas (1996) reported the effect of microwave heating on the migration of di(2-ethylhexyl) adipate from food-grade PVC into olive oil and water. with levels ranging from 49 to 151 mg/kg. 78 and 23 mg/kg. 107 and 25 mg/kg. Di(2-ethylhexyl) adipate residues found in fresh fruits and vegetables were typically < 4 mg/kg. poultry. rewrapped in the home in PVC film and kept for seven days at 5 °C or 30 days at –18 °C were: chicken. Petersen et al. cakes and microwave-cooked foods. Significant quantities of di(2-ethylhexyl) adipate were found in cheeses which had been wrapped at the point of sale. compared with a specific migration limit of 3 mg di(2-ethylhexyl) adipate/dm2 from PVC cling films used in Denmark. with the highest levels found where there was direct contact between the film and food and where the latter had a high fat content in the contact surface (Startin et al. Migration of di(2-ethylhexyl) adipate at levels of 64 and 325 mg/kg was also found in other foods such as sandwiches wrapped in PVC (Kozyrod & Ziaziaris. Di(2-ethylhexyl) adipate levels in unheated film-wrapped ready-to-eat foods were increased by heating. 44 (45%) showed levels of migration of di(2-ethylhexyl) adipate exceeding 30 mg/kg. Of the 98 samples wrapped in PVC films.. Migration was dependent on heating time. salami. only samples in contact with PVC film were found to contain a detectable amount of di(2-ethylhexyl) adipate. migration of di(2-ethylhexyl) adipate increased with both the length of contact time and temperature of exposure. 1987). Overall. Di(2-ethylhexyl) adipate was detected in 36 of 38 samples of cheese wrapped in PVC film. respectively. cheese and ready-to-eat foods at levels as high as 310 mg/kg (cheese). A survey of di(2-ethylhexyl) adipate in Canadian packaging and food sampled during the period 1985–89 was reported by Page and Lacroix (1995). respectively. 1989). of 184 samples of food packaged in a range of plastics. (1997) reported that. fruit and vegetables (except avocado).

. In the United States. The study involved the determination of the urinary metabolite. Loftus et al.. 1994). The probability of a daily intake in excess of 8. .2 mg per day.. A skewed distribution with a median value for the daily intake of 2. WHO.2 mg in the limited population (112 individuals) was calculated to be 3% (Loftus et al. The major urinary metabolite of di(2-ethylhexyl) adipate. 1993.1 mg/dm2 for all PVC films. A limited population study in the United Kingdom was undertaken to estimate the daily intake of di(2-ethylhexyl) adipate following intake of a mean dose of 5. 1994). and as a component of closures with sealing gaskets for food containers..4 mg di(2-ethylhexyl) adipate presented with food. The migration of di(2-ethylhexyl) adipate to non-fatty foods defined as the food simulant water was ≤ 0. 2-ethylhexanoic acid. The United States Environmental Protection Agency (1998) has set a maximum contaminant level (MCL) for di(2-ethylhexyl) adipate in drinking water of 0. 2-ethylhexanoic acid (24-h urine sample) in 112 individuals from five geographical locations.5 Regulations and guidelines The World Health Organization has established an international drinking water guideline for di(2-ethylhexyl) adipate of 80 μg/L (WHO.DI(2-ETHYLHEXYL) ADIPATE 155 were found to be unacceptable. Reformulation of PVC film reflecting the use of less di(2-ethylhexyl) adipate necessitated a more recent evaluation which suggested that the maximum daily intake of di(2-ethylhexyl) adipate in the United Kingdom was 8. as a component of paper and paper-board in contact with aqueous and fatty foods.2 mg (Loftus et al. 2. as a component of cellophane. The maximum daily intake of di(2-ethylhexyl) adipate through the diet in the United Kingdom was estimated in 1987 to be 16 mg (Anon. 1. Studies of Cancer in Humans No data were available to the Working Group. 1994). 1994. 1996). the Food and Drug Administration (1999) permits the use of di(2-ethylhexyl) adipate as a component of adhesives used in food packaging. This value is about one third of the indirectly estimated maximum intake of 8.. 1991. 1993. 1996)..7 mg was determined (Loftus et al. as a plasticizer in polymeric substances used in the manufacture of articles for the food industry. 1993.4 mg/L. has been shown to be an appropriate marker for biological monitoring of dietary di(2ethylhexyl) adipate intake (Loftus et al. 1994).

1 Studies of Cancer in Experimental Animals Oral administration Mouse Groups of 50 male and 50 female B6C3F1 mice. Mean body weights of treated mice of each sex were lower than those of the corresponding controls and the decrease in weight gain was dose-related.001) females. 13/50. 19/50 p < 0. Survival in males was 36/50 (72%). Hepatocellular carcinomas were observed in 7/50 control.. p = 0. respectively. Survival in males was 34/50 (68%) in the control and low-dose groups and 40/50 (80%) in the high-dose group and in females was 29/50 (58%). [The Working Group noted that negative trends were reported for certain tumour types (lymphomas. trend test) and in females (control. 12/49 low-dose and 12/49 high-dose males and 1/50 control. 5/50 and 6/49 in control.. 20/49 and high-dose. pairwise comparisons). The incidences of hepatocellular adenomas and carcinomas combined were also increased in males (control.1.1 3. and that in females was 42/50 (84%). Di(2-ethylhexyl) adipate increased the incidence of hepatocellular adenomas in both males (6/50 control. There was no treatment-related increase in tumours. low-dose and high-dose groups.001. six weeks of age.156 IARC MONOGRAPHS VOLUME 77 3. pairwise comparison. were fed diets containing 0. 32/50 (64%) and 41/50 (82%) in control. > 98%) for 103 weeks and were killed 105–107 weeks after the beginning of treatment. 12 000 or 25 000 mg/kg diet (ppm) di(2-ethyhexyl) adipate (> 98% pure) for 103 weeks and were killed 105–107 weeks after the beginning of treatment. 1982. low-dose and high-dose animals). . 1985). p = 0. low-dose.001) and 12/49 high-dose (p = 0. low-dose and high-dose animals. and 44/50 (88%) in the control. Kluwe et al. Mean body weights of high-dose rats of each sex were lower than those of the controls throughout the study. 2/50 low-dose and 2/50 highdose males and in 0/49. 3/50. and high-dose. 1982. respectively. were fed diets containing 0. 39/50 (78%) and 36/49 (73%) in the control. 8/49 low-dose and 15/49 high-dose (p < 0.1. 3. respectively (National Toxicology Program. 14/50 low-dose (p < 0. respectively. Kluwe et al. five weeks of age. low-dose and high-dose animals. lung and subcutaneous tumours in males and pituitary adenomas in females)] (National Toxicology Program.2 Rat Groups of 50 male and 50 female Fischer 344 rats. 39/50 (78%).003.001. 18/49 p < 0. 27/49.002. 3/50.025) and females (2/50. 1985). 3. Neoplastic nodules or hepatocellular carcinomas were found in 2/49 control. low-dose. 12 000 or 25 000 ppm di(2-ethylhexyl) adipate (purity. and 1/50 females.

Radiolabel from di(2-ethylhexyl) [carbonyl-14C]adipate is distributed to a number of tissues. In rats. blood and kidney had relatively high levels of di(2-ethylhexyl) adipate-associated radiolabel (Takahashi et al.1 In six male volunteers given 46 mg deuterium-labelled di(2-ethylhexyl) adipate [approx.2%) and 2-ethylhexanol (0. The dominant urinary metabolite of di(2-ethylhexyl) adipate (500 mg/kg bw) in male Wistar rats is adipic acid. Some of the biliary-secreted radioactivity (approximately 3% in rats) flows into the enterohepatic circulation. 2-ethyl1. which accounts for 20–30% of the administered oral dose. 1987.7%). Bergman & Albanus..6-hexanedioic acid (0. whereas in cynomolgus monkeys unchanged di(2-ethylhexyl) adipate is also absorbed (BUA.1. the following metabolites were identified (percentage fraction of administered deuterium label): 2-ethylhexanoic acid (8. 34–78% of the dose after 24 h. While most radioactivity was found in the gastrointestinal tract. Passage of di(2-ethylhexyl) adipate (84.5%. 4.6%).65 h.. with maximum levels being reached after 6–12 h. mice. the glucuronide of mono(2-ethylhexyl) adipate and traces of unchanged di(2-ethylhexyl) adipate were found in the urine (BUA. In urine. The other major metabolite which was found only in the stomach is mono(2-ethylhexyl) adipate (Takahashi et al. total radioactivity in the body after 96 h was approximately 0. 1987. with most of the dose appearing in the urine after oral administration to Fischer 344 rats. 47–57%). 1981). 2-ethylhexanoic acid was the only metabolite that could be determined in the plasma.1. A half-life of 6 min for metabolism of di(2-ethylhexyl) adipate has been determined in rat small intestinal mucous membrane homogenates. In rats.1 4.5 h.6%). 75–92%. BUA. B6C3F1 mice and cynomolgus monkeys (rats. 1996). . 1996). 0. Other Data Relevant to an Evaluation of Carcinogenicity and its Mechanisms Absorption. 2-ethyl-5-ketohexanoic acid (0. 1981. monkeys. In cynomolgus monkeys. 2-ethyl-5-hydroxyhexanoic acid (2. 1996).3 μg per animal) through the placenta of pregnant NMRI mice has been described (Bergman & Albanus. 1996). fat. liver.. The half-life for elimination of all metabolites excreted in the urine averaged 1.DI(2-ETHYLHEXYL) ADIPATE 157 4.5 mg/kg bw] in corn oil. BUA. and none of the metabolites could be detected after 36 h (Loftus et al.1%). 1981). It had an elimination half-life of 1.. there is evidence for cleavage of the parent compound and subsequent absorption of the monoester and the acid (Takahashi et al. muscle.2 Experimental systems Di(2-ethylhexyl) adipate is rapidly and completely absorbed from the gastrointestinal tract of experimental animals. distribution. Di(2-ethylhexyl) adipate is rapidly eliminated. metabolism and excretion Humans 4. 1993).

1984). Light microscopic evaluation of liver sections from rats revealed a di(2-ethylhexyl) adipate-dependent loss of glycogen in hepatocytes that progressed from centrilobular to panlobular with increasing dose. plasma cholesterol levels were significantly decreased.2. Consumption of 2. 4.158 IARC MONOGRAPHS VOLUME 77 4. Similar increases in peroxisomal volume density (by morphometric analysis) were reported following dietary administration of di(2-ethylhexyl) adipate (1.5 mg/kg bw per day for 14 days increased peroxisomal cyanide-insensitive palmitoyl-coenzyme A (CoA) oxidation activity from approximately twofold up to 15fold in male rats and female mice. there was dose-related hypertrophy and increased eosinophilia of hepatocytes. In both rats and mice.0 but not ≤ 0. A variety of studies have evaluated the effects of oral administration of di(2ethylhexyl) adipate on rodent liver. administration of di(2-ethylhexyl) adipate by gavage in corn oil at levels of 0.0 or 2. After two weeks and four weeks (but not seven weeks) of feeding.0% (up to 3140 mg/kg bw per day . In male and female Fischer 344 rats and B6C3F1 mice.2 4. although body weight gain was diminished (Lake et al. 1986).2. Hepatic cholesterol synthesis was diminished by di(2-ethylhexyl) adipate consumption (Bell.. 1997). up to fourfold in female rats and ninefold in male mice. Similar dietary administration of di(2-ethylhexyl) adipate (25 000 ppm [2. No evidence of hepatotoxicity was observed by light microscopy. 1982). 1992). 1982). as summarized in Table 1 (Keith et al.5% of diet) to Fischer 344 rats [sex not specified] for 30 days (Reddy et al.1 Toxic effects Humans No data were available to the Working Group. 1984).5%] in diet) did not affect survival in studies of two years’ duration in mice and rats (National Toxicology Program.. The effects of di(2-ethylhexyl) adipate (1% of diet) on plasma lipids were evaluated in male Upjohn:TUC (SD) rats (Bell. 1983.2 Experimental systems The acute oral LD50 values for di(2-ethylhexyl) adipate in Fischer 344 rats were estimated to be 45 (males) and 25 (females) g/kg bw by gavage and in B6C3F1 mice were estimated to be 15 (males) and 25 (females) g/kg bw by gavage (National Toxicology Program.0% di(2-ethylhexyl) adipate in the diet [3 g/kg bw per day] by female Fischer 344 rats was not associated with lethality. plasma triglyceride levels were significantly decreased.. These effects were accompanied by slight but statistically significant increases in catalase activity in mice but not rats. The hepatic effects of di(2-ethylhexyl) adipate were evaluated in female B6C3F1 mice and Fischer 344 rats fed diets containing 0–4.5–2. Morphometric analysis of liver ultrastructure demonstrated increases in peroxisomal volume density. After four weeks (but not two or seven weeks) of feeding.5% di(2-ethylhexyl) adipate in the diet [5 g/kg bw per day] by female B6C3F1 mice and 4.

2% (3075 mg/kg bw per day).4 2.5 2. 1981).9 3.2 4.0 4.6% (1495 mg/kg bw per day) induced dose-dependent increases in relative liver weight and hepatic peroxisome proliferation. This apparent difference in magnitude of response between mice and rats could in part be accounted for by the different rates of di(2-ethylhexyl) adipate intake (see Table 2).8 2. (1992) in rats and 5330 mg/kg bw per day in mice) di(2-ethylhexyl) adipate for one.and 12-hydroxylase activities (CYP4A) were similarly increased at the same or the next lowest dietary concentration (0. as demonstrated by the induction of peroxisomal cyanideinsensitive palmitoyl-CoA oxidation (increases were statistically significant for at least one treatment interval). The effect of di(2-ethylhexyl) adipate administered daily by gavage for 14 days on peroxisomal volume density in Fischer 344 rats and B6C3F1 mice DEHA (mg/kg bw per day) Species Fischer 344 rats B6C3F1 mice Sex Male Female Male Female 0. In mice.8 6.5 Peroxisomal volume density (% of cytoplasmic volume) 1. relative. rats fed di(2-ethylhexyl) adipate had much smaller increases in relative liver weight and peroxisomal palmitoyl-CoA oxidation at doses matching those used in a bioassay (National Toxicology Program. di(2-ethylhexyl) adipate at ≥ 0.3 7.3%.5 1. In contrast to mice. rats were similarly responsive.0 10. there was similar induction of microsomal lauric acid 11. 32%. four and 13 weeks (Lake et al.1 2.4 1. although at even higher dietary concentrations.6% (1495 mg/kg bw per day) and was still elevated at weeks 4 and 13 at doses of ≥ 1. Hepatocellular replication (measured as nuclear 5-bromo-2′-deoxyuridine [BrdU] labelling) was increased during week 1 of di(2-ethylhexyl) adipate treatment of mice at ≥ 0. although the magnitude of response during week 1 was similar to that in mice.2 7. Therefore.0 1.4 1.5 1. While hepatocellular replication (measured as nuclear BrdU labelling) was increased during week 1 of administration in rats.4 1.0 0.5% of . 1997). Microsomal lauric acid 11.and 12hydroxylase activity.3 5. this response was not sustained during weeks 4 or 13. 282 mg/kg bw per day in rats and 808 mg/kg bw per day in mice). Increased liver weights (absolute..4 Not measured 6.1 From Keith et al.DI(2-ETHYLHEXYL) ADIPATE 159 Table 1. In rats. the apparent difference in the results of carcinogenicity testing between mice and rats could be related to differences in intake of di(2-ethylhexyl) adipate and the resulting peroxisome proliferation and related responses in liver. 36% over respective control values) were observed in male Fischer 344 rats fed di(2-ethylhexyl) adipate (2.5 2.0 7.

160 Table 2. NE.30 0.50a 4. (1997) a Dietary levels administered to mice and rats in a carcinogenesis bioassay. Comparison of responses in liver of female mice and rats following four weeks of di(2-ethylhexyl) adipate treatment Diet (%) Intake (mg/kg bw per day) Relative liver weight (% increase over controls) Mouse Rat Peroxisomal palmitoyl-CoA oxidation (increase over control) Mouse Rat Microsomal lauric acid hydroxylase (increase over control) Mouse 11position NC NC 2-fold 3-fold 5-fold NE 12position NC 2-fold 4-fold 8-fold 16-fold NE Rat 11position NC NC NC NC 2-fold 3-fold 12position NC NC NC NC 2-fold 8-fold IARC MONOGRAPHS VOLUME 77 Mouse Rat 0.00 343 808 1495 3075 5330 NE 144 282 577 1135 2095 3140 NC NC 10 50 60 NE NC NC NC 10 30 80 NC NC 2-fold 7-fold 13-fold NE NE NC NC < 2-fold 8-fold 17-fold Adapted from Lake et al.20a 2. resulting in an increase in tumour incidence in mice but not in rats (National Toxicology Program. not evaluated . NC. 1981).15 0. not different from controls.60 1.

in liver but not kidney DNA were reported.2 Experimental systems (a) Developmental toxicity studies Groups of five Sprague-Dawley rats were given intraperitoneal injections of 1. 2-ethylhexanoic acid and 2-ethyl-5-hydroxyhexanoic acid in guinea-pig or marmoset hepatocytes (2-ethylhexanol was evaluated only at ≤ 1 mM in marmoset hepatocytes)..b). The rate of skeletal malformations lay in the same range as in the control groups. Feeding the compound at this dose for 14 days resulted in increased levels of hepatic phospholipids and a decline in phosphatidylcholine:phosphatidylethanolamine ratio (Yanagita et al. mice.3. 1998).DI(2-ETHYLHEXYL) ADIPATE 161 diet) for one week. Primary hepatocyte cultures may be employed to study species differences in hepatic peroxisome proliferation (IARC.3 4. 4. guinea-pigs and marmosets have been studied (Cornu et al. Several studies have evaluated the effects of oral di(2-ethylhexyl) adipate on various aspects of hepatic lipid metabolism. 1990). an indicator of oxidative DNA damage. 5 or 10 mL/kg bw di(2-ethylhexyl) adipate on days 5. fatty acid transporter protein and fatty acid binding protein in the liver (Motojima et al. In hepatocytes from each species. 10 and 15 of pregnancy.3. in rat and mouse hepatocytes. 1992).. Feeding di(2-ethylhexyl) adipate (2% of diet) to male Wistar rats for seven days resulted in increased hepatic fatty acid-binding protein as well as in increased microsomal stearoyl-CoA desaturation activity (Kawashima et al. the rate of visceral malformations was reported . 1987).. The effects of di(2-ethylhexyl) adipate and its metabolites in cultured hepatocytes from rats. Feeding di(2-ethylhexyl) adipate (2% of diet) to male NZB mice for five days resulted in induction of fatty acid translocase. the parent compound di(2-ethylhexyl) adipate had no effect on peroxisomal cyanideinsensitive palmitoyl-CoA oxidation activity. The incidence of externally visible malformations [no further details supplied] was significantly higher in the high-dose group. Fetal weight in the two high-dose groups showed a dose-dependent reduction. 1983a. 2-ethylhexanol..1 Reproductive and developmental effects Humans No data were available to the Working Group. 2-ethylhexanoic acid and 2-ethyl-5-hydroxyhexanoic acid at concentrations ≤ 1 mM induced peroxisomal palmitoyl-CoA oxidation. No induction of peroxisomal palmitoyl-CoA oxidation was seen at concentrations ≤ 1 mM for mono(2-ethylhexyl) adipate or ≤ 2 mM for 2-ethylhexanol. 4. 1995).. and also after two weeks’ administration (Takagi et al. the metabolites mono(2-ethylhexyl) adipate. Slight but statistically significant increases in 8-hydroxydeoxyguanosine (8-OH-dG). However.

intestine and bone marrow during the first 24 h after intravenous or intragastric administration of di(2-ethylhexyl) [carbonyl14C]adipate to pregnant NMRI mice on gestational day 17 (Bergman & Albanus. there was a significant reduction in body weight gain of the females during the last part of the gestation period.and high-dose groups. A single intraperitoneal injection of di(2-ethylhexyl) adipate (12. Radioactivity was observed in fetal liver. When [ethylhexyl-14C]di(2-ethylhexyl) adipate was administered. 1800 or 12 000 ppm di(2-ethylhexyl) adipate for a period of 10 weeks before mating and during the gestation and lactation periods. cited in BUA. liver and intestinal contents of the fetus as well as in the amniotic fluid. In the high-dose group. (c) Reproductive toxicity studies In a dominant lethal study. A remarkably strong uptake of radioactivity was observed in the corpora lutea of the ovary in the pregnant mice.. Correlations with maternally toxic effects were not described (Singh et al. Groups of 24 Alpk:APF50 rats were given approximately 28. in the high-dose group.and high-dose groups than in the control animals. Groups of 30 female and 15 male Alpk:APF50 rats were fed diets containing 300. but increased the aminopyrine-Ndemethylase activity only in pregnant mice. 1987.b). an isoenzyme induced in mouse pregnancy.. the body weight gain of the pups was significantly reduced throughout the postnatal follow-up period (up to day 36 of life) (Tinston. . 1996).162 IARC MONOGRAPHS VOLUME 77 to be higher in the middle. there was a significantly higher incidence of skeletal variations/retardations (Hodge. 1973). The incidence of unilateral ureter kinking was slightly but significantly higher in the middle. 1996). di(2-ethylhexyl) adipate decreased levels of P450-gest. cited in BUA. the number of live births or the survival rate of the pups up to day 22 of life. In the high-dose group. 1983a. 1988. a reduced percentage of pregnancies and an increased number of fetal deaths were observed in Harlan/ICR albino Swiss mice after a single intraperitoneal dose of 10 mL/kg bw was given to male mice before an eight-week mating period (Singh et al. there was a slight reduction of maternal body weight gain. 1975). In the high-dose group. there was very little accumulation of radiolabel but some was found in the urinary bladder. In addition. but increased other P450 isoenzymes (Lamber et al. In the pregnant mice..5 mL/kg bw) on day 15 of gestation increased the cytochrome P450 content in hepatic microsomes in pregnant and non-pregnant NP C57BL/6J mice. 1996). 170 and 1080 mg/kg bw via the feed from days 1 to 22 of gestation. There was no effect on male and female fertility. 1987). (b) Mechanistically oriented developmental toxicity studies No effects on three-day-old chick embryos were found after exposure by injection of 17 μmol di(2-ethylhexyl) adipate per egg into the air chamber of the egg (Korhonen et al. 1991.. cited in BUA.

DI(2-ETHYLHEXYL) ADIPATE 163 In a multigeneration study.2 Experimental systems (see Table 3 for references) Di(2-ethylhexyl) adipate was not mutagenic to Salmonella typhimurium strains TA100.TA1537. For four successive generations. one experiment similarly showed no induction of mutation. chromosomal aberrations or micronuclei after treatment with di(2-ethylhexyl) adipate for either 3 or 51 h. while a second experiment showed an effect but only at a concentration at which precipitation occurred (above 1000 μg/mL). TA1538 or TA98. no substance-specific influence on reproduction rate. typhimurium strains TA100. TA1538 or TA98 in the presence or absence of exogenous metabolic activation in three studies. A single study found no induction of sex-linked recessive lethal mutations after administration of di(2-ethylhexyl) adipate to adult Drosophila melanogaster by feeding or injection. A weak positive result has been reported in a dominant lethal assay in male mice. TA102.1 Genetic and related effects Humans No data were available to the Working Group.4. 1962. A single in-vitro study using rat hepatocytes found no induction of sister chromatid exchanges.4. In bone marrow of mice treated in vivo with di(2-ethylhexyl) adipate. Increased levels of 8-OH-dG were found in the liver but not in the kidney. TA98 or TA97 in the presence or absence of exogenous metabolic activation. formation of 8-OH-dG was measured as an indicator of oxidative DNA damage in liver and kidney of rats exposed to di(2-ethylhexyl) adipate in the diet for two weeks. Urine samples from rats treated by gavage with 15 daily doses of di(2-ethylhexyl) adipate were not mutagenic to S. TA1537. . A separate study found no evidence of covalent binding of di(2ethylhexyl) adipate to mouse liver DNA. It was also not mutagenic to Photobacterium phosphoreum in a single study. With exogenous metabolic activation. One study employing the mouse lymphoma gene mutation assay found no induction of mutations at the Tk locus in L5178Y mouse lymphoma cells following exposure to di(2-ethylhexyl) adipate in the absence of exogenous metabolic activity. rats were given 100 mg/kg di(2-ethylhexyl) adipate per day via the feed. lactation or growth was reported [no further details supplied] (Le Breton. Three putative metabolites of di(2-ethylhexyl) adipate—(mono(2-ethylhexyl) adipate. In one study. chromosomal aberrations were not induced in one study and no effect was seen in a single micronucleus assay.4 4. 4. mono(2-ethyl-5-hydroxyhexyl) adipate and mono(2-ethyl-5-oxohexyl) adipate)—were not mutagenic to S. TA1535. TA1535. typhimurium strains TA100. cited in BUA. 4. 1996).

TA1537. (1982) Zeiger et al. reverse mutation Binding (covalent) to DNA. (1984) Takagi et al. male B6C3F1 mouse bone marrow in vivo Rat (Sprague-Dawley) urine/Salmonella typhimurium TA100. TA1537. TA1537.15 μg/animal. reverse mutation Salmonella typhimurium TA100. (1988) Reisenbichler & Eckl (1993) Reisenbichler & Eckl (1993) Reisenbichler & Eckl (1993) Shelby & Witt (1995) Shelby et al. female NMR1 mouse liver in vivo Oxidative DNA damage (8-OH-dG). TA1538. reverse mutation Photobacterium phosphoreum. bioluminescence assay Mutatox Drosophila melanogaster. TA1535. male B6C3F1 mouse bone marrow in vivo Micronucleus test. 1000c 74 (3 and 51 h incubation) 74 (3 and 51 h incubation) 74 (3 and 51 h incubation) NR 2000 ip × 3 2000 po × 15 1100–1440 po × 1d 2. primary female Fischer 344 rat hepatocytes in vitro Micronucleus assay. TA1535. (1977) Seed et al. (1985) Woodruff et al. (1985) Elmore & Fitzgerald (1990) Woodruff et al. primary female Fischer 344 rat hepatocytes in vitro Chromosomal aberrations. male Fischer 344 rat liver DNA in vivo – – – – – – – – – – – – – – +e With exogenous metabolic system Doseb (LED or HID) Reference IARC MONOGRAPHS VOLUME 77 – – – NT 5000 μg/plate NR 10 000 μg/plate NR 20 000 ppm in feed 1. (1990) – NT NT NT – . primary female Fischer 344 rat hepatocytes in vitro Chromosomal aberrations and micronucleus formation. (1993) DiVincenzo et al. forward mutation Sister chromatid exchange. TA98. L5178Y cells. sex-linked recessive lethal mutations Drosophila melanogaster. inj. TA98. sex-linked recessive lethal mutations Gene mutation. Genetic and related effects of di(2-ethylhexyl) adipate and some derivatives Test system Resulta Without exogenous metabolic system Salmonella typhimurium TA100. TA1535. (1985) McGregor et al. TA98. TA1538.164 Table 3. reverse mutation Salmonella typhimurium TA100. Tk locus in vitro. (1985) Däniken et al.5% diet for 1 and 2w Simmon et al.

TA102. in-vivo tests. in-vitro tests. reverse mutation a Doseb (LED or HID) With exogenous metabolic system 9220 ip × 1 Reference DI(2-ETHYLHEXYL) ADIPATE (+) Singh et al. reverse mutation Mono(2-ethyl-5-oxohexyl) adipate Salmonella typhimurium TA100. TA97. 8-hydroxydeoxyguanosine b 165 . lowest effective dose. oral. TA98. (1991) – – 1000 μg/plate Dirven et al. HID. NT. TA97. negative. (1991) – – 1000 μg/plate Dirven et al. positive. day. μg/mL. highest ineffective dose. TA98. not tested. male Harlan/ICR albino Swiss mice in vivo Mono(2-ethylhexyl) adipate Salmonella typhimurium TA100.Table 3 (contd) Test system Resulta Without exogenous metabolic system Dominant lethal assay. reverse mutation Mono(2-ethyl-5-hydroxyhexyl) adipate Salmonella typhimurium TA100. TA102. (1991) +. TA102. d. TA98. w. –. not reported LED. week c Precipitate formed at doses ≥ 1000 μg/mL d There was no effect of pre-treatment with 10 000 mg/kg in the diet for four weeks. TA97. mg/kg bw/day. e Oxidative damage was not found in rat kidney DNA. NR. po. (1975) – – 1000 μg/plate Dirven et al. 8-OH-dG.

however. summarized below. however. 1995a. Interestingly.b. 1998). Lake.5 Mechanistic considerations Some general considerations about the role of peroxisome proliferation as a mechanism of carcinogenicity are presented in the General Remarks section of this volume. demonstrates that they do not act as direct DNA-damaging agents. 1997). there are no data for mouse liver. however. 1990).166 IARC MONOGRAPHS VOLUME 77 4. indicates that human livers and hepatocytes would be refractory to induction of peroxisome proliferation by di(2-ethylhexyl) adipate. given that the receptor mediates the same response for a variety of other peroxisome proliferators. 1995. there was no effect or changes were much smaller than those that would be produced in rodent hepatocytes at equivalent dose levels (Lake.. Chronic administration of peroxisome proliferators to rodents results in sustained oxidative stress due to overproduction of peroxisomal hydrogen peroxide. which did not respond with liver tumours in the bioassay as did the mice (Lake et al. The induction of peroxisomal fatty acid β-oxidation by di(2ethylhexyl) adipate in vivo under carcinogenicity testing conditions in rats and mice (Lake et al. Studies of this mechanism are reviewed fully in Section 4. a growing body of evidence concerning the molecular basis of peroxisome proliferation. This can theoretically result in increased levels of mutation by increasing the frequency of replicative DNA synthesis as well as increasing the number of hepatocytes at risk. While peroxisome proliferation may be readily demonstrated in cultured rat and mouse hepatocytes.5 of the monograph on di(2-ethylhexyl) phthalate in this volume. 1994. There is clear evidence that di(2-ethylhexyl) adipate causes acute and sustained hepatocellular proliferation under bioassay conditions which resulted in liver tumours in mice. the duration of hepatocellular proliferation was limited in rats. such effects are not observed in hepatocytes from non-responsive species including guinea-pigs. This can theoretically generate reactive oxygen species which can damage DNA and other intracellular targets. Cattley et al. 1997) supports this hypothesis.. Marked species differences in hepatic peroxisome proliferation have been reported (Ashby et al. the modulation of hepatocellular proliferation by peroxisome proliferators has been implicated in the mechanism of carcinogenesis. IARC. Studies of PPARα activation in vitro or in PPARα knock-out mice in vivo have not yet been conducted with di(2-ethylhexyl) adipate. hepatocellular proliferation is likely to be involved in the promotion of growth of preneoplastic hepatocytes. 1998). Similarly. 1995a. No study has yet compared the responsiveness of human versus rodent livers in vivo or hepatocytes in vitro to di(2-ethylhexyl) adipate.b.. The weight of evidence for di(2-ethylhexyl) adipate. Furthermore.. and for other rodent peroxisome proliferators in general. In biopsies from humans receiving hypolipidaemic drugs. Cattley et al. Limited supporting data on induction of oxidative stress (formation of 8-OH-dG in DNA) in rat liver by di(2-ethylhexyl) adipate are available (Takagi et al. primates and humans... it is likely .

including di(2-ethylhexyl) adipate metabolites (Cornu et al. has been attributed to PPARα-mediated regulation of genes encoding lipoprotein lipase and various lipoproteins. 1998). Under conditions of the bioassays. Bell et al.. Tugwood et al. Stated another way. These negative results were significant in that the same metabolites induced typical induction of peroxisomal (cyanide-insensitive) palmitoyl-CoA oxidation activity in rat and mouse hepatocytes. The existence of such a response. 1995). This process is essential for liver hypertrophy and hyperplasia and eventual hepatocarcinogenesis in response to peroxisome proliferators. Rodent peroxisome proliferators exercise their pleiotropic effects due to activation of PPARα.. 4. may be explained by differences in the mechanism of action of PPARα in relation to these hypolipidaemic genes compared to those that regulate hypertrophic and hyperplastic responses: the former may have a lower threshold of activation and may require lower concentrations of receptor due to different binding affinities for different PPREs. No evaluation of peroxisome proliferation in human hepatocytes treated with di(2-ethylhexyl) adipate metabolites in vitro has been published. induction of peroxisome proliferation by a PPARα-independent mechanism would be unprecedented. 1998). in spite of low levels of PPARα. Cultured hepatocytes from non-human primates (marmosets and macaques) and humans have been similarly unresponsive to a variety of peroxisome proliferators (reviewed in Doull et al.. 1995a . Significant responses (peroxisome proliferation. which is not associated with any hypertrophic or hyperplastic response.DI(2-ETHYLHEXYL) ADIPATE 167 to mediate the hepatic effects of di(2-ethylhexyl) adipate. The insensitivity of human hepatocytes towards peroxisome proliferators is reflected in the guinea-pig. 3. to a limited extent. 1992). In summary: 1. 1998. The lack of peroxisome proliferation in hepatocytes from marmosets suggests that human hepatocytes also would be unresponsive (Cornu et al. induction of fatty acid oxidizing enzymes and the stimulation of replicative DNA synthesis) believed to be associated with hepatocarcinogenesis in rodents are not observed in humans and guinea-pigs. Di(2-ethylhexyl) adipate produces liver tumours in mice. .. 1999).. However. Di(2-ethylhexyl) adipate does not show evidence of genotoxicity.. 1992). 1999).. di(2-ethylhexyl) adipate induces peroxisome proliferation and cell replication in liver that are characteristic of a peroxisome proliferator in mice and. Differences in the activation properties for different PPRE-containing promoters have been demonstrated (Hsu et al. in rats. The guinea-pig is also refractory to the hepatic effects of rodent peroxisome proliferators (reviewed in Doull et al. Cattley et al.. 2. This hypolipidaemic response. these species do exhibit hypolipidaemic responses when exposed to some rodent peroxisome proliferators (Lake.. 1998. and like humans expresses similar low levels of PPARα (Bell et al.

1 Summary of Data Reported and Evaluation Exposure data Di(2-ethylhexyl) adipate is a liquid of low volatility. as well as in other plastics and in a number of other minor applications. 5. notably food films. .2 Human carcinogenicity data No data were available to the Working Group. particularly into fatty foods such as cheese and meat. Occupational exposure may occur by inhalation of di(2-ethylhexyl) adipate as an aerosol during its manufacture and its use. studies of di(2-ethylhexyl) adipate or its metabolites regarding peroxisome proliferation in human cells are not available. 5. However. However. Food is the major source of exposure of the general population to di(2-ethylhexyl) adipate because of migration from poly(vinyl chloride) packaging. 6. 7. such as lubricants and cosmetics. 5. In mice. Overall.3 Animal carcinogenicity data Di(2-ethylhexyl) adipate was tested for carcinogenicity by oral administration in one experiment in mice and one experiment in rats. widely used as a plasticizer in flexible poly(vinyl chloride) products. No treatment-related tumours were observed in rats. liver adenomas and carcinomas were produced in both males and females. interspecies comparisons with other peroxisome proliferators. Meat-wrapping workers may be exposed while cutting poly(vinyl chloride) film across a heated cutter.168 IARC MONOGRAPHS VOLUME 77 5. 5. these findings suggest that the increased incidence of liver tumours in mice treated with di(2-ethylhexyl) adipate results from a mechanism that does not operate in humans. The absence of significant response of human liver to induction of peroxisome proliferation and hepatocellular proliferation is explained by several aspects of PPARα-mediated regulation of gene expression. along with the role of PPARα in this response. Hepatic peroxisome proliferation has not been evaluated in studies of human subjects or systems treated with di(2-ethylhexyl) adipate. indicate that humans can reasonably be predicted to be refractory to induction of peroxisome proliferation and hepatocellular proliferation by di(2-ethylhexyl) adipate.

The species differences in carcinogenicity assays of di(2ethylhexyl) adipate (increased hepatocellular tumours in mice. Analyses of mouse bone marrow after treatment with di(2-ethylhexyl) adipate in vivo found no induction of micronuclei in one study and no induction of chromosomal aberrations in one study. One report showed evidence of oxidative damage in rat liver DNA in vivo but not in rat kidney DNA. a single high intraperitoneal dose given to males before mating was associated with a reduced percentage of pregnancies and increased number of fetal deaths. No effects on male or female fertility were found in rats given di(2-ethylhexyl) adipate in the feed. Di(2-ethylhexyl) adipate did not induce gene mutations.DI(2-ETHYLHEXYL) ADIPATE 169 5. No data on reproductive and developmental effects in humans were available to the Working Group. It did not induce sex-linked recessive lethal mutations in Drosophila when administered either by diet or injection. Di(2-ethylhexyl) adipate did not bind covalently to mouse liver DNA in vivo. A weak dominant lethal effect has been reported in male mice. Exposure of rats to di(2-ethylhexyl) adipate during organogenesis caused an increased frequency of variations and retardations in the fetuses at doses below the maternally toxic range. The body weight gain of the pups at the highest dose was reduced throughout the postnatal period. It is hydrolysed in the gastrointestinal tract before absorption. not rats) are consistent with a higher intake of di(2-ethylhexyl) adipate and a greater extent of peroxisome proliferation and associated responses in livers of mice compared with rats fed the same dietary doses. chromosomal aberrations or micronuclei in rodent cells in vitro. sister chromatid exchanges. In mice and rats. In mice. Di(2-ethylhexyl) adipate was not mutagenic to either Photo- . No data on the genetic and related effects of di(2-ethylhexyl) adipate in humans or human cells were available to the Working Group.4 Other relevant data Di(2-ethylhexyl) adipate is rapidly and completely absorbed after oral administration. In hepatocytes isolated from rats and mice. The same treatment of primary cultures of hepatocytes from guinea-pigs and marmosets failed to cause any similar increase in activity. Urine from rats treated with di(2-ethylhexyl) adipate by gavage was not mutagenic to Salmonella typhimurium. di(2-ethylhexyl) adipate induced hepatic markers of peroxisome proliferation (ultrastructural and biochemical) as well as hepatomegaly and increased replicative DNA synthesis. treatment of primary cultures with metabolites of di(2-ethylhexyl) adipate increased peroxisomal palmitoyl-coenzyme A oxidation activity. rapidly and extensively metabolized and rapidly excreted in humans and experimental animals. No data on the toxic effects of di(2-ethylhexyl) adipate in humans were available to the Working Group.

Dickins. A.. N.G. Horely. 25.L. 202 Anon. Hirzel .D. Brady. Biochem.5 Evaluation No epidemiological data relevant to the carcinogenicity of di(2-ethylhexyl) adipate were available. 139–142 Ashby. Inhibition of cholesterolgenesis and modification of phospholipid synthesis.. & Bell. Z. Milwaukee. Lebensm. Elliott. Unters. 313–317 Bell.B. 13. Gray. Ishmael.B.F. References Aldrich Chemical Co. Food chem. S.. 20–26 Bell. J. Kettle. Toxicol. Overall evaluation Di(2-ethylhexyl) adipate is not classifiable as to its carcinogenicity to humans (Group 3). S. 332. R. Odum. 689–693 Bergman... 202. contam. 6. & Kontominas. (1996) Effect of microwave heating on the migration of dioctyladipate and acetyltributylcitrate plasticizers from food-grade PVC and PVDC/PVC films into olive oil and water. (1998) Molecular basis of non-responsiveness to peroxisome proliferators: the guinea pig PPARα is functional and mediates peroxisome proliferator-induced hypolipidaemia. exp. Salter. Lipids.P.J. F. 32.R. & Purchase.... 309–316 BUA (1996) Di-(2-ethylhexyl)adipate (BUA Report 196 by the GDCh-Advisory Committee on Existing Chemicals of Environmental Relevance (BUA)).H.. Stuttgart. F. These data indicate that di(2-ethylhexyl) adipate is not genotoxic.P.R.. There is limited evidence in experimental animals for the carcinogenicity of di(2ethylhexyl) adipate. A. M. D. Toxicol. environ. Hum.. J.170 IARC MONOGRAPHS VOLUME 77 bacterium phosphoreum or Salmonella typhimurium in the presence or absence of exogenous metabolic activation. (1984) Di(2-ethylhexyl)adipate (DEHA): effect on plasma lipids and hepatic cholesterolgenesis in the rat.M. 29. Forsch. A... Elcombe. DOA) on lipid metabolism in the rat: I.. L. Toxicol. B.R. WI. 5. 211–215 Bell. J. Tugwood. (1998) 1998–1999 Aldrich Catalog/Handbook of Fine Chemicals. (1991) Plasticizer migration in foods. C. A. Toxicol. (1987) Di-(2-ethylhexyl)adipate: absorption. M. 18.M.. p. (1983) Effect of the plasticizer di(2-ethylhexyl) adipate (dioctyladipate. A.. S1-S117 Badeka.. Bull. J.. K.J. T. (1994) Mechanistically-based human hazard assessment of peroxisome proliferator-induced hepatocarcinogenesis. J. & Albanus. I. Savory.. Choudhury. autoradiographic distribution and elimination in mice and rats. Food chem.

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1. 3-phenyl-2-propenyl 2-aminobenzoate. new data have become available. Abstr. Reg. 1996). 1995) –177– . 1. October 1982 (IARC.1. 1.1. Since that time. 1978). 1987). brownish powder with a balsamic.2 Structural and molecular formulae and relative molecular mass O C O CH2 CH CH NH2 C16H15NO2 1. Serv.1 1.30 Chemical and physical properties of the pure substance (a) Description: Crystalline solid (Budavari.3 Relative molecular mass: 253. and these have been incorporated in the monograph and taken into consideration in the evaluation.CINNAMYL ANTHRANILATE This substance was considered by previous working groups. 2-aminobenzoate IUPAC Systematic Name: Anthranilic acid. 1983) and March 1987 (IARC. Abstr. in June 1977 (IARC.1 Exposure Data Chemical and physical data Nomenclature Chem. fruity odour (Burdock. cinnamyl ester Synonyms: Cinnamyl alcohol anthranilate. Name: 3-Phenyl-2-propen-1-ol. 3-phenyl-2-propenyl anthranilate 1. No.: 87-29-6 Chem.

5 Analysis Cinnamyl anthranilate can be assayed by a method based on ester hydrolysis. 1. assuming a temperature of 25 °C and a pressure of 101 kPa . diethyl ether and ethanol (National Toxicology Program. baked goods. creams and lotions and as a perfume ingredient in orange blossom. Food and Drug Administration.2 Production Cinnamyl anthranilate can be synthesized by esterification of anthranilic acid with cinnamyl alcohol (Burdock. 1 Calculated from: mg/m3 = (relative molecular mass/24.5 °C (Budavari. 1. 1996) Density: 1. A method has been described for determining the content of this compound in food products by steam distillation followed by paper chromatography and examination under ultraviolet light. 1995). 1991.36 × ppm Technical products and impurities 1. since 1985 (Lucas et al. 1. National Toxicology Program.1. detergents. photooxidation and hydrolysis (National Toxicology Program. neroli. Bulk samples of food-grade cinnamyl anthranilate have been analysed for purity by thinlayer chromatography and high-performance liquid chromatography. 1991) (g) Conversion factor1: mg/m3 = 10. ice cream and ices.1. 1983). sweets.4 Cinnamyl anthranilate is not known to be currently commercially available. 1999.5 °C (Burdock.18 g/cm3 at 15. soluble in chloroform.3 Use Cinnamyl anthranilate was used for nearly 50 years at very low levels as a synthetic flavouring and fragrance agent. It has been used as a fragrance ingredient in various cosmetic products including soaps.45) × ppm. very soluble in acetone and dimethyl sulfoxide. gelatins and puddings and chewing gum. 1996). it has a limit of detection of 1 μg (IARC. 1983. 1999). cologne and other blends (Opdyke. 1991) Melting-point: 61–61. except for research purposes. IARC. 1995) Solubility: Very slightly soluble in water (< 1 mg/mL at 17 °C). 1991) (f) Stability: Sensitive to oxidation in air. 1983).178 IARC MONOGRAPHS VOLUME 77 (b) (c) (d) (e) Boiling-point: 332 °C (National Toxicology Program. Annual production in the United States in the 1970 was in the range of a few hundred kg (IARC. Budavari. It has not been commercially available. It was used as a flavouring agent to impart a grape or cherry flavour in non-alcoholic beverages.. 1975.

001. 1. Food and Drug Administration.1 3. low-dose and high-dose groups were 88. 1981.1 Occurrence Natural occurrence Cinnamyl anthranilate is not known to occur as a natural product. 1.4. National Toxicology Program. The doses were selected on the basis of a subchronic experiment. 82 and 74% in females. Cochran-Armitage . Survival rates at the end of the study (105–107 weeks) in the control. at least five unidentified impurities were found by thin-layer chromatography and two by high-performance liquid chromatography) for 103 weeks. six weeks of age. 82 and 80% in males and 78. 15 or 30 g/kg cinnamyl anthranilate (96% pure.4.4.4 1.2 Occupational exposure No data were available to the Working Group. The Food and Agriculture Organization and the World Health Organization have recommended that cinnamyl anthranilate not be used in food (FAO/WHO. A significant dose-related increase (p < 0. Dose-related reductions in mean body weight were noted in both males and females. Studies of Cancer in Humans No data were available to the Working Group. 1999). 3.5 Regulations and guidelines Although cinnamyl anthranilate was classified in the past as a Generally Recognized As Safe (GRAS) substance by the Food and Drug Administration.1 Studies of Cancer in Experimental Animals Oral administration Mouse Groups of 50 male and 50 female B6C3F1 mice. 3. 1.3 Environmental occurrence No data were available to the Working Group. 2.CINNAMYL ANTHRANILATE 179 1. were fed diets containing 0. 1991. its use in human food has been prohibited in the United States since 1985.1.

05) in highdose males (Stoner et al. respectively. 2. six to eight weeks of age. In the first series.. 37/47 high-dose. 1. 80 and 80% of the males and 78. respectively. No such tumour was observed in the matched controls.70 (p < 0. 1.2 Intraperitoneal administration Mouse: Cinnamyl anthranilate was tested in a lung adenoma screening assay. 15 or 30 g/kg cinnamyl anthranilate (same sample as used above) for 103 weeks. 11/13 high-dose males. seven weeks of age.37 in low-dose females. The dietary levels were selected on the basis of a subchronic study.13 ± 0. 88 and 92% of the females were still alive in the control. 1980). . A few metastases were seen in the lungs of high-dose females (National Cancer Institute. 33/49 high-dose). 1973). as found in subchronic experiments) or 100 mg/kg bw (total doses. 10/15 lowdose males and 10/22 control females. low.69 ± 0. There was a significant dose-related increase in the incidence of liver-cell carcinomas and adenomas combined in males and females (p < 0. 30/50 low-dose.001. lowand high-dose groups. 3.014) and 14/49 (p < 0. respectively).001) in the control.14 ± 0. The numbers of tumours per mouse were 0. Surviving animals were killed at 105–107 weeks. 12 and 2. 7/50 and 12/47 [p = 0. groups of 15 male and 15 female A/He mice.05) in high-dose females.047].and high-dose groups.1.28%] (National Cancer Institute. A control group of 25 females (but no control males) received intraperitoneal injections of the vehicle according to the same schedule.180 IARC MONOGRAPHS VOLUME 77 trend test) in the incidence of hepatocellular carcinomas was found in both males and females: females—1/50. Cochran-Armitage test for trend) (males—14/48 controls. were fed diets containing 0.59 ± 0. 3.4 g/kg bw.75 (p < 0.29 in low dose males and 2.13 in control females. 8/49 (p = 0. there was also a non-statistically significant increase in the incidence of acinar-cell pancreatic tumours (3/45: one carcinoma and two adenomas). Dose-related reductions in mean body weight were noted in both males and females.37% and that that for acinar-cell pancreatic tumours was 0. 7/15 low-dose females. [The Working Group noted that the historical control incidence in male rats for renal tubule tumours was 0. were given thrice weekly intraperitoneal injections of cinnamyl anthranilate [purity unspecified] dissolved in tricaprylin. males—6/48. 1980). as 24 doses of 500 mg/kg bw (the maximal tolerated dose. A non-statistically significant increase in the incidence of renal tubule tumours was observed in high-dose male rats (4/49. two adenocarcinomas and two adenomas). at which time 64. All animals were killed 24 weeks after the first injection and lung tumours were found in 10/15 high-dose females.2 Rat Two groups of 50 male and 50 female Fischer 344 rats.50 ± 0. There was an increased incidence of mineralization and inflammation of the kidneys of treated rats. 20/49 low-dose. females—3/50 controls.

However.2 Experimental systems Bronaugh et al. the major urinary metabolite was hippuric acid (95% of urinary 14C).02 in control females and 0. except that redistilled tricaprylin was used as the vehicle.40 ± 0. the urine contained relatively less hippuric acid (~ 80%) and more benzoic acid (16% of urinary 14C). 24. together with 2. In the rat. 4.12 in low-dose males compared with 0.3% of the dose. After application of 4 μg/cm2 cinnamyl anthranilate.20 0. 1996).8% when the site of application was covered with an occlusive dressing. .01) in high-dose females. 1. which rose to 39.1 4. The metabolism of cinnamyl anthranilate in rats and mice has been studied (Keyhanfar & Caldwell.85 ± 0.05) in low-dose females and 0. 0. metabolism and excretion Humans 4.03 in control males (Stoner et al.23 (p < 0.2% of the dose as unchanged cinnamyl anthranilate. Other Data Relevant to an Evaluation of Carcinogenicity and its Mechanisms Absorption. A further 10% (rat) and 6% (mouse) was recovered in the 24–72-h urine with 10% (rat) and 7% (mouse) in the 0–72-h faeces.0 ± 5.1. Male Fischer 344 rats and male CD-1 mice were given 250 mg/kg bw [3-14C] cinnamyl anthranilate by intraperitoneal injection. 4..1.3 ± 6. 1996).1 ± 2.04% of the dose (Keyhanfar & Caldwell. in mice.001) in high-dose males. using analytical methods able to detect 0. being 0.24 ± 0. When the surface of the skin was occluded. a polyethylene glycol 20 ether) as the receptor fluid.36 (p < 0.47 ± 0. Penetration through excised fullthickness human abdominal skin was determined using diffusion cells with a non-ionic surfactant (6% oleth 20. (1985) determined the percutaneous absorption of cinnamyl anthranilate in vivo in rhesus monkeys and in vitro through human skin. cinnamyl anthranilate was administered to 15 females and 15 males under similar experimental conditions.0 ± 2. The majority of the administered 14C was excreted in the 0–24-h urine (70% of the dose in rats and 78% in mice). the penetration rose to 53. together with much smaller amounts of benzoic acid. 1973).CINNAMYL ANTHRANILATE 181 In the second mouse lung adenoma assay.54 ± 0. Absorption through the shaved abdominal skin of four adult female rhesus monkeys after a 24-h application of 4 μg/cm2 was 26.1 Five human volunteers took a single oral dose of 250 mg cinnamyl anthranilate in water and no unchanged compound was detected in the 0–24-h urine. distribution. The control group consisted of 80 females and 80 males. The numbers of tumours per mouse were increased at the high dose.7% of the dose (n = 7).1% (n = 8) of the dose was absorbed within 48 h.15 (p < 0.

SDS-PAGE examination of the liver microsomes showed marked induction of a P450 isozyme of 53. aniline hydroxylase and para-nitroanisole O-demethylase. the effect of intraperitoneal dose size upon the fate of cinnamyl anthranilate in mice was examined over a range of 5–250 mg/kg bw. Although the P450 marker activities. but at 50 mg/kg bw.182 IARC MONOGRAPHS VOLUME 77 In further studies. 1996). 1000. Cinnamyl anthranilate was detected in the plasma. at 250 mg/kg. 1000. Caldwell et al. in the 9000 × g supernatant were unaltered by cinnamyl anthranilate administration. 1985).2% (Keyhanfar & Caldwell. The peak levels were some 3. but the maximum response (approximately twofold) was the same in both sexes. No intact ester was found in the urine after 5 mg/kg bw. The relative liver weight (% of body weight) and hepatic microsomal cytochrome P450 content increased in a dose-dependent fashion. it was only seen at 5000 ppm and above and the levels were two. 15 000 or 30 000 ppm (mg/kg diet) cinnamyl anthranilate in the diet for four days (Caldwell et al. 4. and correlated . The dose threshold for increased liver weight and microsomal enzyme induction was the same. Male and female B6C3F1 mice were fed diets containing 0. 10. hippuric acid and anthranilic acid within 24-h after removal of the test diet rose with increasing cinnamyl anthranilate dose. 15 000 or 30 000 ppm (mg/kg diet) cinnamyl anthranilate for 19 days.5 times higher in males than in females. 1980). significant above 1000 ppm: this was more evident in males than females.5 h after dosing.2. the percentage was 2. In an earlier study. 1997). The influence of dose size was also examined in male and female B6C3F1 mice given 0. 10. 5000. Similar dietary administration (15 or 30 g/kg diet) did not affect survival in studies of eight weeks’ and two years’ duration (National Toxicology Program. although body weight gain was diminished (Lake et al.2.3–0. rapidly declining from peak levels seen at 0. (1985). 100. In females.2 4. (1985) gave a single oral dose of 500 mg/kg bw to B6C3F1 mice and found 0. 4. accompanied by 17% as anthranilic acid and 35% as hippuric acid. 5000. 3.4% of the dose as unchanged cinnamyl anthranilate in the 0–24-h urine.1 Toxic effects Humans No data were available to the Working Group.to nine-fold lower. Cinnamyl anthranilate was shown to be a hepatic enzyme inducer in mice by Caldwell et al.1 kDa. The urinary excretion of cinnamyl anthranilate. Cinnamyl anthranilate was detected in increasing quantities in the urine of male mice at 1000 ppm and above. 100..1% of the dose was excreted as cinnamyl anthranilate and.2 Experimental systems Dietary consumption (30 g/kg diet) of 11 g/kg bw per day by female B6C3F1 mice and 3 g/kg bw per day by female Fischer 344 rats for up to 13 weeks was not associated with lethality..

there were dose-dependent increases in relative liver weight. cinnamyl alcohol and anthranilic acid.and 12hydroxylase activity (CYP4A) was increased 15-fold at 100 mg/kg bw per day and 17fold at 1000 mg/kg bw per day.15% (439 mg/kg bw per day) resulted in dose-dependent increases in relative liver weight and hepatic peroxisome proliferation.CINNAMYL ANTHRANILATE 183 with that for excretion of unchanged cinnamyl anthranilate noted in the shorter (fourday) experiment (Section 4. respectively).30% (883 mg/kg bw per day). feeding cinnamyl anthranilate at ≥ 0. total cytochrome P450. groups of male CD1 mice were given intraperitoneal injections of 0–200 mg/kg bw cinnamyl anthranilate daily for three days. relative liver weights of mice increased by 22% and 50%. The hepatic effects of cinnamyl anthranilate were evaluated in male CD1 mice and male Fischer 344 rats treated by intraperitoneal injection for three consecutive days (Viswalingam & Caldwell. (1997) have confirmed and extended these findings. While BrdU labelling was increased during week 1 of administration in rats. This apparent difference in magnitude of response was not accounted for by rate of intake (see Table 1). At doses of 100 and 1000 mg/kg bw per day. In contrast to mice. relative liver weights and peroxisomal palmitoyl-CoA oxidation activity were significantly increased only at 1000 mg/kg bw per day (22% and twofold. had a significant effect on the weight or marker enzyme content of mouse liver (Viswalingam & Caldwell. rats fed cinnamyl anthranilate had much smaller increases in relative liver weight and peroxisomal palmitoyl-CoA oxidation. In a separate experiment. this response was not sustained during weeks 4 or 13. . The hepatic effects of cinnamyl anthranilate are apparently due to the intact ester.15% in the diet (439 mg/kg bw per day) and continued to be elevated during weeks 4 and 13 at ≥ 0. Microsomal lauric acid 11. 24 h after the final dose and peroxisomal (cyanide-insensitive) palmitoyl-coenzyme A (CoA) oxidation activity increased fivefold at both levels. Lake et al. 2700 mg/kg bw per day in rats) cinnamyl anthranilate for one. and the magnitude of response during week 1 was again lower in rats. In mice. 1997). Microsomal lauric acid 12hydroxylase activity (CYP4A) was similarly increased. since neither its expected metabolites alone nor an equimolar mixture of the hydrolysis products. At doses of 20 mg/kg bw and above. Limited evaluation indicated that cinnamyl anthrnilate increased the size and number of peroxisomes in electron micrographs of hepatocytes of treated mice. 1997). even at the higher dietary concentrations. there was no induction of microsomal lauric acid 12-hydroxylase activity.2). as demonstrated by the induction of peroxisomal (cyanideinsensitive) palmitoyl-CoA oxidation (see Table 1). respectively. four or 13 weeks. and cyanide-insensitive palmitoyl-CoA oxidation. Hepatocellular replication (measured as nuclear 5-bromo-2′-deoxyuridine [BrdU] labelling) was increased during week 1 of cinnamyl anthranilate treatment at ≥ 0.1. In rats. In rats. Female B6C3F1 mice and female Fischer 344 rats were fed diets containing 0–30 mg/kg diet (11 030 mg/kg bw per day in mice.

15 0. but not in rats (National Toxicology Program. NE.00a 87 439 883 2140 4640 11 030 NE 159 321 763 1370 2700 NC 30 40 60 80 100 NE NC NC 10 15 25 NC 3-fold 4-fold 7-fold 10-fold 15-fold NE NC NC 2-fold 3-fold 6-fold NC 3-fold 4-fold 8-fold 12-fold 16-fold Adapted from Lake et al.03 0. Comparison of responses in liver of female mice and rats following four weeks of cinnamyl anthranilate treatment Diet (%) Intake (mg/kg bw per day) Mouse Rat Relative liver weight (% increase over controls) Mouse Rat Peroxisomal palmitoylCoA oxidation (increase over controls) Mouse Rat Microsomal lauric acid hydroxylase (increase over controls) IARC MONOGRAPHS VOLUME 77 Mouse 11-position 12-position NE NE 6-fold 8-fold 11-fold 13-fold Rat 11-position NE NC NC 2-fold 3-fold 6-fold 12-position NE NC NC NC NC NC 0.75 1.50a 3. not evaluated . NC.184 Table 1.30 0. (1997) a Dietary levels administered to mice and rats in a carcinogenesis bioassay and associated with increased incidence of liver tumours in mice. not different from controls. 1980).

It has been reported to give positive results for mutagenic activity in a bioluminescence assay in Photobacterium phosphoreum with exogenous activation. 1996.2 Experimental systems (see Table 2 for references) Cinnamyl anthranilate was not mutagenic to Salmonella typhimurium when tested in the presence or absence of rat liver S9 fraction.. Some general considerations about the role of peroxisome proliferation as a mechanism of carcinogenicity are presented in the General Remarks section of this volume. CYP4A isozymes and replicative DNA synthesis (Caldwell et al. It has been shown to enhance cell transformation of Syrian hamster embryo cells by SA7 virus. 1997). 1997. Keyhanfar & Caldwell. It was not active in mutation experiments with Drosophila and did not cause sister chromatid exchange or chromosomal aberrations in Chinese hamster ovary cells. Viswalingam & Caldwell.5 of the monograph on di(2-ethylhexyl) phthalate in this volume. It did not induce unscheduled DNA synthesis in rat hepatocytes in an in-vivo/in-vitro assay.4 4. Lake et al. 4. Cinnamyl anthranilate is reported to be active in the mouse lymphoma mutation assay. Hepatic effects were seen in mice only (a) after administration of the intact ester but not an equimolar mixture of its hydrolysis products and (b) at dose levels of cinnamyl anthranilate at which intact cinnamyl anthranilate is excreted in the urine.5 Mechanistic considerations There is a marked species difference in the hepatic effects of cinnamyl anthranilate between mice (both CD-1 and B6C3F1) and Fischer 344 rats. Studies of this mechanism are reviewed fully in Section 4. The species-specificity of peroxisome proliferation has been attributed to differences in the metabolism of cinnamyl anthranilate between rats and mice (Caldwell. In contrast. In mice. which are relatively resistant to its peroxisome-proliferating effect.4. this compound has the characteristic biochemical and morphological effects of a potent peroxisome proliferator. 4. 1985. In rats. do not excrete cinnamyl anthranilate in the urine at any dose level. Viswalingam & Caldwell. rats. It gave negative or equivocal results for the induction of sister chromatid exchanges in Syrian hamster embryo cells.4. these effects are much less evident. 1992.CINNAMYL ANTHRANILATE 185 4.. 1997).3 Reproductive and developmental effects No data were available to the Working Group.1 Genetic and related effects Humans No data were available to the Working Group. . fatty acid oxidation. 4. increasing liver weight.

(1983) +. reverse mutation Salmonella typhimurium TA100. TA1537. TA98. positive. injection. not reported. male Fischer 344 rats in vivo Micronucleus formation. inconclusive. (1986) Dunkel et al. (1987) Elmore & Fitzgerald (1990) Wild et al. in-vivo tests. not tested LED. 5000 in feed 10 30 μg/mL 40 μg/mL 50 μg/mL 40 μg/mL 20. Syrian hamster embryo cells in vitro Cell transformation. NT. (1983) Tennant et al. lowest effective dose. ?. (1986) Mirsalis et al. reverse mutation Drosophila melanogaster. in-vitro tests. (1983) Foureman et al. TA98. (1987) Gulati et al. injc. TA1535. mouse lymphoma L5178Y cells. (1989) Gulati et al. male and female NMRI mouse bone-marrow cells in vivo a Doseb (LED or HID) With exogenous metabolic system Reference IARC MONOGRAPHS VOLUME 77 – – – – – NT ? – – ? – + – ? – – – + 3600 μg/plate 3333 μg/plate NR 1267 in feed 2000 ppm. highest ineffective dose. intraperitoneal c Injection volume not reported b . sex-linked recessive lethal mutations Drosophila melanogaster. TA1535. (1993) Wild et al.3 1000 po × 1 1000 ip × 3 2533 ip × 1 + – – NT NT NT Wild et al. μg/mL. sex-linked recessive lethal mutations Mutation. –. mg/kg bw/day. reverse mutation Photobacterium phosphoreum. Genetic and related effects of cinnamyl anthranilate Test system Resulta Without exogenous metabolic system Salmonella typhimurium TA100. Chinese hamster ovary cells in vitro Chromosomal aberrations. Tk locus in vitro Sister chromatid exchange. HID. C3H/10T½ mouse cells Cell transformation. ip. Chinese hamster ovary cells in vitro Cell transformation. TA1537. SA7 virus/Syrian hamster embryo cells Unscheduled DNA synthesis. (1994) Tennant et al. (1989) Shelby et al.186 Table 2. male B6C3F1 mouse bone-marrow cells in vivo Micronucleus formation. (1989) Tu et al. inj. negative. bioluminescence assay. (1988) Hatch et al. NR.

. 1995a. however.CINNAMYL ANTHRANILATE 187 The weight of evidence for cinnamyl anthranilate and for other rodent peroxisome proliferators in general. Rodent peroxisome proliferators exercise their pleiotropic effects due to activation of PPARα. Cinnamyl anthranilate produces liver tumours in mice.b. demonstrates that they do not act as direct DNA-damaging agents. cinnamyl anthranilate induced peroxisome proliferation and cell replication in the liver that are characteristic of a peroxisome proliferator in mice and. to a limited extent. 1997). a growing body of evidence concerning the molecular basis of peroxisome proliferation indicates that human livers and hepatocytes would be refractory to induction of peroxisome proliferation by cinnamyl anthranilate (Doull et al. Under conditions of the bioassays. which did not respond with liver tumours in the bioassay (Lake et al. the magnitude and duration of hepatocellular proliferation were limited in rats. 2. 1997) supports this hypothesis. Similarly. in rats... 1999). The species difference in peroxisome proliferation in response to cinnamyl anthranilate is associated with a species difference in its metabolism: the compound is completely hydrolysed by rats but not mice. 1998).. Cattley et al. Other data on the induction of oxidative stress are not available for cinnamyl anthranilate. the modulation of hepatocellular proliferation by peroxisome proliferators has been implicated in the mechanism of carcinogenesis. There is clear evidence that cinnamyl anthranilate causes acute and sustained levels of hepatocellular proliferation under bioassay conditions which resulted in liver tumours in mice. The absence of a significant response of human liver to induction of peroxisome proliferation and hepatocellular proliferation is explained by several aspects of PPARα-mediated regulation of gene expression.. Marked species differences in hepatic peroxisome proliferation have been reported (Ashby et al. The only standard genotoxicity assay in which cinnamyl anthranilate is active is the mouse lymphoma mutation assay. 4. hepatocellular proliferation is likely to be involved in the promotion of growth of preneoplastic hepatocytes. Chronic administration of peroxisome proliferators to rodents results in sustained oxidative stress due to overproduction of peroxisomal hydrogen peroxide. 6. 5. . Furthermore. IARC. This process is essential for liver hypertrophy and hyperplasia and eventual hepatocarcinogenesis in response to peroxisome proliferators. 1994. The induction of peroxisomal fatty acid β-oxidation by cinnamyl anthranilate in vivo under bioassay conditions (Lake et al. This can theoretically result in increased levels of mutation by increasing the frequency of replicative DNA synthesis as well as increasing the number of hepatocytes at risk. Lake. Interestingly. No study has yet compared the responsiveness of human versus rodent livers in vivo or hepatocytes in vitro to cinnamyl anthranilate. 1995. 3. In summary: 1.

It has not been commercially available since 1985.3 Animal carcinogenicity data Cinnamyl anthranilate was tested for carcinogenicity in one experiment in mice and in one experiment in rats by oral administration in the diet. 5. This conclusion is further supported by the failure to detect intact cinnamyl anthranilate in the urine of human volunteers given large single doses. indicate that humans can reasonably be predicted to be refractory to induction of peroxisome proliferation and hepatocellular proliferation by cinnamyl anthranilate. Hepatic peroxisome proliferation has not been evaluated in studies of human subjects or systems treated with cinnamyl anthranilate. although studies of human systems have not been performed with this compound. In mice.4 Other relevant data Cinnamyl anthranilate is metabolized by hydrolysis to anthranilic acid and cinnamyl alcohol. 5. a dose-related increase in the incidence of hepatocellular tumours was found.2 Human carcinogenicity data No data were available to the Working Group. along with the role of PPARα in this response. these findings suggest that the increased incidence of liver tumours in mice treated with cinnamyl anthranilate results from a mechanism that is not expected to operate in humans. Overall. but there was no increased incidence of tumours in rats. . an increased multiplicity of lung tumours was found. 8. 5. In a mouse lung tumour bioassay. which is oxidized to benzoic acid.188 IARC MONOGRAPHS VOLUME 77 7. In mice. 5. 5. leading to excretion of unchanged cinnamyl anthranilate in the urine. the hydrolysis is saturated at high doses. but not in rats or humans. However.1 Summary of Data Reported and Evaluation Exposure data Cinnamyl anthranilate was used as a synthetic flavouring and fragrance agent. No information was available on its occurrence in the workplace or in the environment. interspecies comparisons with other peroxisome proliferators.

Marsman. Toxicol. 5. Food chem. Pharmacol. 64/65. Toxicol. D. J. R. 2).L.. pp. Toxicol. exp. B.. S. J..5 Evaluation No epidemiological data relevant to the carcinogenicity of cinnamyl anthranilate were available. CRC Press. References Ashby.. T. (1995) Fenaroli’s Handbook of Flavor Ingredients. Tugwood... Elliott.D. and were not seen after administration of the hydrolysis products. I. Ishmael. Hum. & Ford. Overall evaluation Cinnamyl anthranilate is not classifiable as to its carcinogenicity to humans (Group 3). Lake.. 111–114 Budavari. J. (1985) Comparison of percutaneous absorption of fragrances by humans and monkeys. 559–566 Cattley. C. 47–60 . Sangster..E. A. P.D.A. Pastoor. p.. I. A. & Purchase. S. 12th Ed. Schwetz. (1996) The Merck Index. Brady. These effects are mediated by the intact ester.. Anthony. 651–659 Caldwell.A.. J. R. & Wahli. 13 (Suppl. 3rd Ed. (1992) Problems and opportunities in toxicity testing arising from species differences in xenobiotic metabolism. D. Mailbach. There is limited evidence in experimental animals for the carcinogenicity of cinnamyl anthranilate. Toxicol. J.F.. W.. 116 Caldwell.. Whitehouse Station..R. cinnamyl alcohol and anthranilic acid. J. R.K. Elcombe. Steward. ed.M.... S1–S117 Bronaugh. No relevant data from humans were available. Sutton.G. B.. Vol. Bernard.C..C. 23.. C. J. II. (1998) Do peroxisome proliferating compounds pose a hepatocarcinogenic hazard to humans? Regul. Tugwood.A. (1994) Mechanistically-based human hazard assessment of peroxisome proliferator-induced hepatocarcinogenesis. R. Kettle.... ed.A. Lett. B.. R. Fenner-Crisp. J.I. Elcombe. The corresponding effects on rat liver were very much weaker. Wester.. S. H.. Food chem.. The only standard genotoxicity assay in which cinnamyl anthranilate was active was the mouse lymphoma mutation assay.A. J. (1985) Influence of dose and sex on the disposition and hepatic effects of cinnamyl anthranilate in the B6C3F1 mouse.. & Anderson. NJ. Robinson. Bucks... FL. J..S. DeLuca. Odum. increasing the activity of peroxisomal fatty acid-metabolizing enzymes and microsomal CYP4A and increasing hepatocellular proliferation. 27. Boca Raton. 23.F. J. Merck & Co.. G.. 6. Popp. Toxicol. 387–388 Burdock. D. Cotgreave. B.CINNAMYL ANTHRANILATE 189 Cinnamyl anthranilate has the characteristic effects of a peroxisome proliferator on mouse liver.A..

. 8. mol.. No. G.M... & van Gemert. Putnam.. J. (1994) Chemical mutagenesis testing in Drosophila. 23.. 133–139 IARC (1987) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. 515–531 IARC (1978) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans.. 31.G. Putman. Subpart B. p.L. Toxicol. Cattley. appl. E.. 16. 39. (1999) Flavor and Extract Manufacturers’ Association of the United States: 1995 Poundage and Technical Effects Update Survey. C. M. Nims.E.G. 82/83. Lubet. Section 189. Regul. (1997) Comparison of the effects of cinnamyl anthranilate on hepatic peroxisome proliferation and cell replication in the rat and mouse. Price R.P. 12.. & Shelby. X. Environ. (1988) Interlaboratory evaluation of the C3H/10T1/2 cell transformation assay.W.. B. D..D. Lubet. Pharmacol. J.113. M.W. Toxicol. R. (1986) Chemical enhancement of SA7 virus transformation of hamster embryo cells: evaluation by interlaboratory testing of diverse chemicals. R.A.. J. M. Food chem. Witt. 35. D. & Walters D. Most. J..G. Regul. 673–681 Lake. Geneva. IARCPress Keyhanfar. Flavor and Extract Manufacturers’ Association of the United States. clin.. B. Suppl.. III. J. Lyon. Environ.. 379–387 FAO/WHO (1981) Toxicological Evaluation of Certain Food Additives (WHO Food Add. biol. 340D. R. (1995b) Mechanisms of hepatocarcinogenicity of peroxisome-proliferating drugs and chemicals. Mason. A. Anderson. & Schechtman. L... pp. 549 Foureman.. J. Elcombe. US Code Fed.S. 60 IARC (1995) Peroxisome Proliferation and its Role in Carcinogenesis (IARC Technical Report No. Colouring Agents and Miscellaneous Industrial Chemicals. & Zimmering. Zeiger.D. B. Lake. R. (1989) Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro.M. G. S. Mutag. Spalding. F.... M. Part 189.. (1990) Evaluation of the bioluminescence assays as screens for genotoxic chemicals. 34. V. Tu... Toxicol.C. B.. 13. 327–357 Dunkel. J.G. Anderson. Some Food Additives.. pp. 24). mol. & Caldwell. p. Williams. p. E. Kouri. Toxicol. pp. Pharmacol.. 21–31 Elmore. 241–249 Lake..E. International Programme on Chemical Safety..W. 483–507 Lake. Wilkinson. T. 287–291 IARC (1983) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. C. Cameron. Environ. IARCPress. Lett. R. 133–193 Hatch.G. Cunninghame. 61 . Fundam.. R. Title 21. Rev. & Hallaghan. Results of 70 coded chemicals tested for the National Toxicology Program.. Swenberg. Tennant..190 IARC MONOGRAPHS VOLUME 77 Doull.K. Environ. Ann. Vol. Feed Additives and Naturally Occurring Substances.M.P. Sivak. & Cameron.J. Mutag.W.A.. A. Some Aromatic Amines and Related Nitro Compounds—Hair Dyes. 29. Lyon. 208–227 Gulati. T. L. Lyon.M. (1995a) Peroxisome proliferation: current mechanisms relating to nongenotoxic carcinogenesis.. 16). (1999) A cancer risk assessment of di(2-ethylhexyl)phthalate: application of the new US EPA Risk Assessment Guidelines. Toxicol. 7. B. Overall Evaluations of Carcinogenicity: An Update of IARC Monographs Volumes 1 to 42. B. 60–66 Lucas. Valencia. R. IARCPress. Lyon.M.. Vol. K. (1996) Factors affecting the metabolism of cinnamyl anthranilate in the rat and mouse. IARCPress. J. P. Res. C. Schechtman. & Fitzgerald. Washington DC. Prog.. mol. Mutag..G. 70–73 Food and Drug Administration (1999) Food and drugs. Ser.B. Mutag. Results with 27 chemicals.

D... Tennant. Kniazeff. B. J. A.C... J. (1993) Evaluation of a three-exposure mouse bone marrow micronucleus protocol: results with 49 chemicals.. (1989) Measurement of unscheduled DNA synthesis and S-phase synthesis in rodent hepatocytes following in vivo treatment: testing of 24 compounds. Weisburger.J.B. (1987) Prediction of chemical carcinogenicity in rodents from in vitro genetic toxicity assays. R.D. D.. 13.. Environ. K. B. Environ. 3069–3085 Tennant. M. Steinmetz... Spalding.P.L. Avery. Tyson. (1983) Study of artificial flavouring substances for mutagenicity in the Salmonella/microsome. NC Opdyke. B. Sedita. M.. C. & Eckhardt. 707–719 .. Toxicol.R. E. M.. E.L.. Jones. Environ.. M. 2). No. Ser. G.. A. Caspary. mol.. Erexson.-T. Hook. Gocke. 155–164 National Cancer Institute (1980) Bioassay of Cinnamyl Anthranilate for Possible Carcinogenicity (Tech..L. G. W. Toxicol.. G. Mutag.E. R. C. Weisburger. 236. (Suppl. J. 21. R. Research Triangle Park. Curren. Huberman.. Haseman..CINNAMYL ANTHRANILATE 191 Mirsalis..J. 751–752 Shelby. (1997) Cinnamyl anthranilate causes co-induction of hepatic microsomal and peroxisomal enzymes in mouse but not rat. Government Printing Office National Toxicology Program (1991) NTP Chemical Repository Report: Cinnamyl Anthranilate. R. E. Hamilton..K. King. Pallotta. No. Lubet. Mutag. R. Rep. Basc and micronucleus tests. & Tice. Shelby. Food Cosmet. 196. 160–179 Stoner. J. Shimin. S. mol. R. 8.. Loh. 21. K.. S. 33. Spalding. (1975) Fragrance raw materials monographs: cinnamaldehyde anthranilate.. Sivak...B.. Mutag. 933–941 Tu. appl.K...W.. No.. & Minor. Resnick. & Spalding. 338–347 Wild..H. J. & Kouri. J. Pharmacol.D. Science. Washington DC.. Cancer Res. Zeiger. (1986) An interlaboratory comparison of transformation in Syrian hamster embryo cells with model and coded chemicals. J.W. 14. Hallowell.J.D. & Gori. E.. G. M. J. Anderson. Bakke. W. Stasiewicz.K. (1973) Test for carcinogenicity of food additives and chemotherapeutic agents by the pulmonary tumor response in strain A mice. E. A. A..M.A. R.A. M. Margolin. (NTP) 80-10).H. & Caldwell. C.K.D. Toxicol. Food chem..D. DHEW Publ. 142. (NIH) 80-1752.. 77–98 Viswalingam...

.

1999) (c) Melting-point: 71 °C (Lide.1. Abstr. cis-ortho-coumarinic acid lactone. rectangular plates.: 91-64-5 Chem.7 °C (Lide. 1. Serv. and these have been incorporated into the monograph and taken into consideration in the present evaluation.1 1. 1.2-Benzopyrone. 1999) –193– .COUMARIN This substance was considered by previous Working Groups.2 Structural and molecular formulae and relative molecular mass O O C9H6O2 1. benzo-α-pyrone. fragrant odour resembling that of vanilla beans. coumarinic anhydride. new data have become available. Since that time. 5. pleasant. Name: 2H-1-Benzopyran-2-one IUPAC Systematic Name: Coumarin Synonyms: 1.1 Exposure Data Chemical and physical data Nomenclature Chem. burning taste (Budavari.3 Relative molecular mass: 146. 1987). Reg. No. in October 1975 (IARC.6-benzo-2-pyrone. Abstr. 1999) (d) Density: 0. 1998) (b) Boiling-point: 301.1. 1976) and March 1987 (IARC. ortho-hydroxycinnamic acid lactone 1.15 Chemical and physical properties of the pure substance (a) Description: Orthorhombic.935 g/cm3 at 20 °C (Lide.1.

194 IARC MONOGRAPHS VOLUME 77 (e) Spectroscopy: Infrared (prism [1691]. Budavari. 1 Calculated from: mg/m3 = (relative molecular mass/24. V-225]. Lide & Milne. Various methods can be used to obtain coumarin from each of these starting materials. 1998). phenol (Pechmann reaction) and salicylaldehyde (Perkin reaction). C-13 [242]) and mass spectral data have been reported (Sadtler Research Laboratories. ultraviolet [492]. grating [270]). reading the absorbance or transmittance at 490 nm. 1993). synthetic coumarin must be highly pure (Bauer et al. 1. nuclear magnetic resonance (proton. and comparing against a standard (AOAC International. 1996) (h) Octanol/water partition coefficient (P): log P.98 × ppm 1. undated). three companies each in Japan and the United States.39 (Verschueren. assuming a temperature of 25 °C and a pressure of 101 kPa .5 Analysis Coumarin can be determined in vanilla extract by a photometric method.4 Technical products and impurities Coumarin is commercially available with a minimum purity of 99% (Rhodia.45) × ppm. 1996) (f) Solubility: Slightly soluble in water (100 mg/L at 25 °C) and ethanol. Verschueren.2 Production Until the late 1890s. In order to be suitable for perfumery uses. 1996. Information available in 1999 indicated that coumarin was manufactured by five companies in China.. 1980. 1988. 1996. Synthetic methods of preparation and industrial manufacturing processes were developed starting principally from ortho-cresol (Raschig process). 1. Coumarin is usually sold in the form of colourless shiny leaflets or rhombic crystals (Boisde & Meuly. 1. (i) Conversion factor1: mg/m3 = 5. 0.13 kPa at 106 °C (Verschueren. Boisde & Meuly. 1999). 1998) (g) Volatility: Vapour pressure.1. Hong Kong and India (Chemical Information Services.1. 1993). very soluble in chloroform. [10407. two companies in France and one company each in Germany. coumarin was obtained commercially only from natural sources by extraction from tonka beans. diethyl ether and pyridine (Lide & Milne. 1996).

but this use is declining (Egan et al. coumarin has a significant use in the electroplating industry. It has been used in detergents as a brightener or bleaching agent (Perone. Lake. Coumarin and some of its derivatives have been tested for treatment of schizophrenia. Dexeus et al. cosmetic and related industries. but for commercial use has been mostly produced synthetically for many years. 1999).b. 1999). It is currently undergoing clinical trials for treatment of lymphoedema following breast cancer treatment and in treatment of lung and kidney cancer and of melanoma alone or in combination with cimetidine (Marshall et al. bath products. 1999). 1972). antiperspirant deodorants. Formerly. 1994). mostly associated with vanillin. citrus. mostly in the automotive area..3 Use Coumarin is widely distributed in the plant kingdom. hair sprays. It is normally associated in perfumery with herbaceous odours and enters into the formulation of fern and Chypre-type fragrances. It is widely used in hand soaps. 1990. Coumarin is used in tobacco to enhance its natural aroma. It is also applied in large quantities to give pleasant aromas to household materials and industrial products or to mask unpleasant odours (Egan et al. 1993.8%. 1999).. it is used in rubber and plastic materials and in paints and sprays to neutralize unpleasant odours (Lake. Lake. In other fields. Marshall et al. Lake. 1990.. to provide high polished quality to chrome-plated steel. Because of its unique sweet note and stability. 1990. rosemary and oak moss. Coumarin has been recommended for treatment of a number of clinical conditions. microcirculation disorders and angiopathic ulcers... coumarin has long been recognized as an important raw material in the fragrance industry. 1990. 1972). . 1993).01 to 0. 1989. In industry. face creams. Coumarin possesses both immunomodulatory and direct antitumour activity (Marshall et al. shampoos. baked goods. fragrance creams. coumarin has several other industrial applications.COUMARIN 195 1. for flavouring chocolates. 1993). 1972). It has also been used for prevention of dental caries (Perone. body lotions. Boisde & Meuly.. Boisde & Meuly. In addition to its use in the perfumery. but since 1954 its use as a direct food additive has been suspended in the United States (Boisde & Meuly. 1987a. 1989. and also for treatment of high protein oedemas in animals (Boisde & Meuly. Thornes et al.. including high protein oedema and brucellosis. It is used as an odour-enhancer to achieve a long-lasting effect when combined with natural essential oils such as lavender. and in cream soda-flavoured beverages (Perone. detergents. 1993. Casley-Smith et al. lotions and perfumes at concentrations usually extending from 0. Coumarin has also found use in toothpastes. shower gels and toilet soaps (Cohen. 1993). large quantities of coumarin were used in the food industry.. 1979.

It is found at particularly high levels in some essential oils.4. Its better known occurrences are in sweet clover (Melilotus alba and M. 1972). .2 Occupational exposure According to the 1981–83 National Occupational Exposure Survey (NOES. It was subsequently identified in a large number of plants belonging to many different families. cassia leaf oil and lavender oil (Lake. 1981. 1993. such as cinnamon leaf and bark oil. 1999). peppermint oil (20 ppm). TNO. cassia (Cinnamorum cassia). fruits such as bilberry and cloudberry and other foods such as chicory root (Boisde & Meuly. 1972. Marles et al. sweet woodruff (Asperula odorata). principally as secondary metabolites in green plants (Hoult & Payd. lavender (Lavendula officinalis) and balsam of Peru (Myroxylon pereirae) (Perone. orchids. 1. (a) Foods and fragrances Coumarin is a natural product found at high levels in some essential oils. vanilla leaf (Trilisa odoratissima).3 Environmental occurrence Reports of the release of coumarin to the environment through various waste streams are scant. 1999). 1999). 1999). No other data on occupational exposures were available to the Working Group. other types of cinnamon (900 ppm). National estimates of workers potentially exposed were not available from other countries. woodruff and sweet clover as well as in green tea (0. The maximum total daily human exposure to coumarin has been estimated to be 0. to date at least 1300 have been identified.4.2–1. A broad spectrum of coumarin derivatives (present both in the free state and as glucosides) have also been found in many plants. cinnamon bark oil (7000 ppm). Occupational exposure to coumarin may occur in its production and in its use in the manufacture and formulation of many products. 1993. 1987. Budavari. 1.04 mg/kg bw per day from fragrance use in cosmetic products (Lake.4.7 ppm). comprising 0. lavender oil.06 mg/kg bw. officinalis). 1998). 1987). cassia leaf oil (17 000–87 300 ppm). and 0.196 IARC MONOGRAPHS VOLUME 77 1. vanilla beans (Vanilla planifolia). grasses and citrus fruits (Perone.4 1.02 mg/kg bw per day from dietary exposure.. Coumarin has been isolated from legumes. 1996). Boisde & Meuly. It is also found in Mexican vanilla extracts (Sullivan. Lake. approximately 240 000 workers in the United States were potentially exposed to coumarin. 1996. particularly cinnamon leaf oil (40 600 ppm (mg/kg)). Marles et al..1 Occurrence Natural occurrence Coumarin was first isolated by Vogel in 1820 by extraction from tonka beans (Dipteryx odorata).

1994). The Council of Europe has listed coumarin as an ‘active principle’ and the maximum permitted concentrations of coumarin in foodstuffs are given in Annex II of European directive (88/388/EEC) (European Commission. usual (and maximum) concentrations were found to be for soap. 0. detergent. 1999). 50.1 Studies of Cancer in Experimental Animals Oral administration Mouse Groups of 50–51 male and 50–51 female B6C3F1 mice.2%). were administered coumarin (purity. 100 and . 1988). 3. Coumarin penetrates human skin rapidly and efficiently (see Section 4.046–6. 1998). the permitted limit is 10 mg/kg and in chewing gum it is 50 mg/kg (Lake.5 Regulations and guidelines No occupational exposure limit for coumarin in workplace air has been reported. Studies of Cancer in Humans No data were available to the Working Group. 3.1%) and perfumes.. creams and lotions. while in alcoholic beverages and certain caramel confectionary products.8%) (Cohen. 1999). The general limit for coumarin in food and non-alcoholic beverages is 2 mg/kg.3% (0. 2. coumarin was found in 11 of 22 perfumes at concentrations (w/v) of 0.003% (0.02%). 1979). > 97%) in corn oil by gavage at doses of 0. Food containing any added coumarin as such or as a constituent of tonka beans or tonka extract is deemed to be ‘adulterated under the act’. 0.COUMARIN 197 (b) Medical uses As a consequence of its medical use..043% (Rastogi et al. based upon an order published in the Federal Register of 5 March 1954 (19 FR 1239) (Food and Drug Administration. 0. More recently. many individuals have been exposed to therapeutic doses of coumarin ranging from 100 to 7000 mg per day for periods ranging from two weeks to over two years (Marshall et al. 1.015% (0. 1996) and 40 of 73 deodorants on the European market at concentrations ranging from 1 to 1411 ppm [0.03% (0.1 3.1).. six to seven weeks.14%] (Rastogi et al. (c) Personal care products In personal care products.0001–0. 0.1.

the authors discounted these increases as being within the historical control range for that laboratory (Carlton et al. were administered coumarin (purity. Although there was an increased incidence of hepatocellular tumours (adenomas and carcinomas combined) in low-dose females (0/50 in controls. 86 and 280 mg/kg bw per day. As shown in Table 1.198 IARC MONOGRAPHS VOLUME 77 200 mg/kg bw daily for 103 weeks. six to seven weeks of age. the Working Group was aware of an unpublished company report in which statistical analyses had been applied to mortality-adjusted tumour rates. 8/52 in low-dose. 50 and 100 mg/kg bw daily for 103 weeks. there was significantly increased incidence of alveolar/bronchiolar tumours (adenomas and carcinomas) at the 200 mg/kg bw dose in both males and females and increased incidence of hepatocellular adenomas in females at the low and medium doses. 2/50 and 0/50 respectively) was significantly lower (p < 0. Survival of all dosed groups was similar to that of the controls...1. but not at the highest dose. > 97%) in corn oil by oral gavage at doses of 0. 300. In males. for females. Survival of low-. Evans et al. 1979. 10/52 mid-dose and 20/52 high-dose) and that the incidences were within the historical control range. Griepentrog. increased mortality was attributed . although exact rates were not recorded. 1000 or 3000 mg/kg of diet (ppm) for either 101 weeks (males) or 109 weeks (females). 1976). relative to the controls.001) than that of controls (28/50). 17/52 low-dose. The authors reported that there was no significant increase in the incidence of pulmonary adenocarcinomas in males (11/52 control. for males and 28. respectively. 25..2 Rat Coumarin was tested by oral administration in two early studies in rats (Hagan et al. The Fisher’s exact test for differences between treatment groups and Mantel’s test for dose-related trends showed no treatment-related effect for any tumour type. The achieved intakes of coumarin for the three doses were 26. [The Working Group noted that no information was provided on statistical evaluation in this paper. Groups of 50 male and 50 female Fischer 344/N rats. Body weight gain was reduced in high-dose females. 1996). mid. 1973) and found to produce bile duct carcinomas at high levels of exposure in the latter study (IARC. > 98%) in the diet at concentrations of 0. 1967. 1967.] 3. 1989). However. Bär & Griepentrog. 4/52 in mid-dose and 3/52 in high-dose females). However. as was body weight gain. Body weight gain was significantly decreased by 18% in the high-dose males and by 10% in the mid-dose males. 1993).and high-dose males (9/50. There were marginal increases in the incidence of squamous-cell papillomas and carcinomas of the forestomach in the low-dose males and females (National Toxicology Program. these lesions were deemed to be non-neoplastic cholangiofibrosis and not bile duct carcinomas (Cohen. 91 and 271 mg/kg bw per day. Survival did not differ between groups. were administered coumarin (purity. four weeks of age. respectively. Groups of 52 male and 52 female Swiss CD-1 mice. in a re-evaluation by other pathologists of the slides from the Bär and Griepentrog (1967) study.

logistic regression test a 199 . logistic regression test ** p < 0.Table 1. Incidence of primary tumours in B6C3F1 mice exposed to coumarin Tumour site Animals with tumours Males Control Lung Alveolar/bronchiolar adenomas Alveolar/bronchiolar carcinomas Livera Forestomachb 50 mg/kg bw 100 mg/kg bw 200 mg/kg bw Females Control 50 mg/kg bw 100 mg/kg bw 200 mg/kg bw COUMARIN 14/50 1/50 26/50 2/50 8/50 1/50 29/50 9/50* 14/50 2/50 29/50 4/50 24/51* 1/51 27/51 0/51 2/51 0/51 8/50 1/52 5/49 0/49 26/49** 6/50 7/49 0/49 29/51** 3/51 20/51** 7/51** 12/50 2/51 From National Toxicology Program (1993) Hepatocellular adenomas b Squamous-cell papillomas and carcinomas * p < 0.05.01.

females: 0/65. 3000. There was an increased severity of bile duct hyperplasia and renal tubule hyperplasia in both sexes. 1996). there was no increased incidence of renal tubule adenomas after conventional single-section evaluation. were found in the highest-dose males and females (males: 0/65.] Groups of 50 male and 50 female Sprague-Dawley rats were administered coumarin (purity. although there was no dose–response relationship.] 3.3 Hamster Groups of 11 or 12 male and 10–13 female Syrian golden hamsters. 1000-. in the control. 6/65 and 29/65 and females: 0/65. [The Working Group noted the unusually sharp increase in tumour incidence in the liver at only the highest of five doses and the lack of adequate histopathological description of both tumour types. 1/65.1 and 0. 99%) in the diet at concentrations of 0. 42. 3000 and 5000 mg/kg of diet (ppm) for 104 weeks (males) or 110 weeks (females). 156 and 283 mg/kg bw per day. An accompanying stop-exposure study was carried out in which groups of 20 male rats received 100 mg/kg bw per day of coumarin by gavage in corn oil for nine or 15 months followed by corn oil gavage only until the end of the study at 103 weeks. were administered coumarin (purity. 0/65. 0/65. 2000-. the Working Group was concerned that no descriptive information or illustrations were provided to confirm the diagnosis of cholangiocarcinoma. 0/65. 0/65 and 29/65. was not significantly increased. The achieved intakes of coumarin for the five doses were 13. 87. 3000. 0/65. Significantly increased incidences of cholangiocarcinomas. respectively.200 IARC MONOGRAPHS VOLUME 77 to increased severity of age-related spontaneous chronic progressive nephropathy. 1/65 and 12/65 (Carlton et al. As shown in Table 2. respectively. and . based on single sections. 0/65. 1/65.. Given the issue related to misdiagnosis of bile duct tumours in an earlier study. 107. 1/65 and 37/65. 2000. 0/65. Survival was poor in all groups receiving coumarin. 333-. there was an increase in the nine-month 100 mg/kg bw dose group after step-sectioning of the kidney (National Toxicology Program.5% for up to two years. Whereas hepatic lesions including bile duct hyperplasia were reversible and the incidence of renal tubule adenomas. but the incidence was increased based on the results of step-sectioning of kidney tissue. 0/65. 0/65. Hepatocellular tumours were also found in highdose males: 2/65. for females. [The Working Group noted that the incidence of renal tubule tumours was not increased after conventional examination of the kidney. nor a discussion of the pathology. while the other treated groups were exposed to coumarin diet in utero and throughout the postnatal and chronic periods. some of which were reported as metastasizing. Survival was 9/20 and 2/20 in the two groups. Dose-related decreases in body weight gain in excess of 10–15% occurred in the 2000-. 1000. > 98%) in the diet at concentrations of 0. 0. Groups receiving 3000 and 5000 ppm were 21–28 days of age at the beginning of the study.1. 1993).and 5000-ppm dose groups. 130 and 234 mg/kg bw per day. 0/65. respectively. 2/65. for males and 16. 50.and 5000-ppm dose groups respectively). in which it was between 50 and 70%. eight weeks old. 333. Survival was less than 50% in all groups (including controls) except the groups receiving the two highest doses.

Table 2. Incidence of primary tumours in Fischer 344/N rats exposed to coumarin Tumour site Animals with tumours COUMARIN Males Control Kidneya.05. single section step sections 1/49 0/49 25 mg/kg bw 2/50 4/50 50 mg/kg bw 2/51 5/51* 100 mg/kg bw 1/50 4/50 Females Control 0/49 0/49 25 mg/kg bw 0/50 0/50 50 mg/kg bw 0/50 1/50 100 mg/kg bw 2/49 1/49 From National Toxicology Program (1993) a Renal tubule adenoma * p < 0. logistic regression test 201 .

Coumarin is also extensively absorbed after dermal application. Toxicokinetic studies in humans have demonstrated that coumarin is rapidly absorbed from the gastrointestinal tract after oral administration and extensively metabolized by the liver in the first pass. Egan & O’Kennedy. Lake. 1992a.25 mg/kg bw being 1. 1999). 1997.4-epoxide which degrades spontaneously to form ortho-hydroxyphenylacetaldehyde and may be subsequently converted to ortho-hydroxyphenylethanol and ortho-hydroxyphenylacetic acid. 4-. to yield ortho-coumaric acid (ortho. 1979. with only 2–6% reaching the systemic circulation intact (Ritschel et al. Other Data Relevant to an Evaluation of Carcinogenicity and its Mechanisms Absorption. 1993. 1981). Iscan et al. 1994). 1992. 1997.The elimination of coumarin from the systemic circulation is rapid.or 8-hydroxycoumarin and 6. The first step in the latter pathway is the formation of the unstable coumarin 3. 1981). Fentem & Fry. Ritschel & Hoffmann.. 6.2 and 0. 1992. Fentem et al. 1977.7-dihydroxycoumarin and. 1969. 1993. 5-.hydroxyphenylcinnamic acid) and ortho-hydroxyphenylpropionic acid (Norman & Wood..1 4. Rautio et al. 1997).49 h [109. Born et al. Coumarin may also be metabolized by hydroxylation to yield 3-.. The percutaneous absorption of coumarin has also been demonstrated in vitro with human skin (BeckleyKartey et al. metabolism and excretion Humans 4. There was no evidence of increased bile duct hyperplasia. 1979... .. some 60% of a 2. 1991. 1.. primarily as 7-hydroxycoumarin conjugates.82.] 4. 1999). 1999). [The Working Group noted the small number of animals and the poor survival. 0.. In one study with human subjects.46 and 1.1.1 The absorption. The rapid excretion of coumarin.0-mg dose applied for 6 h was absorbed (reviewed in Lake. the half-lives following intravenous doses of 0. Lake et al. Pelkonen et al. Fentem & Fry. Yourick & Bronaugh.. Lake. distribution. in the urine of human subjects given coumarin orally suggests that there is little or no biliary excretion of coumarin metabolites in humans (Shilling et al. making the study inadequate for evaluation. The pathways of coumarin metabolism are shown in Figure 1. cholangiofibrosis or cholangiocarcinoma due to coumarin exposure or tumours at other sites (Ueno & Hirono.202 IARC MONOGRAPHS VOLUME 77 in the control females. 1984.. by opening of the lactone ring. Coumarin exhibits marked species differences in its metabolism (Cohen. 1979. Cholerton et al. metabolism and excretion of coumarin in humans have been reviewed (Cohen. respectively (Ritschel et al. 88 and 89 min].b.. 1992. 1997). The major primary pathways of coumarin metabolism are 7-hydroxylation or metabolism of the lactone ring by ring opening and cleavage at carbon atom 2 to yield carbon dioxide.1976). distribution.125.

and 8Hydroxycoumarins 7-Hydroxycoumarin OH SG HO O HO O O Glucuronic acid and sulfate conjugates O Coumarin 3-mercapturic acid CH2CH2OH OH ortho-Hydroxyphenylethan-1-ol 6.Figure 1.4-epoxide OH GSH O O HO O O 5-. 6-.7-Dihydroxycoumarin 4-Hydroxydihydrocoumarin glutathione conjugate 203 Adapted from Lake (1999) . Metabolic pathways of coumarin in animals and humans CH2CH2COOH OH ortho-Hydroxyphenylpropionic acid CH OH ortho-Coumaric acid O CHCOOH OH O ? CH2CHOHCOOH OH ortho-Hydroxyphenyllactic acid ? CO2 OH ? 3-Hydroxycoumarin COUMARIN O CH2CHO O CO2 OH ortho-Hydroxyphenylacetaldehyde CH2COOH OH ortho-Hydroxyphenylacetic acid O O O Coumarin O O 4-Hydroxycoumarin Coumarin 3.

While 7-hydroxylation is the major metabolic pathway of coumarin in most subjects. These authors highlighted methodological uncertainties in polymerase chain reactionbased genotyping procedures. Many studies have demonstrated marked interindividual variation in the levels of hepatic CYP2A6 protein. Establishment of the significance of the genetic polymorphism in CYP2A6 must await definitive genotyping and phenotyping procedures. The population frequency of these mutant alleles is uncertain at present. humans also convert coumarin to ortho-hydroxyphenylacetic acid. these were designated CYP2A6*1 (wild type). CYP2A6*2 has a point mutation in codon 160 and the resulting protein product is unable to 7-hydroxylate coumarin (Fernandez-Salguero et al. who found that a monoclonal antibody to CYP2A6 inhibited coumarin 7-hydroxylation by more than 94%. but three others excreted mainly 7-hydroxycoumarin (> 41% of dose) and 4–10% as ortho-hydroxyphenylacetic acid. 1995. Shilling et al. while 7-hydroxycoumarin accounted for 79% of the excreted dose (range.. Gullstén et al. (1998) refer to two individuals (among a population of two hundred) who were totally deficient in 7-hydroxycoumarin excretion after an oral dose of 5 mg coumarin.03% of the dose as 7-hydroxycoumarin and 50% as ortho-hydroxyphenylacetic acid. One subject excreted < 0. The occurrence of variant alleles in the human CYP2A6 gene was shown by Fernandez-Salguero et al. 0. Coumarin 7-hydroxylation activity exhibits a Gaussian distribution in Caucasian populations (Cholerton et al.3–3. The marked interindividual variation in coumarin metabolism to 7-hydroxycoumarin has led to studies to evaluate whether a genetic polymorphism exists in human CYP2A6. Lake. Hadidi et al.5% (range. (1995).. 1995... Hadidi et al. 1997) have been challenged by Oscarson et al. (1999). 1997. (1998). mRNA and associated microsomal coumarin 7-hydroxylase activity (reviewed in Pelkonen et al. but some individuals are deficient in this activity. Rautio et al. 68–92%). a further 4% of the dose (range.. (1969) reported that after an oral dose of 200 mg coumarin per subject..6%) . 1992. After intravenous administration. The role of CYP2A6 in the metabolism of coumarin by human liver microsomes has been confirmed by Sai et al. 1997. Gullstén et al. 1999).204 IARC MONOGRAPHS VOLUME 77 The major pathway of coumarin metabolism in most human subjects is 7-hydroxylation to form 7-hydroxycoumarin. Oscarson et al. 1992).. The functional significance of the rare CYP2A6*3 allele is uncertain. CYP2A6 (cytochrome P450 2A6) has been purified from human liver and CYP2A6 cDNA expression systems are available. (1997) gave members of a family 2 mg coumarin orally and collected their urine for 8 h. 1–6%) was present in the first 24-h urine as ortho-hydroxyphenylacetic acid. which is excreted in the urine as both glucuronic acid and sulfate conjugates. 1997).. 1. initial claims that the incidence of the CYP2A6*2 allele is 4–17% of European populations (Fernandez-Salguero et al. who found the incidence to be 1–3%. CYP2A6*2 and CYP2A6*3. Ten human subjects received coumarin orally (1 g and 2 g) and intravenously (250 mg) in a randomized cross-over study and their 0–48 h urines were assayed for orthohydroxyphenylacetic acid.

1999). the toxicokinetics of coumarin are non-linear at intraperitoneal doses greater than 10 mg/kg bw (Hardt & Ritschel. being around 1–4 h (Lake. 7-hydroxycoumarin. 1999). respectively (Meineke et al. 0. the major metabolic pathway of coumarin in rats is the 3. In rats.8 and 20% of the dose.1.5%) after the 1-g and 2-g doses. 1988). After a 100-mg/kg bw oral dose of [3-14C]coumarin. 1961. 1961). The urine appears to be the major route of coumarin excretion in Syrian hamsters. dogs. Syrian hamsters. Lake et al. 1971. respectively. ortho-hydroxyphenyllactic acid and orthohydroxyphenylacetic acid accounted for 1. oral and topical administration) (Hardt & Ritschel. Waller & Chasseaud.0%) and 5.. Fentem & Fry. 1999). gerbils (intraperitoneal) (Ritschel & Hardt.4. coumarin is rapidly absorbed from the gastrointestinal tract and is distributed throughout the body (Cohen. Generally. with little excretion of unchanged coumarin. The compound is extensively metabolized in all species. Lake.1% (1. 1989a). 1999). most mouse strains. after a 50-mg/kg bw oral or intraperitoneal dose of coumarin to rats. There are important quantitative differences between species in the routes of elimination of coumarin metabolites. 4. while after oral administration the recoveries were 3.. 1961) or intraperitoneal administration to rats (van Sumere & Teuchy. several species including rats. Lake. 1999). 1979. 1988). Ritschel & Hussain. many studies have examined this pathway of coumarin metabolism after oral administration.. guineapigs. 1983). the half-life for the elimination of coumarin is similar in all species examined. Because of the relative ease of measurement of 7-hydroxycoumarin. intravenous. 1998). 1983..8. excreting ≤ 5% of the administered dose as urinary 7-hydroxycoumarin (Cohen. Following oral administration. marmosets and squirrel monkeys are poor 7-hydroxylators. some 50% was excreted in the bile as unknown metabolites within 24 h (Williams et al. 1981). 1997.4epoxidation pathway. urinary 3hydroxycoumarin.8–7. 1981. 1979.. Pelkonen et al. biliary excretion occurs with an appreciable proportion of the dose excreted in the faeces (Cohen. Lake et al.5% (1. . 1990. 1993. but not in marmosets (Kaighen & Williams. rabbits and baboons. Lake.COUMARIN 205 of the dose was recovered as ortho-hydroxyphenylacetic acid. 1983) and rhesus monkeys (intravenous and oral) (Ritschel et al. Lake. For example. No significant tissue accumulation of coumarin and/or coumarin metabolites occurred after oral administration to rats and rabbits (Kaighen & Williams. with urinary 7-hydroxycoumarin accounting for < 1% of the dose (van Sumere & Teuchy.2 Experimental systems The toxicokinetics of coumarin have been studied in a number of species including rats (intraperitoneal. Other studies in vivo have confirmed that rats are poor 7-hydroxylators of coumarin. 1965).. 1979. Unlike in humans. Various metabolites including ortho-hydroxyphenylacetic acid were detected in the faeces (Kaighen & Williams.1–13. dogs (intravenous and oral) (Ritschel & Grummich. In rats. 1971). 0. Overall. 1989a.

orthohydroxyphenylacetic acid. human. but only 7% in cat. marked strain differences have been observed.4-dihydrocoumarin and ortho-hydroxyphenylpropionic acid under anaerobic conditions (Scheline. 1992.b). Studies with radiolabelled coumarin have demonstrated that coumarin can be converted to metabolite(s) that can bind covalently to proteins (Lake. the major pathway of coumarin metabolism in mouse remains 3. 1991). ortho-hydroxyphenylacetaldehyde can be a major metabolite of coumarin in liver microsomes from rats.. Waller & Chasseaud.3% in rat (Pearce et al. It is of interest that studies with cloned human cytochromes P450 expressed in insect SF9 cells have shown that only CYP2A6 catalysed 7-hydroxylation. Syrian hamsters and gerbils (Fentem & Fry. by 225-fold (from 8 to 1800 pmol/mg protein/min for cat and monkey microsomes. 1961.7 to 0. and a coumarin mercapturic acid conjugate has also been reported (Huwer et al.. even in strains with high 7-hydroxylase activity (Lovell et al. In contrast.. In keeping with in-vivo studies. 1999). baboons. 1999). Lake.7-dihydroxycoumarin (Lake.11 nmol/mg protein/min. 1978). Gangolli et al. 1997. 1992). are extensive 7-hydroxylators of coumarin. dog>>cat. However. Species such as rabbits. ortho-hydroxyphenylethanol. 1974.. Coumarin metabolism was examined in hepatic microsomes from eight animal species and humans. rabbit>guinea-pig. Apart from glucuronic acid and sulfate conjugation of hydroxycoumarins. Lake et al. 1984). 1974. Lovell et al. . 7-hydroxycoumarin formation is only a minor pathway of metabolism in most species. in the rank order hamster>rat>mouse. Coumarin may also be metabolized by the gastrointestinal microflora to 3. 1999). excrete up to 26% of an intraperitoneally administered dose of coumarin as 7-hydroxycoumarin (Lush & Andrews. respectively. such as DBA/2 and 129/Rr strains. orthocoumaric acid may be conjugated with glycine (Lake. cats and pigs have been reported to excrete 12–19% of the dose as urinary 7-hydroxycoumarin (Kaighen & Williams. 1968). ortho-hydroxyphenyllactic acid. ortho-coumaric acid and 6. 1989.4epoxidation.206 IARC MONOGRAPHS VOLUME 77 Certain mouse strains. 1999). excreting 60–66% of a dose of coumarin as urinary 7-hydroxycoumarin (Gangolli et al. The contribution of 7-hydroxylation to the metabolism varied even more.. mice. accounting for 84% of total metabolism in human and 73% in monkey.. 1999). 1999). Depending on the substrate concentration employed.. 1A2. ortho-hydroxyphenylpropionic acid.. Coumarin metabolism has been studied in vitro in liver microsomes. Lake. 1992a. 1999). 2B6. In mice.. Metabolites of coumarin detected in such systems include all six hydroxycoumarins. 1981). 2E1 and 3A4 (Zhuo et al. like humans. with high coumarin 7-hydroxylase activity in female mice from strains such as DBA/2 and 129/J (Negishi et al. cynomolgus monkey. The overall rate of metabolism varied from 6. The CYP isoform responsible for coumarin 7-hydroxylation in mouse liver is CYP2A5 and both baboons and cynomolgus monkeys possess hepatic CYP2A isoforms with properties similar to those of human CYP2A6 (Pelkonen et al.. while the formation of ortho-hydroxyphenylacetaldehyde was supported by CYP1A1. other phase II pathways of coumarin metabolism have been identified. For example. ortho-hydroxyphenylacetaldehyde. hepatocytes and liver slices. 3% in mouse and 0.

which was characterized by hepatomegaly and elevated serum enzyme levels. HepG2. (1994) reported one case in which elevated serum aminotransferase levels were measured in a patient given 5 g coumarin per day. 1987b) and 50 (Dexeus et al. 1989).. (1989) in trials with 2173 patients with cancer or chronic infections. Coumarin was not toxic at concentrations up to 100 μg/mL to human peripheral blood mononuclear cells in vitro.U. whereas coumarin concentrations of 25 μg/mL and above produced suppression of murine bone marrow progenitor stem cell activity (Gallicchio et al. No evidence of liver toxicity was observed in 45 renal-cell carcinoma patients given 100 mg coumarin daily in combination with cimetidine (Marshall et al. Casley-Smith and Casley-Smith (1995) reported two cases of hepatotoxicity in five trials involving 1106 lymphoedema patients taking 400 mg coumarin daily for a mean duration of 14. no evidence of liver toxicity was found in groups of 17 (Nolte et al. 1990) cancer patients.1 Toxic effects Humans Recommended doses of coumarin in clinical medicine range from 8 mg per day for the treatment of venous constriction to 7000 mg per day in antineoplastic therapies (Marshall et al. 1994. St 23132. CCRF CEM. gastric carcinoma cell line. 24 (Marshall et al. produced significant suppression of human bone marrow progenitor stem cell activity at concentrations greater than 200 μg/mL. All signs of liver toxicity returned to normal on cessation of treatment.2 4. colon-carcinoma cell line. 1989). 1999)... This incidence is similar to that reported by Cox et al. 1989). Faurschou (1982) reported a case of toxic hepatitis in a patient given coumarin daily for eight weeks. In two lymphoedema patients given 90 mg coumarin per day for five months. In various human tumour cell lines (lymphoblastic cell line.37%) developed elevated serum aminotransferase levels (peak levels of 360–696 I. alterations in liver function have been noted in only a small proportion of patients. Koch et al. 1987a). Marshall et al. Caco-2) coumarin [IC50 values ranging from 232 to 522 μg/mL] and its major metabolite 7-hydroxycoumarin [IC50 values ranging from 110 to 436 μg/mL] inhibited .6 months.. In other studies with similar dosing regimens. 1989). One possible case was reported by Beinssen (1994) and six by Loprinzi et al.. 7-hydroxycoumarin. Some cases of hepatotoxicity have been reported to be associated with exposure to coumarin. Lake... (1997). 22 (Marshall et al. (1997) reported elevated serum alanine aminotransferase activity.2.COUMARIN 207 4. While various mild side-effects have been reported following coumarin treatment. No evidence of liver toxicity was reported in extensive trials with lymphoedema patients given 400 mg coumarin per day in combination with diethylcarbamazine (Jamal et al./L) after total coumarin doses between 1 and 15 g. hepatoma-derived cell line. Coumarin and its metabolite.. Reports of overt toxicity are rare (Cox et al. Only eight patients (0.. 1987). The majority of these patients received 100 mg coumarin per day for one month followed by 50 mg per day for two years.

Twenty-four hours later.71 and/or 2. 1994).57 mmol/kg bw). in some C3H/He mice. 1992. 1998). Fentem et al.2 Experimental systems (a) Single-dose studies The acute oral LD50 of coumarin has been reported to be 420 mg/kg bw in C3H/HeJ mice and 780 mg/kg bw in DBA/2J mice (Endell & Seidel. 1989b. (1996) reported that a single oral dose of coumarin produced liver necrosis in mice. 1984.. but none of the coumarin methyl derivatives examined (intraperitoneally. 1. 3-methylcoumarin or 4-methylcoumarin was administered intraperitoneally at a single dose of 1. 1994). centrilobular hepatic necrosis was observed together with dose-dependent elevation in plasma transaminase activities (Lake. the oral LD50 of coumarin has been reported to range from 196 to 780 mg/kg bw (reviewed in Cohen.. Cottrell et al. Studies with various modulators of cytochrome P450 activity and with depletors of glutathione levels demonstrated that coumarin-induced hepatotoxicity in the rat is likely to be mediated via one or more reactive metabolites generated by cytochrome P450-dependent enzymes and that glutathione conjugation constitutes a detoxification pathway (Lake.4-double bond.. (1989b) showed that dihydrocoumarin. a single dose of coumarin (200 mg/kg bw) or vehicle was administered by gavage.. these compounds had the same order of cytotoxicity in isolated hepatocytes as that observed in vivo (Fernyhough et al. single oral or intraperitoneal doses of coumarin ranging from 125 to 500 mg/kg bw resulted in rapid depletion of hepatic non-protein sulfhydryl groups. mild subcapsular linear hepatocyte necrosis and. Lake et al. In male Sprague-Dawley or Wistar rats. In male Wistar rats. 4. 1956).208 IARC MONOGRAPHS VOLUME 77 cell proliferation after 48 h incubation. Aroclor 1254 (54.71 mmol/kg bw). centrilobular necrosis. Lake et al. Mice were pretreated with β-naphthoflavone (80 mg/kg bw). After 24 h. Coumarin produced histological evidence of centrilobular necrosis. which lacks the 3. 0.. Egan et al. 200 mg/kg bw coumarin was hepatotoxic to both C3H/He and DBA/2 mice. The glucuronide of 7-hydroxycoumarin (tested up to 2315 μg/mL) did not produce this response (Weber et al. 1984. Lake & Evans. while the methyl analogues were much less toxic at equivalent doses.. 1999). Subsequently. In the same study. Lake et al. 125 or 162 mg/kg bw) or vehicle alone by intraperitoneal injection for three consecutive days. (1994) demonstrated that coumarin (intraperitoneally.2.. 1978). Lake et al. produced dose-related hepatic necrosis in male Sprague-Dawley rats. 1993. 1990). The oral LD50 for coumarin in various strains of rats is reported to be 292–680 mg/kg bw (Hazleton et al. In three mouse strains.86 and 1.03 mmol/kg bw and rats were killed 24 h later. primarily glutathione. coumarin. Hepatotoxicity was characterized by an increase in plasma aminotransferase activity. produced little hepatotoxicity in male Sprague-Dawley rats in vivo (127 and 254 mg/kg bw intraperitoneally). 1979. Pretreatment with .

. By seven days after dosing. Coumarin has been reported to be hepatotoxic and nephrotoxic in dogs at an oral dose of 100 mg/kg bw given for periods of 8–22 days (Hazleton et al.. but not in dogs given 10 mg/kg bw coumarin for up to 350 days (Hagan et al. 1967). Clara cell toxicity was not observed in the distal bronchioles.to 3-fold in mice 24 h after treatment (Born et al. Coumarin administration results in increased liver weight and in a variety of morphological changes in treated rats that are dependent . whereas marked centrilobular necrosis was observed in the rat liver. 20. 1967). 10. 1999). Male and female B6C3F1 mice were dosed orally by gavage with 0. coumarin caused generalized epithelial necrosis in the upper airways of rats involving both ciliated and non-ciliated cells. Coumarin produces pulmonary toxicity in mice. In male Fischer 344 rats dosed by gavage (200 mg/kg bw. 100. 150 or 200 mg/kg bw coumarin and lungs were evaluated histologically 24 h later. The toxicity of coumarin has also been assessed in other species..COUMARIN 209 β-naphthoflavone or Aroclor 1254 did not significantly alter coumarin hepatotoxicity in C3H/He mice. At 24–48 h. At doses of 150 mg/kg bw or greater. 1992). Neither 7-hydroxycoumarin nor 3. Hagan et al. single dose) to female mice and male rats resulted in no evidence of hepatocellular necrosis in mice. (b) Multiple-dose studies A number of studies have examined the hepatic biochemical and morphological changes in rats produced by coumarin administration for periods ranging from one week to two years (reviewed in Lake. 1956. Clara cells were observed sloughed into the lumen of the terminal bronchioles along with thinning of the epithelium and flattening of the remaining ciliated cells. 1997). A single intraperitoneal injection of coumarin (125 mg/kg bw) caused centrilobular necrosis in Wistar rats but not in Mongolian gerbils (Fentem et al. In DBA/2 mice. However. while hepatic microsome metabolism of [14C]coumarin doubled following administration of either inducer.4-dihydrocoumarin at a dose of 50 mg/kg bw depleted nasal cytochromes P450 (Gu et al. Clinical markers of hepatic injury (aminotransferase and succinate dehydrogenase activity) were increased nearly 100-fold in rats and 2. 1998). pretreatment with either β-naphthoflavone or Aroclor 1254 did not affect coumarin-induced hepatotoxicity. 50. Hepatotoxic effects were also noted in dogs given 25 or 50 mg/kg bw coumarin for longer periods. Oral administration of coumarin (200 mg/kg bw. A significant reduction in CYP2A and CYP2G levels in the olfactory mucosa of Wistar rats and C57BL/6 mice and suppression of nasal CYP2A in rats occurred within 48 h following a single intraperitoneal injection of coumarin (50 mg/kg bw).. the Clara cells had regenerated and the bronchiolar epithelium appeared normal.. The decrease in cytochrome P450 levels was accompanied by necrosis. coumarin caused selective injury to the Clara cells in the distal bronchiolar epithelium.. The time course of this injury and recovery of the cells was studied up to seven days following a single dose of 200 mg/kg bw coumarin. cell loss and basal cell metaplasia in the olfactory mucosa. single dose).

CYP2B10. this lesion tends to regress with prolonged treatment in favour of the development of bile duct hyperplasia and cholangiofibrosis in the periportal area of the liver lobule (Grasso et al.. . In coumarintreated mice (300 mg/kg bw). respectively (Sparnins et al. by 10 days mouse lungs appeared to be normal. Over a 16-day period. 1993). on five days per week for a total of 12 treatment days. piloerection. centrilobular hepatocellular degeneration and necrosis were observed in rats along with chronic active inflammation and bile duct hyperplasia.. 50. Evans et al. 1996). 150 or 300 mg/kg bw. 300. Carlton et al. 100.5% (Hagan et al. Kidney lesions were not observed in studies with Sprague-Dawley and Osborne-Mendel rats chronically fed coumarin at dietary levels of up to 0. When B6C3F1 mice were treated orally for five days with 200 mg/kg bw coumarin.3-fold). 19. Together with Clara cell necrosis (following a single dose). Such changes include necrosis..210 IARC MONOGRAPHS VOLUME 77 on the magnitude of the dose and the duration of treatment. Three male and three female rats and two male mice receiving 300 mg/kg bw coumarin died. mice became tolerant to the coumarin-induced swelling of Clara cells and necrosis in the terminal bronchioles which is observed after a single dose of coumarin (200 mg/kg bw). 1989. bradypnoea. 1982. 1967. centrilobular hepatocellular hypertrophy was observed (National Toxicology Program. or 600 mg/kg bw. coumarin was administered by gavage to groups of five male and five female Fischer 344 rats and B6C3F1 mice at doses of 0. 1998). there were still areas of bronchiolar epithelial flattening and hyperplasia. While centrilobular hepatic necrosis may be observed in rats treated with coumarin for at least four weeks. A re-evaluation of the kidneys revealed minimal nephropathy with tubular casts in the high-dose male and female rats and minimal to marked focal necrosis of proximal convoluted tubule epithelium was also observed. Clinical findings in mice included inactivity. excessive lacrimation.5%) of male Wistar rats and female ICR/Ha mice for two weeks induced glutathione peroxidase activity in the stomach (1. No histopathological examination was performed in this study (National Toxicology Program. 1974. groups of ten Fischer 344 rats and ten B6C3F1 mice were administered coumarin by gavage at doses of 0. 1993). van Lieshout et al. CYP2C29. In a 13-week study. together with one male and one female in the 300-mg/kg bw groups and one male in the 75-mg/kg bw group. Absolute and relative liver weights increased in male and female rats and mice treated with 150 or 300 mg/kg bw coumarin. 1996). the levels of these enzymes returned to normal (Born et al.. All female and four male rats receiving 400 mg/kg bw coumarin died. Coumarin administration in the diet (0. 200 or 400 mg/kg bw or 0. 75. All the mice receiving 600 mg/kg bw coumarin died. respectively. 25. There were no clinical signs of organ-specific toxicity in rats.. vacuolation. 75. 1999).. ptosis or ataxia. Lake & Grasso. apoptosis. fatty change and bile duct hyperplasia. At the same doses. Although after five days of coumarin treatment.7-fold) and glutathione S-transferase in the liver (5.25–0. In tolerant mice. 150. the levels of cytochromes P450 such as CYP2A4/5. 40.. 38. CYP2E1 and CYP2F2 were decreased.

at doses of 0. In a two-year study in which male and female Syrian hamsters were fed coumarin at dietary levels of 0.5. CD-1 mice and Syrian hamsters.1 and 0. with males affected more severely than females.and 13-week studies. respectively. (c) In-vitro studies and species comparisons Lake et al.5 or 67.5 mg/kg per day coumarin (Evans et al. Histopathological evidence of hepatotoxicity was observed in rats.1.2). Administration of coumarin to mice was associated with centrilobular hypertrophy.COUMARIN 211 In one-.1.. 50. 22. These effects were reduced or not observed in mice and hamsters (Lake & Grasso. 100. Levels of total hepatic glutathione were increased approximately twofold. four.5.2. 25. and there were statistically significant decreases in microsomal cytochrome P450 content and ethylmorphine N-demethylase activity. apoptosis and bile duct proliferation and increases in serum bilirubin content and both serum and hepatic γ-glutamyltranspeptidase activity.0% coumarin. 7. 2. no evidence of any coumarin-induced liver lesion was observed (Ueno & Hirono. including 7-hydroxycoumarin. (1996) which is described more fully in Section 3. Rats were fed 0–0. (1989b) compared the toxicity of coumarin and selected coumarin metabolites. Administration of coumarin to baboons at dose levels of 0.5 and 0–1. haematology or microscopic pathology (Carlton et al. Coumarin was administered in the diet for two years to Sprague-Dawley rats and CD-1 mice in a study by Carlton et al. and 200 mg/kg bw. A sustained stimulation of hepatocyte replicative DNA synthesis was observed in rats treated for four and 13 weeks.5%. The incidence of forestomach ulcers was significantly greater in treated rats than in controls. and 100 mg/kg bw and 0.5% coumarin for 13 weeks. No non-neoplastic lesions of the lungs of dosed mice were considered to be chemicalrelated (National Toxicology Program. No dose-related clinical signs were observed in the CD-1 mice nor were there any changes in clinical pathology. syncytial alteration and eosinophilic foci in the liver. Coumarin was administered by oral gavage for two years to male and female Fischer 344 rats and B6C3F1 mice. the effects of coumarin treatment were compared in male Sprague-Dawley rats. respectively (see Section 3. Coumarin was more toxic than .. 1996). 50. cytological alteration and increased severity of bile duct hyperplasia.5 mg/kg bw per day in the diet for 16–24 months resulted in increased relative liver weight only at the highest dose level. In the rat. ultrastructural examination revealed a dilatation of the endoplasmic reticulum in three of four baboons given 67. While light microscopic examination of liver sections revealed no abnormalities. Mice and hamsters were fed 0–0. 1993). coumarin caused dose-related hepatotoxic effects which included vacuolar degeneration. in metabolically-competent rat hepatocytes. for one. four or 13 weeks. 1996). 1981). fibrosis.75% coumarin for one and four weeks and 0. Hepatic lesions consisted of hepatocellular necrosis. 3-hydroxycoumarin and ortho-hydroxyphenylacetic acid. 1979).

0.3 4. Grote et al.25% coumarin on days 6–17 of gestation.2 Experimental systems Groups of 26–30 female NMRI mice were fed diets containing 0. and the mouse ovarian tumour (MOT) cell assay.25 or 0. When both of these compounds were investigated in metabolically active hepatocytes isolated from male Sprague-Dawley rats.5.1. Coumarin was hepatotoxic in cells prepared from rats. Born et al. with ortho-hydroxyphenylacetic acid being essentially non-toxic. Coumarin toxicity. as judged by cell morphology and by lactate dehydrogenase release. 1 and 2 mmol/L coumarin in 24 h cultured precision-cut male Sprague-Dawley rat. There was no effect on the total number of implantations or on the proportions that were resorptions or fetal deaths. The IC50 value for inhibition of protein synthesis was approximately 0. 4. (1996) compared the toxicity of 0. based on liver protein synthesis and potassium content. The assays were the human epithelial palatal mesenchymal (HEPM) cell assay. nor any reduction in fetal weight (Roll & Bär.5 mmol/L coumarin (and 1 mmol/L for human hepatocytes). 1967).8 mmol/L). which evaluates effects on proliferative potential. Fauve de Bourgogne rabbits and humans. Coumarin was one of a series of chemicals used in an assessment of the predictability of two in-vitro assays for mammalian teratogenesis. Grote & Weinmann. 1973.3. which evaluates . 4. did not cause a change in cell viability or an increase in lactate dehydrogenase activity.. 3-Hydroxycoumarin (0. No embryotoxic or fetotoxic effects were seen in either rats.212 IARC MONOGRAPHS VOLUME 77 any of its metabolites. (2000) demonstrated that ortho-hydroxyphenylacetaldehyde (4 mmol/L) was much more cytotoxic than coumarin (4 mmol/L) to Chinese hamster ovary cells K1. mice and rabbits.1 and 0. ortho-hydroxyphenylacetaldehyde (0.8 mmol/L) caused a greater cytotoxic response compared with coumarin (0. a cell line that does not contain cytochromes P450. whereas monkey and human liver were relatively resistant. Human hepatocytes were sensitive to coumarin toxicity only at a concentration of 1 mmol/L. Ratanasavanh et al. The hepatocytes were incubated with 0. 1971. 0. Dunkin-Hartley guinea-pig. was concentration-dependent in rat and guinea-pig liver. 1977).5 mmol/L for coumarin. DBA/2J mice.3. not a product of coumarin epoxidation. cynomolgus monkey and human liver slices. Price et al. (1996) prepared hepatocytes from male Sprague-Dawley rats.1 Reproductive and developmental effects Humans No data were available to the Working Group. whereas the 7-hydroxy and 3-hydroxy metabolites had IC50 values greater than 1 mmol/L. 0.05. rabbits or miniature pigs given 10–100-fold the human therapeutic dose of coumarin plus rutin (Grote & Günther.8 mmol/L).

coumarin failed to induce gene mutations in other strains of this type.5 Mechanistic considerations Marked inter-species differences have been observed in the metabolism and toxicity of coumarin. It did not induce unscheduled DNA synthesis in human liver slices in vitro. However. 1988). mouse and dog. Goeger et al. Coumarin failed to induce gene mutations at the Hprt locus in Chinese hamster ovary cells.2 Experimental systems (see Table 3 for references) Genotoxicity data on coumarin have been recently reviewed (Lake. micronuclei) occurred only at the highest dose tested. It did not induce micronuclei in rat primary hepatocytes. and has been generally considered to be an antimutagen (Ohta et al. and chromosomal aberrations only in the presence of exogenous metabolic activation. 4.. In contrast. A positive result was also reported in Salmonella strain TA7002. in which 7-hydroxylation is most evident. Sanyal et al. Coumarin induced gene mutations in Salmonella typhimurium strain TA100 with metabolic activation. The two positive chromosome responses (aberrations. 1999). 4. and the authors considered the results to be negative (Steele et al. 1999). although micronuclei were found in established human hepatoma cells treated with coumarin. there are reports that it also acts as a co-mutagen in some assays (Goeger et al. and there was no clear dose–response relationship. 4.1 Genetic and related effects Humans No data were available to the Working Group.4 4.. humans and baboons.. Coumarin induced sister chromatid exchanges in Chinese hamster cells in vitro only without exogenous metabolic activation. 7hydroxylation and ring-opening to ortho-hydroxyphenylacetaldehyde. and of physical agents such as ultraviolet light. However. The IC50 values for coumarin in both assays were in excess of 1 mmol/L. species in which ring-opening predominates. Coumarin modulates the mutagenic effects of other chemicals such as aflatoxin B1 and a range of heterocyclic amines.COUMARIN 213 the effect on attachment of ascites tumour cells to concanavalin-coated beads. 1998). Simic et al.4. Sex-linked recessive lethal mutations were not induced in Drosophila melanogaster. Coumarin did not induce either micronuclei in mice or unscheduled DNA synthesis in rats in vivo. 1997. which responds specifically to reversions at a site where the change is T:A to A:T.. 1983.. Coumarin is hepatotoxic in rat.. The metabolism of coumarin involves two primary pathways.4. 1998. rarely show .

TA98. human liver slices in vitro Unscheduled DNA synthesis. TA7006. TA98. TA7001. rat hepatocytes in vivo Micronucleus formation. (2000) National Toxicology Program (1993) Doseb (LED or HID) Reference IARC MONOGRAPHS VOLUME 77 – – NT (+) NT NT . inj 500 μM 100 73 1600 500 730 320 po × 1 300 po. (1997) Beamand et al. TA7005.214 Table 3. TA1537. TA7004. (1998) Edwards et al. Mixe. 13 w Haworth et al. human Hep-G2 cells in vitro Unscheduled DNA synthesis. (1985) Goeger et al. B6C3F1 mice in vivo – – – – – + – – (+) – – – With exogenous metabolic system +c – – 1000 μg/plate 3333 μg/plated 1000 μg/mL 500 μg/mL. reverse mutation Salmonella typhimurium TA1537. reverse mutation Drosophila melanogaster. (1995) National Toxicology Program (1993) Sanyal et al. Chinese hamster ovary cells in vitro Micronucleus formation. TA7002. (1998) Yoon et al. primary rat hepatocytes in vitro Chromosomal aberrations. (1983) Haworth et al. Chinese hamster ovary cells in vitro Micronucleus formation. reverse mutation Salmonella typhimurium TA1535. TA7003. Genetic and related effects of coumarin Test system Resultsa Without exogenous metabolic system Salmonella typhimurium TA100. (1999) National Toxicology Program (1993) Müller-Tegethoff et al. sex-linked recessive lethal mutations Gene mutation. K1BH4 Chinese hamster ovary cells Hprt locus in vitro Sister chromatid exchange. (1983) Gee et al.

in-vivo test. μg/mL. not tested LED. mg/kg bw/day. –. negative. weak positive. HID. po. w.Table 3 (contd) Test system Resultsa Without exogenous metabolic system Micronucleus formation. highest ineffective dose. lowest effective dose. in-vitro test. injection. oral. NT. week c Only in the presence of hamster S9 d Slightly toxic dose e Mix of strains TA7001-7006 f Treatments on days 1–3 and 5–7 of one week b 215 . (+). positive. inj. ICR mice in vivo a Doseb (LED or HID) With exogenous metabolic system 130 po × 6f Reference COUMARIN – Morris & Ward (1992) +.

Three other studies in rats could not be evaluated.3 Animal carcinogenicity data Coumarin has been adequately tested by oral administration in two experiments in mice and in one experiment in rats. However.4 Other relevant data Coumarin is rapidly and extensively absorbed after topical or oral administration to human subjects. many of which are secondary products from the primary metabolites. is also associated with extensive biliary excretion. a clear relationship between the . It is used for its fragrance in many personal care products (perfumes. cassia. such as cinnamon.2 Human carcinogenicity data No data were available to the Working Group. Coumarin is toxic in the liver of rats and the liver and lung of mice. in some countries. There was no increase in tumour incidences in another strain of mouse. and its several industrial. in the rat at least. while 7-hydroxylation is particularly evident in humans. It has also been used to treat several medical conditions. 5. 5. a few cases of hepatotoxicity have been reported. In one study in rats. its natural presence in many plants and essential oils. deodorants. 5. coumarin produced a low incidence of renal tubule adenomas in males. seen only after step-sectioning of the kidney. in household and industrial products to mask unpleasant odours and.1 Summary of Data Reported and Evaluation Exposure data Coumarin is a natural product occurring in the essential oils of a large number of plants. 5. soaps) and in tobacco. Ring-opening predominates in rodents. as a flavouring agent in food and beverages. It undergoes very extensive metabolism along two major pathways. Susceptibility to liver toxicity. lavender and woodruff. Exposure to coumarin may occur from its production. There are numerous minor metabolites. 5. medical and consumer uses. it produced increases in lung tumours (adenomas and carcinomas) in both males and females and in hepatocellular adenomas in females. In humans exposed to coumarin for treatment of various clinical conditions. 7-hydroxylation and ring-opening to ortho-hydroxyphenylacetaldehyde. In mice of one strain. The relative extent of these two major pathways is highly variable between species.216 IARC MONOGRAPHS VOLUME 77 hepatotoxicity.

rats. A11. 5th rev. 208–209 . whereas monkey and human cells and/or slices appear to be resistant. eds. mice. rabbits or miniature pigs. & Surburg. The target organs for coumarin toxicity are primarily the liver in rats and the liver and lung in mice. with metabolic activation. K. Ed. [CD-ROM]. MD Bär. It was weakly positive in induction of micronuclei in human cells in vitro. Gaithersburg.. 16th Ed.. but also had co-mutagenic properties.. & Griepentrog. rabbits and guinea-pigs. & Schulz. Y.. There is limited evidence in experimental animals for the carcinogenicity of coumarin. Med. F. Coumarin did not induce micronuclei in mice in vivo and was not mutagenic in Drosophila melanogaster. Overall evaluation Coumarin is not classifiable as to its carcinogenicity to humans (Group 3). H. It was mutagenic in only two out of 11 Salmonella typhimurium strains tested. In vitro. Vol.S. F. 244–251 (in German) Bauer. Coumarin is hepatotoxic in rats and mice.F. No data on reproductive and developmental effects in humans were available. Hamsters and gerbils are resistant to acute coumarin-induced hepatotoxicity. Ullmann’s Encyclopedia of Industrial Chemistry. B.COUMARIN 217 dose of coumarin and the hepatotoxic responses observed has not been established. 8. There are marked species differences in these responses. References AOAC International (1998) Official Methods of Analysis of AOAC International. Rounsaville. Coumarin induced sister chromatid exchanges without metabolic activation and chromosomal aberrations with metabolic activation. 5. J. No data were available on the genetic and related effects of coumarin in humans. but failed to induce unscheduled DNA synthesis in human liver cells in vitro. 4th rev. W.5 Evaluation No epidemiological data relevant to the carcinogenicity of coumarin were available. 6... Garbe.. Yamamoto. D. G. coumarin is toxic in either hepatocytes or liver slices from rats. Elvers. Ernähr. pp. New York. but not micronuclei or gene mutations in mammalian cells in vitro. (1967) [Health evaluation of food-flavouring agents].. (1988) Flavors and fragrances. Coumarin was antimutagenic in various assays. No signs of teratogenicity were observed in mice. In: Gerhartz. with the mouse being particularly susceptible to coumarin-induced Clara cell injury. VCH Publishers.

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flash-point (closed-cup). 1987) –227– . 1996) Melting-point: –94.8670 g/cm3 at 20 °C (Lide & Milne.3 (a) (b) (c) (d) (e) Relative molecular mass: 106. phenylethane 1.1 Exposure Data Chemical and physical data Nomenclature Chem.1 1. Abstr. 1996) Density: 0.2 Structural and molecular formulae and relative molecular mass CH2 CH3 C8H10 1. 1. relative vapour density (air = 1). 1996) (g) Volatility: Vapour pressure. nuclear magnetic resonance and mass spectral data have been reported (Lide & Milne.ETHYLBENZENE 1. 1996) (f) Solubility: Slightly soluble in water (152 mg/L at 20 °C) (ECETOC.28 kPa at 25 °C (Lide & Milne.. 3. 1987) Boiling-point: 136. α-methyltoluene. 15 °C (Coty et al. 1.1.17 Chemical and physical properties of the pure substance Description: Colourless liquid with an aromatic odour (Coty et al. 1986) and chloroform.7 (Verschueren. No. 1996) Spectroscopy data: Infrared. ultraviolet [97]. miscible with diethyl ether and ethanol (Lide & Milne.. Name: Ethylbenzene IUPAC Systematic Name: Ethylbenzene Synonyms: EB. Serv.: 100-41-4 Chem. Reg. 1996). 1996).1.1 °C (Lide & Milne. ethylbenzol.1. Abstr.9 °C (Lide & Milne.

1. 0. either in the liquid phase with aluminium chloride catalyst or in the vapour phase with a synthetic zeolite or Lewis acid catalyst (Coty et al. subcutaneous fat. plant foliage. oil/water and oil/blood) have also been measured (Sato & Nakajima. 1996a).. 0.4 Technical products and impurities Because ethylbenzene is used almost exclusively to produce styrene.02 wt% max.01 mmol/L) (WHO. Almost all ethylbenzene is produced commercially by alkylating benzene with ethylene. 1. assuming a temperature of 25 °C and a pressure of 101 kPa .1... 0.228 IARC MONOGRAPHS VOLUME 77 (h) Octanol/water partition coefficient (P)1: log P. isotachophoresis (detection limit. 0. These include derivatization of the acid and GC analysis (detection limit.. toluene. 1996) (i) Conversion factor2: mg/m3 = 4. diethylbenzene. 1979) 2 Calculated from: mg/m3 = (relative molecular mass/24.0 mg/L). A typical sales specification is as follows: purity. n-propylbenzene.5 Analysis Selected methods for the analysis of ethylbenzene in various matrices are given in Table 1. 4 mg/kg max. fish samples) using head-space gas chromatography (GC). 1987. benzene. 1–3 mg/kg max. Ethylbenzene can be determined in biological material (blood. ethyltoluenes and xylenes in ethylbenzene are controlled to meet the required styrene purity specification. meta-xylene + para-xylene.2 wt% max.04 mmol/L). Several methods can be used to determine mandelic acid in urine samples. 1..2 Production Ethylbenzene was first produced on a commercial scale in the 1930s in Germany and the United States. 1987). 3. blood/air.34 × ppm 1.45) × ppm. oil/air.15 (Verschueren.3 wt%. 0. GC with mass spectrometry. Levels of cumene. ortho-xylene + cumene.. The ethylbenzene–styrene industry remained relatively insignificant until the Second World War. Determination of mandelic acid in urine has been recommended as a biomarker of exposure to ethylbenzene. and high-performance liquid chromatography (detection limit. Cannella.. 1. allylbenzene + n-propylbenzene + ethyltoluene. and GC with flame ionization detection (WHO.3 wt%. 99. 1998). when the demand for synthetic styrene–butadiene rubber prompted accelerated technology improvements and tremendous capacity expansion (Coty et al. 1987). total chlorides (as chlorine)..2 wt% max..5 wt% min.1–0. 1 Partition coefficients of ethylbenzene for other media (water/air. 1996a). 20 mg/kg max. and total organic sulfur. the product specification on ethylbenzene is set to provide a satisfactory feedstock for styrene production. 0. (Coty et al. 0.1–0.

01–0. municipal and industrial Purge (inert gas).2 μg/L GC/MS 7. trap (Tenax or OV-1 on Chromosorb W or silica gel). trap (Chromosorb W). desorb into capillary GC column Purge (inert gas). trap (Chromosorb W). ground. and surface water Drinking water and raw source water Wastewater. desorb (carbon disulfide) 1–10 μg/sample Department of Health and Human Services (1994) [NIOSH Method 1501] Environmental Protection Agency (1995a) [Method 524.2 μg/L GC/MS 10 μg/L Solid waste matricesb Purge (inert gas).005 μg/L Environmental Protection Agency (1996a) [Method 8021B] . trap (Tenax or OV-1 on Chromosorb W or silica gel).03–0. trap (Tenax or Chromosorb W) or thermally desorb or headspace sampling or direct injection into capillary GC column GC/PID 0. purge (inert gas). Selected methods for the analysis of ethylbenzene Sample matrix Sample preparation Assay procedurea GC/FID Limit of detection Reference Air Adsorb (charcoal). thermally desorb into GC column Purge (inert gas).06 μg/L GC/PID 0.05 μg/L GC/PID 0. thermally desorb into GC column Add isotope-labelled analogue.ETHYLBENZENE 229 Table 1.2] Environmental Protection Agency (1995b) [Method 502. trap (Tenax or OV-1 on Chromosorb W).2] Environmental Protection Agency (1999a) [Method 602] Environmental Protection Agency (1999b) [Method 624] Environmental Protection Agency (1999c) [Method 1624B] Drinking. desorb into capillary GC column Purge (inert gas). thermally desorb into GC column GC/MS 0.

Germany. gas chromatography/photoionization detection b Includes: groundwater. Information available in 1999 indicated that ethylbenzene was manufactured by nine companies in the United States. four in the Republic of Korea. oily wastes. the Czech Republic. the Netherlands. Canada. Singapore. GC/PID. seven in China. trap (Tenax or Chromosorb W) or thermally desorb or headspace sampling or direct injection into capillary GC column a 0. not reported 5900 380 3820 1630 1470 640 390 14 230 1995 7048 NR 5157 1841 3587 2644 1158 21 435 . polymeric emulsions. the Russian Federation and the United Kingdom and one each in Australia. gas chromatography/flame ionization detection.03–0. spent carbons. mousses. two each in Brazil. sludges. Italy.230 IARC MONOGRAPHS VOLUME 77 Table 1 (contd) Sample matrix Sample preparation Assay procedurea GC/MS Limit of detection Reference Purge (inert gas). GC/MS. Bulgaria. 1999). tars. caustic and acid liquors. Iran. Worldwide capacity for production of ethylbenzene in 1985 and 1995 (thousand tonnes) Location Capacity 1985 North America South America Western Europe Eastern Europe Japan Asia. waste solvents. Cannella (1998) NR. soils. fibrous wastes. Oceania and Far East (except Japan) Other (including the Middle East and Africa) Total From Coty et al. filter cakes. spent catalysts.06 μg/L Environmental Protection Agency (1996b) [Method 8260B] Abbreviations: GC/FID. Table 2. Taiwan and the Ukraine (Chemical Information Services. eight in Japan. gas chromatography/mass spectrometry. Poland. Spain. Slovakia. (1987). Mexico. and sediments Worldwide capacities for the production of ethylbenzene in 1985 and 1995 are presented in Table 2. Romania. Belgium. France.

respectively.4 1. printing inks. 1990)..4..3 Use Ethylbenzene is almost exclusively (> 99%) used as an intermediate for the manufacture of styrene monomer. 1997).. 1995). National estimates of workers potentially exposed in other countries were not available.. is present in many paints and lacquers. Ethylbenzene is also present in commercial mixed xylenes at levels up to 25%. Most occupational exposures to ethylbenzene result from use of products containing technical grades of mixed xylenes. Backer et al. 1985. Measured airborne and blood concentrations of ethylbenzene in several occupational settings are presented in Tables 3 and 4. dried legumes (WHO. 1998) and as a general solvent and diluent (Angerer et al. as it has been found in orange peel. 1988). parsley leaves (Górna-Binjul et al..7–19.7% (Fishbein. 1996). Estimates of ethylbenzene in gasoline have ranged from < 1–2.5 mg/m3] were measured (Lillo et al.. but under higher pressures. ECETOC. and. 1. No ethylbenzene was found from off-gassing of cured paint in a hyperbaric pressure chamber under normal atmospheric pressure.ETHYLBENZENE 231 1. Ethylbenzene emissions during the production of mixed . IARC. 1985. Less than 1% of the ethylbenzene produced is used as a paint solvent or as an intermediate for the production of diethylbenzene and acetophenone. Ethylbenzene has been added to motor and aviation fuels because of its anti-knock properties. 1996a) and other foodstuffs. Cannella.1 Occurrence Natural occurrence Ethylbenzene may occur naturally. insecticides and solvents in the rubber and chemical manufacturing industries (Fishbein. 1987. 1998). including the rubber and plastics industries. 1994).4. 1999). The ethylbenzene present in recovered mixed xylenes is largely converted to xylenes or benzene (Coty et al. 1986). which uses ethylbenzene as a starting material.. levels of 0. Styrene production. as many as 200 000 workers in the United States were potentially exposed to ethylbenzene (see General Remarks). Ethylbenzene may also be present in low-grade toluene preparations (Inoue et al. Ethylbenzene is also used as a negative photoresist solvent in the semiconductor industry (Ladou & Rohm.4–4. as such. 1. Ethylbenzene exposure occurs in the chemical processing industries.2 Occupational exposure According to the 1981–83 National Occupational Exposure Survey (NOES. 1989. Silk screen operations were found to entail exposure levels of less than 4 mg/m3 (Verhoeff et al. consumes approximately 50% of the world’s benzene production.5 ppm [1.

02 ± 3. 49 – – 7. (1992) Kawai et al.27 – – 30 44 38 92d 46 77 16f 16f Holm et al.4 AM. number of measurements d 4-h TWA measurements e 5-min TWA measurements f 30-min TWA measurements taken consecutively during one day a . outside (the control room) operators Gasoline transport drivers Gasoline service station attendants Jet mechanics Coal-tar-free paving Solvent used in histology laboratory 30 121 35 192 23 360 Kawai et al.0 < 0. Levels of occupational exposure to ethylbenzene Source Type of sample Personal Personal Personal Area Personal Personal Concentrationa (mg/m3) AM. 10 ± 4. maximum level found. 48.0 ± 39 AM.08 ± 0.232 IARC MONOGRAPHS VOLUME 77 Table 3. (1987) Rappaport et al. 0.8 ± 14. 3. 4..7 AM. 7...05–0. geometric ± geometric standard deviation. 0. 7.8 GM.07 ± 9. 22 Max.09 AM. Med. 0. (1991) Angerer & Wulf (1985) Angerer & Wulf (1985) Vincent et al..10 AM.. 148 ± 61 Med.9 ± 14 AM.06 GM. (1987) Norseth et al. median b Max. L&UCL. 0. 0. (1991) Angerer & Lehnert (1979) All 8-h time-weighted averages reported except as noted. (1987) Rappaport et al.04–0.11 L&UCL.06 ± 0.08 Max.6 GM.7 GM.08 ± 0. lower and upper confidence limits c No.3 Max.6 Max. 161 L&UCL. 0. GM. arithmetic mean ± arithmetic standard deviation. (1987) e Personal Personal Personal Personal Personal Personal Area Area AM.. 182 AM.c Reference Dip painting Dip painting Varnishing and priming vehicles and metal pieces Varnishing and priming vehicles and metal pieces Spray-painting aircraft Paint or plastic-coated wire production or printing or painting workshops Oil refinery.17 AM. < 0. 0. 1.12 L&UCL.0 ± 6.8–89.5 GM. 9... 8. 178 ± 78 Med.1 ± 16..8 Rangeb (mg/m3) Max. (1995) Rappaport et al.03–1. AM. 191 No. (1994) Inoue et al. 0..03–0.

mainly toluene.8 No. Backer et al. 1979). 1991).082–2. 69 Med. (1997) Angerer & Lehnert (1979) Etzel & Ashley (1994) AM. Unfractionated crude oil contains 1–2. 1997)... 1987.. standard deviation.06–1. (1997) Backer et al. arithmetic mean. Med.64 50–80 0.b Reference Varnishing and priming vehicles and metal pieces Before pumping regular gasoline After pumping regular gasoline Before pumping ethanolblended gasoline After pumping ethanolblended gasoline Solvent use in histology laboratory Nonsmoking firefighters in Kuwaiti oil fields a b 35 26 26 22 22 4 25 Angerer & Wulf (1985) Backer et al. The total emissions of ethylbenzene from the catalytic reformate production of xylene in the United States in 1978 were estimated to have been 970 000 kg.16 Med.40 0. distillation and crystallization vents.. 1985). and oil refining therefore is also likely to result in exposures (Fishbein. so that xylene production workers are likely to have exposure. the xylenes and ethylbenzene.. number of measurements xylenes occur from the reactor. 0.ETHYLBENZENE 233 Table 4. 1996).04–0.. median No. Ethylbenzene has been detected in bitumen fumes during road paving (Norseth et al.5% by weight of C6–C8 aromatics.. from storage units. 0..10 Med. 0. Exposures to ethylbenzene from solvent use also occurred in a histology laboratory (Angerer & Lehnert. .. Ethylbenzene concentrations in the blood of occupationally exposed workers Source Concentration (μg/L)a AM.53 Range (μg/L) – 0.11 Med.06–0. from loading and handling of ethylbenzene and from leaks.. 0.55 0.16 AM. SD. Another source of occupational exposure to ethylbenzene is the production and handling of gasoline and other fuels in which it is a component (Rappaport et al.73 0. A source of ethylbenzene exposure to physicians and nurses in the operating room may be electrocautery smoke generated from dissection of tissues and cauterization of blood vessels (36 μg/m3 maximum detected in an area sample) (Sagar et al. 62.02–0. 61.. (1997) Backer et al.4 (SD. Occupational exposure during the production of styrene was considered to be minimal or unlikely due to the enclosed nature of the styrene production process. (1997) Backer et al. 0.3) Med.

Air levels of ethylbenzene have been measured in Austria. 1987a. Agency for Toxic Substances and Disease Registry. Italy. 1986. 1992. WHO. 1996a)..234 IARC MONOGRAPHS VOLUME 77 1. 1997a). It is also a constituent of asphalt and naphtha (Agency for Toxic Substances and Disease Registry... and an atmospheric half-life of about 65 hours has been estimated in the United States (Agency for Toxic Substances and Disease Registry. 1996a. More than 99% of ethylbenzene can be expected in the air compartment (ECETOC.. Because of its high vapour pressure and low solubility. 1989).. some food samples.c). Wallace et al.. Finland. and it is one of the most commonly found substances at hazardous waste sites. 1986. adhesives. the Netherlands. making it a component of smoke from forest fires and cigarettes. Ethylbenzene is produced by the incomplete combustion of natural materials. liquid process photocopiers and pesticides (Hodgson et al. WHO. generally at very low levels in both ambient and indoor air. and as a fuel additive). cigarette smoke and consumer products and has been detected in human and animal tissues (Fishbein. Ethylbenzene undergoes atmospheric transformations with photolytically generated hydroxyl radicals. 1996a. In an Environmental Protection Agency Total Exposure Assessment Methodology (TEAM) study carried out between . higher levels of ethylbenzene are usually found indoors (De Bortoli et al. 1995. The range of measured indoor air levels overlaps with those measured outdoors. 1988). Other sources of emissions are consumer products such as solvents. WHO. (a) Air Ethylbenzene is ubiquitous in urban and rural atmospheres. 1999d). 1987a. 1996a). 1997a. WHO. 1986. 1985. by dermal contact or ingestion. 1991. use and disposal. The highest levels of ethylbenzene found in the environment are often associated with industrial operations. petroleum and industrial emissions (ECETOC. Measurements in rural areas were lower (< 2 μg/m3) (WHO. Sweden and the United States and were reported to range from < 2 μg/m3 to > 100 μg/m3 in urban areas (Table 5). 1997a. Agency for Toxic Substances and Disease Registry. 1986.4.3 Environmental occurrence Ethylbenzene is widely distributed in the environment (principally due to its use as a solvent alone and as a component of mixed xylenes.b. Wallace et al. Environmental Protection Agency. 1986. Lawryk et al. to a smaller extent. 1997a). Tobacco smoke is a major contributor to indoor air concentrations of ethylbenzene (Wallace & Pellizzari. resulting primarily from vehicle.. Minoia et al. released ethylbenzene will disperse into the atmosphere. Shah & Heyerdahl. fabric and leather treatments. water. Germany. 1995. Large quantities have been emitted during its production.b. Kostiainen. 1996. 1989. Sack et al. Wallace et al. Ethylbenzene is also found in motor vehicle emissions.. sediment soil and biota. 1996a). ECETOC. but when outdoor and indoor levels are compared for a specific house. Human exposure to ethylbenzene occurs mainly via inhalation of vapour and/or mist and.

(1986) Daisey et al.41 (median) 10 (range. (1988) De Bortoli et al.ETHYLBENZENE 235 Table 5.34 3.15 (spring) 4.a Location Reference Urban air 724 8723 ~1050 12 15 54 5 260 NR 30 50 230 15 754 USA USA Netherlands Germany Italy Austria Germany Sweden NR Germany Finland Germany Italy Canada Edgerton et al.9–2. number of measurements 1980 and 1984 in the United States measuring the personal air exposures and exhaled breath concentrations for 198 smokers and 322 non-smokers.3–6. smokers showed significantly higher breath concentrations of ethylbenzene.05) than those . (1985) WHO (1996a) Seifert & Abraham (1982) Rural sites Indoor air (homes) <2 13 3. The indoor air concentrations (geometric means) of ethylbenzene in homes with smokers (n = 345) were 8.6 smokers (breath samples) 6. (1989) Kelly et al. (1996) Wallace & Pellizzari (1986) Wallace & Pellizzari (1986) Wallace et al. (1994) Guicherit & Schulting (1985) Bruckmann et al. 1. (1994) Hodgson et al.98 (autumn) 4.46 (winter) 8.5–161) 27 6.3 μg/m3 in the fall and winter.8 6.4 <2 13 6.8 nonsmokers (breath samples) 2. (1993) De Bortoli et al.1 (median) 0. (1986) Lanzerstorfer & Puxbaum (1990) Seifert & Abraham (1982) Jonsson et al. (1991) Indoor air (office buildings) NR. significantly higher (p < 0.0 (annual means) 7.5–8.3 4–6 0.0–22.5 (median) 1. Occurrence of ethylbenzene in air Source Arithmetic mean concentration (μg/m3) 1.0–19 Gold et al. (1986) Fellin & Otson (1994) 95 783 346 322 198 347 384 4 USA USA Italy USA USA USA USA USA Shah & Heyerdahl (1988) Wallace & Pellizzari (1986) Minoia et al. compared to nonsmokers.5 (geometric mean) 7..35 (summer) 13.2–100 No.2 2. not reported a No.3 (median) (personal air monitoring) 0.

Nonsmokers exposed at work had significantly higher levels of ethylbenzene than unexposed nonsmokers (Wallace et al. 1987. 1981). Hallbourg et al. 1986. Ethylbenzene concentrations averaged over eight parallel commuter trips in an automobile and train in Gothenborg.. Beavers et al. 1995). The levels of ethylbenzene during the spring and summer in homes with smokers (n = 169) and homes without smokers (n = 105) were the same. 3. 1982). The Commission of the European Communities also reported in 1976 that ethylbenzene levels in water were. Sweden were 17.1 μg/m3. Ethylbenzene was listed as one of the 58 most frequently detected chemicals associated with groundwater contamination in the United States. air emissions of ethylbenzene from 1005 industrial facilities were approximately 4 000 000 kg in the United States. 1996a. fuel spillages (Tester & Harker.. (b) Water Ethylbenzene. 1986). Releases of ethylbenzene to water come from a variety of sources including: industrial discharges (Snider & Manning. concentrations of up to 15 μg/L ethylbenzene have been reported (WHO. Surface water discharges of ethylbenzene from 1005 industrial facilities in 1997 in the United States amounted to 2600 kg. which accounted for about 92% of the total environmental releases of ethylbenzene.1 μg/m3. ECETOC. According to the Environmental Protection Agency (1999d) Toxics Release Inventory (TRI) in 1997. 1986). . 1996a. 1985). 1986). is found only infrequently in drinking water from ground or surface sources (Table 6). 1997a). 1991) Ethylbenzene has been shown to be released from volatile organic chemical-laden wastewater in municipal sewer systems in the United States (Quigley & Corsi. 5. which accounted for about 0. according to the Environmental Protection Agency (1999d) Toxics Release Inventory. (c) Soil and sediments Ethylbenzene can be released to soils from a variety of sources (Fishbein. in most cases.236 IARC MONOGRAPHS VOLUME 77 in homes without smokers (n = 164). In industrial and urban areas. usually at < 1 μg/L. WHO.b).. less than 1 μg/L (ECETOC.. Chen & Zoltek. 1992.9 and 2. 1995. Agency for Toxic Substances and Disease Registry. 1985. It was detected in over 4% of the surface water samples and 11% of the groundwater samples analysed at the 1177 National Priority List (NPL) sites (Agency for Toxic Substances and Disease Registry. landfill leachate (Barker.06% of the total environmental releases (4 280 000 kg).5 μg/m3 (Wallace & Pellizzari.1 μg/L in non-industrial areas. The levels of ethylbenzene in surface water are generally less than 0.. leaking petroleum pipelines or leaking underground storage tanks (Cotruvo. 1987c). 1996) and inappropriate disposal of wastes containing ethylbenzene (Eiceman et al. respectively (Löfgren et al.

(1982) Sauer et al.74 μg/L (median) < 1 μg/L 1.5 ng/L 46.87 μg/L (median) 0. 1997a).8 μg/L 0. (1985) Cole et al. The median concentration of ethylbenzene found in sediment samples was 5. (1984) Snider & Manning (1982) Seawater Ambient surface water Polluted surface water Polluted groundwater 12–74 μg/L 7. Sauer & Tyler. 1981. (1979) Staples et al. with ethylbenzene being detected in 11% of the 350 samples collected between 1980 and 1982 in the United States Environmental Protection Agency’s STORET water quality database (Staples et al. (1985) Gomez-Belinchon et al. (1982) Perry et al. (1984) Westrick et al. Releases of ethylbenzene in 1997 to land from 1005 industrial facilities in the United States amounted to 24 590 kg. 1995). (1979) Perry et al. Occurrence of ethylbenzene in water Source Arithmetic mean concentration of positive samples 0. which accounted for about 0. (1991) Storage and Retrieval of Water Quality Information (1986) Barker (1987) Tester & Harker (1981) Van Duijvenboden & Kooper (1981) Stuermer et al.005–15 μg/L 10–26 μg/L Frequency of positive samples 3/466 2/280 3/321 1/186 NR NR 8/8 1/48 110/1101 2/2 NR Location Reference Drinkingwater USA USA USA USA Canada USA USA United Kingdom USA Spain USA Cotruvo (1985) Westrick et al. (1984) Otson et al.ETHYLBENZENE 237 Table 6. (1978) Dawes & Waldock (1994) Staples et al. 1985). cleaning and degreasing solvents. including spillage of gasoline and other fuels (Tester & Harker. 1985).0 μg/L (median) 0.0 μg /L (median) 1–2 μg /L ~35 μg/kg NR.4–4. varnishes and pesticides (Agency for Toxic Substances and Disease Registry. 1987) and disposal of solvents and household products such as paint. leaching from landfill sites (Barker.5–1110 μg/L 30–300 μg/L 92–450 μg/L NR 4/4 NR NR 4/25 2/25 101/1368 59/1475 1/1 Canada United Kingdom Netherlands USA USA USA USA USA USA Industrial effluents 10–100 μg/L > 100 μg/L < 3.3 ng/L < 5.4% of the total . (1982) Gschwend et al. (1984) Westrick et al..8–22 ng/L 0.94 μg/L (median) 0.0 μg/kg dry weight. not reported 1997a). leaking underground storage tanks (Cotruvo.

46 to 104 μg/kg (Lockart et al.257 μg/g dry weight) have been reported (Górna-Binjul et al.8% of paint-related ..6 μg/kg in muscle tissue and from 1. Migration of ethylbenzene from polystyrene containers into dairy products resulted in concentrations of ethylbenzene ranging from 2 to 4 μg/kg in yoghurt and 4 μg/kg for chocolate dessert (Ehret-Henry et al.6 ng/g dry weight) and in parsley leaves (0.45 to 49.2% w/w in 7. in white fish muscle tissue samples.. < 2. greases and lubricants. papaya. adhesive-related products.0 to 9.81 to 46. 1986). Ethylbenzene was detected in 43 of 138 fish samples at 16 of 42 sites in Japan in 1986. An additional estimated 253 500 kg of ethylbenzene or 5.. cleaners for electronic equipment.. (d) Food There are few data on concentrations of ethylbenzene in foodstuffs.. Three days after storage. 2. 1996). maximum 11 μg /kg (Lovegren et al. lentils (5 μg/kg) and beans (mean.9% of total environmental releases were released via underground injection. The concentrations of ethylbenzene in the air of storage sites ranged from 7 to 88 μg/m3. Fishbein. Migration of ethylbenzene from polystyrene into various foods has been reported. 1996). 1979. 1994).. 89–153 μg/kg and wantan soup 9–28 μg/L (ECETOC. ethylbenzene was identified in 157 of 658 (24%) of the products tested. noodle soup. jasmine. Concentrations of ethylbenzene were determined in olives and olive oils exposed to gasoline vapours from gasoline-powered engines. automotive products and miscellaneous products. 1996a). 5 μg /kg.5% of automotive products. levels ranged from 7. 1979). Mean concentrations of ethylbenzene in freshwater fish samples in the Canadian Arctic in 1985 and 1986 ranged from 2. It has been identified as a trace component in the volatiles from honey. olive oil and cheese flavour and in the neutral component of roast beef flavour isolate (Min et al. The highest mean concentrations and percentage of products in each category in which ethylbenzene was found were as follows: 7. oils. 15–21 μg/L. with concentrations ranging from 1.8 μg/kg wet weight (detection limit. 1 μg/kg wet weight) (WHO. (e) Consumer products In a survey of volatile organic chemicals in 1159 household items including household cleaners and polishes.5–6 μg/L. fabric and leather treatments. 1985).4% w/w in 47. paint-related products. 1995). 1992). either on the tree or during storage.3 μg/kg in liver tissue from turbot.238 IARC MONOGRAPHS VOLUME 77 environmental releases according to the Environmental Protection Agency (1999d) Toxics Release Inventory. levels in oil in olives ranged from 15 to 55 μg/kg and in pressed oil from 6 to 60 μg/kg (Biedermann et al. Trace quantities of ethylbenzene have been detected in split peas (13 μg/kg). Ethylbenzene has been shown to migrate at levels of < 6–34 μg/kg from samples of thermoset polyester (containing up to 25 mg/kg ethylbenzene) into pork during cooking (Gramshaw & Vandenburg. The following ethylbenzene levels were found: sour milk beverages. noodle curry. Concentrations of ethylbenzene in orange peel (23.

. the average concentration was not reported (Agency for Toxic Substances and Disease Registry. The median and mean levels of ethylbenzene in non-smokers were 431 and 651 ng/L. 1997a). respectively (range. Ethylbenzene was detected in five of the eight subjects. 1982). 0.11 and 0.12 μg/L in 13 blood samples (Ashley et al. In the United States. Hodgson et al. Russia and the United Kingdom were found to range from 7 to 20 μg/g cigarette weight (typical cigarette weight is 1 g) (Johnstone et al. 1992).. respectively (Ashley et al. with smokers having the highest levels. . the concentrations of ethylbenzene tended to be higher in smokers.7 μg/m3. 1989). at mean and median levels of 0. The average emission factor for ethylbenzene for six brands of cigarettes was 101 μg per cigarette (range.. 175–2284 ng/L). Agency for Toxic Substances and Disease Registry.. (1996) determined the concentration of several volatile organic chemicals related to the environmental tobacco smoke in smoking environments. 1997a).. compared with smokers with median and mean levels of 535 and 837 ng/L. 1987c). (f) Tobacco smoke In a 1962 study. the amount of ethylbenzene in mainstream smoke from a single cigarette containing 16 mg of tar and nicotine was 8 μg (Wallace et al. 1992. (g) Human tissues Ethylbenzene was measured in the blood of 631 non-occupationally exposed people in the United States (as a subset of the Third National Health and Nutrition Examination Survey.8% of fabric and leather treatment products (Sack et al. 1994)..0% w/w in 11.3 to 8. 378–2697 ng/L) (Hajimiragha et al. Conkle et al. respectively (range. levels of ethylbenzene in mainstream cigarette smoke from cigarettes of Argentina. (range. Although a wet tissue concentration range of not detected (detection limit.78–14 μg/h). Ethylbenzene was detected (no concentrations reported) in human milk samples from eight of 17 lactating women in three urban areas in the United States (Pellizzari et al.ETHYLBENZENE 239 products and 1.. Ethylbenzene was measured in 96% of the 46 composite samples analysed for volatile organic chemicals in the 1982 National Adipose Tissue Survey conducted by the Environmental Protection Agency in the United States. In venous blood samples collected from 13 non-smokers and 14 cigarette smokers. In an earlier study. (1975) measured trace quantities of ethylbenzene in the expired air of eight male subjects ranging in age from 23 to 47 years. 1962). the authors reported a mean ethylbenzene concentration of 0. 83–142 μg per cigarette). 2 ng/g) to 280 ng/g was cited.06 μg/L. The average concentration of ethylbenzene in five smoking areas ranged from 1.

BEIs represent the levels of the determinants that are most likely to be observed in biological samples collected from healthy workers exposed by inhalation to air concentrations at the level of the TLV.5 Regulations and guidelines Occupational exposure limits for ethylbenzene are given in Table 7. Occupational exposure limits and guidelines for ethylbenzene Country Year Concentration (mg/m3) 435 545 434 543 200 1000 217 220 435 440 (sk) 100 (sk) 200 435 545 435 215 (sk) 435 100 (sk) 350 435 50 200 1000 200 450 435 1275 435 435 Interpretationa Australia Belgium Czech Republic Denmark Finland France Germany Hungary Ireland Japan Netherlands Philippines Poland Russian Federation Slovakia Sweden Switzerland Turkey United Kingdom 1993 1993 1993 1993 1998 1993 1999 1993 1997 1993 1997 1993 1998 1993 1993 1993 1993 1993 1993 TWA STEL TWA STEL TWA STEL TWA TWA TWA TWA TWA STEL TWA STEL TWA TWA TWA TWA STEL TWA STEL TWA STEL TWA STEL TWA STEL TWA TWA . The determination of mandelic acid in urine is recommended as a biological exposure test for ethylbenzene.5 g/g creatinine [about 10 mmol/L] as a BEI for ethylbenzene exposure.240 IARC MONOGRAPHS VOLUME 77 1. Urine specimens must be collected during Table 7. The Biological Exposure Index (BEI) Committee of the American Conference of Governmental Industrial Hygienists recommends a mandelic acid concentration in urine of 1.

2 years.6 years and their mean length of employment was 12. [The Working Group noted that no precise figures were given to substantiate these assertions. TLV. threshold limit value b The following countries follow the exposure limits suggested by the ACGIH: Bulgaria. The authors stated that cancer incidence among chemical workers in the industrial complex (of comparable age and length of employment) not engaged in ethylbenzene production was about three times the national average. STEL. recommended exposure limit.ETHYLBENZENE 241 Table 7 (contd) Country Year Concentration (mg/m3) Interpretationa United States ACGIH (TLV)b NIOSH (REL) OSHA (PEL) 1999 1999 1999 435 545 435 545 435 TWA STEL TWA STEL TWA From Finnish Ministry of Social Affairs and Health (1998). coexposure to benzene was present. skin designation.7 mg/m3] if the exposure at the TLV of 100 ppm [434 mg/m3] is maintained (American Conference of Governmental Industrial Hygienists. PEL. REL. The concentration in end-exhaled air collected 16 hours after the fourth exposure of the working week should be about 2 ppm [8. in addition. time-weighted average. New Zealand. 2. whereas in the group of ethylbenzene production workers. and the age of the workers and length of follow-up were not sufficient for a proper evaluation of cancer risk in relation to exposure to ethylbenzene. 1988). The BEI Committee recommends the determination of ethylbenzene in end-exhaled air collected before the shift as a confirmatory test for ethylbenzene exposure. 1999). sk. Colombia. short-term exposure limit. Singapore and Viet Nam the last four hours of the last shift of the working week. Republic of Korea. Deutsche Forschungsgemeinschaft (1999) a Abbreviations: TWA. Jordan.] . no tumours had been reported over the 10 previous years. American Conference of Governmental Industrial Hygienists (ACGIH) (1999). Studies of Cancer in Humans Some 200 ethylbenzene-production workers in Czechoslovakia [exact number not stated] between 1964 and 1985 were monitored twice a year for mandelic acid excretion (Bardodej & Círek. The mean age of workers exposed to ethylbenzene was 36. permissible exposure limit.

.1 Studies of Cancer in Experimental Animals Oral administration Rat: Groups of 50 male and 50 female Sprague-Dawley rats. Follow-up covered the period from 1 May 1960 (or the 10th anniversary of employment in the plant) through 31 December 1975.] . 1997). 1978). In a second experiment.. seven weeks of age. 3. since mortality data are not presented separately for this group]. 1985. the rats were permitted to live out their life span. toluene and styrene. one death from leukaemia (0. [The Working Group noted the lack of details on numbers of animals with specific tumours. At 800 mg/kg. including 17 cancer deaths (versus 21. In this second experiment. This study does not permit the evaluation of cancer risk among ethylbenzene-exposed workers.79 expected) and one death from lymphoma (1. The experiment was terminated at 123 weeks.4. and it is reasonable to assume its presence through the remainder of the process. and statistical analysis. 3. because it has been detected in polystyrene food packaging (Section 1. being recorded as an ‘intermediate reduction’ in animal numbers in both males and females.0 expected). there was an increase in the incidence of tumours of the nasal cavity [type unspecified] (2% incidence in females versus 0% in controls) and neuroesthesioepitheliomas (6% in males versus 0% in controls) and a borderline increase in oral cavity cancer (6% in females versus 2% in controls) (Maltoni et al. Among these. A further review of additional death certificates from recent years revealed additional cases of leukaemia and lymphoma. Exposures other than ethylbenzene included benzene. groups of 40 male and 40 female Sprague-Dawley rats received 500 mg/kg bw ethylbenzene per day according to the same regimen.242 IARC MONOGRAPHS VOLUME 77 A mortality study was conducted among 560 styrene production and polymerization workers employed for at least five years on 1 May 1960 at a plant in the United States (Nicholson et al.25 expected) occurred. were administered 0 or 800 mg/kg bw ethylbenzene (purity. up to 145 weeks. historical control data. adjustments for survival. Ethylbenzene was a raw material used in the production of styrene. albeit at low levels. [The Working Group noted that these figures were not formally included in the follow-up period for which the analysis was performed and should be interpreted as a case report. Eighty-three deaths were observed versus 106. Survival was affected by treatment in both experiments.3(d)).4 expected. while 50 male and 50 female Sprague-Dawley rats comprised control groups receiving olive oil only. 99.57%) by stomach tube in 1 mL extra-virgin olive oil solution daily on four days per week for 104 weeks.

.] 3. 1998. [The Working Group noted that the statistically significant increases related only to adenomas in both liver and lung and that these increased incidences were within the historical control range. 31/50.ETHYLBENZENE 243 3. 1999). respectively). Survival was similar among the female groups (31/50. These neoplastic lesions were accompanied by statistically significant increases in the incidence of alveolar epithelial metaplasia in the lungs of high-dose males and of eosinophilic foci of cellular alteration in the livers of high-dose females (Chan et al. 250 or 750 ppm [0. six weeks of age. additional adenomas elevated the increase in the incidence of renal tumours in females also to statistical significance. impurities included 62 ± 3 ppm cumene) by inhalation in whole-body exposure chambers at concentrations of 0. As shown in Table 9. in high-dose males after standard (single section) evaluation and at the high dose in both males and females after step-sectioning of the kidney (Chan et al. were exposed to ethylbenzene (purity. After step-sectioning of the kidney. 1999). 75. > 99%.2 3. There were statistically significant increases in the incidences of alveolar/bronchiolar adenomas in high-dose (750 ppm) males and of hepatocellular adenomas + carcinomas in high-dose (750 ppm) females (see Table 8). 75. National Toxicology Program.2 Rat Groups of 50 male and 50 female Fischer 344/N rats..1 Inhalation exposure Mouse Groups of 50 male and 50 female B6C3F1 mice. 13/50 and 2/50 at 0. 1998. there was a statistically significant increase in incidence at the high dose (750 ppm) in males of renal tubule adenomas and carcinomas combined after standard (single section) evaluation. 75. National Toxicology Program. 325. 250 and 750 ppm. respectively) but was significantly decreased in the high-dose males compared with that of control males (15/50. The mean body weights of exposed males and females were 5–10% lower than those of the control animals. 1083 or 3250 mg/m3] for 6 h per day on five days per week for 103 weeks. Accompanying the neoplastic lesions in the kidneys was a significant increase in the incidence of focal renal tubule hyperplasia. 14/50. > 99%) by inhalation in whole-body exposure chambers at concentrations of 0. 75. The dose levels were selected on the basis of results from 13-week studies. 250 and 750 ppm. . six weeks of age.2. 34/50 and 35/49 at 0. Survival and body weights of the exposed and control groups were similar.2. 250 or 750 ppm for 6 h per day on five days per week for 104 weeks. were exposed to ethylbenzene (purity. judged to be a precursor stage of adenoma.

05. logistic regression test .244 Table 8. Incidence of primary tumours in B6C3F1 mice exposed to ethylbenzene by inhalation Tumour site Animals with tumours Males 0 ppm Liver adenomas Liver carcinomas Lung adenomas Lung adenocarcinomas 0/50 0/50 5/50 2/50 75 ppm 0/50 0/50 9/50 1/50 250 ppm 0/50 0/50 10/50 5/50 750 ppm 0/50 0/50 16/50** 3/50 Females 0 ppm 6/50 7/50 4/50 0/50 75 ppm 9/50 4/50 4/50 2/50 250 ppm 12/50 3/50 5/49 0/49 750 ppm 16/50* 12/50 8/50 0/50 IARC MONOGRAPHS VOLUME 77 From National Toxicology Program (1999) * p < 0.01. logistic regression test ** p < 0.

Incidence of primary tumours in Fischer 344 rats exposed to ethylbenzene by inhalation Tumour site Animals with tumours Males 0 ppm Single sections Kidney adenomas Kidney carcinomas Kidney adenomas and carcinomas combined Step sections Kidney adenomas and carcinomas Combined Kidney adenomas and carcinomas 75 ppm 250 ppm 750 ppm Females 0 ppm 75 ppm 250 ppm 750 ppm ETHYLBENZENE 0/50 0/50 0/50 3/50 0/50 3/50 2/50 1/50 3/50 4/50* 3/50 7/50** 0/50 0/50 0/50 1/50 3/50 3/50 2/50 5/50 8/50 8/50 18/50** 21/50** 0/50 0/50 0/50 0/50 1/50 1/50 7/50* 8/50** From National Toxicology Program (1999) * p < 0.05.Table 9.01. logistic regression test 245 . logistic regression test ** p < 0.

375 and 750 mg/kg bw in corn oil five times per week for 103 weeks. p = 0. Engström. Body weight was decreased in both males (6–18%) and females (8–16%) at the high dose. seven to eight weeks of age.011..] 4. 1978. Fisher’s exact test. distribution. 7/50 low-dose and 10/50 high-dose animals.05. No significant tumour response was observed at any other site in either males or females (National Toxicology Program.246 IARC MONOGRAPHS VOLUME 77 3. 375 and 750 mg/kg bw in corn oil on five days per week for 103 weeks. 1990). Drummond et al. One carcinoma was observed in low-dose males.1 Oral administration Mouse: Groups of 50 male and 50 female B6C3F1 mice. The survival of the high-dose female rats was significantly lower than that of the vehicle controls after week 40. 1999). No significant difference in survival was observed in either sex. nine to 10 weeks of age. The toxicology of these products has been reviewed (WHO. There occurs rapid absorption upon dermal application of .2). Additional step-sections on kidneys resulted in a further increase in the number of adenomas: 1/50 control. 1989). Rat: Groups of 50 male and 50 female Fischer 344 rats.1 Absorption.1. Survival in males in both the low-dose (after week 86) and high-dose groups was significantly lower than that of the controls. metabolism and excretion Humans Ethylbenzene is rapidly absorbed after inhalation exposure of humans.3 Carcinogenicity of metabolites α 1-Phenylethanol [α-methylbenzyl alcohol] 3. 1984a. Xylenes themselves have been evaluted by IARC as not classifiable as to their carcinogenicity to humans (Group 3) (IARC. 1997). Cochran-Armitage trend test).3. 1990) [The Working Group noted the poor survival both in males and females due to accidental deaths related to gavage. Other Data Relevant to an Evaluation of Carcinogenicity and its Mechanisms Ethylbenzene is a significant component of technical xylenes (see Section 1. There was no increase in the incidence of tumours in either sex (National Toxicology Program. 4. Renal tubule adenomas were observed in males: 0/50 control.4.1 4. were administered 1-phenylethanol (food grade) by gavage at doses of 0. 1/50 mid-dose and 5/50 high-dose groups (p < 0. as shown by the excretion of ethylbenzene metabolites and tissue retention of ethylbenzene in exposed workers and volunteers (Engström & Bjurström. were administered 1-phenylethanol (food grade) by gavage at doses of 0.

. including intestine. 1992). After exposure to an atmosphere containing 600 ppm [2. ω-hydroxyacetophenone. Blood concentrations of ethylbenzene in rats after a 2-h inhalation were proportional to its concentration in the atmosphere (Freundt et al. In addition. the time courses of brain. Korn et al.60 g/m3] ethylbenzene for 6 h. This compound undergoes carbonyl reduction giving the chiral diol. Ethylbenzene was detected in brain.. 1-phenyl-1. liver. It is interesting to note that the mandelic acid excreted in human urine after exposure to ethylbenzene is predominantly the R-enantiomer. but there are evident differences in their stereoselectivity between the two compounds and this provides a means for selective monitoring of exposure to these solvents (Drummond et al. 1990). liver and kidney concentrations were broadly similar to those in the blood. yielding 1-phenylethanol (also called αmethylbenzyl alcohol) as the primary product. kidney. phenylglyoxylic acid and benzoic acid (excreted as hippuric acid. falling rapidly thereafter. 1967). radioactivity was found in organs and tissues. The α-carbon of ethylbenzene is a prochiral centre and hydroxylation thus yields a chiral product. 118–215 μg/cm2/h) (Dutkiewicz & Tyras.2:1 mixture of R.. Taken together. 1984a. 22–33 mg/cm2/h) or as an aqueous solution (rate.2). its major fate is oxidation to acetophenone. The further metabolism of ω-hydroxyacetophenone is both complex and obscure. giving phenylglyoxal.. 1992). phenylglyoxal and 1-phenyl-1. 1990). its glycine conjugate). The issue of stereoselectivity has been addressed in animal studies (see Section 4. the absorption rate being 37 ± 31 μg/cm2/min (Susten et al. which involves loss of the chiral centre. Styrene and ethylbenzene share many common metabolic pathways. but the exact interrelationships are uncertain (Engström. but there was considerable retention in adipose tissue (Elovaara et al. these three compounds.. 1989.and meta-hydroxyacetophenone are excreted in the urine. namely mandelic acid.. Korn et al. while the primary alcohol may be oxidized to an aldehyde.2 Experimental systems When rats were kept for 6 h in an atmosphere containing [14C]ethylbenzene.and S-mandelic acid excreted after styrene exposure.1. in contrast to the 1. The metabolism of ethylbenzene in humans occurs along one major pathway which is oxidation at the α-carbon.2-ethanediol are the precursors of three metabolites.. liver and adipose tissue for up to 42 h afterwards (Chin et al.. 4. Ethylbenzene was well absorbed through the skin of HRS/J hairless mice. A metabolic scheme is presented in Figure 1. small amounts of the ringhydroxylation products para.1. 1980).ETHYLBENZENE 247 ethylbenzene as the neat liquid (absorption rate. peak blood levels of ethylbenzene occurred at the end of the exposure. giving the α-keto alcohol. While some 1-phenylethanol is excreted in the urine as its glucuronic acid conjugate. The majority of the acetophenone undergoes oxidation at the ω-carbon.2-ethanediol (also referred to as phenylethyleneglycol). 1989). ω-hydroxyacetophenone. kidney and adipose tissue.

Metabolism of ethylbenzene CH2CH3 Ethylbenzene CHOH CH3 O C CH3 Glucuronide conjugation 1-Phenylethanol URINE HO para-Hydroxy-acetophenone O C CH3 O C CH3 Acetophenone OH meta-Hydroxy-acetophenone O CHOH CH2OH C CH2OH O C CHO 1-Phenyl-1.248 IARC MONOGRAPHS VOLUME 77 Figure 1.2-ethanediol ω-Hydroxy-acetophenone Phenylglyoxal O CHOH COOH C COOH COOH Mandelic acid Phenylglyoxylic acid Benzoic acid Glycine conjugation (hippuric acid) URINE From Engström (1984b) .

60 g/m3] ethylbenzene for 6 h. mandelic. none of the workers showed changes in haematological parameters or serum enzyme levels as a measure of liver function (Bardodej & Círek. 1988). . The metabolic pattern of ethylbenzene was affected by exposure level but not by the duration of administration. 4.b. ultimately leading to excretion of R-mandelic acid and phenylglyoxylic acid as major metabolites. (1990)..60 g/m3] ethylbenzene intermittently for up to 16 weeks. After exposure to atmospheres containing 300 and 600 ppm [1. In contrast. phenylglyoxylic and benzoic acids. acetophenone and para-hydroxyacetophenone (Engström. The R-diol is preferentially converted to R-mandelic acid (30% of the dose in 48 h) with 15% of the dose as phenylglyoxylic acid. which is further oxidized to phenylacetic acid. 1984a. but those of phenylglyoxylic acid and hippuric acid decreased (Engström et al.2-ethanediol converted to phenylglyoxylic and mandelic acids depend upon its stereochemistry. The amounts of 1-phenylethanol and ω-hydroxyacetophenone increased with increasing exposure.1. 1989). When Wistar rats were exposed to 50. 46% was excreted as phenylglyoxylic acid and 47% as R-mandelic acid. 300 or 600 ppm [0. Wistar rats excreted 83% and 59% of the estimated dose as ethylbenzene metabolites in the urine in 48 h.30 and 2.22.2-ethanediol and mandelic acid in rats have been examined by Drummond et al.2 4. together with their glycine conjugates. hippuric and phenaceturic acids (Engström.30 and 2. when racemic mandelic acid was given. ω-hydroxyacetophenone and phenylacetic. respectively.2-ethanediol. 1. after administration of the S-diol. the urinary recovery of metabolites increased with dose but not linearly. 1985). some 80% of the dose was recovered as phenylglyoxylic acid in 48 h with 16% as mandelic acid (R/S 80:20). the principal pathway is αhydroxylation to 1-phenylethanol. The principal metabolites were 1-phenylethanol. When S-mandelic acid was administered. 1984b). Minor metabolites found in rat urine include 1-phenylethanol. the major product is phenylglyoxylic acid (46% of the dose) with 16% as mandelic acid (R/S 80:20). Drummond et al. S-Mandelic acid undergoes a chiral inversion. phenylglyoxal.ETHYLBENZENE 249 The pattern of urinary metabolites in animals is qualitatively similar to that described for humans (see Section 4. Another pathway is oxidation at the ω-carbon of the side-chain giving rise to 2-phenylethanol. However.1). possibly by reversible oxidation to phenylglyoxylic acid.2. In the rat. The stereochemical aspects of the fates of 1-phenyl-1. ω-hydroxyacetophenone and benzoic and phenylacetic acids. The proportions of a dose of 1-phenyl-1.1 Toxic effects Humans In a long-term study (~20 years) of about 200 ethylbenzene production workers exposed to an undefined concentration of this compound. accompanied by smaller amounts of 1-phenyl-1..

1..74.2. No haematological changes were induced by ethylbenzene in any species.2 Experimental systems The acute oral LD50 (lethal dose for 50% of the animals) of ethylbenzene has been estimated to be 3. Ethylbenzene exposure also reduced the secretion of prolactin and increased dopamine turnover within the dopamine–cholecystokinin-8 immunoreactive nerve terminals of the nucleus accumbens (Andersson et al. ethylbenzene caused an increase in kidney and liver weight with slight cloudy swelling in the liver and of the renal tubular epithelium. organ and body weights.43.250 IARC MONOGRAPHS VOLUME 77 4. Smyth et al. 600 or 1250 ppm [1.40 g/m3] ethylbenzene and 0. 136.99 g/m3] ethylbenzene.42 g/m3] for 7–8 h per day respectively. for 6–7 months. (a) In-vivo studies Male New Zealand White rabbits (2200 g) were exposed to 750 ppm [3. Rabbits (1 or 2 per concentration) and guinea-pigs (5–10 per concentration) were exposed to 400. haematological findings.5 g/kg body weight (bw) in rats (Wolf et al.5–5. urinalysis or gross or microscopic changes in any of the species tested that were attributable to exposure to ethylbenzene. Twelve or 24 h following the final day of exposure. There were no alterations in clinical chemistry. 382. In male Sprague-Dawley rats exposed to 2000 ppm [8. Smyth et al..3 g/m3] for a 4-h exposure. 782 or 1610 ppm [0. In a series of studies described by Wolf et al.6.60 or 5. Liver weights were slightly increased in guinea pigs and monkeys exposed to 600 ppm ethylbenzene. female rats and male and female rats (strain not indicated) were administered ethylbenzene by gavage (13. 1981). 1962). with slight histopathological effects noted in the testes of rabbits and monkeys. Male and female Fischer 344 rats (six weeks of age) and B6C3F1 mice (seven to nine weeks of age) and New Zealand White rabbits were exposed by inhalation to 0. 2. blood urea nitrogen. Ethylbenzene depleted both striatal and tubero-infundibular dopamine levels (Mutti et al. 3.66 or 3. (1956). The authors noted that the .70 g/m3] ethylbenzene for 6 h per day for three consecutive days and killed 16–18 h following the last exposure. 408 or 680 mg/kg bw per day) or inhalation (400.40 or 6. Toxicity was evaluated by the following criteria: appearance and behaviour. ethylbenzene increased dopamine and noradrenaline levels and turnover in the hypothalamus and the median eminence. 600 or 1250 ppm ethylbenzene and rhesus monkeys (1 or 2 per concentration) to 400 and 600 ppm ethylbenzene for 6–7 months. 1956. both rats and mice exhibited an increase in mean liver weight and liver-to-body weight ratio.66.. In rats. 1. for 6 h per day on five days per week for four weeks. 0. the rabbits were killed and their brains dissected. At 782 ppm. respectively. histopathological findings and bone marrow counts [no details were provided on these specific measurements of toxicity and the results were reported as indicators of an effect: slight (+) or moderate (++)].. (1962) reported the LC50 (lethal concentration in air for 50% of the animals) in female rats to be 4000 ppm [17.25 g/m3] ethylbenzene for 12 h per day for seven days. 382 or 782 ppm [0. following both gavage and inhalation exposure. 99. 1988).

250. 500. syncytial alterations of hepatocytes. At 600 ppm ethylbenzene. Chronic inhalation exposure to 0. 1982). nine or 16 weeks caused a dose-related increase in hepatic microsomal protein content along with increased NADPH-cytochrome c reductase. 100..70 g/m3] ethylbenzene for 6 h per day for three days. There was no evidence of histological changes in these studies (National Toxicology Program. 1. The latter two activities increased in a concentration-dependent manner in kidney microsomes. 0. 0. lung and kidney weights in exposed rats and an increase in liver weights in exposed mice.ETHYLBENZENE 251 absence of abnormalities in liver histopathology and clinical chemistry indicates that these increases were due to adaptive induction of hepatic mixed-function oxidase rather than toxicity (Crag et al.. Signs of toxicity included increased absolute and relative liver. (1997b) also evaluated the induction pattern of various cytochrome P450s with time following a single intraperitoneal injection of 10 mmol/kg bw ethylbenzene to male Holtzman rats. (1985) demonstrated that exposure of male Wistar rats (~ 342 g) by inhalation to 0. hepatocellular hypertrophy.3 or 4. 1.22. 750 or 1000 ppm [0. Elovaara et al. 0.60 g/m3] ethylbenzene for two.3 g/m3] ethylbenzene for 6 h per day for five days per week. Yuan et al. Exposure to ethylbenzene also caused an . 250 or 750 ppm [0.2. NADPHcytochrome c reductase and 7-ethoxyresorufin O-deethylation activity was increased in both liver and kidney microsomes (Toftgård & Nilsen.43. 3. but slightly increased kidney glutathione levels. 1985). 50. No mortality was observed and the mean body weight gains of the exposed rats and mice did not differ from those of the respective controls. hepatocyte necrosis and thyroid gland follicular-cell hyperplasia. Elovaara et al.1 or 3. 1992).30 or 2. age-related chronic progressive nephropathy in males and females. Yuan et al. This has been supported by the investigations described below. Liver and kidney microsomes were prepared from male Sprague-Dawley rats (200–300 g) exposed by inhalation to 2000 ppm [8. The forms found to be induced were CYP1A1. Ethylbenzene did not deplete liver glutathione. 1989). In one study. 1982. 300 or 600 ppm [0. each of which exhibited different temporal characteristics. 2. (1997a) demonstrated that daily intraperitoneal injections for three days of 10 mmol/kg bw ethylbenzene to male Holtzman rats (seven weeks of age) modulated the levels of various cytochrome P450 isozymes. CYP2B1/2 and CYP2E1.1. five.3 g/m3] ethylbenzene for 6 h per day on five days per week for 104 weeks caused an increased incidence of renal tubule hyperplasia in male Fischer 344/N rats and increased severity of spontaneous. 1.32. demonstrating that ethylbenzene induces rat cytochrome P450 (Toftgård & Nilsen. Male B6C3F1 mice developed an increased incidence of alveolar epithelial metaplasia. there was no increase in serum alanine aminotransferase activity and liver cells showed slight proliferation of smooth endoplasmic reticulum but no necrosis. A 13-week inhalation study of ethylbenzene was conducted by exposing male and female Fischer 344/N rats and B6C3F1 mice to 0. Exposure to ethylbenzene caused an increase in hepatic cytochrome P450 concentration and in the hydroxylation of n-hexane and benzo[a]pyrene. 75. 7-ethoxycoumarin-O-deethylase and UDP-glucuronosyl-transferase activities.

6 or 9 mmol/L.2 Experimental systems (a) Developmental toxicity studies In an inhalation study. In the same astrocyte cultures.252 IARC MONOGRAPHS VOLUME 77 increase incidence of eosinophilic foci of the liver. In New Zealand White rabbits. ATPase activity decreased linearly with log concentration of ethylbenzene (Naskali et al. . In rats (strain not specified) similarly exposed during gestational days 1–19. 4. although the number of implantations per litter and the number of dead or resorbed fetuses per litter did not differ significantly from those of the controls.1 Reproductive and developmental effects Humans No data were available to the Working Group.3. 6 or 9 mmol/L ethylbenzene for 1 h..3. 1999). In CFLP mice exposed to 500 mg/m3 ethylbenzene intermittently 4 h three times per day on gestational days 6–15. increased rates of anomalies (the same as in rats) were found. Exposure to 500 mg/m3 led to lower fetal weight in female offspring (Ungváry & Tátrai.3 4. pituitary gland hyperplasia and thyroid gland follicular-cell hyperplasia in female mice (National Toxicology Program. An increased rate of anomalies (of uropoietic apparatus + extra ribs) and weight retardation were seen in CFY rats exposed to 2400 mg/m3 ethylbenzene for 24 h per day during days 6–15 of gestation.43 or 4. rabbits were exposed to 100 or 1000 ppm [0. 1985). (b) In-vitro studies Neural membranes isolated from primary astrocyte cultures established from newborn Sprague-Dawley rat cerebella were exposed to 3. (b) Reproductive toxicity studies No studies of the effects of ethylbenzene on fertility were available. inhalation exposure to 1000 mg/m3 ethylbenzene for 24 h per day on gestational days 7–20 induced abortions (with decreased maternal weight gain). ethylbenzene (3. 1994).. 1995). 1981). Maternal effects were moderate and dose-dependent (not specified further). A significantly reduced number of live fetuses per litter was found at both exposure levels. 1-h exposure) decreased in a dose-dependent manner the activity of important membrane integral proteins such as Na+/K+-ATPase and Mg2+-ATPase (Vaalavirta & Tahti. there was a significant increase in the incidence of extra ribs at both doses (Hardin et al. 4. Similar exposure to 600 or 1200 mg/m3 ethylbenzene induced skeletal retardation in the fetuses.3 g/m3] ethylbenzene for 6–7 h per day on gestational days 1–24 and sacrificed on the day before term.

34 g/m3] for 6 h per day on five days per week for 13 weeks revealed no changes from normal (National Toxicology Program. It was inactive in inducing sister chromatid exchanges in Chinese hamster embryo cells. At the highest non-lethal concentration. yeast and insects.1 Summary of Data Reported and Evaluation Exposure data Ethylbenzene is a major industrial chemical produced by alkylation of benzene.4 4. inks. 1992).ETHYLBENZENE 253 No testicular abnormalities were reported in Fischer 344 rats and B6C3F1 mice exposed to ethylbenzene concentrations of up to 782 ppm [3. an increase in mutant mouse lymphoma L5178Y cell colonies was induced by ethylbenzene in both the absence and presence of an exogenous metabolic system.4. it was positive in Syrian hamster embryo cells. It also caused cell transformation in these cells at the highest concentration tested. insecticides and in rubber and plastic production. Occupational exposure to ethylbenzene may occur by inhalation during its production and use. 4. Most occupational exposures are related to technical grades of mixed xylenes used as solvents in various paints and coatings. [The Working Group noted the small number of animals (five per sex per group) used]. in vitro. It did not cause chromosomal aberrations in mammalian cells. It is a component of tobacco . 4. 5.1 Genetic and related effects Humans No data were available to the Working Group.2 Experimental systems (see Table 10 for references) Ethylbenzene has been found consistently to be non-mutagenic in bacteria.. It did not induce micronuclei in in-vivo test systems but. The pure chemical is used almost exclusively for styrene production. Sperm or vaginal cytological evaluations of Fischer 344/N rats and B6C3F1 mice exposed to ethylbenzene concentrations of up to 1000 ppm [4. Ethylbenzene from these sources as well as from vehicle emissions is ubiquitous at μg/m3 levels in ambient air. as well as from the production and handling of gasoline and bitumen.4.99 g/m3] for 6 h per day on five days per week for four weeks (Crag et al. 1989). It is also present at up to 25% in technical grades of mixed xylenes and up to 15% in gasoline.40 g/m3] and in New Zealand White rabbits exposed to ethylbenzene at concentrations of up to 1610 ppm [6. but was very weakly positive in cultured human lymphocytes. 5.

mitotic gene conversion Saccharomyces cerevisiae. TA1535. reverse mutation Salmonella typhimurium TA100. TA98. ≤ 40 d NR NR 80 100 125 NG 25 200 1060 Florin et al. WP2 and WP2 uvrA. TA1537. TA98. (1980) Nestmann et al. Syrian hamster embryo cells in vitro Cell transformation. (1985) Gibson et al. TA1537. reverse mutation Pseudomonas putida. TA97. TA1535. National Toxicology Program (1999) Dean et al. Syrian hamster embryo cells in vitro Sister chromatid exchange. mouse lymphoma L5178Y cells in vitro Sister chromatid exchange. (1997) Kerckaert et al. rat liver epithelial cells in vitro Micronucleus formation. reverse mutation Salmonella typhimurium TA100. (1980) Dean et al. TA98. TA98.254 Table 10. Genetic and related effects of ethylbenzene Test system Resulta Without exogenous metabolic system Salmonella typhimurium TA100. (1985) Leddy et al. mitotic gene conversion and reversion Mutation. (1995) Dean et al. (1996) Norppa & Vainio (1983) IARC MONOGRAPHS VOLUME 77 . mutation Saccharomyces cerevisiae. TA1537. (1988). TA1538. human lymphocytes in vitro – – – – – – – – + – – – + + (+) With exogenous metabolic system – – – – – – NT NT – – Doseb (LED or HID) Reference 318 μg/plate 400 μg/plate 2000 μg/plate 1000 μg/plate 2000 μg/plate vapour. Chinese hamster ovary cells in vitro Chromosomal aberrations. TA1538. (1992). TA1535. Chinese hamster ovary cells in vitro Chromosomal aberrations. National Toxicology Program (1999) National Toxicology Program (1999) National Toxicology Program (1999) Dean et al. reverse mutation Salmonella typhimurium TA100. (1985) Nestmann & Lee (1983) McGregor et al. (1985) Zeiger et al. TA1535. reverse mutation Escherichia coli.

intraperitoneal. male and female B6C3F1 mouse peripheral blood erythrocytes in vivo a With exogenous metabolic system Doseb (LED or HID) Reference ETHYLBENZENE – – 650 ip × 2 1000 ppm inh. week 255 . w. negative LED. HID. mg/kg bw/day. weak positive. lowest effective dose. day. d. highest ineffective dose. positive. (1985) National Toxicology Program (1999) b +. –. 5 d/w. inhalation. in-vivo tests. ip. 13 w Mohtashamipur et al. in-vitro tests. inh. × 6 h/d. μg/mL.Table 10 (contd) Test system Resulta Without exogenous metabolic system Micronucleus formation. male mouse bone-marrow erythrocytes in vivo Micronuclei. (+).

No data on reproductive or developmental effects of ethylbenzene in humans were available. In male rats. It is virtually completely metabolized. An increase in the incidence of renal adenomas was seen in females only after step-sectioning. 5. The fate of ethylbenzene is similar in animals and humans. ethylbenzene decreased the activity of several integral membrane enzymes. In mice. A metabolite of ethylbenzene. 5. Liver and kidney weights were increased in rats following exposure to ethylbenzene with no signs of hepatic necrosis. no cancer mortality excess was observed during the follow-up of 15 years.4 Other relevant data Ethylbenzene is well absorbed from the skin. In the first study. .2 Human carcinogenicity data Two studies of workers potentially exposed to ethylbenzene in a production plant and a styrene polymerization plant were available. 1-phenylethanol. it increased the incidence of renal tubule adenomas and carcinomas. These various sources contribute to indoor air levels that are often higher than adjacent outdoor levels. In the second study. 5. Ethylbenzene caused changes in dopamine levels in brain and prolactin secretion in rats exposed for three to seven days. lungs and gastrointestinal tract. Limited data were available to evaluate the toxic effects of ethylbenzene in humans. the primary pathways being hydroxylation of the two carbons of the side-chain.3 Animal carcinogenicity data Ethylbenzene was tested by inhalation exposure in single experiments in mice and rats. In rats and mice. Reduced numbers of live pups per litter or abortions were reported in rabbits exposed during pregnancy. In rat brain cell cultures.256 IARC MONOGRAPHS VOLUME 77 smoke and of several household products. No changes in sperm motility or estrous cyclicity were found in rats or mice exposed to ethylbenzene for 13 weeks. Ethylbenzene is only rarely found in drinking-water but is found at μg/kg levels in a variety of foodstuffs. followed by further oxidation to a range of metabolites that are excreted principally in the urine. it increased the incidence of lung adenomas in males and of liver adenomas in females. increased the incidence of renal tubule adenomas in male rats. developmental retardation and an increased incidence of variations were reported after inhalation exposure during pregnancy. no excess of cancer incidence was found but the description of methods was insufficient to allow proper evaluation of this finding. Cytochrome P450 enzymes were induced in both liver and kidney of ethylbenzene-exposed rats. A study in rats by oral administration could not be evaluated.

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. Health. C. J. & Tátrai. E. 614–631 Toftgård. & Hoogheem.. 10.. Arch.D. (1987a) The TEAM study: personal exposures to toxic substances in air. Sheldon. Zelon. Toxicol. H. Arch.. A. Sparacino.H. & Perritt. Pellizzari E.D..P.. & Hartwell.. B.. R.A.D. Chem. environ. 272–279 . 113–116 Wallace. (1981) Groundwater pollution investigations in the Great Ouse Basin.A. 582–587 Susten. & Sheldon. Van Nostrand Reinhold. L.J. D. 385–393 Wallace. 369–387 Wallace. North Carolina and North Dakota. & Morris. Pellizzari. & Tahti.. Int. (1987b) Emissions of volatile organic compounds from building materials and consumer products.ETHYLBENZENE 265 Staples. Atmos. H. Hartwell..J. (1987c) Exposures to benzene and other volatile compounds from active and passive smoking. Niemeier. Whitmore. 65. I.J. environ. (1985) On the embryotoxic effects of benzene and its alkyl derivatives in mice. W.A. 2223–2230 Van Duijvenboden. Int. Life Sci. Werner. L.D.J. (1985) Assessment of priority pollutant concentrations in the United States using STORET data base. R. occup. Toxicol. D. D. Suk. 16. O.. L. & van Wijnen. Sci. L. Zelon. total Environ. Toxicol. E. 377–380 Wallace. Res. L. Sci... A. & Cicolella. 201–209 Verschueren. int.D. P. 21.. C. Environ. J. 131–142 Storage and Retrieval of Water Quality Information (1986) Water Quality Control Information System.. & Simon. Hartwell.A. kidney and lung. B..D.K+-ATPase and Mg2+-ATPase as targets of organic solvent impact.D. R. 85–92 Verhoeff. 3rd Ed.. T. 21. New York.. G.S. Poirot. H. occup. Arch. Subra. (1982) Effects of xylene and xylene isomers on cytochrome P-450 and in vitro enzymatic activities in rat liver. 24.. A. 197–212 Ungváry. Environ. Rieger. The Netherlands.. Control. Environ. 42. 12. appl.. W. Technol. (1990) In vivo percutaneous absorption studies of volatile organic solvents in hairless mice. Health. and breath levels of volatile organic compounds in New Jersey. 23. & Harker. Ng.W. 35.A. Arch.D. (1995) Astrocyte membrane Na+. & Kooper. & Nilsen. Toxicology. pp. T. Leaderer. drinking water and breath of 400 residents in New Jersey. II.J. indooroutdoor relationships. (1981) Effects on groundwater flow and groundwater quality of a waste disposal site in Noorwijk. 217–225 Tester.F. J.. L. 4.. rats and rabbits. L. E.H. 943–947 Vincent. Toluene. (1982) Organic contaminants in ground water near an underground coal gasification site in northeastern Wyoming. (1988) Residential indoor air contamination by screen printing plants.. Pellizzari. T.D. S. Suppl.. (1994) Occupational exposure to organic solvents during paint stripping and painting operations in the aeronautical industry. Sparacino. Water Pollut. E. environ. (1986) Personal air exposure and breath concentrations of benzene and other volatile hydrocarbons for smokers and nonsmokers. & Zelon H.A. K.G. 290–307 Wallace. & Pellizzari. Toxicol. C. R. Health. L.. R.. E. R. 60. 43. ethylbenzene and aniline. 80. Pellizzari. 8.F... 425–430 Vaalavirta. Environ. Whitmore.. Environmental Protection Agency [database] Stuermer. A.. Washington DC. (1986) Total exposure assessment methodology (TEAM) study: personal exposures.. R. Lett. Environ. T. (1996) Handbook of Environmental Data on Organic Chemicals. C.

G. Davis. 2.. W. F.. & Whitmore.. Environ. Water Works Assoc. Geneva.. D. V. V.. Tyler. E.266 IARC MONOGRAPHS VOLUME 77 Wallace. & Backes.F. (1989) The influence of personal activities on exposure to volatile organic compounds.L. C. Toxicological studies of certain alkylated benzenes and benzene. Biochim. L.D. W.. Anderson. Sequeira.F.M. Arch. R..C.D. 52–59 WHO (1996a) Ethylbenzene (Environmental Health Criteria 186). 37–55 Westrick. T. E. 19. pp.A. W. (1992) Salmonella mutagenicity tests: V. Lawlor.F. International Programme on Chemical Safety Wolf.. D. R. Haworth... Pellizzari. & Thomas..C. R. & Backes. Eyer... Cawley. Geneva. Acta. Health. Health Criteria and Other Supporting Information. Michael.L. Mutagen. Environ. 361–372 Yuan..K. McCollister. (1997a) Ethylbenzene modulates the expression of different cytochrome P-450 isozymes by discrete multistep processes. & Oyen.S.. Vol. Mello. Cawley. B. S.. M. Serron. J. 55–63 Zeiger. Results from the testing of 311 chemicals. 1334. Biochem. Rowe. 339. International Programme on Chemical Safety WHO (1996b) Guidelines for Drinking Water Quality. Am. M. G. C. ind.W. W. Biophys. 76. (1984) The groundwater supply survey. Eyer.J.. D..L. L.J. (1997b) Time course for the modulation of hepatic cytochrome P450 after administration of ethylbenzene and its correlation with toluene metabolism. mol. 50..S. J. Haddican... K. Geneva. & Mortelmans. World Health Organization.A. 14. J. Hollingsworth.. Res.. 2–141 .. biophys. Arch. 481–486 WHO (1997) Xylene (Environmental Health Criteria 190). 2nd Ed. (1956). S. 387–398 Yuan.W.

2-amino-1-methylbenzene. Reg. 1. Serv. No. ortho-methylbenzenamine hydrochloride. 2-aminotoluene.ortho-TOLUIDINE This substance was considered by previous working groups. ortho-tolylamine hydrochloride –267– .1 1. No. 2-methylaniline. Name: 2-Methylbenzenamine hydrochloride IUPAC Systematic Name: ortho-Toluidine hydrochloride Synonyms: 1-Amino-2-methylbenzene hydrochloride.: 636-21-5 Chem. 1982) and March 1987 (IARC. ortho-tolylamine ortho-Toluidine hydrochloride Chem. 2-methyl-1-aminobenzene hydrochloride. and these have been incorporated in the monograph and taken into consideration in the evaluation. Abstr. Since that time. 2-methyl-1-aminobenzene. 1. Abstr. February 1981 (IARC.: 95-53-4 Chem. 2-amino-1-methylbenzene hydrochloride. orthomethylaniline. 2-aminotoluene hydrochloride. 2-methylphenylamine hydrochloride. ortho-methylbenzenamine. ortho-aminotoluene hydrochloride.1. 1978). Name: 2-Methylbenzenamine IUPAC Systematic Name: ortho-Toluidine Synonyms: 1-Amino-2-methylbenzene. 2-methylphenylamine. Serv. new data have become available. Reg. orthomethylaniline hydrochloride. 1987a). in June 1977 (IARC. Abstr. Abstr. 2-methylaniline hydrochloride. ortho-aminotoluene.1 Exposure Data Chemical and physical data Nomenclature ortho-Toluidine Chem.

1.38 × ppm 1 Calculated from: mg/m3 = (relative molecular mass/24. 1980. ultraviolet [442].268 IARC MONOGRAPHS VOLUME 77 1. assuming a temperature of 25 °C and a pressure of 101 kPa . 1996) (h) Stability: Flash-point (closed cup). 0. oxidizes with prolonged exposure to air and light (Budavari. Lide & Milne.45) × ppm. 1996) (g) Volatility: Vapour pressure.. Budavari. 1998) (b) Boiling-point: 200. 1996) (e) Spectroscopy data: Infrared (prism [1543].32 (Hansch et al.HCl 1.1. 85 °C. 1998) (i) Octanol/water partition coefficient (P): log P. 1996).3 Relative molecular mass: 143.9984 g/cm3 at 20 °C (Lide & Milne. miscible with carbon tetrachloride. 1996) (f) Solubility: Slightly soluble in water (15 mg/L at 25 °C) (Verschueren. 1996.62 Chemical and physical properties of the pure substances ortho-Toluidine (a) Description: Colourless to light yellow liquid becoming reddish brown on exposure to air and light (Verschueren. diethyl ether and ethanol (Lide & Milne. 1996) (c) Melting-point: –16. 1995) (j) Conversion factor1: mg/m3 = 4. 1996) (d) Density: 0.16 CH3 HCl C7H9N.2 Structural and molecular formulae and relative molecular mass ortho-Toluidine NH2 CH3 C7H9N ortho-Toluidine hydrochloride NH2 Relative molecular mass: 107.3 °C (Lide & Milne.3 °C (Lide & Milne.013 kPa at 20 °C.72 (Verschueren. 3. relative vapour density (air = 1). grating [8160]). 1. nuclear magnetic resonance (proton [107]. C-13 [225]) and mass spectral data have been reported (Sadtler Research Laboratories.

. National Toxicology Program (1991).. ortho-Toluidine hydrochloride is available from another source as 98% pure (Chemfinder. 1986. 1977). 1974) have been reported. (e) Solubility: Very soluble in water (< 1 g/L at 22 °C). 1993). 1996). 1995.1.1. .5% max. Ward et al. Neurath et al. 1. ortho-Toluidine has also been determined in biological samples (urine.ortho-TOLUIDINE 269 ortho-Toluidine hydrochloride From IARC (1982).. 1995). (a) Description: Colourless to white crystalline solid (b) Boiling-point: 242. orthoToluidine can also be produced by reduction of ortho-nitrotoluene or obtained mixed with para-toluidine by reduction of crude nitrotoluene (Lewis.. 0.1% max. 0.. 1980) and mass spectral data (Mass Spectrometry Data Centre. blood) as the free amine or in derivatized form by gas chromatography (GC) or high-performance liquid chromatography with electrochemical or electron capture detection (El-Bayoumy et al. 1993. grating [29769].. meta-toluidine. 0. nuclear magnetic resonance (proton [10946]) (Sadtler Research Laboratories. except where otherwise indicated. Food and beverage samples have been analysed by GC with flame ionization detection or mass spectrometry (Vitzthum et al.. 1995. insoluble in benzene. Teass et al. 1975.4 Technical products and impurities ortho-Toluidine from one source is available with the following specifications: purity. ultraviolet [1740]. New Jersey Department of Health and Senior Services (1994) and Chemfinder (2000). 1993). Brown et al. sum of meta.5 Analysis Selected methods for the analysis of ortho-toluidine in various matrices are given in Table 1.and para-toluidine. Riffelmann et al.9 °C (g) Stability: Sensitive to exposure to light and moisture (h) Conversion factor: mg/m3 = 5. 0.2 Production Reacting ortho-chlorotoluene with sodium in liquid ammonia generates a mixture of 67% ortho-toluidine and 33% meta-toluidine (Lin & Krishnamurti. 2000). and diethyl ether (f) Volatility: Vapour pressure. 99. and water.13 kPa at 43.4% max. soluble in ethanol and dimethyl sulfoxide. para-toluidine. 0. (Bayer Organic Chemicals.. 1.87 × ppm 1.5% min.2 °C (c) Melting-point: 215 °C (d) Spectroscopy data: Infrared (prism [6330]..1% max..

solid waste matrices. extract with dichloromethane. France. . estimated quantitation limit (the EQL of Method 8270 for determining an individual compound is approximately 660 μg/kg (wet weight) for soil/sediment samples. NR. 1–200 mg/kg for wastes (dependent on matrix and method of preparation). water samples. Information available in 1999 indicated that ortho-toluidine was manufactured by 16 companies in China. gas chromatography. and one company each in Brazil. It has been produced commercially in the United States for over 70 years and commercial production of the hydrochloride salt was first reported in 1956. Selected methods for the analysis of ortho-toluidine Sample matrix Sample preparation Assay procedure GC/FID GC/MS Limit of detection Reference Air Wastewater. 11 companies in India. dry over sodium sulfate. not reported ortho-Toluidine was first produced commercially in the United Kingdom in 1880. production ranged from 6600 to 12 800 tonnes in the early 1990s (Environmental Protection Agency. and that orthotoluidine hydrochloride was manufactured by one company each in Germany and India (Chemical Information Services. six companies in the United States. Mexico. Italy.01 mg/sample 10 μg/L Eller (1994) [Method 2002] Environmental Protection Agency (1999a) [Method 1625] Environmental Protection Agency (1996a) [Method 8260B] Environmental Protection Agency (1996b) [Method 8270C] Environmental Protection Agency (1996c) [Method 8015B] Solid waste matrices GC/MS 13 μg/L Air sampling media. two companies each in Japan and Poland. municipal and industrial Adsorb (silica gel). EQL. concentrate Concentrate by azeotropic distillation 0. three companies each in Germany and Russia. 1999). 1999b). mass spectrometry. and 10 μg/L for groundwater samples). Production of orthotoluidine in the United States in the late 1970s was reported to range from 500 to 5000 tonnes per year (IARC. 1982). MS. FID.270 IARC MONOGRAPHS VOLUME 77 Table 1. Romania and Spain. soil samples Ground and surface water Liquid–liquid extraction or water dilution GC/MS 10 μg/L (aqueous) (EQL) Solvent extraction or direct injection GC/FID NR GC. desorb (ethanol) Add isotope-labelled analogue. flame ionization detector.

National estimates of workers exposed were not available from other countries. as many as 30 000 workers in the United States were potentially exposed to orthotoluidine and its hydrochloride salt (see General Remarks). pharmaceuticals and pesticides (IARC. 1975) and possibly in some fresh vegetables (Neurath et al. Measurements in the 1940s in a United States dye production plant indicated that the concentration of ortho-toluidine was < 0. workers at a plant manufacturing ortho-toluidine from ortho-nitrotoluene were exposed to concentrations of orthotoluidine in the air (n = 215) generally exceeding the maximum permissible concentrations [of 3 mg/m3. levels up to approximately 5 mg/m3 were also reported (the highest levels were observed during extraction and heating of the reduction reaction).4 1. 1970). Concurrently. Patches of cloth placed on the workers’ overalls collected about 0.3 ppm to 1. health-care workers and university teachers were identified as exposed to ortho-toluidine or its salts in the Finnish Register of Employees Exposed to Carcinogens in 1997 (Savela et al. ortho-nitrotoluene levels in the air in 80–90% of the samples exceeded the maximum permissible concentration of 1 mg/m3. including acid-fast dyestuffs. 1977).03 mg ortho-toluidine per square decimetre of skin was measured by collecting 1% acetic acid washes from wrists.01–0. In the former USSR (Khlebnikova et al. optical brighteners. 1999). 1999).4.19 mg/m3] in the workroom air in the breathing zone and area samples and from < 0. 1. 1982.. The highest airborne exposure levels were observed during distillation and extraction processes (25–28. Dermal deposition of 0.ortho-TOLUIDINE 271 1.6 mg/m3). Bayer Organic Chemicals. 1982] by 2–7 times .5 ppm [2. 1999). In addition to inhalatory . IARC.. laboratory workers.. machine operators and cleaners and janitors. Occupational groups included workers in the chemical industry. synthetic rubber and rubber chemicals. dermal levels of ortho-toluidine decreased by a factor of 10.10 mg ortho-toluidine per square decimetre (n = 46).4.. American Conference of Governmental Industrial Hygienists.3 Use The major uses of ortho-toluidine and its hydrochloride salt are as intermediates in the manufacture of over 90 dyes and pigments.7 (mg/L) ppm in the urine of workers engaged in the production of thioindigo.1 Occurrence Natural occurrence ortho-Toluidine has been detected in tea (Vitzthum et al. Ninety laboratory workers. 1995. 1. After post-shift showers.2 Occupational exposure According to the 1981–83 National Occupational Exposure Survey (NOES. chest and back of individuals at the end of work-shifts (n = 168).

Department of Health and Human Services. but no data on exposure levels were provided. hydroquinone and toluene were used to synthesize a rubber antioxidant in a United States chemical plant. 99 μg/L post-shift) in the urine of workers in the rubber chemicals department of the plant. as reported in the Toxics Release Inventory (Environmental Protection Agency. aniline. (a) Air According to the Toxics Release Inventory (Environmental Protection Agency. 1993).3–1 μg/L (Neurath et al..4.3 Environmental occurrence The production of ortho-toluidine and its use as an intermediate in the production of dyes and pigments. 1982) and in wastewaters from . 1996d). 2000). 1996) (see Section 2. 1991. 1984. 1988) and in a plant producing rubber chemicals in the United Kingdom (Sorahan et al. 104 μg/L post-shift). rubber chemicals and other products may result in its release to the environment through various waste streams. 1977). A more recent exposure study showed similar levels of ortho-toluidine (mean. ortho-Toluidine. ortho-Toluidine has been reported in surface water samples taken from three rivers in Germany at levels of 0.. The primary routes of potential human exposure to ortho-toluidine and its hydrochloride salt are inhalation and dermal contact.. 1999)..38 mg/m3]. 1982. Despite low air concentrations (< 1 ppm) [4. there was concern about exposure from ingestion and skin contact (Ott & Langner.. 1982. 1997). 1983). suggesting dermal exposure.2 for further details). 1997. 1997). reduced from a value of 242 kg in 1994 (Environmental Protection Agency.. Consumer exposure may occur from residues present in commercial dyes and on textiles and via smoking (IARC. elevated levels of ortho-toluidine were detected in the urine of exposed workers (mean. 1. Teass et al. in measurements carried out in 1990 (Ward et al. air emissions of ortho-toluidine from 23 industrial facilities were approximately 5260 kg in 1995 in the United States. Department of Health and Human Services.272 IARC MONOGRAPHS VOLUME 77 exposure. Exposed workers also had significantly increased levels of ortho-toluidine–haemoglobin adducts in blood (Ward et al. ortho-Toluidine has been identified in one secondary effluent from seven industrial and publicly owned treatment works (Ellis et al. (b) Water Surface water discharges of ortho-toluidine from 23 industrial facilities in the United States in 1995 amounted to 116 kg. 1982). Environmental Protection Agency. Exposure to ortho-toluidine was reported to occur in an Italian plant producing fuchsin (magenta) and safranine T-based dyes (Rubino et al.. in a German plant producing 4-chloro-ortho-toluidine (Stasik.

1976. 1982).2 μg/24 h in all nine subjects tested.6 μg/L) who were in the control (non-exposed) study group. In a biological monitoring study of occupationally exposed workers. 1984.5 kilogrammes. 1986).0 μg/L) in control groups (Riffelmann et al. respectively. (c) Soil Soil discharges of ortho-toluidine from 23 industrial facilities in 1995 in the United States amounted to only 5. 1986)... 1977). 1. 1982.7 ± 1. . It has also been detected in effluents from refineries and chemical production facilities..1 mg/kg) and carrots (7.3 ± 3. (e) Tobacco smoke ortho-Toluidine has been reported to be present in mainstream cigarette smoke at 32–162 ng per cigarette and at 3 μg per cigarette in sidestream smoke (IARC.. ortho-Toluidine was identified in the urine of both smokers and nonsmokers at levels of 6..0 ± 0. 1995).5 Regulations and guidelines Occupational exposure limits and guidelines for ortho-toluidine are presented in Table 2. significantly higher concentrations of ortho-toluidine were also found in the urine of smokers (1. groundwater and seawater in the United States (Shackelford & Keith. (d) Food and beverages Unspecified isomers of toluidine were found in samples of kale and celery (1.7 μg/24 h in all 10 subjects tested and 4. 1997).ortho-TOLUIDINE 273 synthetic fuel production (Leenheer et al. Department of Health and Human Services. river water. 1975). Stuermer et al. ortho-Toluidine has been identified in the volatile aroma components of black tea (Vitzthum et al. as reported in the Toxics Release Inventory (Environmental Protection Agency..1 ± 3. an estimated 10 000 kg of ortho-toluidine were released via underground injection. 1999).2 mg/kg) (Neurath et al. in process water. Additionally. Environmental Protection Agency. suggesting that sources other than cigarette smoke contribute significantly to ortho-toluidine exposures (El-Bayoumy et al. in comparison with nonsmokers (0.

ca) 9 (sk. New Zealand. ca) 1 none (ca) 9 (sk. Occupational Safety and Health Administration (OSHA) (1999) b TWA. Republic of Korea.5 9 (sk) 4. skin notation. suspected human carcinogen c These countries follow the recommendations of the ACGIH threshold limit values: Bulgaria. ca) 22 (sk) 44 9 (ca) (2. Limited data from animal studies can be supported by evidence that the substance causes cancer by a mode of action that is relevant to man and by results of in vitro tests and short-term animal studies. susp. Deutsche Forschungsgemeinschaft (1999). 2 (Germany). susp. A3 (ACGIH). sk).h. time-weighted average.9 (sk) 9 (sk.274 IARC MONOGRAPHS VOLUME 77 Table 2. A3) lfc (sk. 0. confirmed animal carcinogen with unknown relevance to humans. short-term exposure limit. ca) 22 (sk) 3 (sk) 9 0. ca. lfc. lowest feasible concentration. United Nations Environment Programme (1999). Occupational exposure limits and guidelines for ortho-toluidinea Country Australia Belgium Canada Czech Republic Denmark Finland France Germany Ireland Japan Philippines Poland Russian Federation Sweden Switzerland Turkey United Kingdom United States ACGIHc NIOSH OSHA a Year 1993 1993 1994 1993 1993 1998 1993 1999 1997 1998 1993 1998 1993 1993 1993 1993 1993 1999 1999 1999 Concentration (mg/ m3) 9 (sk.h. Substances that are considered to be carcinogenic for man because sufficient data from long-term animal studies or limited evidence from animal studies substantiated by evidence from epidemiological studies indicate that they can make a significant contribution to cancer risk. technical exposure limit.4 (sk.ca) 5 20 9 (sk. ca) 9 (sk. ca) 22 (sk) Interpretationb TWA TWA TWA TWA STEL TWA TWA Ceiling TWA TRK TWA TWA TWA TWA STEL TWA STEL TWA TWA TWA TWA TWA TWA From Finnish Ministry of Social Affairs and Health (1998). carcinogen. STEL. ca) 22 (sk) 0.5 (sk. Jordan. TRK (Germany). Singapore and Viet Nam. American Conference of Governmental Industrial Hygienists (ACGIH) (1999). sk.ca. Colombia. .

No quantitative exposure measurements or data on smoking were available.ortho-TOLUIDINE 275 2. 4-chloro-ortho-toluidine and 4-chloroacetyl-ortho-toluidine. One of these areas. Thirty-one of these deaths occurred among 610 men with exposure to benzidine. . 1993) and safranine T. Conso and Pontal (1982) reported three cases of bladder cancer occurring among 50 workers employed in a factory where para-toluenediamine (IARC. aniline.8% complete and. ortho-aminoazotoluene. ortho-toluidine and 4. 1975. Thirty-six deaths from urinary bladder cancer were observed. involving exposure either to a combination of toluene. [The Working Group noted that the excess of bladder cancer could not be attributed with certainty specifically to exposure to ortho-toluidine or to any one of the other compounds involved.6]. [The Working Group noted that although a formal estimate of expected numbers of bladder cancer cases was not provided. fuchsin and safranine T (two deaths from bladder cancer). only the study by Rubino et al.5 [95% confidence interval (CI). involved potential exposure to ortho-toluidine. Mortality was compared with that of the Italian male population as a whole.] Three pertinent epidemiological studies have been published since the last IARC review. three bladder cancers among 50 persons is likely to represent a substantial excess. The relationship of these bladder cancers to ortho-toluidine exposure cannot be determined because of co-exposure to ortho-aminoazotoluene. 1987c). among those traced (868).] 2. ortho-nitrotoluene.23 expected.4′-methylene bis(2methylaniline) (three deaths from bladder cancer) or to a combination of orthotoluidine. 2. which has been classified as possibly carcinogenic to humans (IARC.2 Cohort studies (see Table 3) Among the epidemiological studies of cancer in humans identified in the previous IARC review (1982). 906 men first employed in a dyestuff factory in northern Italy between 1922 and 1970 were followed from 1946 to 1976. 1987b) was synthesized from ortho-toluidine and ortho-aminoazotoluene (see IARC.1 Case reports Studies of Cancer in Humans One case report pertaining to ortho-toluidine has been published since the previous review (IARC. The standardized mortality ratio (SMR) for bladder cancer in this group was 62.08 expected) occurred among 53 men engaged solely in the manufacture of fuchsin (magenta. ortho-nitrotoluene. (1982) provides adequate information on the study population and methods. 2. see IARC.5-diaminotoluene. the bromoindigo and thioindigo production area. 20. 1-naphthylamine. 2-naphthylamine or several of these. 1982). but five (with 0. 1987c). Ott and Langner (1983) studied the mortality of 342 employees assigned to three aromatic amine-based dye production areas from 1914 to 1958 in the United States.3–145. with 1. 4. Follow-up was 95.4′-methylene bis(2-methylaniline). 260 (30%) had died. In that study.

5 [1.02 (0. (1991).3] 1131 1.4–1.276 Table 3.001 98 1.4–143.7 (31.1 (0.8–2.001 36 29.6] 17 SMR.8–1. 1.9] [0.2] 23 1. (2000) UK Rubber chemicals IARC MONOGRAPHS VOLUME 77 Size of the total study Cohort definition Period of follow-up Deaths All causes N SMR (95% CI) All cancers N SMR (95% CI) Bladder cancer mortality N SMR (95% CI) 906 men First employed 1922–70 1946–76 275 men Employed any time during 1940–58 1940–75 1749 (1643 men) Employed any time during 1946–88 1973–88 2160 men Employed six months between 1955–84 1955–96 (mortality) 1971–92 (incidence) 260 1.2 expected [2] [2.7] 96 2.7–1.6 [1. 1.12 (0.0 [0.4 (0.4 (0. Prince et al.92–6.0] [49] [1.7) 5 1.3 [0. (1982) Italy Dyestuff production Ott & Langner (1983) USA Dye production Stasik (1988) Germany 4-Chloro-ortho-toluidine production and processing 116 men Employed before 1970 < 1967–86 Ward et al.1) 0 1.01 (0.2) Bladder cancer incidence N SIR (95% CI) 8 72.8–1.6–1.1] [0.68–1.6 p < 0.5–3.3) 13 3.7) . (2000) USA Rubber chemicals Sorahan et al.96–1.0] 19 1.8–2.3 p < 0.91–1.0] [0.4) [190] [0.2] 19 SIR.1) 305 1. Summary of cohort studies of workers exposed to ortho-toluidine Reference Country Industry Rubino et al.3–7.

0–59. standardized incidence ratio 277 .4] RR (internal analysis) 1–4 years: n = 2.6–13.3–145. Prince et al.6] 0 3 SMR.6 (1. n = 1.0] 6 27.4–20. 1. number..7 (1.3–46.8] [3.4] Aniline (3) 2-Mercaptobenzothiazole Phenyl-β-naphthylamine (3) ortho-TOLUIDINE Bladder cancer incidence N SIR (95% CI) Co-exposuresa 4.8 [0. 7.99 ≥ 10 708 584 51 73 Sorahan et al.2 [10. (2000) _______________________________________ Duration of exposure (y) Total <5 5–9.5-Diaminotoluene (3) Aniline (3) ortho-Aminoazotoluene (2B) Multiple exposures including 4-chloroortho-toluidine and 4chloroacetyl-orthotoluidine N-Acetyl-ortho-toluidine 6-Chloro-ortho-toluidine 4-Chloro-ortho-toluidine (2A) Aniline (3) Hydroquinone (3) Toluene (3) Carbon disulfide Sulfur Benzothiazole 4-Aminobiphenyl (contaminant) (1) 2-Mercaptobenzothiazole (Ward et al. 95% CI.Table 3 (contd) Reference Subgroup exposed to orthotoluidine Size Bladder cancer mortality N SMR (95% CI) Rubino et al. 1996) (Proprietary chemical) a Previous IARC overall evaluations of carcinogenicity are given in parentheses.4′-Methylene bis (2methylaniline) (2B) Magenta (2B) Safranine T ortho-Nitrotoluene (3) 2.2–49.3] ≥ 5 years.0–56. (1991).6–28. N. [15. SMR.5 [2. (2000) 53 117 Same as full cohort 53 5 62. SIR.9) [SIR 7.3] 0 – 1 8. standardized mortality ratio. (1982) Ott & Langner (1983) Stasik (1988) Ward et al.5 [20.0. 6.4) 7 6.

11.9%) were obtained.278 IARC MONOGRAPHS VOLUME 77 ortho-Toluidine concentrations measured in breathing zone and area samples were consistently below 0. Among 98 deaths identified. [95% CI.19 mg/m3] and urinary levels measured in 1948 ranged from undetected (< 0. 1. Germany. 1. employees had to be currently working as of 1 January 1940 or hired after that date. No deaths due to bladder cancer were observed.7 (95% CI.0]). The study was initiated at the request of the union representing workers at the plant. between 1967 and 1985. It was also unclear in what year the follow-up started. Individuals not known to be deceased based on company records and social security follow-up were assumed to be alive. Urothelial carcinomas were noted in eight of the employees.8–2. Quantitative exposure data were not available. with 1. Mortality was analysed separately for 275 individuals not exposed to arsenicals.5 ppm [2. 1988). 6-chloro-ortho-toluidine.] In a historical mortality study of 335 male employees involved in the production and processing of 4-chloro-ortho-toluidine between 1929 and 1982 in Essen. 0. no deaths from bladder cancer were identified. SMR. The excess of bladder cancer could not be attributed with certainty specifically to ortho-toluidine or to any one of the other compounds present. [The Working Group noted that the definition of the subcohort was made a posteriori. [The Working Group noted that the conclusions of this study were limited by the small size of the population exposed to ortho-toluidine. 10 of which were coded to digestive organs (5.7 mg/L in workers engaged in the production of thioindigo. [95% CI. Mortality was followed from 1 January 1940 through 31 December 1975.2 deaths expected from malignant neoplasms of the urinary organs. In order to be included in the study.2]). vinyl chloride or asbestos. exposure to 4-chloro-ortho-toluidine was reported to be predominant. an incidence study was initiated and the vital status ascertainment for a subcohort of 116 subjects who had been employed in the 4-chloro-ortho-toluidine production plant before 1970 was extended through 1986. US white male referent rates were used to calculate expected number of deaths.5 expected. SMR. 31. was 0. 94 death certificates (95.3 mg/L) to 1. There were 23 deaths due to malignant neoplasms (17. but this was justified by the comment that improvements in industrial hygiene were introduced in 1970.] A bladder cancer incidence study was conducted among workers exposed to orthotoluidine at a plant manufacturing rubber chemicals in New York State.4–143) (Stasik.3. United States. The expected number of bladder cancers in the subcohort of 117 workers exposed to ortho-toluidine in the production of bromoindigo and thioindigo was not provided. The standardized incidence ratio (SIR) based on eight observed cases was 72. who had noted a number of bladder cancers among workers in a department where an . The expected number of incident bladder cancers in the cohort of 116 men. two of whom had died as of December 1986. 0. There were no data on the smoking habits of the cohort.7 expected. calculated based on the 1983 rates from the Saarland Cancer Registry.8. As a result of this discovery. All eight had been employed in the 4-chloroortho-toluidine production plant before improvements in industrial hygiene were made in 1970. orthotoluidine and 4-chloro-ortho-toluidine. Four monocyclic amines had been used at the plant: N-acetyl-ortho-toluidine.8–3.

all-cancer or bladder cancer mortality in the total cohort (Table 3). A study cohort was identified from personnel records of the chemical plant. 708 of whom had been assigned to the department where the accelerator and antioxidant were produced (and were considered to be definitely exposed to ortho-toluidine and aniline). other reactants and intermediates included 2-mercaptobenzothiazole (Ward et al. Among the major reactants used in these processes were two primary aromatic amines. Person–years at risk began on 1 January 1973 (the first year in which matching with the registry through social security number was possible) or on the date of starting employment. 4Aminobiphenyl was identified as a potential low-level contaminant (< 1 ppm) [4. 1. It was estimated. 1. or through matching with the cancer registry in the state where the plant was located. The antioxidant was produced in a separate building that opened in 1957 and in 1970 an expansion of this building was completed and production of a new accelerator was begun.6. 0. Six of the seven bladder cancer cases occurred among workers employed in the exposed department for over 10 years. using a method of indirect adjustment (Axelson & Steenland. carbon disulfide. benzothiazole and a proprietary chemical which was introduced into the process in 1970 (Ward et al.9–6. 1988). that differences in smoking habits between the cohort and the United States general population would account for an SIR of 1. sulfur.38 mg/m3] in some bulk samples of process chemicals in 1990 (Ward & Dankovic. SIR. the SIR for this group was 27. Bladder cancer cases were identified from company and union records and confirmed through medical records. Within the exposed department. 6. 10. 3. . [95% CI. seven of which occurred in the definitely exposed group (SIR. 1991).05 for bladder cancer. A subsequent mortality analysis of the same cohort followed through 1994 (Prince et al. 2. which opened in 1946 for production of poly(vinyl chloride).0–59. Data on smoking were available for 143 members of the cohort and showed that cohort members were slightly more likely to be current or former smokers than the general United States population.0–9. 1. toluene.4]).7. whichever occurred later..5. The SIR was not elevated among workers in the probably unexposed group (two observed. Cancer incidence rates were compared with bladder cancer incidence rates in the same state. [95% CI. ortho-toluidine and aniline.17–5.6–13. hydroquinone. 3. [95% CI. There were 13 cases of bladder cancer in 1973–88 in the cohort overall (SIR. it was not possible to separate individuals with exposure to ortho-toluidine from individuals with exposure to other chemicals. among whom 91% were white.4.2 [95% CI. 1991). Race was recorded in the personnel records for only 670 subjects. Follow-up was from 1973 to 1988.2].3]) and four in the possibly exposed group (SIR. 1996). 2000) showed no elevation in all-cause..2]).. [95% CI.ortho-TOLUIDINE 279 antioxidant and an accelerator were produced. Vital status was identified from company records and records of the United States social security administration. There were 1749 (1643 male) individuals employed in the plant between 1946 and 1988. and 753 of whom had never worked in either definitely or possibly exposed jobs.0]). 288 had been assigned to departments such as maintenance in which possible exposure was considered to have occurred.

Potential for 4-aminobiphenyl contamination arose from use of diphenylamine as a reactant intermittently from 1972 to 1985 (Ward et al. 95% CI. 2. 1987d).8. 95% CI. 1. (2000) updated a study of workers exposed to several aromatic amines in a factory manufacturing chemicals for the rubber industry in the United Kingdom (Sorahan & Pope.25 expected among the 605 individuals (SMR. 2mercaptobenzothiazole. five occurred among 94 individuals who had been exposed to phenyl-β-naphthylamine [SIR.7. some evidence for an exposure–response relationship with duration of exposure to ortho-toluidine (RR internal analysis: 1–4 years. seven occurred among individuals exposed to aniline [SIR. Mortality was examined for the period 1955–96 and cancer incidence was examined for the period 1971–92.5.1] and three occurred among 53 individuals who had been exposed to ortho-toluidine [SIR. 1987e). n = 2. All of the bladder cancer cases among workers exposed to ortho-toluidine had also had exposure to one or more of the other chemicals. 95% CI. An exposure assessment study was conducted at the plant in February–March 1990 (Ward et al. 7. 2. Haemoglobin adducts reflect only recent exposures (Hemminki. phenyl-β-naphthylamine or ortho-toluidine. 2-Mercaptobenzothiazole has not been reviewed by IARC.8]. 605 of whom had been exposed to aniline.2–8. 1996). for which the Working Group had no data on carcinogenicity. 1988). . Aniline is not known to induce bladder cancer in humans or animals (IARC. [The Working Group noted that. 3.6. RR. There were nine bladder cancer deaths observed and 3..9]. Poisson regression analyses revealed no association between estimated duration of mercaptobenzothiazole exposure and risk of bladder cancer. 0. 95% CI. 1993).4].6–3. 6. but has shown some evidence of carcinogenicity in rats and equivocal evidence of carcinogenicity in mice (National Toxicology Program. of which nine occurred among [357] individuals who had been exposed to mercaptobenzothiazole [SIR. 1994) (commercial diphenylamine was contaminated with low levels of 4-aminobiphenyl in the 1970s and earlier (Safe et al. 1. 1. 1. intermediates and end-products present in the previous cohort has been evaluated by IARC. 1.. This study demonstrated substantially higher urinary concentrations and levels of haemoglobin adducts of aniline and ortho-toluidine among exposed workers compared with in-plant controls. The updated study included 2160 male production workers.] Sorahan et al.4–20. 1992). while ortho-toluidine was a plausible cause of the bladder cancer excess. the contribution of other chemicals cannot be ruled out.3–5.8. A total of 30 incident bladder cancers were identified.. The presence of a proprietary chemical was noted. 95% CI. 95% CI. All subjects had at least six months’ employment in the factory and some employment in the period 1955–84.2–4. is classified by IARC in Group 1 and is known to be a highly potent human bladder carcinogen (IARC.280 IARC MONOGRAPHS VOLUME 77 The carcinogenicity of some of the reactants. 1972. and it is therefore possible that higher levels of 4-aminobiphenyl contamination existed in the past.3). 1977)) and from its possible formation in one of the reactions in the antioxidant process.0. Levels of 4-aminobiphenyl adducts were much lower than those of ortho-toluidine and aniline and did not differ between the exposed and unexposed groups (Table 4). 4-Aminobiphenyl. which was present as a potential contaminant.

RR. 2.3. Air and urine levels and haemoglobin adduct levels measured in 1990 among chemical workers employed at a plant where excess bladder cancer incidence was observed Exposure matrix Exposure group Exposed Exposed Unexposed Exposed Unexposed Exposed Unexposed Exposed Unexposed Exposed Unexposed Exposed No. Negative results on mortality from bladder cancer might be caused by limited power due to high survival from this disease.0–56.5). 1.1) 0. (1996) Hb: haemoglobin 28 28 25 42 25 42 27 46 27 46 27 42 1. Mean of total group (SD) 187 (181) 412 (366) 3.7 (106. 95% CI.0001 0.8 (1.6–28.7 (119. ≥ 5 years.7. 1. n = 1.ortho-TOLUIDINE 281 Table 4. 7.0001 0. Individual smoking habits had not been taken into account in the analysis of most of the reported studies.4.9 (2. [The Working Group made two observations relevant to the interpretation of all five cohort studies. and the strongest evidence for an exposure–response relationship with duration of exposure to phenyl-β-naphthylamine (RR internal analysis: 1–4 years. ≥ 5 years.17–9.5.3.8 (25. 0. n = 4. 95% CI. the excesses of bladder cancer reported in the four positive studies were much too large to have been due to smoking alone. however. Therefore.4) 98.6.8) 81.0001 0.8) 29.9).5 (63.4) 3163 (1302) 17 441 (8867) 3515 (6036) 40 830 (32 518) 74.48 p value Aniline levels in air (μg/m3) ortho-Toluidine levels in air (μg/m3) Aniline levels in postshift urine (μg/L) ortho-Toluidine in post-shift urine (μg/L) Aniline adducts (pg/g Hb) ortho-Toluidine adducts (pg/g Hb) 4-Aminobiphenyl adducts (pg/g Hb) From Ward et al. n = 1. differences between results of analyses based on mortality and morbidity data might reflect the lower sensitivity of the former.6–21. RR. RR.] .0001 0.7) 2. 95% CI.

001 trend test) in control. four to six weeks of age. pooled controls. respectively.1. Groups of 50 male and 50 female B6C3F1 mice. plus additional controls used for the other compounds tested in the study. p = 0.1. the incidence of vascular tumours (haemangiomas and haemangiosarcomas combined.025. 97–99%) in the diet at dose levels of 16 000 or 32 000 mg/kg diet (ppm) for three months and then at levels of 8000 or 16 000 ppm for a further 15 months. 3. 5/99. Fisher’s exact test) in concurrent controls. In female mice. respectively. mainly observed in the abdominal viscera) was increased: 1/19. In male mice. 1978). low-dose and high-dose groups. the incidence of hepatocellular adenomas or carcinomas (combined) was increased: 0/20. four to six weeks of age. were treated with ortho-toluidine hydrochloride (purity. and tumour incidences of matched and pooled controls were compared statistically (both separately and together) with those of treated groups.007. 97–99%) in the diet at dose levels of 8000 or 16 000 ppm for three months and then at levels of 4000 or 8000 ppm for a further . Fisher’s exact test) and 9/21 (p < 0. Mean body weights of both treated males and females were lower than those of the corresponding controls. six weeks of age. 9/102. observed in abdominal viscera) was 0/15. 5/18 (p < 0. The doses were chosen on the basis of preliminary tests. respectively [the separate incidences for haemangiomas and haemangiosarcomas were not reported] (Weisburger et al. the higher being the maximum tolerated dose. low-dose and highdose groups. Fisher’s exact test. the incidence of haemangiomas or haemangiosarcomas (combined.2 Rat Groups of 25 male Sprague-Dawley CD rats.. respectively (National Cancer Institute. Cochran–Armitage trend test) in control. Fisher’s exact test) in concurrent controls. Concurrent control groups consisted of 20 male and 20 female untreated mice. low-dose and high-dose groups. observed in abdominal viscera) was 0/14.002. In male mice. Mortality was not significantly related to treatment in either sex. > 99%) in the diet at 1000 or 3000 ppm for 102–103 weeks. A simultaneous control group of 25 untreated mice of each sex was used. 3.025.1 3. 2/50 and 12/50 (p < 0. Fisher’s exact test) and 9/11 (p < 0. were administered ortho-toluidine hydrochloride (purity. the incidence of vascular tumours (haemangiomas and haemangiosarcomas combined. 1979).282 IARC MONOGRAPHS VOLUME 77 3. Animals that died during the first six months of the study were discarded without necropsy. all sites.025. 5/14 (p < 0. Animals were kept without treatment for an additional three months and then killed. pooled controls.05. were treated with ortho-toluidine hydrochloride (purity.1 Studies of Cancer in Experimental Animals Oral administration Mouse Groups of 25 male and 25 female Swiss CD-1 mice. In female mice. 4/49 and 13/50 (p < 0. low-dose and high-dose groups.

025. pooled controls. The doses were chosen on the basis of preliminary tests. respectively (Hecht et al. 18/111. 3/50 and 21/49 (p = 0. recrystallized product] in the diet at a concentration of 4000 ppm (0. low-dose and high-dose groups. respectively. were administered ortho-toluidine hydrochloride (purity. > 99%) in the diet at concentrations of 3000 or 6000 ppm for 101–104 weeks. In females. A control group of 30 untreated male rats was used. The incidence of fibromas of the skin was 1/27 and 25/30 [p < 0.025. the incidence of transitional cell carcinomas of the urinary bladder was 0/20. 3/23 and 4/24 in simultaneous controls.. and that of peritoneal sarcomas was 0/27 and 9/30 [p < 0. 5/111.001. and tumour incidences of matched and pooled controls were compared with those of treated groups. and that of mesotheliomas of multiple organs or tunica vaginalis was 0/20. Fisher’s exact test].01. that of fibromas of the spleen was 0/27 and 10/30 [p < 0.ortho-TOLUIDINE 283 15 months. fibrosarcomas.001. Fisher’s exact test) and 9/49 (p = 0.and high-dose groups.001. six weeks of age. osteosarcomas or angiosarcomas (combined) of multiple organs (mainly subcutis and spleen or bone) was 0/20.028. 17/50 (p < 0. low-dose and high-dose groups. 28/50 (p < 0. Fisher’s exact test) and 37/49 (p < 0.003. respectively. Fisher’s exact test) and 21/24 (p < 0. A concurrent control group of 25 untreated male rats was used. that of subcutaneous integumentary fibromas was 0/20.001. Fisher’s exact test). Fisher’s exact test). Fisher’s exact test) and 27/49 (p < 0. 1982). Mortality was significantly affected by treatment of male and female rats (p < 0. respectively (Weisburger et al. the higher being the maximum tolerated dose. Groups of 50 male and 50 female Fischer 344 rats. Fisher’s exact test. Mean body weights of treated male and female rats were lower than those of the corresponding controls. 1978). low.001. Fisher’s exact test) in control. The experiment was terminated at 93 weeks. compared with all controls) in simultaneous controls.001. that of mammary fibroadenomas was 0/27 and 11/30 [p < 0. Matched control groups consisted of 20 male and 20 female untreated rats. Fisher’s exact test) and 22/47 (p < 0. Animals that died during the first six months of the study were discarded without necropsy. Fisher’s exact test]. and that of mammary adenomas and fibroadenomas (combined) was 7/20. 9/45 (p = 0. The incidence of subcutaneous fibromas and fibrosarcomas (combined) was 0/16.001. plus additional controls used for the other compounds tested in the study. Mean body weights were lower in the treated than in the control group. that of sarcomas. Groups of 30 male Fischer 344 rats. In males.036. pooled controls.001. were treated with orthotoluidine hydrochloride [purity not specified..001. 20/50 and 35/49 . angiosarcomas or osteosarcomas (combined) of multiple organs (mainly subcutis and spleen or bone) was 0/20. Animals were kept without treatment for an additional six months and then killed. A nonstatistically significant increase in the incidence of transitional-cell carcinomas of the urinary bladder was also observed: 0/16. Tarone test for positive trend). 15/50 (p = 0.001. the incidence of sarcomas. Fisher’s exact test] in control and treated groups. Fisher’s exact test). fibrosarcomas. 18/23 (p < 0. Fisher’s exact test]. Fisher’s exact test).028 mol/kg of diet) for 72 weeks. eight weeks of age.

] 3. 1979). The experiment was terminated at 93 weeks. The incidence of tumours in the treated groups was not significantly different from that in the control groups [details on the incidence of specific tumours not reported] (Hecht et al.and high-dose groups.001. Mean survival times were shorter in the treated groups. respectively (National Cancer Institute. [The Working Group noted the small number of animals. No mesotheliomas were seen in concurrent controls (0/10) (National Toxicology Program. 1983).3 Carcinogenicity of metabolites Rat: Groups of 30 male Fischer 344 rats. Fisher’s exact test] in control and treated groups. Fisher’s exact test]. low. 1982). 45 days of age. were treated with ortho-nitrosotoluene [purity not specified. recrystallized product] in the diet at a concentration of 3380 ppm (0. and that of urinary bladder tumours was 0/27 and 15/29 [p < 0. recrystallized product] in peanut oil once per week for 52 weeks.. compared with 75..028 mol/kg of diet) for 72 weeks.284 IARC MONOGRAPHS VOLUME 77 (p = 0. were given subcutaneous injections of 1.5 and 68.001. being 61.8 weeks in male and female treated hamsters.3 and 57. respectively.001. A control group of 30 untreated male rats was used. 1996). Fisher’s exact test]. that of hepatocellular carcinomas was 0/27 and 18/29 [p < 0. Fisher’s exact test]. Mean body weights were lower in the treated than in the control group.006) in control. . that of fibromas of the spleen was 0/27 and 14/29 [p < 0. Mesotheliomas in the epididymis were observed in 2/20 male rats exposed to ortho-toluidine hydrocloride. Mean body weights in the treated groups were similar to those of the control groups. eight weeks of age.2 Subcutaneous injection Hamster: Groups of 15 male and 15 female Syrian golden hamsters. respectively.7 weeks in male and female controls. Control groups of 15 male and 15 female hamsters were given 52 subcutaneous injections of peanut oil vehicle.01. eight weeks of age. were administered ortho-toluidine hydrochloride at a concentration of 5000 ppm in the diet for 13 weeks and then kept for observation for a further 13 weeks. The experiment was terminated at 87 weeks. respectively (Hecht et al. when animals were killed. Male Fischer 344/N rats.9 mmol/kg bw (2 mg/kg bw) orthotoluidine (free base) [purity not specified. 3. low dose and short duration of treatment. The incidence of fibromas of the skin was 1/27 and 19/29 [p < 0.

1 4... Fischer 344 rats . while not able to determine the relative contributions of airborne versus dermal exposure.. which might in part explain the marked variability (~ 100-fold) in ortho-toluidine–haemoglobin adducts reported in unexposed groups between nonsmokers and smokers in three studies (Stillwell et al. 1986). 1987. distribution. 1980).. 4.82 mmol/kg [88 mg/kg bw] (Kulkarni et al. The amount of unchanged compound appearing in the urine within 24 h varied according to the isomer of toluidine administered at 500 mg/kg bw: 21% for ortho-toluidine. The authors suggested that nitrobenzene.. (1986) found that the levels in urine of smokers and nonsmokers were not significantly different (see Section 1. kidney.1. adduct levels were similar in smokers (40 494 pg/g Hb) and nonsmokers (41 028 pg/g Hb) in the exposed group. The major excretory route was via the urine. 2. While ortho-toluidine is a component of cigarette smoke (IARC. (1996). Ward et al. (1996). 1996).. However. Approximately 1% of the 400-mg/kg bw dose was eliminated via the lungs and ~ 3% in the faeces (Son et al. Ward et al. found strong evidence that ortho-toluidine was absorbed in an exposed group of workers at a chemical manufacturing facility (see Section 2. El-Bayoumy et al..4. metabolism and excretion Humans 4. in contrast to this finding. in the study by Ward et al. > 92% of the administered radiolabel was eliminated in the urine within 72 h. Following oral administration of a single 50-mg/kg bw dose of ortho-[methyl-14C]toluidine to male Sprague-Dawley rats. as well as in the unexposed group in smokers (3510 pg/g Hb) and nonsmokers (3518 pg/g Hb). Other Data Relevant to an Evaluation of Carcinogenicity and its Mechanisms Absorption. smoking significantly increased the level of orthotoluidine–haemoglobin adducts. They concluded that the possibility of some exposure to ortho-toluidine in the ‘unexposed’ workers could not be ruled out. after subcutaneous doses of 400 mg/kg bw (Son et al. colon and bladder. with ~ 84% of the 400-mg/kg bw dose being eliminated via this pathway within the first 48 h. 1983). In two of these studies (Stillwell et al. fumes or vapour (ILO.1 ortho-Toluidine is absorbed via the respiratory tract and skin. Bryant et al. 1980). 1988.. 1988). 1971). lung..3(e)). nitrotoluenes and dietary factors may be major contributors to ortho-toluidine levels in human urine. It can be inhaled as dust.5% for para-toluidine. 1980) or 0. whereas. Such differences may help to explain why only the ortho-isomer causes tumours in the urinary bladder of rats (Cheever et al.2 Experimental systems The tissue distribution of radioactivity in male Fischer 344 rats 48 h after subcutaneous injection of 50 or 400 mg/kg bw ortho-[methyl-14C]toluidine was mainly in the liver.2). Bryant et al.ortho-TOLUIDINE 285 4.1. 1987.5% for metatoluidine and 2. spleen.

1987). 1983). N-acetyl-ortho-aminobenzyl alcohol... The latter metabolite could undergo non-enzymatic breakdown to yield a highly reactive nitrenium ion.1%). The secondary metabolite ortho-azoxytoluene is postulated to be formed by an interaction between orthonitrosotoluene and N-hydroxy-ortho-toluidine (Son et al. 1980). binding to hepatic DNA and RNA appeared to be maximal 24–48 h after dosing (Brock et al. 1983.. However. N-acetyl-4-amino-meta-cresol (2. kidney and liver. after a single oral dose of 500 mg/kg bw ortho-toluidine. 1980. which could undergo further acetylation to form N-acetoxy-N-acetyl-orthotoluidine. spleen. Gupta et al.5% dose) and 2-amino-meta-cresol (2. ortho-nitrosotoluene. ortho-azotoluene. 1987). 1980). Son et al. N-Hydroxy-ortho-toluidine is also a urinary metabolite of male Fischer 344 rats (Kulkarni et al. The highest levels of binding were found in the blood. Gupta et al.. The major ether-extractable urinary metabolites of a 400-mg/kg dose of ortho-toluidine are azoxytoluene. A double acid conjugate of 4-amino-meta-cresol was also identified. The same authors also postulated that N-acetyl-ortho-toluidine can be metabolized to produce N-acetyl-N-hydroxy-orthotoluidine. anthranilic acid and N-acetylanthranilic acid (Son et al. but no data on its in-vivo formation exist (Gupta et al. with sulfate conjugates of 4-aminometa-cresol (27... nitrene or free radical that can covalently bind to tissue macromolecules (Figure 1). In male Crl:CD rats. On the basis of data from the Salmonella/mammalian microsomal mutagenicity assay.8%) and N-acetyl-ortho-aminobenzyl alcohol by a ratio of 6:1. 1990). (1988) have shown that administration of ortho-toluidine to male Sprague-Dawley rats induces various metabolic activities associated with the cytochrome P450 system. . respectively. N-Acetylation of ortho-toluidine is also a major metabolic pathway in male Fischer 344 rats. 1980).8% of the dose).6%).. Cheever et al. 4-amino-meta-cresol. The metabolism of ortho-toluidine in rats proceeds primarily through ring hydroxylation with subsequent conjugation (Cheever et al. After a 400-mg/kg bw dose of ortho-[methyl-14C]toluidine administered subcutaneously to male Fischer 344 rats. the suggestion that they are formed is based on results with other aromatic amines and amides (Thorgeirsson et al. 51% of urinary metabolites were conjugates. is proposed to be formed through a reaction of ortho-toluidine with ortho-nitrosotoluene. (1987) proposed that N-hydroxy-ortho-toluidine undergoes further metabolic activation via an acetylation reaction catalysed by N-acetyltransferase to form N-acetoxy-ortho-toluidine. Another putative secondary metabolite. N-acetyl-4-amino-meta-cresol (8.. N-acetyl-ortho-toluidine.286 IARC MONOGRAPHS VOLUME 77 excreted only approximately 5% of the dose in the urine as ortho-toluidine over 48 and 6 h. N-acetyl-4-amino-meta-cresol. Leslie et al. (1980) also reported finding 4-amino-3-methylphenol in the urine of rats fed ortho-toluidine.. dominating over glucuronides of 4-amino-meta-cresol (2. ortho-Toluidine can be oxidized to yield N-hydroxy-ortho-toluidine and ortho-nitrosotoluene (Son et al. there are no in-vivo data to substantiate the existence of N-acetylN-hydroxy-ortho-toluidine or N-acetoxy-N-acetyl-ortho-toluidine in rats fed orthotoluidine. 1980) (see Figure 1)..

47–0. A linear dose–response relationship was seen for [14C]ortho-toluidine binding to haemoglobin.6 mmol/kg bw [50. ortho-Toluidine forms haemoglobin adducts in female Wistar rats and female B6C3F1 mice after administration of a single oral dose of 0. (1987) Brackets indicate postulated proximate or reactive metabolites of ortho-toluidine. Son et al. The route of [14C]ortho-toluidine administration . (1980). The biological half-lives of [14C]ortho-toluidine bound to albumin and haemoglobin were calculated to be 2. 1988). N CH3 NH CH3 Nitrenium ion Nitrene Free radical Adapted from Cheever et al. In male Sprague-Dawley rats given intraperitoneal doses of [14C]ortho-toluidine ranging from 10 to 100 mg/kg bw. respectively. peak albumin binding occurred after 4 h at 50 mg/kg bw and peak haemoglobin binding at 24 h following a dose of 100 mg/kg bw (DeBord et al...6 and 12.4–64.ortho-TOLUIDINE 287 Figure 1. (1980). 1992). based on data from the metabolism of other aromatic amines. (1983) and Gupta et al.3 days.3 mg/kg bw] (Birner & Neumann. Metabolic disposition of ortho-toluidine in rats NH2 CH3 NH2 NHCOCH3 CO2H CO2H ortho-Toluidine Anthranilic acid N-Acetyl-anthranilic acid NH2 CH3 N O CH3 NHOH CH3 HO NH2 CH3 OH NHCOCH3 CH3 NHCOCH3 CH2OH N-Acetyl-o-toluidine N-Acetyl-o-amino benzyl alcohol COCH3 NOH 4-Amino-m-cresol o-Nitrosotoluene N-Hydroxy-o-toluidine 2-Amino-m-cresol NHCOCH3 O CH3 Sulfate glucuronide Sulfate N OH H 3C CH3 N CH3 N-Acetyl-N-hydroxy-o-toluidine N-Acetyl-4-amino-m-cresol o-Azoxytoluene COCH3 NOCOCH3 CH3 NOCOCH3 Sulfate glucuronide CH3 N-Acetoxy-o-toluidine N-Acetoxy-N-acetyl-o-toluidine H N+ CH3 . Kulkarni et al.

kidney and lung. 900 mg/kg bw) to male Fischer 344 rats for either five. 1978). 1976. 1996. in the liver. 1984).288 IARC MONOGRAPHS VOLUME 77 significantly affected binding to haemoglobin. including capsular and parenchymal fibrosis (Goodman et al. 4. 1983).. with rats injected intraperitoneally having approximately twofold higher [14C]ortho-toluidine–haemoglobin levels than animals treated orally. 3000 and 6000 ppm (mg/kg) ortho-toluidine hydrochloride given in the diet over 104 weeks induced proliferative mesenchymal lesions in the spleen. respectively. [The Working Group noted that many aromatic amines induce methaemoglobinaemia (Watanabe et al. 164 mg/kg bw in male rats and 246 mg/kg bw in female rats (Weisburger et al. 1984).. Histopathological examination of the livers did not reveal any overt hepatotoxic effect (Short et al. but may be associated with suboptimal fetal outcome (Fan & Steinberg. 1999).4dinitrobenzene. presumably reflecting CYP1A isoenzymes. Coleman & Coleman. haemosiderosis and bone marrow hyperplasia (after 10 days) consistent with enhanced erythrocytic destruction. 10 or 20 days led to splenic congestion. 113 mg/kg bw in female mice. as substrates (Gnojkowski et al. The effect of methaemoglobinaemia on fetal development has not been well studied. increased haematopoiesis. The activity of NADPH-cytochrome c reductase and the content of cytochrome b5 were enhanced only in the liver. ortho-Toluidine given to male Wistar rats by intraperitoneal injection (75 mg/kg bw for three consecutive days) increased the microsomal arylhydrocarbon hydroxylase activity. No significant effect was observed on epoxide hydrolase or glutathione S-transferase activity using benzo[a]pyrene 4.. 4. Administration of 225 mg/kg bw ortho-toluidine per day by gavage (estimated oral LD50..3 Reproductive and developmental effects No data were available to the Working Group. 1996). Kilpatrick & Laros.2 Experimental systems The following intraperitoneal LD50 values have been reported for ortho-toluidine hydrochloride: 179 mg/kg bw in male mice. 4.2.1 Toxic effects Humans No data were available to the Working Group.5-oxide and 1-chloro-2.2.2 4.] . In both male and female Fischer 344 rats..

including the use of the fluctuation protocol. TA1538. It was a mitochondrial ‘petite’ mutagen..1 Genetic and related effects Humans No data were available to the Working Group. There are some difficulties in evaluating the significance of much of the available data. it failed to cause intrachromosomal recombination in the same yeast strain.4 4. Although ortho-toluidine caused a recombinogenic event leading to deletion (in either the presence or absence of exogenous metabolic activation). or incorporating the addition of norharman or lithocholic acid.ortho-TOLUIDINE 289 4. At very high concentrations (20 mg per plate). in the absence of S9 mix. 1981. However. but failed to give a positive response for forward mutation in a nuclear gene. Where positive response have been seen in microbial assays. Uniformly negative results were found for reverse mutation in E. TA102. However. one positive result was reported only when exogenous metabolic activation was present and another only when it was absent. The genetic toxicology of ortho-toluidine has also been reviewed more briefly. Of eight assays for gene conversion carried out in different laboratories. TA1535 and TA1538. By far the majority of the data from bacterial or bacteriophage assay systems show negative or. ortho-Toluidine gave positive results for forward mutation in recombination-deficient strains of Bacillus subtilis. TA1537. 1991) summarizes the conclusions of these trials. they have generally required variations to the standard test procedures. this result was not reproduced in two further studies carried out in other laboratories. TA1535.4. it was differentially toxic towards Escherichia coli strains differing in capacity for recombinational repair. High concentrations of S9 mix. inconsistent data were seen in all other assays with this species of yeast. treatment with ortho-toluidine resulted in differential toxicity between repair-proficient and -deficient strains. TA98 and TA97. although there are sporadic reports of positive responses. A review (Danford. ortho-Toluidine gave positive results for induction of bacteriophage lambda. Almost all of the results were negative. TA98. but only when tested in the presence of exogenous metabolic activation. coli strains WP2 or WP2 uvrA. 1985). typhimurium strains TA100. However. only in the presence of S9 mix. it failed to induce SOS activity in Salmonella typhimurium TA1535/PSK1002. 4.4. since there seems to be substantial variation in results between different laboratories and minor variations in protocols. with strains TA100. . In Saccharomyces cerevisiae. A large series of studies have been reported using S. by Sellers and Markowitz (1992). Ashby et al. weakly positive results. in the context of carcinogenesis. at most. using lower concentrations. or special types of S9 mix may also be important.2 Experimental systems (see Tables 5 and 6 for references) The genetic toxicology of ortho-toluidine was extensively studied in two international collaborative trials for evaluation of short-term tests for carcinogens (de Serres & Ashby.

forward mutation Salmonella typhimurium TM677. TA98. TA1535. forward mutation Salmonella typhimurium TM677. DNA strand breaks or cross-links (Salmonella typhimurium TA1535/pSK1002) Escherichia coli pol A/W3110-P3478. (1981) Rosenkranz & Poirier (1979) Green (1981) Tweats (1981) Kada (1981) Skopek et al. TA1537. TA1538. SOS repair. DNA strand breaks or cross-links Prophage induction. TA98. TA1537. TA1538. TA1537. TA1537. reverse mutation Salmonella typhimurium TA100. TA1535. reverse mutation Salmonella typhimurium TA100. differential toxicity Bacillus subtilis rec strains. TA92. (1975) Simmon (1979) Tanaka et al. TA98. (1981) Liber (1985) McCann et al. TA1535. TA98. TA98. TA98. TA98. TA1537. reverse mutation Salmonella typhimurium TA100. Genetic and related effects of ortho-toluidine Test system Resultsa Without exogenous metabolic system Prophage induction. (1980) Baker & Bonin (1981) Brooks & Dean (1981) MacDonald (1981) Nagao & Takahashi (1981) Doseb (LED or HID) Reference IARC MONOGRAPHS VOLUME 77 . differential toxicity Escherichia coli rec strains. differential toxicity (liquid suspension tests) Escherichia coli rec strains. reverse mutation Salmonella typhimurium TA100.290 Table 5. TA1535. reverse mutation Salmonella typhimurium TA100. reverse mutation Salmonella typhimurium TA100. TA1537. (1987) Rosenkranz et al. TA1538. reverse mutation NT – – + ? – + NT – – – – – – – – With exogenous metabolic system + – – NT – – + – – – – – – – – – 2500 1670 250 μg/plate 20 μL/disc 2500 1000 20 μL/disc 500 500 10 000 μg/plate 1000 μg/plate 1000 μg/plate 10 μL/plate 2000 μg/plate 5000 μg/plate 1000 μg/plate Thomson (1981) Nakamura et al. SOS repair. differential toxicity Escherichia coli rec strains. forward mutation Salmonella typhimurium TA100.

reverse mutation Salmonella typhimurium TA100. TA98. TA98. TA98. forward mutation Salmonella typhimurium TA1535. reverse mutation Salmonella typhimurium TA100. TA1538. (1982) Thompson et al. reverse mutation Salmonella typhimurium TA100. reverse mutation – – NT – – – – – – NT – – – – With exogenous metabolic system – – – – – – – – +c + – – – – 10 000 μg/plate 2000 μg/plate 200 μg/plate 1000 μg/mL agar 10 000 μg/plate 1000 μg/mL 2000 μg/plate 5000 μg/plate 2000 μg/plate 480 μg/plate 250 μg/plate 300 μg/mL 1000 μg/plate 100 μg/plate Richold & Jones (1981) Rowland & Severn (1981) Sugimura et al. (1983) Baker & Bonin (1985) Falck et al. TA1537. TA1538. (1977) Doseb (LED or HID) Reference ortho-TOLUIDINE 291 . TA1537. TA1537. TA98. G46. TA1535.Table 5 (contd) Test system Resultsa Without exogenous metabolic system Salmonella typhimurium TA100. reverse mutation Salmonella typhimurium TA1535. TA98. reverse mutation Salmonella typhimurium BA13 (L-arabinose resistance). reverse mutation Salmonella typhimurium TA100. TA1538. TA1535. TA1537. TA102. C3076. TA97. reverse mutation Salmonella typhimurium TA100. TA102. TA1537. TA1538. TA1535. TA97. TA98. reverse mutation Salmonella typhimurium TA1538. TA1538. reverse mutation Salmonella typhimurium TA1535. reverse mutation Salmonella typhimurium TA100. TA1537. (1985) Matsushima et al. TA98. TA1538. (1985) Rexroat & Probst (1985) Zeiger & Haworth (1985) Dorado & Pueyo (1988) Rosenkranz & Poirier (1979) Gatehouse (1981) Zeiger & Haworth (1985) Ferreti et al. TA1535. reverse mutation Salmonella typhimurium TA100. TA98. TA1535. reverse mutation Salmonella typhimurium TA100.

genetic crossing-over Saccharomyces cerevisae. (1983) Zeiger & Haworth (1985) Gatehouse (1981) Thompson et al. reverse mutation Salmonella typhimurium TA98. reverse mutation – – NT NT NT – – – – – – + – + NT – With exogenous metabolic system – +d – – + – – – – NT NT + – NT – – 100 μg/plate 10 μg/mL 200 μg/plate 50 μg/plate 2. reverse mutation Escherichia coli WP2 and WP2 uvrA. gene conversion Saccharomyces cerevisiae.5 μg/plate 1000 μg/plate 1000 1000 1000 504 2520 300 333 μg/plate 50 2 μL/mL 500 Garner & Nutman (1977) Gatehouse (1981) Nagao et al. gene conversion. (1983) Falck et al. reverse mutation Salmonella typhimurium TA1538. reverse mutation Salmonella typhimurium TA98. reverse mutation Escherichia coli WP2 uvrA. reverse mutation Escherichia coli WP2 uvrA. (1978) Kawalek et al. gene conversion Saccharomyces cerevisiae.292 Table 5 (contd) Test system Resultsa Without exogenous metabolic system Salmonella typhimurium TA1538. (1977) Nagao et al. (1981) Sharp & Parry (1981b) Zimmermann & Scheel (1981) Arni (1985) Doseb (LED or HID) Reference IARC MONOGRAPHS VOLUME 77 . (1985) Carere et al. (1985) Carere et al. reverse mutation Aspergillus nidulans. forward mutation Aspergillus nidulans. (1985) Sharp & Parry (1981a) Jagannath et al. reverse mutation Salmonella typhimurium TA98. TA97. reverse mutation Salmonella typhimurium TA98. differential toxicity Saccharomyces cerevisiae. gene conversion Saccharomyces cerevisiae. DNA repair-deficient strains.

(1985) Fujikawa et al. (1985) Parry & Eckardt (1985a) Carls & Schiestl (1994) Carls & Schiestl (1994) Ferguson (1985) Mehta & von Borstel (1981) Harrington & Nestmann (1985) Mehta & von Borstel (1985) Mehta & von Borstel (1985) Parry & Sharp (1981) Parry & Eckardt (1985b) Zimmermann et al.94 mM in feedf 10 700 1 mM in feed Brooks et al. reverse mutation Saccharomyces cerevisiae. genetic crossing-over. aneuploidy Drosophila melanogaster. gene conversion. gene conversion.5 μL/mL 2 mM in feed 0. forward ‘petite’ mutation Saccharomyces cerevisiae. gene conversion Saccharomyces cerevisiae. genetic recombination Drosophila melanogaster. gene conversion Saccharomyces cerevisiae strain XV185-14C & RM52. somatic mutation (and recombination) Drosophila melanogaster. aneuploidy Saccharomyces cerevisiae. somatic mutation (and recombination) – – – + – + – – – + + + – + + (+) + With exogenous metabolic system – – – + – NT – – +e +e + + NT 2000 1000 500 1000 5000 2500 2222 21.2 378 378 50 NR 1.Table 5 (contd) Test system Resultsa Without exogenous metabolic system Saccharomyces cerevisiae. deletion assay Saccharomyces cerevisiae. forward/reverse mutation Saccharomyces cerevisiae. aneuploidy Saccharomyces cerevisiae. interchromosomal recombination Saccharomyces cerevisiae. reverse mutation Saccharomyces cerevisiae. strain XV185-14C. reverse mutation Saccharomyces cerevisiae. (1985) Vogel (1985) Würgler et al. reverse mutation Saccharomyces cerevisiae strain D7-144. somatic mutation or recombination Drosophila melanogaster. (1985) Vogel (1985) Doseb (LED or HID) Reference ortho-TOLUIDINE 293 . (1985) Inge-Vechtomov et al.

(1985) Barfknecht et al. golden Syrian hamster primary hepatocytes in vitro + – + + + – – – – – – – NT + (+) NT NT NT NT NT NT With exogenous metabolic system Doseb (LED or HID) Reference IARC MONOGRAPHS VOLUME 77 1 mM in feed 2 mM in feed 5 mM in feed 319 4280 2140 54 10. cross-links or related damage. (1994) Batiste-Alentorn et al. Chinese hamster ovary cells in vitro Unscheduled DNA synthesis. rat primary hepatocytes in vitro Unscheduled DNA synthesis. wing-spot test DNA strand breaks. cross-links or related damage. (1987) Kornbrust & Barfknecht (1984) . somatic mutation Drosophila melanogaster. (1985) Lakhanisky & Hendrickx (1985) Thompson et al.7 10. cross-links or related damage.294 Table 5 (contd) Test system Resultsa Without exogenous metabolic system Drosophila melanogaster. Sprague-Dawley rat primary hepatocytes in vitro Unscheduled DNA synthesis.7 Batiste-Alentorn et al. rat primary hepatocytes in vitro Unscheduled DNA synthesis. (1995) Bradley (1985) Douglas et al. (1991) Batiste-Alentorn et al. Chinese hamster ovary cells in vitro DNA strand breaks.7 53. (1983) Kornbrust & Barfknecht (1984) Probst & Hill (1985) Williams et al. rat hepatocytes in vitro DNA strand breaks. Sprague-Dawley rat primary hepatocytes in vitro Unscheduled DNA synthesis. rat primary hepatocytes in vitro Unscheduled DNA synthesis. somatic mutation (and recombination).5 10 10. somatic mutation (and recombination) Drosophila melanogaster.

(1985) Garner & Campbell (1985) Styles et al. (1985) Lee & Webber (1985) Kuroki & Munakata (1985) Amacher & Turner (1985) Knaap & Langebroek (1985) Lee & Webber (1985) Myhr et al. mouse lymphoma L5178Y cells. ouabain or thioguanine resistance in vitro Gene mutation. oubain resistance in vitro Gene mutation. ouabain resistance. Tk locus in vitro Gene mutation. Chinese hamster lung V79 cells. Balb/c 3T3 cells.Table 5 (contd) Test system Resultsa Without exogenous metabolic system Unscheduled DNA synthesis. Hprt locus. Chinese hamster cells in vitro – – – (+) – – – – – + – (+) – – NT + With exogenous metabolic system NT – NT – – – – – – NT – – + – (+) – 10. Chinese hamster lung V79 cells. (1985) Oberly et al. (1985) Matthews et al. Tk locus and Hprt locus in vitro Gene mutation. Chinese hamster ovary cells in vitro Gene mutation. mouse lymphoma L5178Y cells. Chinese hamster lung V79 cells Hprt locus in vitro Gene mutation. (1985) Styles et al. (1987) Zdzienicka & Simons (1985) Fox & Delow (1985) Kuroda et al. (1985) Perry & Thomson (1981) Doseb (LED or HID) Reference ortho-TOLUIDINE 295 . Chinese hamster lung V79 cells Hprt locus in vitro Gene mutation. in vitro Gene mutation. mouse lymphoma L5178Y cells. golden Syrian hamster primary hepatocytes in vitro Gene mutation. Tk locus in vitro Gene mutation. mouse lymphoma L5178Y cells. Tk locus in vitro Gene mutation.7 500 2000 500 NR 535 800 1. mouse lymphoma L5178Y cells. Tk locus in vitro Gene mutation.3 μL/mL NR 300 500g 1004 200 1004 250 300 Barfknecht et al. ouabain resistance in vitro Sister chromatid exchange. Hprt locus in vitro Gene mutation. mouse lymphoma L5178Y cells. Tk locus in vitro Gene mutation. mouse lymphoma L5178Y cells. mouse lymphoma L5178Y cells. ouabain or trifluorothymidine resistance.

Chinese hamster cells in vitro Chromosomal aberrations. (1985) Lane et al. Chinese hamster ovary cells in vitro Micronucleus formation. (1985) Palitti et al. Chinese hamster cells in vitro Sister chromatid exchange. BALB/c3T3 mouse cells Cell transformation. RL4 rat liver cells in vitro Micronucleus formation.8 1070 NR 12 250 1000 2140 300 700 60 600 150 500 Douglas et al. Chinese hamster cells in vitro Sister chromatid exchange. (1985) Priston & Dean (1985) Danford (1985) Lawrence & McGregor (1985) Matthews et al. C3H/10T½ mouse cells Cell transformation. Chinese hamster cells in vitro Chromosomal aberrations. Chinese hamster cells in vitro Chromosomal aberrations. Chinese hamster cells in vitro Chromosomal aberrations. (1985) Natarajan et al. Syrian hamster embryo cells in vitro Chromosomal aberrations. (1985) Nesnow et al. (1985) van Went (1985) Priston & Dean (1985) Douglas et al. Chinese hamster cells in vitro Sister chromatid exchange. Chinese hamster CH1-L liver fibroblasts in vitro Cell transformation. RL4 rat liver cells in vitro Aneuploidy. (1985) .296 Table 5 (contd) Test system Resultsa Without exogenous metabolic system Sister chromatid exchange. Chinese hamster ovary cells in vitro Sister chromatid exchange. C3H/10T½ mouse cells – + + – + + – + + + – – – + + – – + With exogenous metabolic system Doseb (LED or HID) Reference IARC MONOGRAPHS VOLUME 77 – + – – + NT – NT NT + + – (+) NT NT (+) +h NT 1070 50 500 2140 268 21. (1985) Fritzenschaf et al. (1985) Gulati et al. (1993) Danford (1985) Gulati et al. Chinese hamster CH1-L liver fibroblasts in vitro Chromosomal aberrations. (1985) Ishidate & Sofuni (1985) Natarajan et al. Chinese hamster cells in vitro Sister chromatid exchange.

(1985) Obe et al. human TK6 cells in vitro Gene mutation. baby hamster kidney BHK-21 cells Cell transformation. (1999) Crespi et al. (1985) Suk & Humphreys (1985) Hatch & Anderson (1985) Martin et al. clonal assay Cell transformation. SA7/Syrian hamster embryo cells DNA strand breaks (Comet assay). Syrian hamster embryo cells. Syrian hamster embryo cells. Chinese hamster ovary cells Cell transformation. human AHH-1 cells in vitro Sister chromatid exchange. clonal assay Cell transformation. (1985) Doseb (LED or HID) Reference ortho-TOLUIDINE 297 . clonal assay Cell transformation. baby hamster kidney BHK-21 cells Cell transformation. Syrian hamster embryo cells.Table 5 (contd) Test system Resultsa Without exogenous metabolic system Cell transformation. RLV/Fischer rat embryo cells Cell transformation. (1985) Crespi et al. (1998) Daniel & Dehnel (1981) Styles (1981) Zdzienicka et al. MCL-5 cells Gene mutation. human lymphocytes in vitro + + + + NT – (+) (+) + – + ? With exogenous metabolic system NT NT NT + + – NT NT NT + NT – 1 100 750 NR 25 500 10 965 454 450 300 600 Barrett & Lamb (1985) Sanner & Rivedal (1985) Kerckaert et al.

298 Table 5 (contd) Test system Resultsa Without exogenous metabolic system Sister chromatid exchange. RNA or protein. (1989) Brock et al. (1980) Darroudi & Natarajan (1985) Cesarone et al. (1989) McFee et al. (1989) Salamone et al. cross-links or related damage. Chinese hamster ovary cells in vitro DNA strand breaks. Crl:CD rat liver in vivo + + – + + (+) + – – – – + + With exogenous metabolic system NT – +c 21. B6C3F1 mouse bone marrow cells in vivo Sister chromatid exchange. B6C3F1 mice bone marrow cells in vivo Micronucleus test. typhimurium TA98) Body fluids from WAG/Rij rats (plasma).4 214 300 po × 1 400 ip × 1 100 200 600 338 ip × 2 160 ip × 2 300 ip × 1 300 ip × 1 20 μg/mL 500 po × 1 Lindahl-Kiessling et al. (1982) Neal & Probst (1983) McFee et al. animal cells in vivo Sister chromatid exchange. (1989) Vian et al. (1993) Tanaka et al. (1990) Doseb (LED or HID) Reference IARC MONOGRAPHS VOLUME 77 . B6C3F1 mice bone marrow cells in vivo Micronucleus formation. animal cells in vivo Micronucleus formation. (1981) Tsuchimoto & Matter (1981) McFee et al. CD-1 mice in vivo Chromosomal aberrations. B6C3F1 mice in vivo Micronucleus formation. Pleurodeles waltl in vivo Binding (covalent) to DNA. microbial mutagenicity (S. human lymphocytes in vitro Body fluids from Sprague-Dawley rats (urine). sister chromatid exchange. human lymphocytes in vitro Micronucleus formation. (1989) Fernandez et al.

highest ineffective dose. ip. –. positive. HID. negative. intraperitoneal. lowest effective dose. in-vivo test. (CBA × BALB/c)F1 mice in vivo Sperm morphology. Chinese hamster V79 cells rodent in vitro Inhibition of intercellular communication. (CBA × BALB/c)F1 mice in vivo a Doseb (LED or HID) With exogenous metabolic system NT NT NT 5 5 535 250 ip × 5 400 ip × 5 Reference + + – ? – Elmore et al. in-vitro test. μg/mL. ?. (1985) Topham (1981) Topham (1980) +. NT. (1985) ortho-TOLUIDINE Scott et al. not with rat liver S9 d S9 from phenobarbital-treated rats e Activity detected in YEPD medium f Acute feeding g Toxicity higher in the presence of S9 h Activation by co-cultivation with X-irradiated primary rat hepatocytes i Growth of V79 (T2-14) 6-thioguanine-resistant cells b 299 . NR. weakly positive. po. not tested. (+).Table 5 (contd) Test system Resultsa Without exogenous metabolic system Inhibition of intercellular communication. oral c Active only with 30% hamster liver S9. Chinese hamster V79 cells rodent in vitroi Sperm morphology. (1985) Umeda et al. Chinese hamster V79 cells rodent in vitro Inhibition of intercellular communication. mg/kg bw/day. not reported LED. inconclusive.

62 μg/plate 3. (1987) Gupta et al. reverse mutation Salmonella typhimurium TA98. TA98. reverse mutation N-Acetoxy-N-acetyl-ortho-toluidine Salmonella typhimurium TA100.75 μg/plate 4. highest ineffective dose . HID. (1987) Gupta et al. (1987) Gupta et al. TA98.2 μg/plate Gupta et al. reverse mutation N-Acetyl-N-hydroxy-ortho-toluidine Salmonella typhimurium TA100. –.16 μg/plate 0.1 μg/plate 2. (1987) +. lowest effective dose. reverse mutation N-Acetyl-ortho-toluidine Salmonella typhimurium TA100. reverse mutation a b Doseb (LED or HID) With exogenous metabolic system Reference IARC MONOGRAPHS VOLUME 77 – – – – – – + – – – + – 0. positive.300 Table 6.1 μg/plate 5. (1987) Gupta et al. negative LED. reverse mutation Salmonella typhimurium TA98. (1987) Gupta et al. Genetic and related effects of metabolites of ortho-toluidine Test system Resultsa Without exogenous metabolic system N-Hydroxy-ortho-toluidine Salmonella typhimurium TA100.

although two other similar studies gave negative results. Only one out of four studies in mice. there are isolated reports of ortho-toluidine increasing gene mutations at loci other than Tk in L5178Y cells or in other animal cells in vitro. apparently in the absence of exogenous metabolic activation. as well as in primary cultures of cells isolated from human breast milk. ortho-Toluidine caused aneuploidy in mammalian cells in vitro. in either animal or human cells. ortho-Toluidine caused DNA strand breakage in various animal cell lines in vitro. However. On the basis of these data. Metabolites of ortho-toluidine Gupta et al. but one study in a newt model. they proposed a metabolic . Assays for forward mutation or genetic crossing-over in Aspergillus nidulans gave completely negative results. Studies on sperm morphology have given equivocal data. even in the absence of exogenous metabolic activation. Manifestation of these effects seems to require incubation times longer than 3 h. The response was substantially increased when the cells were incubated in the presence of the DNA repair inhibitors. and enhanced sister chromatid exchanges in rodent models. A number of in-vivo studies have been conducted. Only one out of eight studies showed that treatment with ortho-toluidine could lead to unscheduled DNA synthesis. although it should be noted that the cell types have some endogenous metabolic capability. In some of these studies. have revealed positive results. but only in the presence of exogenous metabolic activation. as did a forward mutation assay in Schizosaccharomyces pombe. suggested that it enhanced micronucleus frequency. Most studies of effects on sister chromatid exchanges. A single study suggested a weak positive effect in gene mutation at the Hprt locus in V79 Chinese hamster cells. MCL-5. hydroxyurea and cytosine arabinoside. The latter effects did not generally appear to require exogenous metabolic activation. ortho-toluidine inhibited intracellular communication. while in others it appeared to reduce the effect.ortho-TOLUIDINE 301 It gave a positive result in one out of seven assays for reverse mutation. ortho-Toluidine gave a positive result in a host-mediated assay for bacterial mutagenesis. and increased cell transformation in all but one of 11 studies. S9 mix was required. In two out of three studies. The alkaline single-cell gel electrophoresis (‘comet’) assay revealed DNA breakage after ortho-toluidine treatment in a metabolically competent human mammary cell line. It increased somatic mutation but not genetic crossing-over in Drosophila melanogaster. There have been occasional reports of ortho-toluidine causing chromosomal aberrations or micronuclei in various cultured cell lines. Two out of five studies suggested a positive response at the Tk locus but not usually at other loci in mouse lymphoma L5178Y cells. However. ortho-Toluidine failed to cause mutation to ouabain resistance in V79 Chinese hamster cells. it caused aneuploidy in two out of three assays. (1987) synthesized the various N-oxidized putative metabolites and esters of ortho-toluidine and tested them for mutagenic activity in the Salmonella microsome mutagenicity assay.

optical brighteners. In each case. it induced an increased incidence of haemangiomas and haemangiosarcomas and hepatocellular carcinomas .5 Mechanistic considerations ortho-Toluidine undergoes extensive metabolism in vivo and. 5. In none of these studies. although co-exposures differed between studies. pharmaceuticals and pesticides. The fifth study had limited power to detect a risk. however. 5. 4.2 Human carcinogenicity data Five studies were available for evaluation. rubber chemicals. N-hydroxylation is thought to be the first step in its metabolic activation.302 IARC MONOGRAPHS VOLUME 77 pathway involving nitrenium ion/nitrene/free radicals which could bind covalently to DNA (see Table 6). Two recent cohort studies in Germany. like other aromatic amines. Human exposure has been reported during its use in production of dyestuffs and rubber chemicals. the United Kingdom and a larger study in the United States among workers in 4-chloro-ortho-toluidine production and in rubber chemical manufacturing looked at bladder cancer incidence. the subgroups of workers exposed to ortho-toluidine were small. the highest risk was observed in the subgroup with the longest duration of exposure. 5. Of these five studies. Two mortality studies were conducted in the 1980s among dye production workers in Italy and in the United States. pigments. After oral administration to mice. Nonoccupational exposure to ortho-toluidine may result from its occurrence in certain foods and from exposure to tobacco smoke. In the two studies with data on duration of exposure to ortho-toluidine. four observed a very high excess of bladder cancer among ortho-toluidine-exposed workers. 5.3 Animal carcinogenicity data ortho-Toluidine was tested for carcinogenicity as its hydrochloride salt in two experiments in mice and in three experiments in rats and as the free base in one limited experiment in hamsters. could confounding by concomitant exposure to various other potential bladder carcinogens be ruled out with confidence.1 Summary of Data Reported and Evaluation Exposure data ortho-Toluidine and its hydrochloride salt have been widely produced commercially throughout the twentieth century for use in manufacture of dyestuffs.

mammary fibroadenomas and transitional-cell carcinomas of the urinary bladder.4 Other relevant data ortho-Toluidine undergoes extensive metabolism in vivo. repeated administration of ortho-toluidine led to haemosiderosis. 5. orthotoluidine caused reverse mutation at some loci and occasionally recombinational events. In the yeast Saccharomyces cerevisiae. Bacterial or bacteriophage assay systems showed negative or inconsistent data or. 5. Overall evaluation ortho-Toluidine is probably carcinogenic to humans (Group 2A). It has been demonstrated to be a mutagen but not a recombinogen in Drosophila melanogaster. sarcomas. In rats. Evidence that ortho-toluidine undergoes metabolic activation in vivo is supported by the fact that it forms both haemoglobin and albumin adducts in laboratory animals and haemoglobin adducts in humans. including fibromas. weakly positive results. mesotheliomas.ortho-TOLUIDINE 303 or adenomas.5 Evaluation There is limited evidence in humans for the carcinogenicity of ortho-toluidine. bone marrow and splenic proliferation and splenic fibrosis consistent with a response to erythrocyte destruction. It caused gain or loss of whole chromosomes and mutation of mitochondrial DNA. at most. There is sufficient evidence in experimental animals for the carcinogenicity of ortho-toluidine. In rats. it generally caused sister chromatid exchanges and sometimes also increased gene mutations. orthoToluidine may inhibit intercellular communication. leading to covalent binding to tissue macromolecules. splenic congestion. In cultured mammalian cells. In rodent models in vivo. it enhanced sister chromatid exchanges but gave equivocal results for micronuclei and sperm morphology. It induced aneuploidy and increased cell transformation in such cells. with the bulk of the dose being excreted in the urine within 24 h. oral administration of ortho-toluidine increased the incidence of tumours in multiple organs. Like other aromatic amines. it is thought to undergo metabolic activation initially via N-hydroxylation. . chromosomal aberrations and micronuclei.

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B. Evaluation of Short-Term Tests for Carcinogens. Elsevier Shackelford.D. & Shelby.S. D. Everett. Finnish Institute of Occupational Health. W. In: Ashby..J. Evaluation of Short-Term Tests for Carcinogens. 3. 998–1002 Sorahan. D. Athens.R.M. 893–899 Skopek. T. J. Med. Hamilton. P. Evaluation of Short-Term Tests for Carcinogens. (1999) ASA 1997 [Reviews 140]. phenyl-beta-naphthylamine and otoluidine. F. W. Ishidate. & Nelmes.. 47–48 (in Finnish) Scott. T. Kaden. Report of the International Programme on Chemical Safety’s Collaborative Study on in vitro assays. C. pp. 62. F.. C. Xenobiotica.. 502–516 Sharp. & Parry. & Thilly. 613–618 Sellers. & Jackson.. GA.. J. & Parry. Amsterdam. Volume 5.J. D.. pp. Occup.. 301–316 de Serres. B. aniline.M. 371–375 Son. M. eds. L.. Toxicol. D. Evaluation of Short-Term Tests for Carcinogens. E. (2000) A further cohort study of workers employed at a factory manufacturing chemicals for the rubber industry. J.. (1981) Mutagenic activity of 42 coded compounds using 8-azaguanine resistance as a genetic marker in Salmonella typhimurium. Andon. J.C. Progress in Mutation Research. O.. Br. Elsevier Science. B. 50. pp. J. 665–671 Savela. J. 35. (1992) Reevaluating the carcinogenicity of ortho-toluidine: a new conclusion and its implications. natl Cancer Inst. eds.C.E. Pharmacol.J. eds. pp. & Ashby.W.G.. Amsterdam. Regul. In: de Serres. & Markowitz. F. L. J. & Rivedal. (1979) In vitro mutagenicity assays of chemical carcinogens and related compounds with Salmonella typhimurium. Volume 5.. 16. de Serres.D. J.M. 57. (1981a) Use of repair-deficient strains of yeast to assay the activity of 40 coded compounds. Report of the International Collaborative Program. M. A. M. pp. Med. T. Evaluation of Short-Term Tests for Carcinogens. with special reference to the chemicals 2-mercaptobenzothiazole (MBT). Ishidate. & Ashby.A. J.W. M.J.J.J.. (1981b) Induction of mitotic gene conversion by 41 coded compounds using the yeast culture JD1 (Chapter 44). T. 491–501 Short. F. Volume 1. Jr.M. H. Progress in Mutation Research. (1976) Frequency of Organic Compounds Identified in Water (EPA-600/4-76-062). Matter. J.. Elsevier Science. B. Fundam. J. & Fiala. R. appl. & Ashby.. & Keith. Davidson.. pp. Environmental Research Laboratory.. 457–468 Sorahan.H. Jr... (1980) Metabolism of o-[methyl-14C]toluidine in the F344 rat.. & Ashby.. F.. C. environ. Report of the International Collaborative Program. D.F. eds. Margolin. Progress in Mutation Research. Elsevier Science. B. & Kauppinen. Amsterdam. J. Matter. Report of the International Programme on Chemical Safety’s Collaborative Study on in vitro assays.S.. Amsterdam. K. Report of the International Collaborative Program. S. Draper. Elsevier Science.H.R. 285–292 Simmon. Vuorela.M. Amsterdam. (1985) Assays for inhibition of metabolic cooperation between mammalian cells in culture. In: de Serres. V. Helsinki. Amsterdam. Volume 1.K.H. (1985) Tests with the Syrian hamster embryo (SHE) cell transformation assay. de Serres. eds (1981) Progress in Mutation Research. Elsevier Science.. pp. T.E. Progress in Mutation Research.. 106–115 .J. Margolin. E. (1983) Subacute toxicity of several ringsubstituted dialkylanilines in the rat.. eds. King.R.. Progress in Mutation Research. ind. M. Draper. & Pope. & Kerr. Report of the International Collaborative Program. M.318 IARC MONOGRAPHS VOLUME 77 Sanner. Evaluation of Short-Term Tests for Carcinogens. & Shelby. Sistrunk. F. 10. A. In: de Serres. Toxicol. Volume 1.. In: Ashby. (1993) Mortality study of workers employed at a plant manufacturing chemicals for the rubber industry: 1955–86. 226 Sharp. Volume 1.

Volume 5. Int. 136B. mutagenicity and carcinogenicity of aromatic amines. Arch.E. J. F. F... K. D. Nagao.F. 16. E. T. Environ. & Shelby.. 5. & Ashby. J.. & Mii....W. & Humphreys.W.K. F. Hill. & McManus...B.E.. J. J. D. A. Matter. 803–811 Thorgeirsson. & Ward. M.S.S. J. 349–387 Topham. Med. B. Draper. & Wishnok. pp.. Environ. & Morris. Marui. In: Ashby. Dankovic.H.ortho-TOLUIDINE 319 Stasik. (1983) The induction of bacterial mutation and hepatocyte unscheduled DNA synthesis by monosubstituted anilines. 5. Mutagen. (1981) Mutagenic activity of 42 coded compounds in the lambda induction assay. W. 79...A.E.. Progress in Mutation Research. Amsterdam. 221–227 Stuermer... In: de Serres. B. Biomed.A. T. 14. (1982) Organic contaminants in groundwater near an underground coal gasification site in northeastern Wyoming.J.. (1980) Do induced sperm-head abnormalities in mice specifically identify mammalian mutagens rather than carcinogens? Mutat. (1985) Assays for the induction of gene mutations at the thymidine kinase and the Na+/K+ ATPase loci in two different mouse lymphoma cell lines in culture. M. Biochem. occup. (1983) Metabolism. Evaluation of Short-Term Tests for Carcinogens. J. Exp. J. Report of the International Programme on Chemical Safety’s Collaborative Study on in vitro assays.A.J. Elsevier Science.H.. W. & Cross. Amsterdam. C. environ.S. 21–24 Stillwell. Stettler. In: de Serres. I. Ishidate. Application to human dosimetry.. Margolin. 60. Technol. C. Report of the International Collaborative Program. eds. & Ashby. Progress in Mutation Research. Volume 1. pp.J.. M. 582–587 Styles. M. pp. M. D. Amsterdam. M.J. Report of the International Collaborative program. Volume 1.Z. Int. In: Ashby.. M. (1993) Biological monitoring for occupational exposures to o-toluidine and aniline.. pp. B. 1011–1025 Suk.. & Shelby. Elsevier Science.E... (1982) Metabolic aspects of the comutagenic action of norharman. J. Progress in Mutation Research. Health. Res.. (1980) Mutagenicity of extracts of urine from rats treated with aromatic amines. Evaluation of Short-Term Tests for Carcinogens. Toxicol. Glowinski.E. & Probst. Draper. Progress in Mutation Research. S. 65. eds. S115–S118 Thomson. F. W. 73–176 Teass. P. Cheever.S.J. occup. Savage. 224–235 Thompson. K. & Wakabayashi.. Adv. 74.E. Elsevier Science. Amsterdam.. de Serres. M. Biol. DeBord.. Weigel. (1988) Carcinomas of the urinary bladder in a 4-chloro-o-toluidine cohort.K.G.C.. de Serres.D. (1981) Activity of 42 coded compounds in the BHK-21 cell transformation test. Bryant. R. environ. J.. Epp. K. Arch. eds.. 673–683 Tanaka... Brown. Volume 5.G. J. Evaluation of Short-Term Tests for Carcinogens. 638–646 Styles. M. Margolin. Mutat. Res. Jr.. 379–387 .J.D. D. Clay. M. Jr.A. B. L.H. S.E. Ishidate. G. environ. Mass Spectrom..L. Rev. M.J. Sci. L. Ng. eds. K. Report of the International Programme on Chemical Safety’s Collaborative Study on in vitro assays. Health. (1985) Assay for the carcinogenicity of chemical agents using enhancement of anchorage-independent survival of retrovirus-infected Fischer rat embryo cells. Elsevier Science. Matter. (1987) GC/MS analysis of biologically important aromatic amines. 587–596 Sugimura. Evaluation of ShortTerm Tests for Carcinogens. J.

G. Volume 1. Ishidate. (1985) Assays for inhibition of metabolic cooperation by a microassay method. F. Draper. Elsevier Science. M. B. eds. Markowitz. Ruder. 93–102 Vitzthum. 501–506 Ward. Roberts. J.. (1996) Handbook of Environmental Data on Organic Chemicals. Elsevier Science. Progress in Mutation Research. 619–622 United Nations Environment Programme (1999) Recommendations and Legal Mechanisms [http://dbserver.. D.. Dankovic. Noda. Roberts..E. B. Progress in Mutation Research.J.. & Streicher. F. Amsterdam. (1981) Activity of coded compounds in the micronucleus test.G.. Teass. (1993) The in vitro micronucleus test on isolated human lymphocytes. Progress in Mutation Research. M. S.W. Food Chem. G. (1981) Activity of 42 coded compounds in a differential killing test using Escherichia coli strains WP2. Matter. 291.D. Elsevier Science.H. E. N.W. Volume 1.J. & Ashby...D. Sabbioni. O.. Margolin. D.. Jr. Res. eds. 999–1003 Vogel. pp. natl Cancer Inst..get_search] Verschueren. (1981) Evaluation of some chemicals by the sperm morphology assay. A.K. Mutat. Evaluation of Short-Term Tests for Carcinogens.. de Serres. (1991) Bladder cancer in workers exposed to aniline (Letter to the Editor). Progress in Mutation Research.H. Werkhoff. B.J.J. M. M. Bichet. Report of the International Programme on Chemical Safety’s Collaborative Study on in vitro assays. M. Elsevier Science.. 313–317 Ward.. Reed. J. 3rd Ed. Van Nostrand Reinhold. B. & Ashby.. E. J. Draper. Talaska.P. J.J. E. In: de Serres. K. Elsevier Science. L. Amsterdam.R. Matter.J.E.320 IARC MONOGRAPHS VOLUME 77 Topham. W. D.. J. & Gouy D.. In: de Serres.. K. 83. F.. 88. 705–711 Tweats. (1975) New volatile constituents of black tea aroma.irptc. Report of the International Programme on Chemical Safety’s Collaborative Study on in vitro assays.. 83. & Halperin.E. 1735–1737 Vian. J. natl Cancer Inst. WP67 (uvrA polA).. F. Volume 5. & Ashby..C. A. Carpenter. Amsterdam.. Margolin. eds. M... In: Ashby. E. J. B. Volume 1. P. 86. D. (1994) A re-examination of the cause of excess bladder cancers in chemical plant workers (Letter to the Editor). P. de Serres. Evaluation of Short-Term Tests for Carcinogens.. 1507–1508 Ward. & Tanaka..M.M. DeBord.A. pp. 60–62 Ward.. Evaluation of Short-Term Tests for Carcinogens.. natl Cancer Inst. & Fajen. In: de Serres.. & Shelby. J. (1991) Excess number of bladder cancers in workers exposed to ortho-toluidine and aniline. K. 1046–1052 .. D. Amsterdam. J. J. Volume 5.. D. & Hubert. eds. & Dankovic. 718–720 Tsuchimoto & Matter. Evaluation of Short-Term Tests for Carcinogens. pp. J. K.M. & Shelby. natl Cancer Inst. Progress in Mutation Research. G. F.. Brown. E. New York. agric... J. A. M. Report of the International Collaborative Program. (1996) Monitoring of aromatic amine exposure in workers at a chemical plant with a known bladder cancer excess. L. D.G. In: Ashby. 199–209 Umeda. J.ch:8887/irptc/owa/lg. Report of the International Collaborative Program.unep. Evaluation of Short-Term Tests for Carcinogens. and CM871 (uvrA lexA recA). Roberts.. pp.. Ishidate. pp. eds. Jr. R. Flesch. 23.. Amsterdam. pp. Report of the International Collaborative Program.. (1985) The Drosophila somatic recombination and mutation assay (SRM) using the white-coral somatic eye color system.

.D.J. & Shelby. Margolin... Evaluation of Short-Term Tests for Carcinogens. F.K. F.I.B. C.E.E.E. B. B. & Shelby. B.. Volume 5. Margolin... B.. (1985) Tests with the rat hepatocyte primary culture/ DNA-repair test. Draper. C. Jr.H.C. O’Donnell. E.. eds. (1985) The test for sister-chromatid exchanges in Chinese hamster V79 cells in culture. M..H. R. Progress in Mutation Research. & Chu. Elsevier Science. Margolin. M.. (1985) Assays for the induction of cell transformation in Chinese hamster ovary (CHO) cells and in Syrian hamster embryo (SHE) cells. Matter. pp. A. Ishidate. Draper. M.. Volume 5. In: Ashby. Ishidate.. Draper. M. (1976) Water-soluble components of four fuel oils: chemical characterization and effects on growth micro algae. Elsevier Science. 341–345 Winters. J.H. M... Report of the International Programme on Chemical Safety’s Collaborative Study on in vitro assays.G.I. J. (1985) Somatic mutation and recombination test in wings of Drosophila melanogaster. H. G. J..M. Ishidate.J.. B. M. & Ikeda. & Shelby. & Simons.. Volume 5. J.D. Elsevier Science. M. Progress in Mutation Research. eds.H. J... pp.. & Shelby. J. Matter.. & Shelby. Volume 5. E.W. (1978) Testing of twenty-one environmental aromatic amines or derivatives for long-term toxicity or carcinogenicity. Int. Matter. Report of the International Programme on Chemical Safety’s Collaborative Study on in vitro assays. J. environ. B. S. B. eds. 36.... J. pp. Volume 5. Jr.6-trinitrobenzene and other nitro-amino derivatives of benzene and chlorobenzene. 269–276 Würgler. T. K. Progress in Mutation Research. K. U.Z.. Homburger.J... Draper. & Ved Brat. E.E..J. 2. Evaluation of Short-Term Tests for Carcinogens. Elsevier Science. Matter. M. 157–168 Weisburger. environ..J. Amsterdam. 583–586 Zdzienicka. Marine Biol. Health. & Van Baalen. 325–340 Zdzienicka. de Kok.ortho-TOLUIDINE 321 Watanabe. Elsevier Science. Russfield. Weisburger. M..D. Progress in Mutation Research. J.F.. Amsterdam.. M. Ishidate. Amsterdam. M. B. M. Toxicol. (1985) Assays for the induction of mutations to 6-thioguanine and ouabain resistance in Chinese hamster ovary (CHO) cells in culture. 469–477 Williams. F. Jr. (1985) Tests with a preincubation modification of the Salmonella/ microsome assay... In: Ashby. Arch. de Serres. In: Ashby. 685–688 Zeiger. S. Report of the International Programme on Chemical Safety’s Collaborative Study on in vitro assays.. eds. B. B. Ishidate. Jr. 187–199 . M. pp. G. Volume 5... Progress in Mutation Research.. M.. Pathol. Report of the International Programme on Chemical Safety’s Collaborative Study on in vitro assays.E. Jr. 37. Batterson. Report of the International Programme on Chemical Safety’s Collaborative Study on in vitro assays. C. Amsterdam.Z. & Frei. eds. In: Ashby.. A. Margolin. pp. Ishidate. de Serres. M. de Serres. B. Matter. Elsevier Science. Evaluation of ShortTerm Tests for Carcinogens. de Serres. B.. eds.D. Graf.4...E. In: Ashby.D.J.W.J.H. Van Dongen. Report of the International Programme on Chemical Safety’s Collaborative Study on in vitro assays. Ishihara. F. M.3diamino-2.. Margolin.H. In: Ashby.. Tong.M. M. F. (1976) Toxicity of and biological monitoring for 1. Amsterdam.. pp. Evaluation of Short-Term Tests for Carcinogens. Evaluation of Short-Term Tests for Carcinogens. N.D.M. Amsterdam. M. Margolin. Evaluation of Short-Term Tests for Carcinogens. M. F.C. Jr. Boger.H. & Shelby. F. de Serres. F.E. M. 325–356 van Went. Draper. J. J. de Serres. & Haworth. Progress in Mutation Research. Matter. occup... & Simons. M. Draper.

Volume 1. & Scheel. J. B. Elsevier Science. Report of the International Collaborative Program. Draper.. I. M.. Heinisch. F. Report of the International Programme on Chemical Safety’s Collaborative Study on in vitro assays. 235–242 . Evaluation of Short-Term Tests for Carcinogens. eds.. F.K.K. B.M. J. Margolin.E. Progress in Mutation Research. F..H.J. Matter. & Scheel. Progress in Mutation Research. J. Volume 5. M.. pp. Evaluation of Short-Term Tests for Carcinogens. Elsevier Science. & Shelby. F. In: de Serres.. 481–490 Zimmermann. & Ashby. Ishidate. pp. M. (1985) Tests for the induction of mitotic aneuploidy in the yeast Saccharomyces cerevisiae strain D61. I.322 IARC MONOGRAPHS VOLUME 77 Zimmermann..J. Amsterdam.D. Amsterdam. Jr. In: Ashby. eds.. (1981) Induction of mitotic gene conversion in strain D7 of Saccharomyces cerevisiae by 42 coded chemicals. de Serres.

Serv. Abstr. para-chloro-ortho-toluidine hydrochloride –323– . Reg. 1978). para-chloro-ortho-toluidine. No. Reg.I. Azoic Diazo Component 11. No. 4-chloro-6-methylaniline.4-CHLORO-ortho-TOLUIDINE This substance was considered by previous working groups in June 1977 (IARC. March 1987 (IARC. 1.1 1. and these have been incorporated in the monograph and taken into consideration in the evaluation. new data have become available. 1987a) and February 1989 (IARC. 3-chloro-6-aminotoluene.1 Exposure Data Chemical and physical data Nomenclature 4-Chloro-ortho-toluidine Chem. Abstr.: 3165-93-3 Chem. Serv. June 1982 (IARC. 1983). Abstr. Name: 4-Chloro-2-methylbenzenamine hydrochloride IUPAC Systematic Name: 4-Chloro-ortho-toluidine hydrochloride Synonyms: 4-Chloro-2-methylaniline hydrochloride. 2-methyl-4-chlorobenzeneamine 4-Chloro-ortho-toluidine hydrochloride Chem. 1990).1.: 95-69-2 Chem. 5-chloro-2-aminotoluene. 4-chloro-2-methylaniline. Since that time. Name: 4-Chloro-2-methylbenzenamine IUPAC Systematic Name: 4-Chloro-ortho-toluidine Synonyms: 2-Amino-5-chlorotoluene. 4-chloro-2-toluidine. Abstr. 2-methyl-4-chloroaniline hydrochloride. C. 1. 2-methyl-4-chloroaniline.

slightly soluble in carbon tetrachloride (Lide. Sanyo Fast Red TR Base. Fast Red TR Base.1.3 Relative molecular mass: 141.2 Structural and molecular formulae and relative molecular mass NH2 CH3 Cl C7H8ClN Hydrochloride C7H8ClN. ultraviolet [5411].3 °C (Lide. Fast Red TRO Base.HCl 1. 1993) Boiling-point: 244 °C (Lide. 1999) (b) Conversion factor1: mg/m3 = 7. nuclear magnetic resonance (proton [558]) and mass [NIST 69505] spectral data have been reported (Lide & Milne.1.4 Technical products and impurities (a) (b) (c) (d) Trade names for 4-chloro-ortho-toluidine include: Daito Red Base TR. Kako Red TR Base. 1999) Melting-point: 30. Red TR Base. 1999) Spectroscopy data: Infrared (grating [669].79 × ppm 4-Chloro-ortho-toluidine hydrochloride (a) Description: Buff-coloured or light-pink powder (Department of Health and Human Services. Red Base NTR.07 Chemical and physical properties of the pure substance 4-Chloro-ortho-toluidine Description: Crystalline solid (Lewis. 1 Calculated from: mg/m3 = (relative molecular mass/24. Fast Red 5CT Base. 1999) (f) Conversion factor1: mg/m3 = 5. Mitsui Red TR Base.324 IARC MONOGRAPHS VOLUME 77 1. 1996) (e) Solubility: Soluble in ethanol.1.45) × ppm. Fast Red TR-T Base. Fast Red Base TR.28 × ppm 1. assuming a temperature of 25 °C and a pressure of 101 kPa .6 Relative molecular mass: 178.

1987).N-dimethylformamide. 1988). Environmental Protection Agency.2 Occupational exposure According to the 1981–83 National Occupational Exposure Survey (NOES. 1. It has been sold as both the free amine and the hydrochloride salt (Tariff Commission. acetate and nylon and as intermediates in the production of Pigment Red 7 and Pigment Yellow 49 (Society of Dyers and Colourists. Information available in 1999 indicated that 4-chloro-ortho-toluidine was manufactured by two companies in China (Chemical Information Services.4 1.1 Occurrence Natural occurrence 4-Chloro-ortho-toluidine and its hydrochloride salt are not known to occur as natural products. see IARC. 1998).3 Use 4-Chloro-ortho-toluidine and its hydrochloride salt have been used to produce azo dyes in situ on cotton. GC/ECD and reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet detection have been used to determine 4chloro-ortho-toluidine in human urine samples (Geyer & Fattal. silk.2 Production Commercial production of 4-chloro-ortho-toluidine began in Germany in 1924 (Uebelin & Pletscher. 1999). 1971. Liquid chromatography with electrochemical detection has been used to determine haemoglobin adducts of 4-chloro-ortho-toluidine (Riffelmann et al. 1940). 1971. 1999). Production of both forms has been discontinued in most countries (Stasik. 1945).1.. 1995). 1983]. 4-Chloro-ortho-toluidine was also used from the 1960s until the 1980s in the manufacture of chlordimeform [N′-(4-chloro-2-methylphenyl)-N. 1988). 250 chemists employed in health services in the United States were potentially exposed to 4-chloro-ortho-toluidine (see General Remarks).4. Only three laboratory workers were .4. 1988.. 1982). Fan & Ge. 1. Environmental Protection Agency.4-CHLORO-ortho-TOLUIDINE 325 1. 1940. thin-layer chromatography and GC with electron capture detection (ECD)/flame ionization detection have been used to determine 4-chloro-ortho-toluidine in food and plant materials (Kossman et al. an acaricide and insecticide that is believed to be no longer produced or used worldwide (WHO. 1954) and was first reported in the United States in 1939 (Tariff Commission.5 Analysis Gas chromatography (GC) with alkali flame ionization. 1. 1.

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identified as exposed to 4-chloro-ortho-toluidine or its salts by the Finnish Register of Employees Exposed to Carcinogens in 1997 (Savela et al., 1999). No data on the extent of exposure in the manufacture of 4-chloro-ortho-toluidine, textile dyes, pigments or insecticide chlordimeform or in the use of 4-chloro-ortho-toluidine-based dyes were available to the Working Group. 4-Chloro-ortho-toluidine is a major metabolite of chlordimeform and it has been detected in the urine of workers exposed to chlordimeform in packaging and agriculture (Folland et al., 1978; Geyer & Fattal, 1987). Exposure to 4-chloro-ortho-toluidine and chlordimeform were measured in the urine of chlordimeform production workers and was reported to be minimal after substantial improvement of working conditions in a chlordimeform-manufacturing plant in 1980 (Popp et al., 1992). Stasik (1991) reported that 4-chloro-ortho-toluidine was detected in the urine of two workers at a chemical plant 12 and 24 h after exposure at concentrations of 1.7 and 2.1 mg/L, respectively. 1.4.3 Environmental occurrence

The principal source of 4-chloro-ortho-toluidine in the environment was as an impurity and as a decomposition (metabolic) product of chlorodimeform. This pesticide is no longer produced (WHO, 1998). (a) Water

4-Chloro-ortho-toluidine occurred in water as a result of the hydrolysis of chlordimeform via hydrolysis of the intermediate N-formyl-4-chloro-ortho-toluidine (WHO, 1998). (b) Soil

The microbial degradation of chlordimeform in soils by a number of bacterial and fungal species has led to formation of 4-chloro-ortho-toluidine (Johnson & Knowles, 1970). When 14C-labelled 4-chloro-ortho-toluidine was incubated at a concentration of 5 ppm [mg/kg] in non-autoclaved soil, almost 20% of 4-chloro-ortho-toluidine was metabolized to carbon dioxide within 40 days (Bollag et al., 1978). (c) Plants and foods

4-Chloro-ortho-toluidine has been identified in field samples of plant materials treated with chlordimeform, e.g., in young bean leaves at concentrations of less than 0.1– 0.2 ppm [mg/kg], in grape stems at 0.02–0.3 ppm [mg/kg], in a mixture of grape stems and berries at 0.02–0.5 ppm [mg/kg] and in prunes and apples at less than 0.04 ppm [mg/kg] (Kossmann et al., 1971). In an experimental field application (one to three treatments with chlordimeform, harvesting 42 days after last treatment), 4-chloro-orthotoluidine was found in rice grains at 3–61 ppb [μg/kg] and in straw parts at 80–7200 ppb [μg/kg] (Iizuka & Masuda, 1979). 4-Chloro-ortho-toluidine was detected as a metabolic product in cotton plants following treatment with chlordimeform (Bull, 1973).

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1.5

Regulations and guidelines

4-Chloro-ortho-toluidine is listed as a carcinogen in Finland, Germany (Class 1 substances that cause cancer in man) and Switzerland. Switzerland has set an occupational exposure limit (time-weighted average) [8 h time-weighted average] of 12 mg/m3 with a skin irritation notation for 4-chloro-ortho-toluidine (American Conference of Governmental Industrial Hygienists, 1999; Deutsche Forschungsgemeinschaft, 1999).

2.
2.1 Cohort studies

Studies of Cancer in Humans

Two epidemiological studies (Ott & Langner, 1983; Stasik, 1988) concerning workers exposed to both 4-chloro-ortho-toluidine and ortho-toluidine are more fully described in the monograph on ortho-toluidine in this volume. Ott and Langner (1983) studied the mortality of 342 employees assigned to three aromatic amine-based dye production areas from 1914 to 1958 in the United States. One of these areas, the bromoindigo and thioindigo production area, involved potential exposure to ortho-toluidine, 4-chloro-ortho-toluidine and 4-chloro-acetyl-orthotoluidine. In a separate analysis of 275 individuals not exposed to arsenicals, vinyl chloride or asbestos, no deaths due to bladder cancer were observed, with 1.2 deaths expected from malignant neoplasms of the urinary organs. There were 23 deaths due to malignant neoplasms (17.5 expected; standardized mortality ratio (SMR), 1.3; [95% confidence interval (CI), 0.8–2.0]), 10 of which were coded to digestive organs (5.7 expected; SMR, 1.8; [95% CI, 0.8–3.2]). [The Working Group noted that the conclusions of this study were limited by the small size of the population exposed to 4-chloro-ortho-toluidine and the ascertainment of only deceased and not incident bladder cancer cases.] In a historical mortality study of 335 male employees involved in the production and processing of 4-chloro-ortho-toluidine between 1929 and 1982 in Essen, Germany, no deaths from bladder cancer were identified. Four monocyclic amines had been used at the plant: N-acetyl-ortho-toluidine, 6-chloro-ortho-toluidine, ortho-toluidine and 4-chloro-ortho-toluidine; exposure to 4-chloro-ortho-toluidine was reported to be predominant. Urothelial carcinomas were subsequently recorded in eight of the employees between 1967 and 1985, two of whom had died as of December 1986. All eight had been employed in the 4-chloro-ortho-toluidine production plant before improvements in industrial hygiene were made in 1970. As a result of this discovery, an incidence study was initiated and the vital status ascertainment for 116 subjects with exposure before 1970 was extended through 1986. The expected number of incident bladder cancers in the cohort of 116 men was 0.11. The standardized incidence ratio (SIR) based on eight

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observed cases from 1967 to 1985 was 72.7 (95% CI, 31.4–143) (Stasik, 1988). [The Working Group noted that the definition of the subcohort was made a posteriori, but this was justified by the authors’comment that improvements in industrial hygiene were introduced in 1970. It was also unclear in what year the follow-up started. The excess of bladder cancer could not be attributed with certainty specifically to 4-chloro-orthotoluidine or to any one of the other compounds present.] Popp et al. (1992) reported the results of a bladder cancer incidence study conducted among a group of 49 male workers exposed to 4-chloro-ortho-toluidine in the synthesis of chlordimeform (evaluated as not classifiable as to its carcinogenicity to humans, Group 3 (IARC, 1987b)) from 1950 to 1986 in a German chemical plant. The period of follow-up was 1950–90. Seven cases of bladder cancer were identified between 1982 and 1990 in workers exposed to 4-chloro-ortho-toluidine while synthesizing chlordimeform before 1976, when working conditions were improved. Expected numbers of bladder cancers were calculated based on cancer registry data from the former German Democratic Republic, Denmark and Saarland. SIRs based on the three estimates of expected numbers of deaths were 89.7 (95% CI, 35.6–168.6) (German Democratic Republic), 35.0 (95% CI, 13.9–65.7) (Denmark) and 53.8 (95% CI, 21.3–101.1) (Saarland). No bladder tumours were noted among individuals exposed only to the final product, chlordimeform; however, the size of this group is unknown. The only other aromatic amine to which there was potential exposure was 4-chloroaniline (classified as possibly carcinogenic to humans, Group 2B (IARC, 1993)), which was used for appreciably shorter periods and in smaller quantities than 4-chloro-ortho-toluidine. [The Working Group noted that the nature of the facility and the methods of this study were not fully described. Concomitant exposure to chlordimeform and 4-chloroaniline could not be excluded as confounders. The Working Group considered that the excesses of bladder cancer reported were too large to have been due to smoking alone.]

3.
3.1 3.1.1

Studies of Cancer in Experimental Animals

Oral administration Mouse

Groups of 25 male and 25 female Swiss CD-1 mice, four to six weeks of age, were fed 4-chloro-ortho-toluidine hydrochloride (97–99% pure) in the diet at dose levels of 0, 750 or 1500 mg/kg diet (ppm) (males) or 0, 2000 or 4000 ppm (females) for 18 months. Animals were kept without treatment for three further months and then killed. The doses were chosen on the basis of preliminary tests, the higher being the maximum tolerated dose. Additional control groups were prepared for the other compounds tested in the study, and tumour incidences of concurrent and pooled controls were compared statistically (both separately and together) with those of treated groups. Animals that

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died during the first six months of the study were discarded without necropsy. In male mice, the incidence of vascular tumours (haemangiomas and haemangiosarcomas combined, observed mainly in the spleen and in the subcutaneous or subperitoneal adipose tissue) was 0/14, 5/99, 12/20 (p < 0.025, Fisher’s exact test) and 13/20 (p <0.025, Fisher’s exact test) in concurrent controls, pooled controls, low-dose and high-dose groups, respectively. In female mice, the incidence of vascular tumours (haemangiomas and haemangiosarcomas combined) was 0/15, 9/102, 18/19 (p < 0.025, Fisher’s exact test) and 12/16 (p < 0.025, Fisher’s exact test) in concurrent controls, pooled controls, low-dose and high-dose groups, respectively [separate incidences for haemangiomas and haemangiosarcomas were not reported] (Weisburger et al., 1978). Groups of 50 male and 50 female B6C3F1 mice, six weeks of age, were administered 4-chloro-ortho-toluidine (purity, > 99%) in the diet at concentrations of 3750 or 15 000 ppm (males) and 1250 or 5000 ppm (females) for 99 weeks, except high-dose females which had all died by 92 weeks. Concurrent controls consisted of 20 male and 20 female untreated mice. Mean body weights of all treated groups were lower than those of the corresponding controls and were dose-related. Mortality was dose-related for both males and females (p < 0.001, Tarone test for dose-related trend). In male mice, the incidence of haemangiosarcomas (originating in the adipose tissue adjacent to the genital organs) was 0/20, 3/50 and 37/50 (p < 0.025, Fisher’s exact test) in control, lowand high-dose groups, respectively. In female mice, the incidence of haemangiosarcomas was 0/18, 40/49 (p < 0.025, Fisher’s exact test) and 39/50 (p < 0.025, Fisher’s exact test) in control, low- and high-dose groups, respectively (National Cancer Institute, 1979). 3.1.2 Rat

Groups of 25 male Sprague-Dawley CD rats, four to six weeks of age, were treated with 4-chloro-ortho-toluidine hydrochloride (97–99% pure) in the diet at dose levels of 2000 or 4000 ppm for three months and then at levels of 500 or 1000 ppm for a further 15 months. Animals were kept without treatment for an additional six months and then killed. The doses were chosen on the basis of preliminary tests, the higher being the maximum tolerated dose. A concurrent control group of 25 untreated male rats was used, plus additional controls used for the other compounds tested in the study, and tumour incidences of matched and pooled controls were compared with those of treated groups. Animals that died during the first six months of the study were discarded without necropsy. There was no treatment-related increase in the incidence of tumours at any site (Weisburger et al., 1978). [The Working Group noted the nonstandard protocol and that no information on survival was given.] Groups of 50 male and 50 female Fischer 344 rats, six weeks of age, were administered 4-chloro-ortho-toluidine (purity, > 99%) in the diet at concentrations of 1250 or 5000 ppm for 107 weeks. Concurrent controls consisted of 20 male and 20 female untreated rats. Mean body weights of the high-dose males and females were

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lower than those of the corresponding controls. Mortality was not significantly affected by treatment in rats of either sex. At the end of the study, survival in all groups was more than 50% in males and more than 70% in females. No tumours occurred at incidences that could be related to the treatment (National Cancer Institute, 1979).

4.

Other Data Relevant to an Evaluation of Carcinogenicity and its Mechanisms
Absorption, distribution, metabolism and excretion Humans

4.1 4.1.1

No data were available to the Working Group. 4.1.2 Experimental systems

In Sprague-Dawley rats treated orally with 4-chloro-ortho-[14C]toluidine, 71% of a 3 μCi (specific activity, 1.43 mCi/mmol) dose was excreted in the urine and 24.5% in the faeces after 72 h. In addition to 4-chloro-ortho-toluidine, the metabolites 5-chloroanthranilic acid and 4-chloro-2-methylacetanilide were isolated from the urine. A number of other metabolites were isolated but their chemical structures were not determined. The highest levels of 4-chloro-ortho-[14C]toluidine equivalents were found in the fat, liver and kidney (Knowles & Gupta, 1970). 4-Chloro-ortho-toluidine was given orally to female Wistar rats and female B6C3F1 mice (0.5 mmol [71 mg]/kg bw) and found to bind to haemoglobin. It was concluded that binding of such monocyclic aromatic amines occurs via a reaction of the respective nitrosoarene metabolites with haemoglobin (Birner & Neumann (1988). In Osborne-Mendel rats administered 4-chloro-ortho-[methyl-14C]toluidine intraperitoneally at a dose of 14 mg/kg bw, the level of adducts bound to protein, DNA and RNA was significantly higher in the liver than in 10 other tissues examined. In in-vitro studies, the major and minor microsomal metabolites were identified as 5-chloro-2-hydroxyaminotoluene and 4,4′-dichloro-2,2′dimethylazobenzene, respectively. Binding to microsomal protein was increased in Osborne-Mendel rats pretreated with phenobarbital intraperitoneally at a dose of 100 mg/kg bw for two days, implying that cytochrome P450 is involved in the reaction (Hill et al., 1979). Following oral administration of 25 mg/kg bw 4-chloro-ortho-toluidine to male Sprague-Dawley-derived [Tif:RAIf (SPF] rats and male mice [Tif:MAGf (SPF)] both from CIBA-GEIGY, covalent binding to both DNA and protein occurred (Bentley et al., 1986a). Administration of 4-chloro-ortho-toluidine to male Sprague-Dawley rats induces a number of enzymes involved in xenobiotic metabolism: cytochrome P450, ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase, glutathione S-transferase and epoxide hydrolase (Leslie et al., 1988).

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4.2 4.2.1

Toxic effects Humans

Toxic effects due to 4-chloro-ortho-toluidine result from inhalation or skin contact (Stasik, 1991). The first symptom of 4-chloro-ortho-toluidine toxicity is macroscopic or microscopic haematuria. Further symptoms include dysuria, reduced bladder capacity and generalized pain in the lower abdomen. Haemorrhagic cystitis is the leading symptom of acute toxicity. Acute intoxications with 4-chloro-ortho-toluidine have occurred in workers in the chemical industry involved in the production and use of the compound. A single dermal exposure was sufficient to produce signs of toxicity. Endoscopic inspection of the urinary bladder revealed necrotic epithelial damage, bleeding and oedema. Methaemoglobinaemia was observed in about 50% of the intoxicated individuals (reviewed by Stasik, 1991). [The Working Group noted the incomplete reporting of these data.] 4.2.2 Experimental systems

The following LD50 values have been reported for intraperitoneally administered 4-chloro-ortho-toluidine: 720 mg/kg bw in male mice, 680 mg/kg bw in female mice, 560 mg/kg bw in male rats and 700 mg/kg bw in female rats (Weisburger et al., 1978). Administration of 4-chloro-ortho-toluidine in the diet for 99 weeks to B6C3F1 mice (3750 and 15 000 ppm [mg/kg diet] for male mice; 1250 and 5000 ppm for female mice) led to a high incidence of haemosiderin deposit in the renal tubular epithelium, particularly in mice with haemangiosarcoma (National Cancer Institute, 1979). 4.3 Reproductive and developmental effects

No data were available to the Working Group. [The Working Group noted that many aromatic amines, including 4-chloro-orthotoluidine, induce methaemoglobinaemia (Watanabe et al., 1976; Sachsse et al., 1980, cited in WHO 1998; Stasik, 1991; Coleman & Coleman, 1996). The effect of methaemoglobinaemia on fetal development has not been well studied, but may be associated with suboptimal fetal outcome (Fan & Steinberg, 1996; Kilpatrick & Laros, 1999).] 4.4 4.4.1 Genetic and related effects Humans

No data were available to the Working Group.

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4.4.2

Experimental systems (see Table 1 for references)

The data available up to 1990 and 1993 have been reviewed by IARC (1990) and Jackson et al. (1993), respectively. 4-Chloro-ortho-toluidine has been tested in a number of laboratories with a range of Salmonella strains, and in general the results have been negative. There were single positive results with TA100 and TA98 (with metabolic acivation) and with TA1535 (without metabolic activation). Mutation tests (with and without activation) and DNA repair assays (without activation only) in Escherichia coli were negative, while a positive response was observed for differential toxicity in S. typhimurium, indicating DNA damage. However, positive results have been obtained in several tests in mammalian systems, including induction of DNA strand breaks in vitro, unscheduled DNA synthesis in rat primary hepatocytes, sister chromatid exchanges and chromosomal aberrations (only in the presence of an external metabolizing system) in Chinese hamster cells in vitro, and transformation of BALB/c 3T3 mouse cells. The mouse spot test was also positive. 4-Chloro-ortho-toluidine bound to hepatic DNA and RNA in mice and rats in vivo, with a greater extent of binding in mice than in rats. In contrast, it gave negative results in both the sister chromatid exchange and chromosomal aberration assays in human lymphocytes in vitro and in the mouse heritable translocation assay in vivo. 4.5 Mechanistic considerations

Like other aromatic amines 4-chloro-ortho-toluidine has been shown to undergo metabolic activation resulting in covalent binding to tissue proteins, DNA and RNA both in vivo and in vitro (Hill et al., 1979; Bentley et al., 1986a,b; Birner & Neumann, 1988).

5.
5.1

Summary of Data Reported and Evaluation

Exposure data

4-Chloro-ortho-toluidine and its hydrochloride salt were produced commercially in substantial amounts as intermediates in the manufacture of azo dyes and chlordimeform, an insecticide. Since the 1980s, production and use of 4-chloro-ortho-toluidine have been discontinued in most countries. 5.2 Human carcinogenicity data

Three small cohort studies of workers exposed to 4-chloro-ortho-toluidine, one each among dye, 4-chloro-ortho-toluidine and chlordimeform production workers, were

Table 1. Genetic and related effects of 4-chloro-ortho-toluidine
Test system Resulta Without exogenous metabolic system Salmonella typhimurium, repair-deficient strains, TA1538, TA1978, differential toxicity Escherichia coli rec strains, differential toxicity Salmonella typhimurium TA100, reverse mutationc Salmonella typhimurium TA100, reverse mutationc Salmonella typhimurium TA1535, TA1537, TA98, reverse mutationc Salmonella typhimurium TA100, TA1537, TA1538, TA98, reverse mutationc Salmonella typhimurium TA100, reverse mutation Salmonella typhimurium TA1535, reverse mutationc Salmonella typhimurium TA1535, TA1537, reverse mutation Salmonella typhimurium TA1537, TA98, reverse mutationc Salmonella typhimurium TA98, reverse mutation Escherichia coli WP2 uvrA, WP2, other strains, reverse mutationc DNA strand breaks, cross-links or related damage, Chinese hamster V79 lung cells in vitroc Unscheduled DNA synthesis, rat primary hepatocytes in vitro Sister chromatid exchange, Chinese hamster ovary cells in vitroc Chromosomal aberrations, Chinese hamster ovary cells in vitroc Cell transformation, BALB/c 3T3 mouse cellsc Sister chromatid exchange, human lymphocytes in vitro + + – – – – – + – – – – (+) + + – + – With exogenous metabolic system NT NT + – – NT + NT – – + – NT NT + + NT – 250 mg/disc 1000 mg/disc 17.8 μg/plate 333 μg/plate 1000 μg/plate 325 μg/plate 100 μg/plate 200 μg/plate 1500 μg/plate NR 375 μg/plate 2000 μg/plate 534 14.2 50 400 75 283 Rashid et al. (1984) Rashid et al. (1984) Zimmer et al. (1980) Haworth et al. (1983) Haworth et al. (1983) Rashid et al. (1984) Göggelmann et al. (1996) Rashid et al. (1984) Göggelmann et al. (1996) Zimmer et al. (1980) Göggelmann et al. (1996) Rashid et al. (1984) Zimmer et al. (1980) Williams et al. (1989) Galloway et al. (1987) Galloway et al. (1987) Matthews et al. (1993) Göggelmann et al. (1996) Doseb (LED or HID) Reference

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Table 1 (contd)
Test system Resulta Without exogenous metabolic system Chromosomal aberrations, human lymphocytes in vitro Mouse spot test, C57BL6J×T micec Mouse heritable translocation test, NMRI/SPF micec Binding (covalent) to DNA, rat and mouse liver in vivoc Binding (covalent) to RNA or protein, rat and mouse liver in vivoc
a

Doseb (LED or HID) With exogenous metabolic system (+) 283 100 po × 3d 200 po; 7 d/w, 7 w 25 po × 1 25 po × 1

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(+) + – + +

Göggelmann et al. (1996) Lang (1984) Lang & Adler (1982) Bentley et al. (1986a) Bentley et al. (1986b)

+, positive; (+), weak positive; –, negative; NT, not tested LED, lowest effective dose; HID, highest ineffective dose; in-vitro tests, μg/mL; in-vivo tests, mg/kg bw/day; NR, not reported c Test performed with the hydrochloride salt of 4-chloro-ortho-toluidine d Treatments on days 8, 9 and 10 of embryonic development
b

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available. Two of them showed high relative risks of bladder cancer. Despite problems in the cohort definitions in these two studies, the high relative risks observed for bladder cancer most likely represent a true excess. However, confounding cannot be excluded due to the presence of other exposures including potential bladder carcinogens. The third study had limited power to detect a risk due to use of mortality data only in a small cohort. 5.3 Animal carcinogenicity data

4-Chloro-ortho-toluidine or its hydrochloride was tested for carcinogenicity by oral administration in two experiments in mice and in two experiments in rats. The compounds increased the incidence of haemangiosarcomas in the spleen and adipose tissue in both male and female mice, but no increase in the incidence of tumours was observed in rats. 5.4 Other relevant data

4-Chloro-ortho-toluidine undergoes extensive metabolism in rodents in vivo. Like other aromatic amines, it undergoes metabolic activation via initial formation of the N-hydroxy derivative. The further metabolic processing of this metabolite has not been investigated. In humans, 4-chloro-ortho-toluidine induces acute toxicity in the urinary bladder and causes methaemoglobinaemia. In rodents, 4-chloro-ortho-toluidine and/or its metabolites bind to macromolecules in liver cells. 4-Chloro-ortho-toluidine gave variable results in the majority of bacterial tests for mutagenicity. While most of the mammalian tests were positive, chromosomal aberration assays gave conflicting results. These data overall indicate that 4-chloroortho-toluidine causes DNA damage in mammalian cells. 5.5 Evaluation

There is limited evidence in humans for the carcinogenicity of 4-chloro-orthotoluidine. There is sufficient evidence in experimental animals for the carcinogenicity of 4-chloro-ortho-toluidine. Overall evaluation 4-Chloro-ortho-toluidine is probably carcinogenic to humans (Group 2A).

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6.

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American Conference of Governmental Industrial Hygienists (1999) TLVs and other Occupational Exposure Values—1999 [CD-ROM], Cincinnati, OH, ACGIH® Bentley, P., Waechter, F., Bieri, F., Stäubli, W. & Muecke, W. (1986a) Species differences in the covalent binding of p-chloro-o-toluidine to DNA. Arch. Toxicol., Suppl. 9, 163–166 Bentley, P., Bieri, F., Muecke, W., Waechter, F. & Stäubli, W. (1986b) Species differences in the toxicity of p-chloro-o-toluidine to rats and mice. Covalent binding to hepatic macromolecules and hepatic non-parenchymal cell DNA and an investigation of effects upon the incorporation of [3H]thymidine into capillary endothelial cells. Chem.-biol. Interact., 57, 27–40 Birner, G. & Neumann, H.-G. (1988) Biomonitoring of aromatic amines. II: Hemoglobin binding of some monocyclic aromatic amines. Arch. Toxicol., 62, 110–115 Bollag, J.-M., Blattmann, P. & Laanio, T. (1978) Adsorption and transformation of four substituted anilines in soil. J. Am. Food Chem., 26, 1302–1306 Bull, D.L. (1973) Metabolism of chlordimefon in cotton plants. Environ. Entomol., 2, 869–871 Chemical Information Services (1999) Directory of World Chemical Producers (Version 99.1.0) [CD-ROM], Dallas, TX Coleman, M.D. & Coleman, N.A. (1996) Drug-induced methaemoglobinaemia. Drug Saf., 14, 394–405 Department of Health and Human Services (1999) Report on Carcinogens, 8th Ed., Research Triangle Park, National Toxicology Program, pp. 80–81 Deutsche Forschungsgemeinschaft (1999) List of MAK and BAT Values 1999 (Report No. 35), Weinheim, Wiley-VCH, pp. 41, 113 Environmental Protection Agency (1988) Benzenamine, 4-chloro-2-methyl-; benzenamine, 4-chloro-2-methyl-, hydrochloride; benzenamine, 2-chloro-6-methyl-; proposed significant new use of chemical substances. Fed. Regist., 53, 36076–36080 Fan, D.-F & Ge, S.-D. (1982) Gas-liquid chromatographic determination of chlordimefon and its metabolites in cargo rice and husks. J. Assoc. off. anal. Chem., 65, 1517–1520 Fan, A.M. & Steinberg, V.E. (1996) Health implications of nitrate and nitrite in drinking water: an update on methemoglobinemia occurrence and reproductive and developmental toxicity. Regul. Toxicol. Pharmacol., 23, 35–43 Folland, D.S., Kimbrough, R.D., Cline, R.E., Swiggard, R.C. & Schaffner, W. (1978) Acute hemorrhagic cystitis. Industrial exposure to the pesticide chlordimeform. J. Am. med. Assoc., 239, 1052–1055 Galloway, S.M., Armstrong, M.J., Reuben, C., Colman, S., Brown, B., Cannon, C., Bloom, A.D., Nakamura, F., Akmed, M., Duk, S., Rimpo, J., Margolin, B.H., Resnick, M.A., Anderson, B. & Zeiger, E. (1987) Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: evaluation of 108 chemicals. Environ. mol. Mutag., Suppl. 10, 1–175 Geyer, R. & Fattal, F. (1987) HPLC determination of the metabolite 4-chloro-o-toluidine in the urine of workers occupationally exposed to chlordimeform. J. anal. Toxicol., 11, 24–26 Göggelmann, W., Bauchinger, M., Kulka, U. & Schmid, E. (1996) Genotoxicity of 4-chloro-otoluidine in Salmonella typhimurium, human lymphocytes and V79 cells. Mutat. Res., 370, 39–47

935–963 Knowles. (1971) Specific determination of chlorphenamidine [N′-(4-chloro-o-tolyl)-N. Lyon. Mortelmans. 57... H.. D. (1993) The genetic toxicology of putative nongenotoxic carcinogens. R. Occupational Exposures of Hairdressers and Barbers and Personal Use of Hair Colourants: Some Hair Dyes.L. C. Suppl. K. Lyon. & Resnik. Mutat. T. R. V. (1982) Studies on the mutagenic potential of the pesticide chlordimeforme and its principal metabolites in the mouse heritable translocation assay. Contam. 5.. 135.. & Knowles. Suppl. 39. Cancer Res. IARCPress. pp. 59 IARC (1990) IARC Monographs of the Evaluation of Carcinogenic Risks to Humans. 277–285 IARC (1983) IARC Monographs on the Evaluation of Carcinogenic Risks of Chemicals to Humans. E.N′-dimethylformamide. S.. Lyon. its principal metabolites and the drug lisuride hydrogen maleate. (1979) Macromolecular binding and metabolism of the carcinogen 4-chloro-2-methylaniline. Entomol. 2528–2531 IARC (1978) IARC Monographs on the Evaluation of Carcinogenic Risks to Man. 22. environ. 856–859 Kossmann. eds. Res. 16. Vol. (1970) Microbiological degradation of the acaricide N-(4chloro-o-tolyl)-N. (1970) N′-(4-Chloro-o-tolyl)-N. environ. & Boyd. Industrial Dyestuffs and Aromatic Amines.-W. p. Cosmetic Colourants. & Adler.K.K. & Gupta. (1999) Maternal hematologic disorders. 3–142 Hill. M. C.O. pp.. & Zeiger. 305–321 Iizuka. T.D.. 63.F. pp. & Waters. & Struck. 123–137 IARC (1993) IARC Monographs on the Evaluation of of Carcinogenic Risks to Humans.N-dimethyl formamidine] in plants and soil materials by colorimetry and thin-layer and electron capture gas chromatography. (1983) Salmonella mutagenicity test results for 250 chemicals. 296.F. agric. Bull. Lyon. Mutat.. 60 IARC (1987b) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Econom. Some Aromatic Amines and Related Nitro Compounds—Hair Dyes. 243–248 . I. pp. W. Lyon. 7. Vol.T. Bull. 19. 158–170 Kilpatrick. J.K. p.O. Vol. 241–277 Johnson. M.. 7. Lyon. Saunders. Geissbühler. 219–224 Lang. Stack. Mutat. pp. Overall Evaluations of Carcinogenicity: An Updating of IARC Monographs Volumes 1 to 42. B.B. K. 61–72 IARC (1987a) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. (1979) Residual fate of chlorphenamidine in rice plant and paddy soil. W. 1. Res. IARCPress.A. and Exposures in the Textile Manufacturing Industry.. 745–749 Jackson. H. R. T.S. IARCPress. 30. R. (1984) The mammalian spot test and its use for testing of mutagenic and carcinogenic potential: experience with the pesticide chlordimeform. Lawlor. IARCPress. Overall Evaluations of Carcinogenicity: An Updating of IARC Monographs Volumes 1 to 42. R. S. 48. A.. Suppl. Philadelphia.F. Res. 92. Mutag. Toxicol. Shih. Toxicol.-D. J. & Laros. Speck. 360–364 Lang. R.. Environ. Some Flame Retardants and Textile Chemicals. IARCPress. Food Chem. Colouring Agents and Miscellaneous Industrial Chemicals. & Masuda. Miscellaneous Pesticides. IARCPress. H. Maternal–fetal Medicine.N-dimethylformamidine-14C (Galecron) and 4-chloro-ortho-toluidine-14C metabolism in the white rat. In: Creasy.. Contam.J. Vol.4-CHLORO-ortho-TOLUIDINE 337 Haworth...

165. M. 917–928 (in German) Tariff Commission (1940) Synthetic Organic Chemicals. 153.R. M. Environ. Health Perspect. 84. & Tennant. K. Centers for Disease Control.A. 37. Unpublished data as of July 1999. 4042. Second Series). Government Printing Office.. 4037. Transformation responses of 168 chemicals compared with mutagenicity in Salmonella and carcinogenicity in rodent bioassays. Int. Schmieding. 116.. Vol. p. 529–531 Rashid. occup. pp. Wschr. 12th Ed. G. K.. R.0 [CD-ROM].J.R. & Mumma. B19. 4025. M.. G.J.. Med.G. (1954) Aetiology and prophylaxis of industrial tumours in the dye industry.R. US Production and Sales.D. Schmieding. Schweiz. 3165-93-3) (Tech. E. Müller. Bradford. D.. Ercegovich. Jr (1993) Hawley’s Condensed Chemical Dictionary. & Stacey. & Pletscher. (1992) Incidence of bladder cancer in a cohort of workers exposed to 4-chloro-ortho-toluidine while synthesizing chlordimeform. F. environ.. Washington DC. (1988) Carcinomas of the urinary bladder in a 4-chloro-o-toluidine cohort. FL.. Department of Health and Human Services.J... (1995) Biomonitoring of urinary aromatic amines and arylamine hemoglobin adducts in exposed workers and nonexposed control persons. occup....O. Second Series).Yorkshire. 60. W. & Langner. MD NOES (1999) National Occupational Exposure Survey 1981–83. 1941–43 (Report No. Dtsch. CRC Press Lide. J. J.. (1999) ASA 1997 (Reviews 140). G. p. med. environ... OH. Washington DC. Sci.H. (1991) Urinary bladder cancer after 4-chloro-o-toluidine. US Production and Sales. 101 (Suppl. ind. R. & Kauppinen. 2529–2535 Lewis. W. 2). M. T. Rep. Spalding. Finnish Institute of Occupational Health (in Finnish). Public Health Service. Health. M. 140. Health. Biochem. Government Printing Office. 763–768 Popp. National Institute for Occupational Safety and Health Ott.. 3rd ed. Vuorela. 1939 (Report No. 95–110 Riffelmann.W. 1444–1447 (in German) Uebelin. & Norpoth. p. M. 4. New York. R. DHEW Publ. Br. & Norpoth. Med. 68. J.338 IARC MONOGRAPHS VOLUME 77 Leslie. J. Murray. Arch. Popp. & Milne. environ. (1983) A mortality survey of men engaged in the manufacture of organic dyes. (1993) Transformation of BALB/c-3T3 cells: V. Version 5. Speck. W. 73 . (1999) CRC Handbook of Chemistry and Physics. 25. Series No. A.W... (NIH) 79-1721). 55 Lide. (1984) Evaluation of chlordimeform and degradation products for mutagenic and DNA-damaging activity in Salmonella typhimurium and Escherichia coli. 42 Society of Dyers and Colourists (1971) Colour Index. FL.J. Boca Raton. Wochenschr. N. 9 Tariff Commission (1945) Synthetic Organic Chemicals.. Van Nostrand Reinhold. Boca Raton. Reidy. 4348 Stasik. R. (1988) Induction of xenobiotic biotransformation by the insecticide chlordimeform. Bethesda. CRC Press Matthews.A. K. 36–43 Savela. No. [CD-ROM].W. occup. 49. Health. A. 79th Ed. Cincinnati. R. C. Arch. p. (1996) Properties of Organic Compounds. Int. 21–24 Stasik. C. 347–482 National Cancer Institute (1979) Carcinogenesis Bioassay of 4-Chloro-o-toluidine Hydrochloride for Possible Carcinogenicicity (CAS No. C. med. Helsinki. a metabolite 4-chloro-o-toluidine and a structurally related chemical o-toluidine. W. Vahrenholz. D. Pharmacol. ed..F.

M. K. & Ikeda. 325–356 WHO (1998) Chlordimeform (Environmental Health Criteria 199).. J. Ishihara. Boger. World Health Organization Williams. 263–286 Zimmer..3diamino-2. Arch. 221.. Van Dongen.. Mazurek. E.K. Toxicol. B. (1978) Testing of twenty-one environmental aromatic amines or derivatives for long-term toxicity or carcinogenicity. Geneva..A. 77. & McQueen. C. (1989) Structure-activity relationships in the rat hepatocyte DNA-repair test for 300 chemicals. J. N.4. Mutat. Int. environ. & Bhuyan. Res. (1980) Bacterial mutagenicity and mammalian cell damage by several substituted anilines.6-trinitrobenzene and other nitro-amino derivatives of benzene and chlorobenzene. Mori.. Russfield.K. Pathol.. E. 317–326 . T. J. (1976) Toxicity of and biological monitoring for 1. F. C. Health.. 37.G.H. G. 157–168 Weisburger. occup.. Homburger. M. Petzold. D. H.4-CHLORO-ortho-TOLUIDINE 339 Watanabe. G..B... A. 2. environ.C. & Chu. Res. Mutat. Weisburger.

.

Serv. 5-chloro-2-toluidine.3 Relative molecular mass: 141. Abstr. 1. 1996) –341– .1. 1996) (d) Spectroscopy data: Infrared [COB.2 Structural and molecular formulae and relative molecular mass NH2 CH3 Cl C7H8ClN 1. 71121] spectral data have been reported (Lide & Milne. 2315]. Name: 5-Chloro-2-methylbenzenamine IUPAC Systematic Name: 5-Chloro-ortho-toluidine Synonyms: 2-Amino-4-chlorotoluene. 4-chloro-2-aminotoluene. Reg. 3-chloro-6-methylaniline.1. Abstr.5-CHLORO-ortho-TOLUIDINE 1. ultraviolet [1302] and mass [NIST.: 95-79-4 Chem. 2-methyl-5-chloroaniline 1. No.1 1.1 Exposure Data Chemical and physical data Nomenclature Chem. 1996) (e) Solubility: Very soluble in ethanol (Lide & Milne. 1993) (b) Boiling-point: 239 °C (Lide & Milne. 1996) (c) Melting-point: 26 °C (Lide & Milne.1. 5-chloro-2-methylaniline.6 Chemical and physical properties of the pure substance (a) Description: Off-white solid or light brown oil which tends to darken on storage (Lewis.

Spectrolene Red KB. assuming a temperature of 25 °C and a pressure of 101 kPa . Pharmazoid Red KB. India. Fast Red KB amine. 1. Fast Red KB salt.1. 1999) 1. 1. Genazo Red KB soln. Red KB base. Fast Red KB-T Base. Metrogen Red Former KB soln.45) × ppm.4 Conversion factor1: mg/m3 = 5. Hiltonil Fast Red KB base. 1. It is also used as a dye for cotton.4. 1. Japan and the United Kingdom (Chemical Information Services.1 Occurrence Natural occurrence 5-Chloro-ortho-toluidine is not known to occur as a natural product.4 1. Ansibase Red KB. silk and nylon (National Library of Medicine.2 Production Information available in 1999 indicated that 5-chloro-ortho-toluidine was manufactured by three companies in China and by one company each in Germany. 1 Calculated from: mg/m3 = (relative molecular mass/24.79 × ppm Technical products and impurities Trade names for 5-chloro-ortho-toluidine include: Acco Fast Red KB base.2 Occupational exposure No data were available to the Working Group.3 Use 5-Chloro-ortho-toluidine is used as a dye intermediate in the synthesis of Pigment Red 11.5 Analysis No methods have been reported for the analysis of 5-chloro-ortho-toluidine in environmental matrices.4.342 IARC MONOGRAPHS VOLUME 77 (f) 1. Pigment Yellow 77 and for Azoic Coupling Component 21. Azoene Fast Red KB base. 1999).1. Fast Red KBS salt. Stable Red KB base. Fast Red KB salt Supra. Naphthosol Fast Red KB base.

Fisher’s exact test. the incidence of hepatocellular carcinomas was 4/20. p < 0.001 and p = 0.001. distribution and use.001. p = 0. respectively. p < 0. Concurrent controls consisted of 20 male and 20 female untreated mice. 3.001. followed by an observation period of 13 weeks. However. 19/50 and 25/47 (p = 0. Studies of Cancer in Humans No data were available to the Working Group.4. the incidence of haemangiosarcomas (mostly originating in the adipose tissue adjacent to the genital organs) was 0/20. Cochran-Armitage trend test) in control. In female mice. 1999). respectively. 6/50 and 22/43 (p < 0. Mortality was dose-related for each sex (p < 0. chromatography showing a single homogeneous peak) in the diet at concentrations 2000 or 4000 mg/kg diet (ppm) for 78 weeks. were administered 5-chloro-ortho-toluidine (technical grade.1 3. trend . no data on its occurrence in the environment were available that would permit assessment of exposures by the Working Group. 1. 11/50 and 37/47 (p < 0. trend test) in control.1 Studies of Cancer in Experimental Animals Oral administration Mouse Groups of 50 male and 50 female B6C3F1 mice. Fisher’s exact test.011.5 Regulations and guidelines 5-Chloro-ortho-toluidine is listed as a Class 3 carcinogenic substance in Germany. low. lowand high-dose groups. these are substances which cause concern that they could be carcinogenic for man but which cannot be assessed conclusively because of lack of data (Deutsche Forschungsgemeinschaft. respectively. the incidence of haemangiosarcomas (mostly originating in the adipose tissue adjacent to the genital organs) was 1/20.1.039 for male and female groups. Mean body weights of treated groups of each sex were lower than those of the corresponding control groups.007. Tarone test for dose-related trend). In male mice. 2. In male mice.and high-dose groups. 3.5-CHLORO-ortho-TOLUIDINE 343 1. six weeks of age.3 Environmental occurrence 5-Chloro-ortho-toluidine is produced in relatively small volume as a dye intermediate and thus may be released into the environment in various waste streams as a result of its production. Fisher’s exact test.001.

low.344 IARC MONOGRAPHS VOLUME 77 test) in control. respectively. In female mice. Fisher’s exact test) and 26/43 (p < 0.and high-dose groups.2 Experimental systems 5-Chloro-ortho-toluidine was given orally to female Wistar rats and female B6C3F1 mice (0. 19/50 (p < 0. It was concluded that binding of such monocyclic aromatic amines occurs via a reaction of the respective nitrosoarene metabolites with haemoglobin.001.1. Although a positive association between dose and the incidence of phaeochromocytomas of the adrenal gland was observed in male rats (control. neither of the Fisher’s exact tests for comparison of the treated groups with the control group showed statistical significance (National Cancer Institute. low-dose. 4.and high-dose groups.2 Rat Groups of 50 male and 50 female Fischer 344 rats. distribution. 1979).1. metabolism and excretion Humans 4. The haemoglobin binding index of 5-chloro-ortho-toluidine was 28-fold higher in rats than in mice. 3. [The Working Group noted the small numbers of animals in the control group.1 No data were available to the Working Group. 7/48. chromatography showing a single homogeneous peak) in the diet at concentrations of 2500 or 5000 ppm for 78 weeks.] 4. low. respectively (National Cancer Institute. 1979).1. six weeks of age. p < 0. followed by an observation period of 25–26 weeks.019. Mortality was not affected by treatment in either sex.and high-dose females were lower than those of the corresponding control group. . trend test). 0/20. were administered 5-chloro-ortho-toluidine (technical grade. This difference may reflect the proportions of the dose of 5-chloro-orthotoluidine that undergo N-oxidation in rats compared with mice (Birner & Neumann. 1988). 2/49.001. Fisher’s exact test. the incidence of hepatocellular carcinomas was 0/20. trend test) in control. Concurrent controls consisted of 20 male and 20 female untreated rats. Other Data Relevant to an Evaluation of Carcinogenicity and its Mechanisms Absorption. Mean body weights of low. high-dose.1 4. p = 0.5 mmol [71 mg]/kg bw) and found to bind covalently to haemoglobin.001.

] 4.4. 4. it did not induce mutagenicity in Salmonella typhimurium.2 Experimental systems 5-Chloro-ortho-toluidine given orally to male mice at a dose of 200 mg/kg bw inhibited testicular DNA synthesis. Ashby and Tennant (1988) have identified this chemical as showing structural features predictive of genotoxicity. 4. but the data-set is inadequate to allow firm conclusions.2 Experimental systems (see Table 1 for references) There are few data available on the genetic toxicology of 5-chloro-ortho-toluidine. 4.1 Genetic and related effects Humans No data were available to the Working Group. Kilpatrick & Laros. as measured by tritiated thymidine incorporation (Seiler. 4.2. 1996).3 Reproductive and developmental effects No data were available to the Working Group. 1996. In single assays. 1999).4 4. prophage lambda in Escherichia coli or unscheduled DNA synthesis in cultured rat hepatocytes. but may be associated with suboptimal fetal outcome (Fan & Steinberg.5 Mechanistic considerations Like other aromatic amines. The effect of methaemoglobinaemia on fetal development has not been well studied. 1977).4. .5-CHLORO-ortho-TOLUIDINE 345 4. No genotoxic effects were seen in three different assays. Coleman & Coleman.2 4. probably N-oxidation. [The Working Group noted that many aromatic amines induce methaemglobinaemia (Watanabe et al. 5-chloro-ortho-toluidine undergoes an initial metabolic activation step. to form a nitrosoarene that can bind covalently to haemoglobin.1 Toxic effects Humans No data were available to the Working Group..2. 1976.

SOS repair. lowest effective dose. HID. NT. BALB/c 3T3 mouse cells Unscheduled DNA synthesis. TA1537. DNA strand breaks or cross-links Salmonella typhimurium TA100. (1988) –. TA98. reverse mutation Cell transformation. μg/mL .2 IARC MONOGRAPHS VOLUME 77 Reference – – + – DeMarini & Brooks (1992) Haworth et al. not tested LED. TA1535.346 Table 1. (1993) Yoshimi et al. in-vitro test. highest ineffective dose. Genetic and related effects of 5-chloro-ortho-toluidine Test system Resultsa Without exogenous metabolic system Prophage induction. (1983) Matthews et al. rat primary hepatocytes in vitro a b Doseb (LED or HID) With exogenous metabolic system – – NT NT 4.4 666 μg/plate 160 14. negative.

Overall evaluation 5-Chloro-ortho-toluidine is not classifiable as to its carcinogenicity to humans (Group 3). to form a nitrosoarene that can bind covalently to haemoglobin. No data were available on human exposure.2 Human carcinogenicity data No data were available to the Working Group. 5.3 Animal carcinogenicity data 5-Chloro-ortho-toluidine was tested for carcinogenicity by oral administration in one experiment in mice and in one experiment in rats. In rats. no carcinogenic effect was observed. probably via N-oxidation.1 Summary of Data Reported and Evaluation Exposure data 5-Chloro-ortho-toluidine is an aromatic amine which is produced in relatively small quantities as an intermediate in the manufacture of some pigments and azo dyes. . 5.5 Evaluation No epidemiological data relevant to the carcinogenicity of 5-chloro-ortho-toluidine were available.5-CHLORO-ortho-TOLUIDINE 347 5. it increased the incidence of haemangiosarcomas (mostly of adipose tissue) and of hepatocellular carcinomas in both males and females. 5.4 Other relevant data 5-Chloro-ortho-toluidine undergoes an initial metabolic activation step. The few available genotoxicity test results on 5-chloro-ortho-toluidine were negative. 5. 5. There is limited evidence in experimental animals for the carcinogenicity of 5-chloro-ortho-toluidine. In mice.

Toxicol. Arch.A.0) [CD-ROM].0 [CD-ROM]. (1996) Drug-induced methaemoglobinaemia. Iwata. & Laros. Saunders. Maternal–fetal Medicine. Mutat. 157–168 Yoshimi. 187. & Zeiger. E. Mutag. R..G. G. CAS No. H. 12th Ed.. Lawlor.B.W. 19. D. pp. & Coleman N. Sugie.M. pp. New York.J.M. 394–405 DeMarini. H. Bethesda. 110–115 Chemical Information Services (1999) Directory of World Chemical Producers (Version 99.... R. & Milne. S. S. Rep.. Mutat. & Neumann. H. 935–963 Lewis. 2062] Seiler. (1976) Toxicity of and biological monitoring for 1. & Brooks. eds. Jr (1993) Hawley’s Condensed Chemical Dictionary. occup. Mori.. V. Niwa. In: Creasy. 17–115 Birner.P. M. N. DHEW Publ.G. 95-79-4 (Tech.. (1999) Maternal hematologic disorders. K. 14.. Int. No. p.K. 62. (1996) Health implications of nitrate and nitrite in drinking water: an update on methemoglobinemia occurrence and reproductive and developmental toxicity. Regul. Res. 206. 305–310 Watanabe. & Tennant. 46. 5 (Suppl. W.J.. H. 98–111 Deutsche Forschungsgemeinschaft (1999) List of MAK and BAT Values 1999 (Report No. Health. & Ikeda. A. 41.R. Mortelmans. D.. Wiley-VCH. 35–43 Haworth. FL.4. mol. Toxicol. N. & Steinberg.K. Pharmacol. R.. Arch. S. (1988) Biomonitoring of aromatic amines.6-trinitrobenzene and other nitro-amino derivatives of benzene and chlorobenzene. & Shimizu.. Hashida. T.. References Ashby. K. Preliminary results in the validation of a novel short term test. (1996) Properties of Organic Compounds. Mutag. Ishihara.. (1983) Salmonella mutagenicity test results for 250 chemicals. J. Weinheim.D. II: Hemoglobin binding of some monocyclic aromatic amines..W. Dallas. 55 Lide. (NIH) 79-1743). J. A. CRC Press National Cancer Institute (1979) Bioassay of 5-Chloro-o-toluidine for Possible Carcinogenicity. Drug Saf. G. (1992) Induction of prophage lambda by chlorinated organics: detection of some single-species/single-site carcinogens. 1). & Resnik.. Mutat. environ.E. Environ. R.. Bethesda.. Van Nostrand Reinhold. TX Coleman M. 204. Series No. 35). Res. H. Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the US NCI/NTP. Boca Raton.3diamino-2. Version 5. T. (1977) Inhibition of testicular DNA synthesis by chemical mutagens and carcinogens. Environ. W..1. 37. Philadelphia. (1988) Chemical structure. (1988) The genotoxicity of a variety of aniline derivatives in a DNA repair test with primary cultured rat hepatocytes. Speck. 118 Fan. 23. 3–142 Kilpatrick.348 IARC MONOGRAPHS VOLUME 77 6. R. Res. 183–191 . C. MD [Record No. MD National Library of Medicine (1999) Hazardous Substances Data Bank (HSDB).

1980. iminodiethanol. Dow Chemical Company.14 Chemical and physical properties of the pure substance (a) Description: Deliquescent prisms.N-diethanolamine. 2. 1996) (f) Solubility: Very soluble in water (954 g/L) and ethanol.8 °C (Lide & Milne.2′-Iminobis[ethanol] IUPAC Systematic Name: 2.1.1.DIETHANOLAMINE 1.2′-iminodi-1-ethanol 1. 1996) (e) Spectroscopy data: Infrared (proton [5830]. Abstr. 2-(2-hydroxyethylamino)ethanol. 1996) (d) Density: 1.1 1.N-bis(2hydroxyethyl)amine. colourless. Lide & Milne. No. N. di(β-hydroxyethyl)amine. 1996. Serv. Abstr. slightly soluble in benzene and diethyl ether (Lide & Milne. C-13 [2936]) and mass spectral data have been reported (Sadtler Research Laboratories.: 8033-73-6 Chem. 1998. di(2-hydroxyethyl)amine. DEA. 1999) (b) Boiling-point: 268.1. No.2′-Iminodiethanol Synonyms: Bis(hydroxyethyl)amine. Verschueren.2′-dihydroxydiethylamine.N′-iminodiethanol. N. bis(2-hydroxyethyl)amine. 1.0966 g/cm3 at 20 °C (Lide & Milne. Reg.1 Exposure Data Chemical and physical data Nomenclature Chem.3 Relative molecular mass: 105. nuclear magnetic resonance (proton [6575]. 2. 1996) (c) Melting-point: 28 °C (Lide & Milne. 1996) –349– .: 111-42-2 Deleted CAS Reg. Name: 2. diolamine.2 Structural and molecular formulae and relative molecular mass CH2 H N CH2 CH2 OH CH2 OH C4H11NO2 1. N. viscous liquid with a mild ammonia odour (Budavari. grating [33038]).

4 Technical products and impurities Diethanolamine is commercially available with the following specifications: purity. and by spectrophotometry (Kenyon et al. Sollenberg. –2. Since the mid-1970s.01 mm Hg [1. < 0.3% min. halogenated organics..1. 1992.2 Production Ethanolamines became available commercially in the early 1930s. 0. nitrites. and by reversed-phase HPLC (Fukui et al.30 × ppm 1. Pietsch et al. by isotachophoresis. 1994) with excess ammonia. and water content. 1994). relative vapour density (air = 1). 1999) (i) Octanol/water partition coefficient (P): log P.6.5 Analysis Diethanolamine can be determined in workplace air by drawing the air sample through aqueous hexanesulfonic acid and analysing by ion chromatography..25% max. Diethanolamine can be determined in water samples by gas chromatography (GC) and by high-performance liquid chromatography (HPLC) with fluorescence detection (Melnick et al.18 (Verschueren.45% max. The limit of detection for this method is 13 μg per sample (Eller.33 Pa] at 20 °C. by ion-exclusive chromatography. 1. 99.15% max.. 0. assuming a temperature of 25 °C and a pressure of 101 kPa .. An 1 Calculated from: mg/m3 = (relative molecular mass/24. 1996) (h) Stability: Incompatible with some metals. strong acids and strong oxidizers (Dow Chemical Company. Maurer et al. in metalworking and cutting fluids by GC–mass selective detection of silylated derivatives. 1. 1995. monoethanolamine. flash-point. 3.350 IARC MONOGRAPHS VOLUME 77 (g) Volatility: Vapour pressure. Schubert et al. 1996) (j) Conversion factor1: mg/m3 = 4. they assumed steadily growing commercial importance as intermediates after 1945. by capillary zone electrophoresis with indirect ultraviolet detection. Ethanolamines are produced on an industrial scale exclusively by reaction of ethylene oxide (see IARC.. 1993. Fernando. 1997). 1998). 1996. because of the large-scale production of ethylene oxide. colourless ethanolamines has been possible. Chou. 1996. Diethanolamine is also available as a blend of 85% diethanolamine and 15% deionized water which is a low freeze-grade product for use in colder temperatures (Dow Chemical Company. 149 °C (Verschueren. (Dow Chemical Company.45) × ppm. triethanolamine (see monograph in this volume).. economical production of very pure. 1998a). 1998b).b. 0.. and in cosmetics and pharmaceuticals by GC with flame ionization detection.1.. 1997). 1994a.. This reaction takes place slowly but is accelerated by water.

40. Dow Chemical Company. Canada. 1983). as a dispersant in agricultural chemical formulations. three companies each in China and Germany. the concentration of diethanolamine may range from 1 to 25% (National Toxicology Program. the Russian Federation.. printing inks. 1999a). petroleum and coal production. South America.. polymers and polymer production. 30–35% diethanolamine and 15–20% triethanolamine (Hammer et al.. Bollmeier. Table 1. Mexico.DIETHANOLAMINE 351 anhydrous procedure uses a fixed-bed ion-exchange resin catalyst (Hammer et al. electroplating. Brazil... wetting agents and detergents (Beyer et al. 220. About 50% of world production of ethanolamines in 1985 was monoethanolamine. coatings. as a corrosion inhibitor. and one company each in Belgium.3 Use Diethanolamine is used as surface-active agent in metal-cutting fluids and oils (see General Remarks). Worldwide production of ethanolamines in 1985 was approximately (thousand tonnes per year): United States. paper. In the cosmetic formulations. 1. soldering flux. 1997. Knaak et al. eastern Europe. natural gas treatment. two companies each in France and India. Table 2 presents estimates of percentages used in major applications in the United States (Knaak et al. Other applications of diethanolamine are in adhesives. and as an intermediate in the production of other compounds such as fatty acid condensates of diethanolamine which are extensively used in soaps and cosmetics as emulsifiers. a fuel-gelling agent. 18. Japan. . mining. western Europe. south-east Asia. Estimated annual production of diethanolamine in the USA (thousand tonnes) Year Production 1960 24 1965 35 1970 42 1975 39 1980 56 1985 76 1989a 92 From Bollmeier (1992) a National Toxicology Program (1992) Information available in 1999 indicated that diethanolamine was manufactured by seven companies in the United States. metal cleaning and lubricating. 1997). paint and pigments.. 1999). Estimated annual production of diethanolamine in the United States is presented in Table 1. Sweden and the United Kingdom (Chemical Information Services. 4. thickeners. an epoxy hardener. antistatic agents. 145. 1987). 1992. a pharmaceutical intermediate and an ointmentemulsifier. 1987). Netherlands. 1998b). Spain. textile finishing and polyurethane production and use (Hammer et al. 1987. cement and concrete work. rubber processing. Iran.

. Diethanolamine also occurs as a contaminant in triethanolamine products (National Toxicology Program.2 Occupational exposure Diethanolamine is present in machining and grinding fluids and has been detected in workplace air in the metal manufacturing industry.352 IARC MONOGRAPHS VOLUME 77 Table 2. 1999e). 1..1 Occurrence Natural occurrence Diethanolamine is not known to occur as a natural product. 1994). In a German study (1992–94). 1999b.4 1. diethanolamine was measured in samples of metalworking fluids in a range of 0–44% (n = 69). A level of 0. 1999). (1997) Percentage of production 39 30 15 10 8 2 2 Free diethanolamine is reported to be a contaminant in fatty acid-diethanolamine condensates (amides of coconut oil acid. as many as 800 000 workers (many of whom were metalworkers) in the United States were potentially exposed to diethanolamine (see General Remarks). All personal exposures were below the detection limit (0.2% of the amine. It was present in bulk cutting fluids at levels ranging from 4 to 5% (Kenyon et al.c. According to the 1981–83 National Occupational Exposure Survey (NOES.02 mg/m3) (Levin et al. 1993). 1.. Major uses of diethanolamine in the United States Applications Surfactants Gas purification Textile processing Metalworking fluids Miscellaneous Laundry detergents Agricultural chemicals From Knaak et al.4. .d). Diethanolamine has also been reported to be present in wetting fluids used in road paving.05 mg/m3 was detected in a stationary sample at a slurry machine discharging a bitumen emulsion containing 0. The number of samples with diethanolamine present steadily declined from 90% to 60% over the study period (Pfeiffer et al.4. 1996). oleic acid and lauric acid) at levels ranging from < 1% to nearly 10% (National Toxicology Program.

Knaak et al.DIETHANOLAMINE 353 1. Environment Canada.4. 1996). 1995).. respectively. 1995. Mathews et al. The Food and Drug Administration permits the use of diethanolamine as a component of adhesives in food packaging.3 Environmental occurrence Production of diethanolamine and its wide use in industrial and consumer products may result in its release to the environment (Yordy & Alexander. 1995).. According to the National Pollutant Release Inventory (NPRI) of Canada. 1996). (a) Air According to the Environmental Protection Agency Toxics Release Inventory. . 1983. 1995). as reported in the Toxics Release Inventory (Environmental Protection Agency.. (c) Soil Releases of diethanolamine to land and underground from 358 industrial facilities in the United States in 1994 (as reported to the Toxics Release Inventory) amounted to 77 050 kg and 36 850 kg respectively (Environmental Protection Agency. as an indirect food additive. 1. (b) Water Surface water discharges of diethanolamine from 358 industrial facilities in 1994 in the United States amounted to 100 350 kg. 1995). Because of the spectrum of industrial and consumer uses of diethanolamine and its miscibility with water.5 Regulations and guidelines Occupational exposure limits and guidelines for diethanolamine are presented in Table 3. 1996). Beyer et al. as a component of the uncoated or coated food contact surface of paper and paperboard for use with dry solid foods with no free fat or oil on the surface. as reported to the NPRI (Environment Canada. 1999). air emissions of diethanolamine from 358 industrial facilities in 1994 were approximately 149 200 kg in the United States (Environmental Protection Agency. 1995. and for use only as an adjuvant to control pulp absorbance and pitch content in the manufacture of paper and paperboard or for use only in paper mill boilers in the United States (Food and Drug Administration. large amounts of the chemical can be discharged into wastewater and sewage in an unaltered form (Yordy & Alexander. as reported to the NPRI (Environment Canada. On-site releases of diethanolamine (and its salts) to water from 74 facilities in Canada amounted to about 26 000 kg. on-site releases of diethanolamine to air from 74 facilities amounted to about 40 000 kg (Environment Canada.. Mathews et al. Canadian on-site releases of diethanolamine (and its salts) to land and underground amounted to about 118 000 kg and 497 000 kg. 1981. 1997). 1981.

1. short-term exposure limit. In the light of these concomitant exposures. time-weighted average. Jordan. ethanolamines have been used as additives for metalworking fluids since the 1950s and are present in wetting fluids used in asphalt paving. Occupational exposure limits and guidelines for diethanolaminea Country Year Concentration (mg/ m3) 15 15 15 15 15 2 5 (sk) 15 15 2 15 Interpretationb Australia Belgium Denmark France Ireland Netherlands Russian Federation Switzerland United Kingdom United States ACGIHc NIOSH a 1993 1993 1993 1993 1997 1997 1993 1993 1997 1999 1999 TWA TWA TWA TWA TWA TWA STEL TWA TWA TWA TWA From American Conference of Governmental Industrial Hygienists (1999). therefore. There are three major types of metalworking fluid. non-melanoma skin cancer and leukaemia (reviewed by Partanen & Boffetta. straight (generally mineral oils). New Zealand. 1999) and coal-tar pitches (Group 1) (IARC. did not make a detailed evaluation of these studies. Colombia. STEL. skin notation c These countries follow the recommendations of the ACGIH threshold limit values: Bulgaria. Singapore and Viet Nam 2.3-butadiene (Group 2A) (IARC. These groups of workers are also exposed to known or suspected carcinogens present in road paving and roofing materials (see Table 4). Results from cohort and case–control studies of asphalt and road-maintenance workers suggest elevations in the risk of several cancers. The Working Group. 1994). stomach. However. 1987b). any observed risk elevations cannot be specifically attributed to diethanolamine or to any other constituent of the complex mixtures. including lung. National Library of Medicine (1999) b TWA. 1987a). These compounds include benzene (Group 1) (IARC. sk. Studies of Cancer in Humans The Working Group was not aware of any study that specifically examined the risk of cancer among persons exposed to diethanolamine.354 IARC MONOGRAPHS VOLUME 77 Table 3. soluble (straight oils diluted with water and additives) and synthetic (water and addi- . Republic of Korea.

. undiluted. Overall evaluation: 1. 3.DIETHANOLAMINE 355 Table 4. limited evidence. air refined (air-blown) Extracts of steam-refined bitumens Extracts of air-refined bitumens 1.] Asbestos [1332-21-4] Benzene [71-43-2] Bitumens [8052-42-4]. crystalline [7631-86-9] Solar radiation Styrene [100-42-5] Human S S I I I I I L S S L I I I I I I I I I I I I I I I I L S I Animal S S L L L S S S S S S L I I I I I I I L S S I S S S L S S L Overall evaluation 1 1 3 3 3 2B 2B 2A 1 1 2A 2B 2B 3 3 3 3 3 3 3 2A 2A 3 2B 2B 2B 3 2A 1 2B From Partanen and Boffetta (1994) a I. carcinogenic to humans. S. undiluted.3-Butadiene [106-99-0] Coal-tars [8007-45-2] Coal-tar pitches [65996-93-2] Diesel engine exhaust Gasoline Gasoline engine exhaust Kerosene [8008-20-6] Petroleum solvents Polyaromatic hydrocarbons Anthracene [120-12-7] Phenanthrene [85-01-8] Fluoranthene [206-44-0] Pyrene [129-00-0] Chrysene [218-01-0] Benzo[a]pyrene [50-32-8] Benz[a]anthracene [56-55-3] Perylene [198-55-0] Benzo[b]fluoranthene Benzo[j]fluoranthene Benzo[k]fluoranthene [207-08-9] Anthanthrene [191-26-4] Silica. 2A. not classifiable as to its carcinogenicity to humans. possibly carcinogenic to humans. inadequate evidence. sufficient evidence. cracking-residue Bitumens [64742-93-4]. steam-refined (straight-run) Bitumens [92062-05-0]. Degrees of evidence for carcinogenicity in humans and experimental animals and overall evaluation of carcinogenicity to humans for agents to which asphalt workers and roofers may be or may have been exposed. undiluted. L. as evaluated by IARC as of 1993a Agent [CAS No. 2B. probably carcinogenic to humans.

This was an extension of an earlier study reported by Järvholm et al. either diethanolamine or triethanolamine.4. The use of ethanolamines and nitrites together as additives to metalworking fluids can lead to the formation of N-nitrosodiethanolamine. [The Working Group noted that part of this cohort was also studied in relation to exposure to N-nitrosodiethanolamine. There were 209 deaths (standardized mortality ratio (SMR). Ethanolamines were introduced as additives in the metalworking fluids used in the department in the mid-1950s. [0.4% were lost to followup). Mortality and cancer incidence follow-up was conducted from 1958 until 1983 and expected numbers were calculated using reference rates from the same city. with the only notable excess being for stomach cancer (SIR. [1. Only studies which stated that ethanolamines (no studies indicated diethanolamine alone) were used as additives or that presented results for workers primarily exposed to soluble or synthetic fluids were considered by the Working Group. in particular. A total of 792 employees met the entrance criteria (4. existed and no . (1981) in which a two-fold excess of stomach cancer morbidity was reported among workers in the grinding department during 1958–76. 0. there were 41 incident cancers (SIR. 95% CI.69]. Järvholm and Lavenius (1987) examined the risk for cancer among Swedish men employed for at least five years and any time between 1950 and 1966 in the grinding or turning departments of a company producing bearing rings. The other studies are described in detail below. 1986) are reported in the monograph on N-nitrosodiethanolamine in this volume. 0.7–3. Among the subcohort of 559 workers in grinding departments. 95% CI. Studies stating that ethanolamines and nitrites were used as additives or which presented results for exposure to nitrosamines are described in detail in the monograph in this volume on N-nitrosodiethanolamine. the last two introduced in the 1940s. containing ethanolamines)). Of these. These mixtures may contain many potential carcinogens and.] Eisen et al. Metalworking fluids are complex mixtures that may vary considerably depending on the type of fluid and the additives used. Ethanolamines. [0. The results of this investigation (Järvholm et al. (1992) performed a cohort mortality study of 46 384 workers employed for three or more years before 1985 in three United States auto parts manufacturing facilities..356 IARC MONOGRAPHS VOLUME 77 tives) (see General Remarks). 559 men had been employed in the grinding department where soluble and some synthetic oils (acid-refined from 1940–75 and solvent-refined mineral oils from 1975) were used. 95% confidence interval (CI). there is potential for exposure to N-nitrosodiethanolamine (see monograph in this volume) in all of the studies considered.87) in the full cohort.2).86). 0. soluble oils (water-miscible or emulsifier oils) and synthetic oils (chemical fluids. 0. 95% CI. [0.94) and 67 incident cancers (standardized incidence ratio (SIR).45–0. are very common additives to both soluble and synthetic metalworking fluids (see Sections 1.83].5]. A number of studies have examined the risk of cancer among workers exposed to metalworking fluids. The characteristics of these studies are presented in Table 5 and a summary of the results for specific cancer sites is presented in Table 6.54–0. Exposure to all three types of metalworking fluid (straight oils (insoluble or cutting oils).0).63].3 and 1.71–0.

any time 1950–66 in the grinding and turning departments of a bearing rings company (may overlap with Järvholm et al.. duration of exposure to metalworking fluid and other components. but it is included because it forms the study base of the nested case–control studies considered.. incl. (1992)a USA Tolbert et al. (1992) USA Eisen et al. (1998) USA Cohort 792 men employed > 5 years.Table 5. Characteristics of studies on diethanolamine exposure Study/country Study design Study population Follow-up period 1958–83 Potential exposures Järvholm & Lavenius (1987) Sweden Eisen et al. duration of exposure to metalworking fluid and other components. 538 controls (study base: Eisen et al.. (1994) USA Sullivan et al.. no analysis by sub-group Analysis of three sub-groups exposed to each type of metalworking fluid by years of exposure Cumulative exposure to straight and soluble types of metalworking fluid and metalworking fluid particulate exposure during grinding. nitrosamines. 1986) 46 384 employed for > 3 years before 1985 at three auto parts manufacturing facilities 33 619 (two of the three facilities in Eisen et al. 971 controls (study base: Eisen et al. DIETHANOLAMINE Cohort 1941–84 Nested case– control of laryngeal cancer Nested case– control of oesophageal cancer 1941–84 53 fatal cases. Cumulative exposure to the three types of metalworking fluid. 357 . 1992 cohort) 1941–84 a The results of this study were not considered by the Working Group. 1992) 108 fatal and incident cases. 1992 cohort) Analysis of the subgroup of 559 grinders exposed to soluble or synthetic oils Cohort 1941–84 All three types of metalworking fluid.

1–7.7 (0. (1994) (mortality) Soluble fluids (mg/m3–years) 0 0.97–1.5–2.8) NR NR NR NR NR NR 41 33 [0.7–3.0) 1.2) 16 1.6) 8 0.45–0.0–2.0) 1.03 (0.5] (0.22 (0. SMR/ PMR All cancer Obs.5) 1.4) 1.2) [2.00 1. not applicable. not reported .0) [1.0 (0.2 (0.6–1.7) NA NA NA NA NA – – 2.66] (0.0) 1632 1.1) 0.7–3. SMR/ PMR Järvholm & Lavenius (1987) (incidence) All grinders (SIR) > 20 years latency (SIR) Tolbert et al. SMR/ PMR/OR Leukaemia Obs.5) 2 2 [2.3) 8 1.0–6. SMR/ PMR Oesophagus Obs.2–7.87–1.0–1. SMR/ PMR All mortality Obs.46–0.8 (0.0–1.1) 1.6–1.0 > 6.7–1.0–2.9) 19 1.63] (0.358 Table 6. (1998) (20-year lag) (mortality) 5 mg/m3–years synthetic oil 5 mg/m3–years soluble oil 8 7 [1.5–3.5 (0.8–2.4 (1.5) 333 0.02 (0.1) NA NA NA NA NA NA.9) 1.1–2. NR.6–1.7] (0.0] (0.86) [0.3–8.3 (1.1) 1.78–1.34 (0.0–1.6) 0. SMR/ PMR/OR Larynx Obs.96–1.90 (0.00 (0.98–1.8 (1.0 Sullivan et al.03) 0.7) 0.87) 99 17 35 10 30 6 75 4 61 19 1479 200 7287 922 NA NA 9 41 29 29 1.6–3.0 (1.93) IARC MONOGRAPHS VOLUME 77 21 1.0) 1.6 (0.4–1.76–0.4] (0.81 (0.7–2.2–1.1) 1.5–2. SMR/ PMR Pancreas Obs.01 (0.16 (0.4) 0.3–1.7 (0.0) 1.7–3.99 (0.0) 1.2 (1.3 (0.03 (0. Results of epidemiological studies of cohorts exposed to soluble and synthetic metalworking fluids Reference Stomach Obs. (1992) (mortality) Synthetic oils White males Soluble oils White males Black males Eisen et al.9) 1.6 (1.97 (0.0 > 2.

0–2. 95% CI.4. 95% CI. 0.006) (for soluble oils. 95% CI.2. Among black men exposed to soluble oils (n = 4964). Results for black men exposed to synthetic fluids or women exposed to any fluids were not presented because of small numbers. no strong dose–response relationship was observed. p = 0. 95% CI. there were 7287 deaths (SMR. 0. This cohort formed the study base for the three subsequent studies in this monograph. Indices of exposure were developed for duration and cumulative . Slightly larger excesses were observed among persons exposed to synthetic fluids for eight or more years for colon cancer (rate ratio. With the exception of statistically significantly negative associations between lung cancer and synthetic fluids (p = 0.8–3. 1. 95% CI.0–2.6.5–3. 0.0) and larynx (SMR.2. 0.5) and laryngeal cancer (SMR. 1.7). Among white men exposed to synthetic fluids (n = 8446). Eisen et al. length of follow-up. Potential cases were individuals who had.0).81) and small excesses were observed for pancreatic cancer (SMR. 2. 1. 95% CI. 95% CI. 1. 0. 1.3. 95% CI.DIETHANOLAMINE 359 separate analyses for subgroups were presented (Tolbert et al.0–1.4) and pancreatic cancer (rate ratio. 1.0) and brain (SMR. Exposure was assessed based on air sampling data. 1. plant records and interviews with plant personnel. 0.7).5.00) and small excesses were observed for cancers of the stomach (SMR.7). larynx (SMR.6. 9349 deaths were identified and death certificates were obtained for 92%.1) and pancreatic cancer (rate ratio. 1. Plant records and industrial hygiene data were used in combination with detailed work history records to identify which persons were exposed to different types of machining fluid and their duration of exposure.2. Mild excesses were observed among persons exposed to soluble fluids for 20 or more years for stomach cancer (rate ratio. (1992) where metalworking fluids were used extensively. Among white men exposed to soluble oils (n = 23 488).0. or died from.. 95% CI. 0. 1. Tolbert et al. year of birth and age at risk. 0. 0.7) and leukaemia (SMR. Poisson regression analyses were performed to examine the relationships between duration of exposure to each of the three types of metalworking fluid and specific cancer sites after adjustment for plant.9–1. 1. sex. race.0–1. 1.3.7–3. 95% CI. 95% CI.2. there were 922 deaths (SMR.6. 1.4. 1. 1.09). (1994) reported the results of a nested case–control study of laryngeal cancer among the members of the cohort studied by Eisen et al.1) and leukaemia (SMR. (1992) reported the results of a cohort study of 33 619 persons who had worked for at least three years before 1985 in two of the three facilities studied by Eisen et al.8–2. (1992). 1.01) and small excesses were observed for cancers of the stomach (SMR. plant. 95% CI. 1.9–4.7–2.2). race and sex.7–2. Mortality was followed from 1941 to 1984 and vital status could be determined for 94% of the cohort at the end of follow-up. there were 1632 deaths (SMR. 1992).4). 1. 0. In total. laryngeal cancer between 1941 and 1984 and people with laryngeal cancer identified using regional tumour registries or based on other information included on death certificates.5–3. Cases were verified using tumour registry or hospital records and a total of 108 cases were eligible for inclusion (all but one being squamous-cell carcinomas). 95% CI. 1. Incidence density sampling was used to select five controls for each case matched on the basis of year of birth.

with the addition of duration and cumulative exposure to synthetic fluids and duration of exposure to nitrosamines. Matched analyses were performed using conditional logistic regression models with additional adjustment for time since hire. The Working Group was also aware of a number of population-based case–control studies that reported risks associated with exposure to unspecified metalworking fluids or employment in occupations with potential exposure to metalworking fluids. [The Working Group noted that the mixed and varying exposures may explain the variability of the results of the different studies and also make it very difficult to ascribe the excesses of cancer observed to any single agent. 95% CI. [The Working Group noted that in the last studies.1–7. The relationship with exposure to synthetic fluids or ethanolamines was not presented. The risk for laryngeal cancer was not found to be associated with either cumulative level (mg/m3–years) or duration of exposure to soluble metalworking fluids. 53 cases and 971 controls remained. plant.] The Working Group was aware of several other cohort and proportionate mortality studies which included workers exposed to metalworking fluids but did not include analyses of sub-groups of workers exposed to soluble or synthetic fluids. However. Potential cases were 60 individuals who died of oesophageal cancer between 1941 and 1984. 1. (1992).360 IARC MONOGRAPHS VOLUME 77 exposure to the straight and soluble metalworking fluids and duration of exposure to biocides. 3. Analyses for exposure specifically to ethanolamines and the risk for oesophageal cancer were not presented.1–9. race and sex. 95% CI. (1998) conducted a nested case–control study of oesophageal cancer among the members of the cohort studied by Eisen et al. sulfur and various metals. Matched analyses were performed using conditional logistic regression with additional adjustment for time since hire.3. Sullivan et al. data on tobacco smoking and alcohol drinking were not directly presented. Incidence density sampling was used to select 20 controls for each case matched on the basis of year of birth.6 for five years). Work history data and an exposure matrix developed for the study were used to assign exposure. After allowing for a 20-year latency. (1994). but because of missing data.8. 1. The same indices of exposure were used as those described for Eisen et al. 2.5 for 5 mg/m3–years) and duration of exposure to synthetic fluids (odds ratio.] . Lagging was used to account for latency. these studies were not considered informative for the evaluation because of the unknown probability of exposure to ethanolamines and the potential for confounding from exposure to other known or suspected carcinogens. oesophageal cancer was associated with cumulative exposure to synthetic fluids (odds ratio.

33/50.05. Poly-3 trend test) for the control. Survival rates for dosed male and female groups were similar to those of corresponding vehicle control groups. 48/50 and 48/50 (p < 0.004) and 5/50 (p = 0. mid. 38/50 and 42/50 (p < 0. Survival of dosed male mice was similar to that of the vehicle control group. respectively). hepatocellular carcinoma: 12/50. 32 or 64 mg/kg bw and females 0. 4/50.and high-dose males were lower than those of the vehicle controls after weeks 88 and 77. the incidences of hepatoblastoma in the mid. 42/50. 6/50 and 6/50 (p = 0. respectively). Poly-3 trend test) in the control. mid.1. low-.and high-dose groups. 49/50 and 45/50 (p < 0. were administered 0.1 3. respectively). 3. 2/50. > 99%) in 95% ethanol by dermal application on five days per week for two years. low-. Poly-3 trend test). 8/50 (p = 0. but survival of dosed female mice was reduced (44/50. In male mice. 1999a). The mean body weights of the low.001. 3. The mean body weights of the mid.and high-dose groups. When combining single with extended step-sectioning. 50/50. for the control. 17/50.DIETHANOLAMINE 361 3. respectively (National Toxicology Program. the incidences of hepatocellular adenoma and of hepatocellular adenoma and carcinoma (combined) in all dosed groups were significantly greater than those in the vehicle control group (hepatocellular adenoma: 31/50. hepatocellular carcinoma: 5/50. low-. low-. Males received 0. Poly-3 trend test). low-. Renal tubule adenomas in males showed a marginal increase after standard single-section examination (1/50. 16. respectively). > 99%) in 95% ethanol by dermal application on five days per week for two years. 33/50 and 34/50 (p < 0. the incidences of hepatocellular neoplasms were significantly higher than those in the vehicle control group (hepatocellular adenoma: 32/50. 8/50 and 7/50 (p = 0. six weeks of age. pairwise comparisons) in the control.2 Rat Groups of 50 male and 50 female Fischer 344/N rats.and high-dose groups were significantly increased compared with the vehicle control (0/50. In the female mice. mid.and high-dose groups.and mid-dose females were lower than those of the vehicle controls from week 73.001. mid. respectively.and high-dose groups. the incidences were: 1/50. In addition. 6/50. but those of the high-dose females were reduced compared with the vehicle controls from week 53. respectively). 16 or 32 mg/kg bw. The mean body weight .001.1 Studies of Cancer in Experimental Animals Skin application Mouse Groups of 50 male and 50 female B6C3F1 mice. 80 or 160 mg/kg bw diethanolamine (purity. low-.001. six weeks of age. 40. Poly-3 trend test) in the control.and high-dose groups. Poly-3 trend test).055. mid. 8. 33/50 and 23/50 for the control.028. were administered diethanolamine (purity. 19/50.1.and high-dose groups. mid.

which carry a zeta-globin promoted v-Haras gene on an FVB background. The same three condensates were also tested in the transgenic Tg. and the fact that these studies were not designed as. The concurrent negative control groups were treated with 200 μL 95% ethanol.c. 10 or 20 mg diethanolamine per mouse per application (higher than the MTD). Six weeks after the last application. 1999a). Survival was high in both the control (90%) and treated groups (80–95%).. The diethanolamine was administered in 200-μL volumes.2 Genetically modified mouse Groups of 15–20 female Tg. five times per week for 20 weeks. approximately 99% pure) twice per week for 20 weeks. lauric acid and oleic acid diethanolamine condensates (National Toxicology Program. and did not represent. This judgement was based on the fact that the substances tested were complex mixtures of imprecise composition. 3. The Working Group concluded that these studies could not be used in the evaluation of the carcinogenicity of diethanolamine per se.AC mice. [The Working Group was aware of three carcinogenicity bioassays (dermal application studies) in B6C3F1 mice and Fischer 344/N rats of fatty acid-diethanolamine condensates conducted by the National Toxicology Program. There were no increases in tumours in treated groups compared with the vehicle controls (National Toxicology Program. These were coconut oil acid.. that the actual diethanolamine content had not been measured in any of the three studies and therefore the precise levels of exposure were indeterminable. In contrast to the positive controls. 2000). There was no evidence of chronic irritation or ulceration at the site of application.362 IARC MONOGRAPHS VOLUME 77 of the high-dose male group was lower than that of the vehicle controls from week 8 and the mean body weight of the high-dose female group was lower than that of the vehicle controls from week 97.25 μg 12-O-tetradecanoylphorbol 13-acetate (TPA. there was no increase in the incidence of skin tumours in diethanolamine-treated animals in this model (Spalding et al. which developed multiple papillomas in 18/20 animals. were administered diethanolamine topically in 95% ethanol (the diethanolamine used was from the same chemical batch as that used in the mouse National Toxicology Program study (National Toxicology Program. 2000). The positive control group was treated with 1.AC and p53+/– mouse models (Spalding et al. 1999a). Animals that did not survive until the end of week 10 were not included in the data summaries or calculations.d).] . 1999b. all surviving mice were killed. conventional or adequate carcinogenesis bioassays of diethanolamine. Lesions were diagnosed as papillomas when they reached at least 1 mm in diameter and persisted for three weeks. The doses of diethanolamine selected were based on the maximum tolerated dose used earlier (National Toxicology Program. 14 weeks of age. 1999a) and were 5.

78 mL/kg bw per 4-cm2 area) was the same as in the carcinogenicity bioassay (Section 3. oral + dermal (grooming allowed) and oral gavage treatment. The radioactivity was found in carcass. liver or kidneys but very little in urine (0...68%) than that from undiluted material (0. Other Data Relevant to an Evaluation of Carcinogenicity and its Mechanisms Absorption. (2000). 1.11%). The dermal dosing method for diethanolamine (90 mg/mL in ethanol. Human skin proved to be the best barrier against aqueous diethanolamine (37%. 1999a). the blood levels of diethanolamine were 5. 1996). The total absorbed dose from aqueous diethanolamine was greater (0. Absorbed [14C]diethanolamine was determined in 48-h urine and faeces and from sampled tissues. respectively. . while the majority of [14C]diethanolamine was recovered in the occlusive wrappings (80%) and in skin of the dose site (3.7 μg/g for dermal (collared mice). 1997)..1 4.DIETHANOLAMINE 363 4. mice. 15 μL volume. [14C]Diethanolamine was applied to 19. and protected non-occlusively by a dome of wire mesh) were more efficiently absorbed (27–58%) than the test doses (2–28 mg/kg bw. rabbits and humans (female mammoplasty patients). faeces or blood (Waechter et al. 1990. Gillner & Loeper.1 No data were available to the Working Group.. Evidence of dermal absorption and the effect of grooming were reported by Stott et al. Melnick & Tomaszewski. distribution.4% and washed animals 0. 1997).1..1. w/w) followed by rat.5 cm2 of the dorsal skin (20 mg/cm2. cited by Knaak et al. Skin penetration rates and permeability constants (kp) for 14C-labelled diethanolamine (Table 7) were determined in vitro using full-thickness skin preparations from rats. Knaak et al.64% of the dose. Unwashed rats absorbed 1. 1500 mg/kg bw) and covered for 48 h (no washing) or for 6 h before it was removed by washing. 6. metabolism and excretion Humans 4. Dermal doses of [14C]diethanolamine applied in 95% ethanol (for 48 h) to a 1-cm2 area of B6C3F1 mouse skin (8–81 mg/kg bw.1. 4. 1983.02–1. Dermal absorption was also studied in rats.2 Experimental systems (a) Absorption and distribution Data on the toxicokinetics of diethanolamine have been reviewed (Beyer et al. rabbit and mouse skin when the chemical was applied as an ‘infinite dose’ (20 mg/cm2 to cm2 of skin for 6 h). After the final dose (1–2 h).6%).3%) (Sun et al.23–6. 25 μl volume) applied to a 2-cm2 area of Fischer 344 rat skin (3–16%) (Mathews et al. 1995.1..6 and 7. 1997). Diethanolamine was administered (160 mg/kg bw per day) to B6C3F1 mice by dermal application (with or without access to the application site) or by oral gavage for two weeks. 1995. National Toxicology Program.

respectively.09e From Sun et al. Single oral doses (0. Non-radiolabelled diethanolamine was applied to the dorsal skin of rats (1500 mg/kg bw.62 3.113–45.43d Mouse 6.01d 0.08 ± 0. Liver.364 IARC MONOGRAPHS VOLUME 77 Table 7.7 23. The tissue-to-blood . fat or heart (Waechter et al. [14C]Diethanolamine (1500 mg/kg bw) was then applied to the skin for a 48-h penetration test. 1997).01d 1.6 0.81 ± 2. with less than 3% in faeces and only 0.01 0.42 0. About 20–30% of oral and intravenous doses (7 mg/kg bw) was found in urine (mainly as unchanged diethanolamine).15d 0.3 3.2 12. and less than 0. kpc (cm/h × 10–4) Undiluted diethanolamine Rat Mouse Rabbit Human 0.03e 0. ca.60 7. Animals in the three-day and six-day pretreatment groups absorbed 21% and 41% of the applied dose.34 Aqueous diethanolamine (37% w/w) Rat 0. urine from three-day and six-day groups contained 4.39d Human 0. Most of the diethanolamine was retained in tissues at high concentrations. 1997).2% or less was exhaled (CO2) within 48 h.8 46. 20 mg/cm2 to 25 cm2 of skin and covered) once per day for 6 h per day for three or six days.05 0.7–200 mg/kg bw) were well absorbed but excreted very slowly. 1995.3% and 13%. derived from the slope of the linear segment of a plot of the cumulative mg/cm2 absorbed versus time c kp = Steady-state penetration rate (mg/cm2/h) Initial concentration (mg/cm3) d Mean ± SE (n = 3) e Mean ± SE (n = 6) Combined data from several studies (cited above) showed that in rats the absorption rate increased linearly with the [14C]diethanolamine dose (single). cited by Knaak et al.68 ± 5.2 0. and that a 100-fold increase in the dose of diethanolamine (188–19 720 μg/cm2) resulted in a 450-fold increase in absorption rate (0. [14C]Diethanolamine (7 mg/kg bw) was given orally to male Fischer 344 rats once or by daily repeat dosing for up to eight weeks.8 0.9 1. kidney or carcass contained the majority of absorbed radioactivity..5 2.23 ± 0.4 132.7 0.56 ± 0.0 294.02 0..9 5..28d Rabbit 2.3 0. respectively.4 1. (1996) a Extrapolated from the intercept of the linear segment (regression) line with the abscissa b Penetration rate at steady state.0 μg/cm2 per h) (Knaak et al.3% was found in brain.42 0. Skin penetration characteristics of undiluted and aqueous solutions of [14C]diethanolamine Species Cumulative dose absorbed (%) Lag time (h)a Steady-state penetration rateb (μg/cm2/h) Permeability constant.30 ± 1.02 ± 0.8 1.04 ± 0.

(b) Metabolism and excretion Diethanolamine is incorporated into membrane phospholipids (Artom et al. In contrast. After single oral and intravenous administrations of diethanolamine to Fischer 344 rats.. The synthesis of liver phospholipids in vitro was competitively inhibited by diethanolamine (Ki ∼ 3 mM). Diethanolamine is conserved and metabolized by biosynthetic routes common to ethanolamine. brain and muscle. 1997). 1979a). These levels were inversely related to the blood diethanolamine levels (uptake) after the final dose.. The blood was a notable exception in that it continued to bioaccumulate diethanolamine throughout the eight-week dosing period. 84 and 70%. 2000). 1997). 1958. 1958) and interacts with lipid metabolism in vivo. The catabolism of diethanolamine-containing lipids was slower than that of the corresponding cholineand ethanolamine-containing derivatives (Artom et al..N-dimethylated derivatives that are incorporated as polar head groups into aberrant phospholipids which are.3 mg equivalent/g). Functional and structural alterations induced by diethanolamine in liver mitochondria may ensue from its adverse effects on lipid metabolism in subcellular membranes (Barbee & Hartung.. respectively in male B6C3F1 mice after two weeks’ administration of diethanolamine (160 mg/kg bw per day) via oral gavage or skin painting. 1995. The metabolism of diethanolamine leading to urinary elimination is illustrated in Figure 1. resulting in O-phosphorylated. 1949. phosphocholine and glycerophosphocholine were reduced as much as 64. the hepatic levels of sphingomyelin were increased relative to those in control mice. The highest concentrations of diethanolamine-associated radioactivity measured 72 h after the final dose given in the eight-week period were found in the liver (0. Diethanolamine was a less effective precursor (Km = 12 mM) in phospholipid synthesis than the natural substrates choline (Km = 0. 1995. only a small . 1997). Hepatic levels of choline. the bioaccumulation of diethanolamine to plateau levels at between four and eight weeks was accompanied by an increasing degree of methylation and accumulation of aberrant sphingomyelinoid lipids in tissues. and were directly correlated with blood diethanolamine levels (Stott et al. incorporated into critical membranes (Mathews et al. the compound is excreted predominantly unchanged in urine. Uptake. 1979a). Tissue radioactivity was found mainly in aqueous extracts (up to 90%) and 5–10% was organic-extractable (Mathews et al.054 mM) (Barbee & Hartung. 1979b). 30–40 for the lung and spleen and 10–20 for the heart. After repeated administration (7 mg/kg bw on five days per week for eight weeks). for example by inhibiting incorporation of ethanolamine and choline into phospholipids in rat liver and kidney. About 30% of the diethanolamine-derived phospholipids in rat liver were ceramides (sphingomyelins) and about 70% were phosphoglycerides following a single oral dose of diethanolamine (7 mg/kg bw). in turn. retention and metabolism of diethanolamine in human and rat liver slices are reported to be similar (Mathews et al. Barbee & Hartung..076 mM) and ethanolamine (Km = 0.. N-methylated and N.DIETHANOLAMINE 365 ratios were 150–200 for the liver and kidney.

. 2000). (1997) After four weeks’ repeated administration of [14C]diethanolamine (7 mg/kg bw per day on five days per week) to rats. the relative amounts of N-methyldiethanolamine and N. 1995.N-dimethyl-2-oxomorpholinium appearing together with unchanged diethanolamine in urine increased markedly..N-Dimethyldiethanolamine O CH3 O OH CH3 d c N CH3 d b O a HO N CH3 H2O N. based on urinary excretion data from an eight-week oral dosing study (7 mg/kg bw per day. 1987).N-Dimethyl-2oxomorpholinium From Mathews et al.and dimethylated derivatives. Figure 1. No evidence of formation of N-nitrosodiethanolamine in vivo was found.. Excretion of N-nitrosodiethanolamine in urine was evident in Sprague-Dawley rats receiving diethanolamine via the skin (100–400 mg/animal) and sodium nitrite in the drinking water (2000 ppm [mg/L]) for six days but not in the absence of sodium nitrite (Preussmann et al. The log-linear response with time was a first-order process with a whole-body elimination half-life of about six days (Mathews et al. Proposed pathway of diethanolamine metabolism in the rat. As a result of progressive methylation after repeated oral administration (eight weeks). 1981). 1997).N-dimethyldiethanolamine in vivo and in incubations with rat liver microsomes in vitro (Mathews et al. five days per week for eight weeks) H HO N OH HO CH3 N CH3 OH Diethanolamine N. 40 mg/kg bw) (Stott et al.. The quaternized lactone was formed from N. .. 1997). in B6C3F1 mice treated with diethanolamine (160 mg/kg bw per day) and sodium nitrite (140 ppm in drinking-water.366 IARC MONOGRAPHS VOLUME 77 portion being found as its mono. however. the urinary excretion of radiolabel (during the ‘washout phase’) was followed for another four weeks. N-Nitrosodiethanolamine was detected in a gastric rinse of rats treated with 100 μmol diethanolamine [59 mg/kg bw] and 400 μmol sodium nitrite [153 mg/kg bw] by gavage (Konishi et al.

the effect was present even at the lowest dose after 13 weeks in both males and females. 2500 or 5000 ppm [mg/L] diethanolamine in the drinking-water (equivalent to 25–440 mg/kg bw per day).and triethanolamine) has been reviewed (Knaak et al. In Swiss Webster mice. 1997). B6C3F1 mice were given to 0. 2500.b).2 4. a dosedependent increase in liver weight was observed after two weeks. Kidney toxicity. 630. Melnick et al. 320. 1994a. no vacuoles were visible in hepatocytes and fatty droplets were reduced in number (Blum et al. 630.15% diethanolamine and 0. were observed 4 h after dosing. By 24 h. marked liver changes. 320. 1250. Extensive information is available on toxic effects following oral and dermal application of diethanolamine in a 13-week subchronic study (National Toxicology Program. Two male rats died in the highest-dose group. 4. increased kidney weight. Moreover. including tubular necrosis. 1250.. 160.2. tubular necrosis and loss of kidney function occurred after two weeks. Epithelial cell necrosis in kidney tubules was seen only at the highest dose in both sexes. At the three higher dose levels. At this dose. Some mild changes in the liver were observed. Hepatocellular necrosis was found with doses ≥ 2500 ppm. as well as after a 15-min exposure to pure diethanolamine at aerosol concentrations of 0.. In a concurrent study. Poorly regenerative microcytic anaemia developed within two weeks. Demyelination in the medulla oblongata (brain) and spinal cord was found after 13 weeks in both males and females (Melnick et al. 1994a). The positive reaction was observed in a 39-year-old male metal worker after a 30-min or 45-min inhalation exposure to aerosols from a warmed cutting fluid (40 °C) containing 0. 1992. without observed changes in bone marrow.2. 1998). exposures were equivalent to 100– 1700 mg/kg bw per day for males and 140–1100 mg/kg bw per day for females. 1972). such as weight increase.DIETHANOLAMINE 367 4. the mice lost weight and all males and females in the two highest-dose groups died before the end of the study. both male and female rats lost weight in a dose-dependent fashion. while groups of 10 females were given 0.0 mg/m3 (Piipari et al. was seen .32% triethanolamine.. Groups of 10 male Fischer 344/N rats were given 0. including extensive vacuolization and fat droplets. the LD50 for diethanolamine (by intraperitoneal injection) was 2.75 and 1. 630. 5000 and 10 000 ppm [mg/L] in the drinking-water.2 Experimental systems The toxicity of diethanolamine (as well as of mono. Diethanolamine-induced occupational asthma was diagnosed following specific bronchial provocation tests in an exposure chamber..3 g/kg bw. In both males and females.. 1250 or 2500 ppm (equivalent to 15–240 mg/kg bw per day).1 Toxic effects Humans The only experimental data available on human exposure to airborne diethanolamine come from clinical provocation tests. Cytological changes in hepatocytes were found at all doses after 13 weeks.

.368 IARC MONOGRAPHS VOLUME 77 only in male mice after 13 weeks. degeneration of cardiac myocytes was seen at doses of 2500 ppm and above (Melnick et al. including tubular necrosis. 1982). Diethanolamine treatment caused a marked reduction in hepatic choline metabolite concentrations in mice following two weeks of dermal dosing. Kidney toxicity. hyperkeratinosis and acanthosis of the epidermis. In the same study (Melnick et al.. 1990). the effects of dermal exposure were observed during a 13-week study. the pattern by which choline metabolites were altered was similar to the pattern of change that has been observed following dietary choline deprivation in rodents (Pomfret et al. The most pronounced reduction was in the hepatic concentration of phosphocholine. 2000). and cardiac myocyte degeneration were found in both males and females. 1997). Kidney toxicity. 1994b). but no histopathological changes were observed in the liver. but hepatocellular necrosis occurred only in male mice. 4.1 Reproductive and developmental effects Humans No data were available to the Working Group. irritation of the skin was moderate. Liver weights were increased in both males and females. accompanied by inflammation. Demyelination in the medulla oblongata (brain) and spinal cord also occurred. was observed..b).3. 1994a. including tubular necrosis and mineralization. some rats died during the study period. 2000). In both males and females. Microcytic anaemia also developed. the highest dose induced a decrease in body weight compared with controls.. Skin toxicity was observed at the site of application and liver weight increased. Moreover. whereas irritation of the eye was severe (Dutertre-Catella et al. Excess choline also prevented diethanolamine-induced inhibition of phosphatidylcholine synthesis and incorporation of diethanolamine into SHE cell phospholipids (Lehman-McKeeman & Gamsky. after skin application of doses of 80–1250 mg/kg bw on five days per week. 4.3 4. competitive and reversible manner (Lehman-McKeeman & Gamsky.. Irritation of the eye and skin after application of pure (98%) diethanolamine was investigated in New Zealand White rabbits. At the highest dose. In mice. Groups of 10 male and 10 female rats received applications of 32–500 mg/kg bw on five days per week. especially in females. 1999. After 72 h. 2000)..3. . Diethanolamine has been shown to inhibit choline uptake into cultured Syrian hamster embryo (SHE) and Chinese hamster ovary cells and to inhibit the synthesis of phosphatidylcholine in in-vitro systems in a concentration-dependent. similarly to that observed after oral exposure.2 Experimental systems The reproductive and developmental toxicity of diethanolamine tested has been reviewed (Knaak et al. Ulcerative skin lesions at the site of application developed. the intracellular storage form of choline (Stott et al.

4. 500. cited by Knaak et al. No treatment-related malformations were observed (Gamer et al. 1999). 4. 35. There was no effect of any treatments on development or on the incidence of external. but delayed ossification of the axial skeleton and distal appendages was observed in fetuses of the 1500-mg/kg bw group (Marty et al. In a 13-week subchronic study in male Fischer 344/N rats. 1994a). There was no effect of any treatments on fetal weight or on the incidence of external. 1983) and by Knaak et al. TA1538 or TA98 in three studies. The two higher dose levels produced severe skin irritation. Inhalation exposure of pregnant Wistar rats to 0. Reduced sperm count and motility as well as degeneration of the seminiferous tubules were found at a dose of 2500 ppm.. Maternal body weight gain was reduced in the 200-mg/kg bw group. 50.... 800 or 1200 mg/kg bw per day.DIETHANOLAMINE 369 Diethanolamine was administered by gavage to Sprague-Dawley rats on days 6–15 of gestation at dose levels of 0. TA1535.1 Humans No data were available to the Working Group. (1997). cited in Marty et al. or to Escherichia coli WP2 uvrA in a single study. testis and epididymis weights were decreased at diethanolamine doses of 1200 ppm or more in the drinking water (Melnick et al. 150. 1990. 200. Rats receiving 500 mg/kg bw or higher doses either died or were in a moribund condition and were killed. but none of the gestational parameters in the treated groups was significantly different from those of the controls (Environmental Health Research & Testing..4 Genetic and related effects The genetic toxicity of diethanolamine has been reviewed by an expert panel for the cosmetic ingredient review (Beyer et al. visceral or skeletal abnormalities (Marty et al.2 mg/m3 diethanolamine aerosols for 6 h per day on days 6–15 of gestation caused an increased incidence of cervical ribs in the fetuses. Diethanolamine was applied as an aqueous solution to the skin of New Zealand White rabbits on days 6–18 of gestation at dose levels of 0. The highest dose level produced marked skin irritation.4. It did . 1993. 500 and 1500 mg/kg bw per day. in the presence or absence of exogenous metabolic activation. visceral or skeletal abnormalities. 1997). 4. The rats were killed on day 20 and the uteri examined for number of implantation sites and for live and dead implantations. 1999). TA1537. Diethanolamine was painted as an aqueous solution on the skin of CD rats on days 6–15 of gestation at dose levels of 0. 4. 100 or 350 mg/kg bw per day.. 1999).2 Experimental systems (see Table 8 for references) Diethanolamine was not mutagenic to Salmonella typhimurium strains TA100..

370 Table 8. (1983) National Toxicology Program (1999a) Dean et al. TA1538. (1988) National Toxicology Program (1999a) National Toxicology Program (1999a) Dean et al. TA1535. TA1535. Syrian hamster embryo cells (8-day treatment) Cell transformation. (1985) Dean et al. reverse mutation Salmonella typhimurium TA100. Chinese hamster ovary CHO cells in vitro Sister chromatid exchange. Genetic and related effects of diethanolamine Test system Resulta Without exogenous metabolic system Salmonella typhimurium TA100. Tk locus in vitro Sister chromatid exchange. Syrian hamster embryo cells (24-h treatment) Cell transformation. TA1535. (1996) Kerckaert et al. (1985) Dean et al. TA1537. rat liver RL cells in vitro Cell transformation. TA1537. (1982) Kerckaert et al. Chinese hamster ovary CHO cells in vitro Chromosomal aberrations. mouse lymphoma L5178Y cells. Syrian hamster embryo cells (7-day treatment) Cell transformation. reverse mutation Salmonella typhimurium TA100. reverse mutation Escherichia coli WP2/WP2uvrA. mitotic gene conversion in stationary and log-phase cultures Gene mutation. Chinese hamster ovary CHO cells in vitro Chromosomal aberrations. (1985) National Toxicology Program (1999a) Sorsa et al. TA1538. reverse mutation Saccharomyces cerevisiae JD1. Syrian hamster embryo cells (7-day treatment) – – – – – – – – – – – + + +c With exogenous metabolic system Dose (LED or HID) Reference IARC MONOGRAPHS VOLUME 77 – – – – – – – – – – NT NT NT NT 3333 μg/plate 3333 μg/plate 4000 μg/plate 4000 μg/plate 5000 330 2176 1500 3010 0. (1996) Lehman-McKeeman & Gamsky (2000) . TA98.5 × GI50 500 4500 250 500 Haworth et al. (1985) Inoue et al. TA98. TA1537.

in-vivo tests.Table 8 (contd) Test system Resulta Without exogenous metabolic system Micronucleus formation. 12 d Reference DIETHANOLAMINE – – Fernandez et al. negative. positive. NT. concentration causing 50% growth inhibition b 371 . not tested LED.6 and 5 GI50. lowest effective dose. 12 d 75 ppm. mg/kg bw/day c Negative in the presence of 30 mM choline d In the presence of sodium nitrite or nitrate at pH 8. in-vitro tests. HID. newt larvae (Pleurodeles waltl) blood cells in vivo Micronucleus formation. (1993) L’Haridon et al. newt larvae (Pleurodeles waltl) blood cells in vivod a Dose (LED or HID) With exogenous metabolic system 75 ppm. –. (1993) +. highest ineffective dose. μg/mL.

Stott et al. 5. (2000) showed that diethanolamine induced choline deficiency and depleted several choline-containing compounds in B6C3F1 mice. 1996). It did not induce sister chromatid exchanges in Chinese hamster ovary cells in two studies with or without exogenous metabolic activation. A single study using cultured rat liver cells found no induction of chromosomal aberrations and one study in Chinese hamster ovary cells also found no induction of chromosomal aberrations in either the presence or absence of exogenous metabolic activation. diethanolamine alters choline homeostasis in a manner resembling choline deficiency. Diethanolamine did not induce mutations in mouse lymphoma L5178Y cells at the Tk locus in the presence or absence of exogenous metabolic activation in one study. Occupational exposure may occur by inhalation and dermal contact. A much larger study revealed induction of cell transformation at a similar dose after a seven-day treatment and at a much higher dose after a 24-h treatment with diethanolamine.372 IARC MONOGRAPHS VOLUME 77 not induce gene conversion in Saccharomyces cerevisiae strain JD1 in the presence or absence of exogenous metabolic activation.5 Mechanistic considerations In mice. inks. 4. Diethanolamine-induced choline deficiency thus provides a mechanism for the tumorigenesis noted in mice but not in rats. 2000) found that diethanolamine inhibited the uptake of choline into mammalian cells. oils. It is known that deprivation of choline in the diet of rodents predisposes to the appearance of hepatocellular carcinomas (Zeisel. Exposure of the larvae of the newt Pleurodeles waltl to diethanolamine did not induce micronuclei in their blood cells and this result remained unaffected by changing the pH or by the addition of sodium nitrite or nitrate.1 Summary of Data Reported and Evaluation Exposure data Diethanolamine is a viscous liquid widely used as a chemical intermediate and as a corrosion inhibitor and surface-active agent in various products including metalworking fluids. diethanolamine had no effect after an eight-day treatment. while Lehman-McKeeman & Gamsky (1999. fuels. paints. cosmetic formulations and agricultural products. No data were available on environmental exposure to . particularly in metal-machining occupations. In one study of cell transformation in the Syrian hamster embryo clonal assay. A further seven-day treatment cell transformation study demonstrated a positive dose-related response to diethanolamine up to 500 μg/mL that was abolished by coadministration with 30 mM choline. 5.

4 Other relevant data Diethanolamine is metabolized by biosynthetic routes common to endogenous alkanolamines (ethanolamine and choline) and incorporated into phospholipids. oesophagus and larynx. 5. In the absence of sodium nitrite. 5. Oral or dermal exposure of rats to diethanolamine during organogenesis was not associated with any sign of developmental toxicity. Small excesses were observed for cancers at various sites. consistent with choline deficiency. The general population may be exposed through contact with a variety of personal care products. In the mouse study. Diethanolamine competitively inhibits the cellular uptake of choline in vitro and hepatic changes in choline homeostasis. It is excreted predominantly unchanged with a half-life of approximately one week in urine. while inhalation exposure to diethanolamine aerosols caused signs of developmental toxicity. It is difficult to draw conclusions regarding diethanolamine using data from studies of exposures to these complex mixtures. as well as an increase in the incidence of hepatoblastomas in males.2 Human carcinogenicity data Two cohort studies and two nested case–control studies looked at cancer mortality or incidence among workers using metalworking fluids with ethanolamines as additives.DIETHANOLAMINE 373 this substance.3 Animal carcinogenicity data Diethanolamine was tested for carcinogenicity by dermal application in one study in mice and in one study in rats. In most of these studies. only associations with use of soluble oils or synthetic fluids were presented and no results were given specifically in relation to diethanolamine exposure. in particular the stomach. 5. there was no treatment-related increase in the incidence of skin tumours after skin application. are observed in vivo. In rats. In a Tg. there was a treatment-related increase in the incidences of both hepatocellular adenomas and carcinomas in both males and females. Dermal exposure of rabbits during organogenesis caused no sign of developmental toxicity. no conversion to N-nitrosodiethanolamine is observed. No data on reproductive and developmental effects in humans were available. There was also a marginal increase of renal tubule adenomas in males. no treatment-related increase in the incidence of tumours was seen in either males or females. . with or without sodium nitrite.AC transgenic mouse model using similar doses to the first mouse study.

There is limited evidence in experimental animals for the carcinogenicity of diethanolamine. & Hartung..E. appl. J. R.. 431–440 Beyer. . (1979b) Diethanolamine-induced alteration of hepatic mitochondrial function and structure. Chem. Diethanolamine induced cell transformation in Syrian hamster embryo cells in vitro in two studies but not in another. Jr (1958) In vivo incorporation of diethanolamine into liver lipides. Berndt W. 495–503 Artom. & Oates..374 IARC MONOGRAPHS VOLUME 77 Testicular effects have been found after exposure of rats to diethanolamine in the drinking water. Pharmacol...L.. Toxicol. References American Conference of Governmental Industrial Hygienists (1999) TLVs and other Occupational Exposure Values—1999 [CD-ROM]. 6. R. Pharmacol. ACGIH® Artom. Diethanolamine did not induce micronucleus formation in larval newt blood cells in either the absence or presence of sodium nitrite or nitrate.O. No data on genetic and related effects of diethanolamine in humans were available to the Working Group. Toxicol. appl. Schroeter.. W. (1979a) The effect of diethanolamine on hepatic and renal phospholipid metabolism in the rat. It was without effect on gene conversion in yeast and was not mutagenic in bacteria.F. Hoffmann. 421–430 Barbee.. diethanolamine.A... J.J. Am.. OH. Cincinnati. M. W. R. Boutwell. A. sister chromatid exchanges or chromosomal aberrations.K. Coll.H.B. W. Lofland. S. 180.. 5. Overall evaluation Diethanolamine is not classifiable as to its carcinogenicity to humans (Group 3)1.. H. Carlton. 183–235 1 Dr Mirer dissociated himself from the conclusions of the Working Group. & Crowder. It did not induce gene mutations. D. S. C. 47. Cornatzer. and monoethanolamine. biol. Bergfeld. 47. The limited data available to the Working Group do not indicate that diethanolamine is genotoxic. J. 833–837 Barbee. J.K. biol. Toxicol. C. 233. K.W. (1949) The action of an analogue of ethanolamine (diethanolamine) on the formation of liver phospholipides. Chem. (1983) Final report on safety assessment of triethanolamine..5 Evaluation There is inadequate evidence in humans for the carcinogenicity of diethanolamine. & Hartung.J. 2.

. 701–704 Food and Drug Administration (1999) Food and drugs. (1992) Mortality studies of machining fluid exposure in the automobile industry. MI Dow Chemical Company (1998b) The Specifier’s Guide to Buying and Applying Ethanolamines.. 7. Tolbert..N. J.. & Tanaka. J. L’Haridon. M. Toxicol. No. R. (1998) The Merck Index. Forensic Sciences.170. MI Dow Chemical Company (1999) Material Safety Data Sheet: Diethanolamine. 83–99 Fernando. 4th Ed. 12th Ed. Dallas. Tolbert. N. 175–185 Bollmeier. 292. John Wiley. Hodson-Walker. 138–165.105. (1998) Determination of diethanolamine and N-nitrosodiethanolamine in fatty acid diethanolamides. US Code Fed. In: Kroschwitz.. 455–460 Eisen.. S. Hallock.. Vol. 2.J. BASF Aktiengesellschaft.. NJ. Initial assessment. (1993) Study of the Prenatal Toxicity of Diethanolamin in Rats after Inhalation. R. Huizenga. 57–77 Dow Chemical Company (1998a) Sales Specification: Diethanolamine. Regul. Engin. (1993) Amphibian micronucleus test(s): a simple and reliable method for evaluating in vivo effects of freshwater pollutants and radiations. Chemical Information Services (1999) Directory of World Chemical Producers (Version 99. Konishi. Ohta. V. (1982) Comparative study of skin and eye irritation by ethanolamines (mono.F. Brooks.1. (1994) Mortality studies of machining fluid exposure in the automobile industry.O. Drugs.F.A.G..A. I: A standardized mortality ratio analysis. D. MI Dutertre-Catella. (1985) Genetic toxicology testing of 41 industrial chemicals. Midland. 176. Lubric.. appl. M. Bunseki Kagatu... di.. 51.I.. B. 43. Office of Pollution Prevention and Toxics. 22... Hellwig.. p. S. P. & Hutson.J. Cincinnati. Am. 176. L. Pharmacol. prof. Tribol. J. M. ind. & Hildebrand. H. Smith. Arch. 26. pp. Project No. Canada Environment Protection Act. 4th Ed. Huyen. Mutat.K. L. 94113). (1992) Ion-exclusion chromatography with UV detection for the determination of alkanolamines in cosmetics using water-glycerine as an eluent. I.. Ryback. mal.S. 809–824 Eisen. H. (1995) Spectrophotometric determination of ethanolamines in lubricating emulsions©. ed. 81. J.. Merck & Co..J. Ontario. Version 12:2 [CD-ROM].180.. P.A. Parts 175. & Woskie. Ludwigshafen. M. tri and poly). H. Lich.J. Midland.0). Monson. T27–T31 Gamer. G. Med. 185–202 Eller. Johnson. C..H.. (1972) Toxicity of diethanolamine in mice. B. & Zoll-Moreux. ind.P. New York. 244 Fernandez.DIETHANOLAMINE 375 Blum. & Smith. 186–211 Fukui. 943–947 Dean.... AOAC Internat. J. Mutat.. C. K. & Truhaut. Soc.. R. 14 Environmental Protection Agency (1996) 1994 Toxics Release Inventory (EPA 745-R-96-002). T.R.M. TX [CD-ROM] Chou.. J. Monson. E.M. R. pp. Washington DC. Med. Gauthier.R. ed. A.. 22. T. Am. Res. J.E. P. & Howe-Grant. E. p. K. Germany . D. (1994) NIOSH Manual of Analytical Methods (DHHS (NIOSH) Publ. Title 21. & Geller. Midland. 31R0233/90010).. 153.. 1–20 Budavari. T. Kirk-Othmer Encyclopedia of Chemical Technology. Res.Whitehouse Station.E. Cosmetics.R. National Institute for Occupational Safety and Health [Method 3509] Environment Canada (1995) National Pollutant Release Inventory (NPRI) Summary Report 1995. III: A case–control study of larynx cancer. (1992) Alkanolamines. 41. eds. OH.. A. K.

H. Speck. Sunakawa.F. R. Nakajima. (1996) Use of Syrian hamster embryo cell transformation assay for carcinogenicity prediction of chemicals currently being tested by the National Toxicology Program in rodent bioassays. T. In: Bartsch.. (1993) Ethanolamine exposures of workers using machining fluids in the automotive parts manufacturing industry. & Isfort. R. 305–313 Järvholm. Weber.. 53.376 IARC MONOGRAPHS VOLUME 77 Gillner. Environ. (1982) Mutagenicity tests and in vitro transformation assays on triethanolamine. IARCPress.B. 7. Rigas. 73–159 IARC (1999) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. A. Y. S. (1995) Health effects of selected chemicals. Some Industrial Chemicals.. 250–252 . IARCPress. & Cornish..A. 1). Stott. & Tanaka. R. Yamamoto. & Bilsky. Health. & Loeper. Woskie. H. G. T. environ. L. S. & Schulte-Hermann. Lyon.. 1–19 Hartung. 84). R. Yokose. eds. I. O’Neill. 101.. 71. W. H. E.. Mutat. Brauninger. Ullmann’s Encyclopedia of Industrial Chemistry.M. K. K.F. Hammond.T. Lillienberg.. & Smith. pp. (1981) Cancer morbidity among men exposed to oil mist in the metal industry. 1–86 Konishi. pp. pp. G.. Okamoto. Mori.. Yamasaki. Vol. I. Med. O. IARCPress. 1075–1084 Knaak. (1987) Ethanolamines and propanolamines.. Körnig. & Denda. B. B. Ed. 23. pp..K. Lyon. & Zeiger. Vol.. K. J. Overall Evaluations of Carcinogenicity: An Update of IARC Monographs Volumes 1 to 42. (1997) Toxicology of mono-. Res. pp. eds. Rev.. IARCPress. (1978) Mutagenicity screening of industrial chemicals: seven aliphatic amine and one amide tested in the Salmonella/microsome assay. environ. 5 (Suppl.. T. G.K. H. A10. occup. G.. 3–142 Hedenstedt. 3. Contam. appl. T.. L. pp. Lyon. Re-evaluation of Some Organic Chemicals. Res.. Hallock. Y. Lawlor.. 109–225 Inoue.. Diethanolamine. Health Perspect.A. 5th rev. Mortelmans. 5). di-. J. Suppl. J. Relevance of N-nitroso Compounds to Human Cancer: Exposures and Mechanisms (IARC Scientific Publications No. & Lavenius...J. Y. H. M.. (1987) Lung carcinogenesis by N-nitrosobis(2-hydroxypropyl)amine-related compounds and their formation in rats. IARCPress. Lyon.. W. Mutag. Appl.-W.K. Busch. R. A. M.. (1983) Salmonella mutagenicity test results for 250 chemicals. 149. Y. 198–199 IARC (1987a) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans... Sallsten. 120–122 IARC (1987b) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. A. Suppl. Kaudy. W. Shatkin. K. B. J. 42.. and triethanolamine.H. 7. 361–366 Järvholm.. Overall Evaluations of Carcinogenicity: An Update of IARC Monographs Volumes 1 to 42. Thiringer. S. Mutat. & Schulz. (1987) Mortality and cancer morbidity in workers exposed to cutting fluids... (1970).. occup. environ.S. 28. Toxicol. Environ. E.. 308 Haworth.. L. H.J. 8. Arch. 17. 52–75 Hammer. Toxicol..R. In: Gerhartz. J. Rounsaville.. Y. & Axelson. 60. New York... & Kieczka. 655–661 Kerckaert. 104 (Suppl. Yamamoto. W. Leung. Lyon.. 174–175 IARC (1994) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Hyg. J. LeBoeuf. 333–337 Kenyon. Vol.. mol. Acute and chronic toxicity of diethanolamine (Abstract). Hydrazine and Hydrogen Peroxide. Pharmacol. VCH Publishers. Nord.

. M. 11–19 Myhr. H. A.. (1994a) Toxicity of diethanolamine: 1. Hyg.D. pp.J..L.. T. M. Boca Raton. L. 625–633 Mathews. & Milne. J. Amsterdam. 401–410 Melnick. Research Triangle Park. Thompson. R. J. Toxicol.H. W. W. M... and Mezza. H.B. & Tomaszewski. J. Mahler. 262–266 (in German) Melnick.. 27. Black.. (1999) Developmental toxicity of diethanolamine applied cutaneously to CD rats and New Zealand White rabbits. (1994b) Toxicity of diethanolamine: 2.E. C. L. Water Res. appl. Ser. Singer. & Bellan. R. Hejtmancik. Garner. 855–862 Lehman-McKeeman. Hejtmancik.R. & Gamsky..S. 7 (Suppl. 1–9 Melnick. Chem.0 [CD-ROM].. B. Res.J.A. L. D. K. Toxicol.. E. NIH Publication No. 30. Garner. & Hallgren.A. J. Drinking water and topical application exposures in F344 rats. Version 5. 60–94 Marty. biophys. 92-3343). (1994) Exposure to low molecular polyamines during road paving. NC .. N-nitrosodiethanolamine and their precursors in vivo using the newt micronucleus test. D.. 58 National Library of Medicine (1999) Registry of Toxic Effects of Chemical Substances (RTECS) Database. Hohaus.. II.L.. R. & Carney.DIETHANOLAMINE 377 L’Haridon. Biochem. 77. K. Results with 20 chemicals. Pharmacol.. appl. 13. Toxicol.R. M.R. (1996) Properties of Organic Compounds. & Matthews. 14.. & Gamsky. Ryan.. Sci. J. No. D. Drinking water and topical application exposures in B6C3F1 mice... II. Xenobiotica. 8.L. 20. Anderson. 3). M.W.. V. Department of Health and Human Services. & Persing. (1989) Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells in vitro.R. Andersson. Vol. Nitrogen and Phosphorus Solvents. Bucher. C. Comm.. 733–746 Maurer.. M. 38. Mutag. B.S.E. occup. intravenous and dermal administration. (1997) Diethanolamine absorption. 262. (1993) Evaluation of the genotxicity of N-nitrosoatrazine. S.A. Toxicol. & Matthews.L.M. (1995) Metabolism. Elsevier. Fernandez. 27. E. L.E. J. Ethel Browning’s Toxicity and Metabolism of Industrial Solvents. Resnick. Environ. mol. 600–604 Lehman-McKeeman. Bethesda. FL. 169–181 Mathews. 111-42-2) Administered Topically and in Drinking Water to F344/N Rats and B6C3F1 Mice (Tech. & Zeiger..E.m. Mutagen. K. Bowers. M. Res. M. (1999) Diethanolamine inhibits choline uptake and phosphatidylcholine synthesis in Chinese hamster ovary cells. Regul.M. Neptun.J. B.E. Parfü. 14. 35205] National Toxicology Program (1992) Toxicity Studies of Diethanolamine (CAS No.A.. 55. & Reed.B. G.O. (1996) [Analysis of alkanolamines in cosmetics and pharmaceutics]... CRC Press Loveday..L. Ann. Mahler.. & Schubert. mol. and incorporation of diethanolamine into phospholipids. bioaccumulation. (2000) Choline supplementation inhibits diethanolamine-induced morphological transformation in Syrian hamster embryo cells: evidence for a carcinogenic mechanism. metabolism and disposition in rat and mouse following oral.A.R. (1990) Diethanolamine.. J. J. Rep. Toxicol. Bucher. J. E. In: Buhler..L. (1986) Results from the testing of coded chemicals in the L5178Y TK+/– mouse lymphoma mutagenesis assay.. C. Environ. R.. D. 303–310 Levin. E.. J. Neeper-Bradley. J. Lugo. 257–264 Lide. Ferrier.W. Kosmet. eds. & Caspary. MD [Record No.D. E.C.

358–362 Pomfret. 393–399 . 479. (1994) Cancer risk in asphalt workers and roofers: review and metaanalysis of epidemiologic studies. Med. National Institute for Occupational Safety and Health Partanen. Reinhalt. Tuppurainen. Blome. 111-42-2) in F344/N Rats and B6C3F1 Mice (Dermal Studies) (Tech. Ser.. NIH Publication No. W. 99-3971). 721–740 Pfeiffer... Deininger. Research Triangle Park.-U. Breuer. H. 99-3970). R. Vom Wasser. E.. Department of Health and Human Services. 1. NC National Toxicology Program (1999e) Toxicology and Carcinogenesis Studies of Triethanolamine (CAS No.. (1996) Kühlschmierstoffe [Cutting Oils]. Am.. NIH Publication No. Luft. 88. E. T.A. & Sonnenschein. S. G. Germany (BIAReport 7/96) (in German) Pietsch.H. Pflaumbaum. & Nordman. Ser. No. Nutr. p. (1981) Urinary excretion of N-nitrosodiethanolamine in rats following its epicutaneous and intratracheal administration and its formation in vivo following skin application of diethanolamine... PA. 227–231 Sadtler Research Laboratories (1980) Sadtler Standard Spectra. C.. Nies. 28. E.. Public Health Service. 480. Centers for Disease Control. Spiegelhalder. 533–541 Preussmann. D. Clin. Sankt Augustin.. Schmidt. Cincinnati. Stockmann. (1998) Diethanolamine-induced occupational asthma: a case report.. Keskinen.. Rep.. Riepe. 481. F. Department of Health and Human Services. daCosta. R. Mäntylä. Rep. Willer. 99-3968). H. Ser. Sacher. H. J. Hauptverband der gewerblichen Berufsgenossenschaften. & Worch.-J. Kleine. (1997) Liquid chromatographic determination of polar organic nitrogen compounds and their behaviour during drinking water treatment. Unpublished data as of July 1999. 1980 Cumulative Index (Molecular Formula Index).. No. W. J.. 478. W. OH. Henriks-Eckerman. & Boffetta. Research Triangle. & Zeisel.S. R. Cancer Lett. Gefahrst. NIH Publication No.-L. No. Department of Health and Human Services. 99-3969). I.A. No. E.. NC National Toxicology Program (1999d) Toxicology and Carcinogenesis Studies of Oleic Acid Diethanolamine Condensate in F344/N Rats and B6C3F1 Mice (Dermal Studies) (Tech. exp. NC [draft] National Toxicology Program (1999c) Toxicology and Carcinogenesis Studies of Lauric Acid Diethanolamine Condensate in F344/N Rats and B6C3F1 Mice (Dermal Studies) (Tech.. Research Triangle Park. M. K.. Ser. NC [This report has not been peer-reviewed] National Toxicology Program (1999b) Toxicology and Carcinogenesis Studies of Coconut Oil Acid Diethanolamine Condensate (CAS No.378 IARC MONOGRAPHS VOLUME 77 National Toxicology Program (1999a) Toxicology and Carcinogenesis Studies of Diethanolamine (CAS No. J. G. NC NOES (1999) National Occupational Exposure Survey 1981–83.. M. B. (1990) Effects of choline deficiency and methotrexate treatment upon rat liver. 449. Research Triangle Park. Rep. No.A. Rep. Allergy. Rep. Biochem. 68603-42-9) in F344/N Rats and B6C3F1 Mice (Dermal Studies) (Tech. 102-71-6) in F344/N Rats and B6C3F1 Mice (Dermal Studies) (Tech. No.. 56. G. Hahn. B. Ser. Research Triangle Park.. Brauch. 13. P. Eisenbrand. H. W. Würtele. 51 Schubert.. 119–135 Piipari. G. 26. & Hofmann. H. NIH Publ. Tuomi. J. & Maurer.. Hohaus. T. Philadelphia.. NIH Publication No.. Dengel. H.. W. 00-3365). (1996) Determination of alkanolamines in water-miscible cooling lubricants by capillary zone electrophoresis. ind. L.

J. W.. S. Brzak.J. and humans.R. 213–223 Stott.L.. M. E.DIETHANOLAMINE 379 Sollenberg.F. & Frantz.A. J. Toxicol. Dietary Fats. 53. Qual. 18. Tallant. K.A. & Stewart. S. 10.W. Lett. In: Hebver & Kritchevsky. T..J. & Tennant. S.H. J. M. & Alexander.E. Work Environ. 114. S. New York. J..cut.A. Toxicol. 3rd Ed.. L. Am. (1996) In vitro skin penetration of monoethanolamine and diethanolamine using excised skin from rats.. Pyy. R. E. J. C. 131–146 Tolbert.S.W. H. & Monson. Thornton. Salomaa. Proc. Lipids.H.R.. Conner. Bartels..M..E.. J. D. Wegman. cell death and cell transformation. Health. K.. (1995) Diethanolamine: Pharmacokinetics in Sprague-Dawley Rats Following Dermal or Intravenous Administration. Risks associated with specific fluid types.... pp.F..A. M. 266–270 Zeisel.. (1997) Isotachophoretic determination of ethanolamines in metalworking fluids and in air samples from workrooms.. J.. mice.J. Tolbert. New York. pp. Med. R. (1992) Mortality studies of machining fluid exposure in the automobile industry.M. Beskitt. (1998) Mortality studies of metalworking fluid exposure in the automobile industry: VI.H. Woskie. rabbits. L. (2000) Responses of transgenic mouse lines p53± and Tg·AC to agents tested in conventional carcinogenicity bioassays. M.. 753–756 Waechter. French. Furefi-Machacek. D. Eisen. Plenum Press. M. G. Kriebel.. R... (1996) Handbook of Environmental Data on Organic Chemicals. T.A. 313–317 Sorsa. A nutrient that is involved in the regulation of cell proliferation.. 36–48 Sun. Bormett. J. Sci.-H.W. Hammond.. 131–141 . J. 67–75 Sullivan.R. (1996) Choline.J. Mar... J. 465–479 Spalding..K. Mutat.. & Smith. Pothier.. M. II. MI. ind.. 204.R. M. S. environ. Res. Control Quality.E. S. Monson. P. M. Dow Chemical Company Yordy. Van Nostrand Reinhold. S.. Smith. F. P. ocular Toxicol. Stasiewicz. J. & Yager. 351–360 Verschueren. 34.R. eds. Hallock. D.D. (1981) Formation of N-nitrosodiethanolamine from diethanolamine in lake water and sewage.A.J.. (2000) Potential mechanisms of tumorigenic action of diethanolamine in mice. Hallock.W. P. (1988) Biological and environmental monitoring of occupational exposure to cyclophosphamide in industry and hospitals. Nylund.T. L. Hormones and Tumorigenesis. 15. Eisen. & Zeisel. Toxicol. Markham.. Midland. A case–control study of esophageal cancer. J.. 10... Scand. Tice. R.

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36549-54-9. TEOA.3 Relative molecular mass: 149.2′.2′′-Nitrilotriethanol Synonyms: Alkanolamine 244.1. C-13 [1871]) and mass spectral data have been reported (Sadtler Research Laboratories.19 Chemical and physical properties of the pure substance (a) Description: Hygroscopic crystals. TEA (amino alcohol).1. 1996) (c) Melting-point: 20. Budavari. 1996) –381– . grating [18547]). or colourless.2 Structural and molecular formulae and relative molecular mass CH2 N CH2 CH2 CH2 CH2 OH CH2 OH OH C6H15NO3 1.1 Exposure Data Chemical and physical data Nomenclature Chem. TEA.4 °C (Lide & Milne. tris(2-hydroxyethyl)amine 1. 1996. Serv.2′.: 102-71-6 Deleted CAS Reg. 1998) (b) Boiling-point: 335. 1996) (d) Density: 1. triethanolamin. nuclear magnetic resonance (proton [7209].1 1. Abstr.1. 1980. Abstr. tris(β-hydroxyethyl)amine. 1. viscous liquid with a mild ammoniacal odour (Lide & Milne. 36659-79-7. Name: 2. No.5 °C (Lide & Milne.2′′-Nitrilotris[ethanol] IUPAC Systematic Name: 2. 10565527-4. Lide & Milne. Nos: 36549-53-8. 1996) (e) Spectroscopy data: Infrared (proton [10636]. Reg. nitrilotriethanol.1242 g/cm3 at 20 °C (Lide & Milne. 126068-67-5 Chem. 36549-55-0.TRIETHANOLAMINE 1.

1996. slightly soluble in benzene. 1996) (j) Conversion factor1: mg/m3 = 6. Trade names for triethanolamine include Daltogen. (see monograph in this volume). a low freeze-grade blend (85% TEA 85 and 15% deionized water) for use in colder temperatures. Calculated from: mg/m3 = (relative molecular mass/24.45) × ppm. 5. 1998) (h) Stability: Incompatible with metals such as aluminium and copper. 1998). and Trolamine. by isotachophoresis..1.. 0. 1996). Thiofaco T-35. 1999a) (i) Octanol/water partition coefficient (P): log P. assuming a temperature of 25 °C and a pressure of 101 kPa 1 . and a blend of 85% triethanolamine and 15% deionized water [TEA 99 Low Freeze Grade] (Dow Chemical Company. oxidizing materials and absorbent materials (cellulose. and by spectrophotometry (Kenyon et al. ethanol and methanol.382 IARC MONOGRAPHS VOLUME 77 Solubility: Miscible with water. sawdust) (Dow Chemical Company. 1993. halogenated organics. Sting-Kill.05% max.. 1992. 1996. (Dow Chemical Company. including a blend of 85% triethanolamine and 15% diethanolamine [TEA 85]. Schubert et al. Triethanolamine can be determined in metalworking and cutting fluids by gas chromatography–mass selective detection of silylated derivatives. Budavari. monoethanolamine. 99. 185 °C (Budavari.. by capillary zone electrophoresis with indirect ultraviolet detection. 1998) (g) Volatility: Vapour pressure. and in cosmetics and pharmaceuticals by ion-exclusion chromatography and by reversed-phase high performance liquid chromatography (Fukui et al.20% max. strong acids. and water content. 1995. Sterolamide. 1994).40% max. 1996). 0. 1. 1997). 1999b). soluble in chloroform.0% min.3 Pa at 20 °C. Sollenberg.5 Analysis Triethanolamine can be determined in workplace air by drawing the air sample through aqueous hexanesulfonic acid and analysing by ion chromatography. < 1.3 (Verschueren. Maurer et al.4 Technical products and impurities (f) Triethanolamine is commercially available with the following specifications: purity. The limit of detection for this method is 20 μg per sample (Eller. diethyl ether and lignans (Lide & Milne. relative vapour density (air = 1).14 (Verschueren. diethanolamine. 0.. –2.. Triethanolamine is also available in several other grades. flash-point.1. Fernando.10 × ppm 1. acetone.

West & Gonsior. metal-cleaning and lubricating. 1983. in electroless and electroplating.. 1994) and excess ammonia. lithography. Ethanolamines are produced on an industrial scale exclusively by reaction of ethylene oxide (see IARC. It is also extensively used in emulsifiers. they assumed steadily growing commercial importance as intermediates after 1945. Since the mid-1970s. Czech Republic. because of the large-scale production of ethylene oxide. a curing agent for epoxy and rubber polymers. Other applications of triethanolamine include: adhesives. Table 1. 1996. Estimated annual production of triethanolamine in the United States is presented in Table 1. two companies each in Italy and the Russian Federation and one company each in Australia. coatings. Iran. cement and concrete work.. France. but is accelerated by water. Japan. 1999). mining. in fuels. Brazil. 1.3 Use Triethanolamine is used as a corrosion inhibitor in metal-cutting fluids (see General Remarks). as a copper–triethanolamine complex to control freshwater algae on lakes and ponds and as a neutralizer–dispersing agent in agricultural herbicide formulations. five companies in the United States. three companies each in China. paint and pigments.. shampoos and other personal products (Beyer et al. colourless ethanolamines has been possible. petroleum and coal production. An anhydrous procedure uses a fixed-bed ion-exchange resin catalyst (Hammer et al. This reaction takes place slowly. 2000). Spain and the United Kingdom (Chemical Information Services. 1996). as a pharmaceutical intermediate and an ointment-emulsifier. antistatic agents. thickeners and wetting agents in the formulation of consumer products such as cosmetics. detergents. Santa María et al. Estimated annual production of triethanolamine in the United States (thousand tonnes) Year Production 1960 13 1965 25 1970 38 1975 41 1980 56 1985 71 1990 98 From Bollmeier (1992) Information available in 1999 indicated that triethanolamine was manufactured by six companies in India. economical production of very pure. in . Belgium. Worldwide production has been estimated at 100 000–500 000 tonnes per year and European production at 50 000–100 000 tonnes per year (United Nations Environment Program Chemicals. printing inks. 1987).2 Production Ethanolamines became available commercially in the early 1930s.TRIETHANOLAMINE 383 1. Germany and Mexico.

4. It was present in bulk cutting fluids at levels ranging from 0. Concentrations were generally higher for workers engaged in transfer operations and lowest for assembly workers (who did not use machining fluids themselves).3 Environmental occurrence The broad utility of triethanolamine in a large number of industrial applications and consumer products may result in its release to the environment through various . Triethanolamine is present in machining and grinding fluids and has been measured in the metal manufacturing industry.4. (1997) Percentage of production 33 25 20 8 6 3 2 1. polyurethane production and use and wood pulping (Dow Chemical Company. Table 2.1 Occurrence Natural occurrence Triethanolamine is not known to occur as a natural product..2 Occupational exposure No data on the number of workers exposed to triethanolamine were available from the 1981–83 National Occupational Exposure Survey (NOES.02 to 244 μg/m3 (n = 110) (Kenyon et al. In a German study (1992–94). textile finishing. 1999) conducted by the National Institute for Occupational Safety and Health (NIOSH). rubber processing..4. Table 2 presents estimates of the percentages used in major applications (Knaak et al. 1996). 1998).3 to 40%. triethanolamine was measured in metalworking fluid samples (n = 69).. soldering flux. Major uses of triethanolamine in the United States Applications Metalworking fluids Concrete/cement Surfactants Textile processing Miscellaneous Agricultural chemicals Cosmetics From Knaak et al. Personal air exposures ranged from 0. 1997) in the United States.384 IARC MONOGRAPHS VOLUME 77 polymers and polymer production. 1993).4 1. 1. 1. The proportion of samples containing triethanolamine varied over time between 50 and 85% (Pfeiffer et al.

. Occupational exposure limits and guidelines for triethanolaminea Country Year Concentration (mg/ m3) 5 5 3 (aerosol) 5 (vapour aerosol) 5 10 5 Interpretationb Ireland Netherlands Russian Federation Sweden United States ACGIH a 1997 1997 1988 1993 TWA TWA Ceiling Ceiling TWA STEL TWA 1999 From American Conference of Governmental Industrial Hygienists (ACGIH) (1999). eyes. 1983. time-weighted average. Product formulations containing also monoethanolamine (triethanolamine–ethanolamine) may be in contact with the skin for variable periods of time following each application. nails. In 1981. mucous membrane and respiratory epithelium. 1996. Daily or occasional use may extend over many years (Beyer et al. Dermal exposure to triethanolamine-containing products (principally personal care products) is the primary route of general population exposure to triethanolamine (Jones & Kennedy. 1983). United Nations Environment Programme (1999) b TWA.. Table 3.. short-term exposure limit The Food and Drug Administration (1999) permits the use of triethanolamine as a component of adhesives in food packaging as an indirect food additive.5 Regulations and guidelines Occupational exposure limits and guidelines for triethanolamine are presented in Table 3. Santa María et al. 1. and to adjust pH during the manufacture of amino resins permitted for use as components of paper and paper board in the United States. 1994). Batten et al. as a component of the uncoated or coated food contact surface of paper and paper board for use with dry solid foods with no free fat or oil on the surface. 1988.. 1996). STEL.TRIETHANOLAMINE 385 waste streams (Beyer et al. triethanolamine was reported to be an ingredient (generally at a concentration of less than or equal to 5%) in 2720 out of 22 572 cosmetic products which may be applied to or come into contact with skin. hair.. West & Gonsior. Small amounts may be ingested from lipsticks.

There was a statistically significant (p < 0.2). but no increase in the incidence of tumours at any site in male mice (Hoshino & Tanooka. were fed diets prepared by adding 0. The use of ethanolamines and nitrites together as additives to metalworking fluids can lead to the formation of N-nitrosodiethanolamine.05. 7/37. six weeks of age. straight (generally mineral oils). 1978). test unspecified) increase in the incidence of lymphomas in female mice (controls. 3. 3. soluble (straight oils diluted with water and additives) and synthetic (water and additives). 1/36. 9/36). Studies of Cancer in Humans The Working Group was not aware of any studies that specifically examined the risk of cancer among persons exposed to triethanolamine. either triethanolamine or diethanolamine.1 3.1 Studies of Cancer in Experimental Animals Oral administration Mouse Groups of 40 male and 40 female ICR-JCL mice. The characteristics of these studies are presented in Table 4 of the monograph on diethanolamine in this volume and a summary of the results of these studies for specific cancer sites is presented in Table 5 of the same monograph.4. as well as the possibility that heating of the triethanolamine in the diet may have produced degradation products. A number of studies have examined the risk of cancer among workers exposed to metalworking fluids. However. high dose. Control animals received untreated diet [heating not specified]. There are three major types of metalworking fluid. Only studies which stated that ethanolamines (no study indicated triethanolamine alone) were used as additives or that presented results for workers primarily exposed to soluble or synthetic fluids were considered by the Working Group.386 IARC MONOGRAPHS VOLUME 77 2. Studies stating that ethanolamines and nitrites were used together as additives or which presented results for exposure to nitrosamines are described in detail in the monograph in this volume on N-nitrosodiethanolamine.3% w/w (high dose) triethanolamine (analytical grade) to a powdered diet (heated for 40 min at 100 °C and formed into pellets) throughout their lifespan. The survival rate was 50% at 85 weeks in females and at 65 weeks in males for both treated and control animals.03 (low dose) or 0. [The Working Group noted the lack of historical control data on the incidence of lymphomas in female mice.] .1. Ethanolamines. The other studies are described in detail in the monograph on diethanolamine.3 and 1. are very common additives to both soluble and synthetic metalworking fluids (see Sections 1. low dose. ethanolamines have been used as additives for metalworking fluids since the 1950s (see General Remarks).

and 16. high-dose. The percentage mortality in the control. No treatment-related increase in the incidence of tumours was observed (Maekawa et al. at which time the study was terminated. dietary concentrations in both female treatment groups were reduced by half. 3. 100%. 1999). a dose-dependent increase in mortality due to nephrotoxicity was seen..1 Skin application Mouse Groups of 60 male and 60 female B6C3F1 mice. 200. low. 92%.2 3. Administration of triethanolamine was therefore ceased in both female treatment groups at week 68 for one week and thereafter. After week 104. Male mice received 0. At approximately experimental week 60.1. the authors noted that the mice were chronically infected with Helicobacter hepaticus. containing 1.9% diethanolamine as a contaminant) at concentrations of 0% (control). 86%.TRIETHANOLAMINE 387 Groups of 50 male and 50 female B6C3F1 mice. 96%. The high dose was estimated to be the maximum tolerated dose. 100. 1% (low dose) or 2% (high dose) for two years. six weeks of age. from week 69. loss of body weight gain and mortality increased in the female 2% group. There was no treatment-related increase in tumour incidence in either sex (Konishi et al.9% diethanolamine as a contaminant) at concentrations of 0% (control). containing 1. an organism that is known to induce hepatitis. were given drinking-water containing triethanolamine (reagent grade. six weeks of age.2. 1992). were administered triethanolamine (purity. in females. untreated drinking-water was given to all rats and observation was continued until week 113. were given drinking-water containing triethanolamine (reagent grade. 32 and 42 in females. so that interpretation of any relationship between triethanolamine and liver neoplasms was inconclusive (Fox et al. low-dose. 1998. Treatment with triethanolamine produced a reduction in weight in both males and females and. 1% (low dose) or 2% (high dose) for 82 weeks. 99%) topically in acetone on five days per week for 103 weeks. National Toxicology Program. 1986). [The Working Group did not consider this study in its evaluation of carcinogenicity since it was considered inadequate by the National Toxicology Program. 300 or 1000 mg/kg bw triethanolamine. respectively.. 32 and 34 in males. 630 or 2000 mg/kg bw and female mice received 0. 3. There was no significant difference between the body weights of treated and untreated mice.and high-dose groups was 32.2 Rat Groups of 50 male and 50 female Fischer (F344/DuCrj) rats. when all surviving animals were killed. The percentage of mice surviving at week 82 was: females—all groups.] . males—control. six weeks of age.. Although there was an apparent association with hepatocellular tumours in both sexes and hepatoblastomas in males.

2000).1 4. approximately 99% pure) three times per week or with 1. There was no significant increase in the incidence of tumours at any site (National Toxicology Program. The survival rates of males were 21/50. 11/50. Lesions were diagnosed as papillomas when they reached at least 1 mm in size and persisted for at least three weeks. 10 or 30 mg triethanolamine per mouse per application.2 Genetically modified mouse Groups of 15–20 female Tg. 18/49 and 19/50 and of females were 25/50. 32.388 IARC MONOGRAPHS VOLUME 77 3. The mean body weight of females receiving 250 mg/kg bw ranged from 9% to 12% lower than that of the vehicle controls from weeks 73 to 93. Triethanolamine was administered in 200-μL volumes. low-. 14 weeks of age. were administered triethanolamine in acetone topically (the triethanolamine used was from the same chemical batch as that used in the National Toxicology Program mouse study (National Toxicology Program. 3. distribution. was 7% lower than that of the vehicle control group.1.1 No toxicokinetic data related directly to triethanolamine were available. which carry a zeta-globin v-Ha-ras gene on an FVB background.3 Rat Groups of 60 male and 60 female Fischer 344/N rats. were administered triethanolamine (purity. Animals that did not survive until the end of week 10 were not included in the data summaries or calculations.2. six weeks of age.and high-dose rats respectively.. The positive control group was treated with 1. 4. 1999)). and by the end of the study. 63.5 μg twice per week for 20 weeks. There was no evidence of chronic irritation or ulceration at the site of application during the exposure period. 125 or 250 mg/kg bw triethanolamine. five times per week for 20 weeks.Ac mice. 63 or 125 mg/kg bw and females received 0.2. The doses of triethanolamine selected were based on the maximum tolerated dose (MTD) used earlier (National Toxicology Program. Six weeks after the last application. mid. Other Data Relevant to an Evaluation of Carcinogenicity and its Mechanisms Absorption. 29/50 and 18/50 in the control. there was no increase in the incidence of skin tumours in triethanolamine-treated animals (Spalding et al. which developed multiple papillomas in 19/20 animals. The concurrent negative control groups were treated with 200 μL acetone. metabolism and excretion Humans 4. 99%) topically in acetone on five days per week for 103 weeks. 1999) and were 3.25 μg 12-O-tetradecanoylphorbol 13-acetate (TPA. . Male rats received 0. In contrast to the positive controls. 1999). all surviving mice were killed.

1993.. and about 5000-fold higher than that after a 1. The biotransformation of [14C]triethanolamine to monoethanolamine and diethanolamine was specifically investigated in mice after both intravenous and dermal treatments. were about fivefold higher than those after application of 1000 mg/kg [14C]triethanolamine to 1 cm2 of skin.2-h half-life). 1. 63% of the dose disappeared from intestines within 65 min (Kohri et al. Skin absorption rates (as blood concentration–time curves) after dermal application of aqueous and neat [14C]triethanolamine to mouse skin (2000 mg/kg bw..0 × 10–2 cm/h) were similar (Waechter & Rick. 1997).85 × 10–2 cm/h) and mice (1. Gillner & Loeper. with less than 10% found in the skin at the site of application. the peak blood levels of [14C]triethanolamine were observed 2 h after its application in C3H/HeJ mice (2000 mg/kg bw). Knaak et al. 1997). 1997. 1999).1. distribution. In dermal toxicity studies. 1983. blood levels. cited in Knaak et al.75 cm2 area). cited in Knaak et al. After topical application of 2000 mg/kg bw [14C]triethanolamine in acetone to 2 cm2 of mouse back skin. whereas in Fischer 344 rats (1000 mg/kg bw). enclosed by a glass ring) showed no significant change with the use of water as the vehicle (Waechter & Rick. About 60% of the radioactivity in [14C]triethanolamine applied to mouse skin (1000 mg/kg bw) was excreted in 48 h in urine and 20% in faeces..58-h halflife) and a slow phase (10.0 mg/kg bw intravenous dose (Waechter & Rick. The sixfold difference in the absorption rates was explained by the sixfold higher concentration of triethanolamine applied to rat skin rather than by a greater permeability (kp) of rat skin.0 mg/kg bw intravenously showed first-order biphasic kinetics with a rapid (0. This was evaluated by analysis of [14C]triethanolamine-equivalents in the blood. 1990. Neither of the hypothetical metabolites . National Toxicology Program. The slow phase half-lives for elimination of triethanolamine in mice after dermal exposure to 1000 and 2000 mg/kg bw in acetone were 9. the blood levels (expressed as radioactivity) indicated that triethanolamine was absorbed less rapidly than by mice.6 h. Triethanolamine thus enhances its own penetration. 1988.. 1. Data from various studies in mice and rats (1000–2000 mg/kg bw) suggest that absorption of dermally administered triethanolamine is almost complete in 24 h (Waechter & Rick.8–2. The elimination of [14C]triethanolamine from the blood of mice administered 1.4 mg/cm2/h). 1988. metabolism and excretion Toxicokinetic data for triethanolamine have been reviewed (Beyer et al.2 Experimental systems (a) Absorption. 1997).7 h and 18..TRIETHANOLAMINE 389 4. Melnick & Tomaszewski. the absorption rate through rat skin (2. After a single application of [14C]triethanolamine for 48 h to the skin of rats (1000 mg/kg bw. 1982).75 cm2 area) and mice (2000 mg/kg bw. since the kp values for rats (1. cited in Knaak et al. 1988. 1988.4 mg/cm2/h) was estimated to be greater than that through mouse skin (0. Absorption in the gastrointestinal tract of triethanolamine administered orally to Wistar rats is rapid. expressed as radioactivity/mL. cited in Knaak et al.. 1997).

41 had positive reactions. In vitro. with formation of diethanolamine.. Calas et al.. cited in Melnick and Thomaszewski. 1994. 29 had applied lotion-based medicaments locally for some time (Scheuer. In some volunteers. Herman. When a total of 1357 patients suspected of having allergic eczematous contact dermatitis were patch-tested with triethanolamine. Many case reports on people exposed to cosmetics and investigations in groups of workers exposed to cutting fluids (containing triethanolamine among many other components) indicate that contact dermatitis and allergic reactions to triethanolamine occur (e.4–2. 1991). Triethanolamine caused no significant increase in arachidonic acid and prostaglandin concentrations in suction blister fluid samples. The cumulative percentage recoveries reported for unchanged triethanolamine were 53 and 57% in urine and 20 and 23% in faeces at day 1 and day 3. respectively. The urinary and faecal excretion ratio of unchanged triethanolamine remained constant throughout the treatment period (for five to six days) in both males and females. 1990). ethanolamine and glyoxylate as reaction products (Fattakhova et al..g.. in .. Hamilton & Zug. whereas more than 95% of the radioactivity detected in urine was identified as unchanged triethanolamine (Waechter & Rick. skin irritation was observed at lower concentrations of triethanolamine. 1996. 1970). 1978. Triethanolamine has been clinically tested with other model irritant compounds for potency to stimulate signal release of proinflammatory mediators in human skin in order to find biomarkers of irritancy. triethanolamine had an inhibitory effect on the incorporation of [32P]phosphate into phospholipids from rabbit and human tissues (Morin & Lim. A small amount of triethanolamine (1. Jones & Kennedy. 1994. 1990).7%) was excreted as glucuronide conjugates (Kohri et al. Batten et al. however. 1983). suction blister fluid specimens were taken from the site of treated skin. Shrank. The disposition of triethanolamine in male Wistar (180–250 g) rats after administration of a single oral dose (350 mg/rat) was measured in a four-day follow-up study. triethanolamine was mainly excreted unchanged. Hüner et al. above this concentration (mild) skin irritation is observed. Also.. after 24 h. 1983). 1985.2.390 IARC MONOGRAPHS VOLUME 77 was detected in urine (by mass spectral analysis). particularly in persons who had a history of contact dermatitis and scarified skin (Beyer et al.. 1997). 1988. 1982). Neat or aqueous triethanolamine was applied to the lower arm of 12 male volunteers.1 Toxic effects Humans Triethanolamine appeared to be without irritating effects below a concentration of 5% in most people. 1983. Cytochrome P450 monooxygenasedependent oxidative N-dealkylation of triethanolamine does occur in microorganisms. triethanolamine was found to be the most frequent sensitizer (Tosti et al. Of these. 4. Blum & Lischka. Among patients with contact dermatitis. after multiple oral administration to male and female rats.2 4.. 1988.

1. no histopathological changes were observed in Fischer 344 rats exposed to 2% triethanolamine in the drinking-water. various haematological changes were considered not to be dose-related. In a 13-week study in Fischer 344 rats given 125–2000 mg/kg bw dermally per day on five days per week. 1997).. and major histopathological changes were observed in the kidneys. rather high doses of triethanolamine are well tolerated by rats and mice. [The mice consumed up to 3000 mg/kg bw per day at the high dose. In a 14-day study. compared with controls. Mineralization and necrosis of the renal papilla were found. 1985. In general. Irritation to the eye and skin was minimal to slight 72 h after application of pure (98%) triethanolamine to New Zealand White rabbits (Dutertre-Catella et al. chronic– active inflammatory process on the skin as observed in several studies and reported extensively in a National Toxicology Program (1999) study. 1997). but no histopathological changes were apparent. of undiluted triethanolamine to rats and mice in (sub-)chronic studies led to a necrotizing. Body weight gain was depressed by 10–14% after two years on this regimen. In mice. Maekawa et al. 1997). 4.. 1992). 1998). animals receiving 2000 mg/kg bw per day showed body weight gain reductions and . Subchronic inhalation exposure to triethanolamine aerosols has been studied in both male and female Fischer 344 rats and B6C3F1 mice at concentrations of 125– 2000 mg/m3 for 6 h per day on five days a week over a 16-day period (an unpublished study reported by Mosberg et al. Kidney weights were greatly increased.] Dermal application of high concentrations and..TRIETHANOLAMINE 391 contrast to the irritants sodium lauryl sulfate. (1986) reported that exposure to triethanolamine in the drinking-water led to kidney toxicity at a concentration of 1–2% (approximately 1000 mg/kg bw per day intake) in both male and female Fischer 344 rats... In rats. equivalent to an intake of approximately 2500–2800 mg/kg bw per day (an unpublished study reported by Hejtmancik et al. which is common in ageing Fischer 344 rats.. In a chronic study in B6C3F1 mice given 1 or 2% triethanolamine (containing 2% diethanolamine) in the drinking-water (see Section 3. cited by Knaak et al. 1985. and reviewed in Knaak et al. especially when undiluted triethanolamine is applied.2. Battelle Columbus Division Laboratories. Signs of kidney and liver toxicity were observed by Kindsvatter (1940) in guineapigs and albino rats during oral administration of triethanolamine (200–1600 mg/kg bw per day). little toxicity was observed in either sex (Konishi et al. in particular.2 Experimental studies The toxicology of triethanolamine has been reviewed (Knaak et al. suggesting a dose-related acceleration of chronic nephropathy.. several minor changes were observed such as a decrease in body weight and an increase in kidney weight.1). 1982). Major sites of toxicity in rats and mice are liver and kidney. while skin toxicity occurs after dermal application. benzalkonium chloride and Tween 80 that gave positive test results (Müller-Decker et al.. indicating little toxicity under these conditions.

the internal organs showed no change in weight and no histopathological signs were observed (DePass et al. 4. although the incidence of hyperplasia was the same. In the 13-week study (250–4000 mg/kg bw per day on five days per week). [This study was not reviewed in detail by the Working ... and liver and kidney weights were increased at that dose. 7. acanthosis and inflammation at the site of application were observed in both males and females at 4000 mg/kg bw per day. 1000.2 Experimental systems Triethanolamine was administered to groups of 10 male and 10 female Fischer 344/N rats and B6C3F1 mice for 13 weeks by topical application at dose levels of 0. 16 and 19 (Burnett et al.3 4. 4.1 Reproductive and developmental effects Humans No data were available to the Working Group. respectively. 1998) complicates the interpretation of the results.1–1. Infection with Helicobacter hepaticus in these mice (Fox et al. 2000 or 4000 mg/kg bw per day. 13. No embryotoxic or teratogenic effects were produced when pregnant rats were exposed by topical administration to their shaved skin of semipermanent hair-dye preparations containing 0. 4. Body weight gains were significantly lower in the highest-dose group of male and female rats. 1995).5% triethanolamine on gestational days 1.3.3. inflammation and ulceration were present at the site of application in both males and females. At the 15-month interim evaluation. 500. doses of 32–125 mg/kg bw per day were applied to the skin of males and of 63–250 mg/kg per day to the skin of females. Another subchronic study of triethanolamine administered dermally to C3H/HeJ mice (140–2000 mg/kg bw in males and 160–2300 mg/kg bw in female mice. In neither rats nor mice was there any significant change in sperm motility. 1999). The National Toxicology Program (1999) study also included B6C3F1 mice. In a subsequent 103-week study. The effects were dose-related and were similar in both males in females. In females. three times per week for 95 days) found no signs of toxicity except for slight epidermal hyperplasia at the application site. 1000 or 2000 mg/kg bw per day and 0. 10.392 IARC MONOGRAPHS VOLUME 77 kidney weight increases. females received doses of 100–1000 mg/kg bw per day and males 200–2000 mg/kg bw per day. morphology or number and there was no change in the mean duration of the estrous cycle (National Toxicology Program. which was more severe in males than in controls. At the end of the study. 1976). hyperplasia in the kidney was observed. but there was no change in mice at any dose level.. Hypertrophy of the pars intermedia of the pituitary gland also occurred. In the subsequent 103-week study. Skin inflammation was found in both males and females at the site of application. kidney weights increased after application of 250 mg/kg bw per day.

] The reproductive and developmental toxicity of triethanolamine tested without the complication of many other accompanying substances has been reviewed (Knaak et al. this activity was lost in the presence of exogenous metabolic activity. mutations were induced in this system without exogenous metabolic activation.4. However. In a single study. triethanolamine was not mutagenic to Bacillus subtilis strains carrying uvrA or uvrA and polA mutations in the presence or absence of exogenous metabolic activation. 4. fertility or offspring growth and survival was observed. In a single study. 4. TA1537 or TA1538 in the presence or absence of exogenous metabolic activation in a number of studies. TA100. Triethanolamine was administered as a solution in acetone to the skin of male and female Fischer 344 rats at dose levels of 0 or 500 mg/kg bw per day for 10 weeks before mating and then to the females during gestation and lactation. TA1535. .1 Humans No data were available to the Working Group.. 1997).2 Experimental systems (see Table 4 for references) Triethanolamine was not mutagenic to Salmonella typhimurium strains TA98. 4. In a similar study with CD-1 mice administered triethanolamine doses of 0 or 2000 mg/kg bw per day.TRIETHANOLAMINE 393 Group because of the low proportion of triethanolamine in the complex mixtures tested. sex-linked recessive lethal mutations were not induced in Drosophila melanogaster by treatment with triethanolamine either by diet or injection. no effect of treatment was observed. This review is used as the reference source to the following studies because they have not been reported in the open literature (Battelle reports). 1983). Triethanolamine did not induce mutations in Escherichia coli WP2 uvrA and WP2 try– in the presence or absence of exogenous metabolic activation in two studies. (1997) and by the National Toxicology Program (1999).4. Triethanolamine did not induce gene conversion in Saccharomyces cerevisiae in the presence or absence of exogenous metabolic activation in one study. when triethanolamine was mixed with sodium nitrite. by Knaak et al.. Triethanolamine did not induce sister chromatid exchanges in Chinese hamster ovary cells in either the presence or absence of exogenous metabolic activation. Unscheduled DNA synthesis was not induced in rat primary hepatocytes exposed to triethanolamine in two studies. No effect on mating.4 Genetic and related effects Triethanolamine has been reviewed by an expert panel for cosmetic ingredient review (Beyer et al.

(1985) Yoon et al. TA1537. TA98. (1987) . Chinese hamster ovary CHO cells in vitro – – – – – – – + + + – – – – – With exogenous metabolic system Doseb (LED or HID) Reference IARC MONOGRAPHS VOLUME 77 – – – – – – – – – – NT – NT – 100 mg/plate 20 000 μg/plate 4000 μg/plate 3333 μg/plate 10 000 μg/plate 4000 μg/plate 10 000 8580 8580 8580 4000 μg/disk 5000 30 000 ppmc 14 900 10 100 Beyer et al. reverse mutation Bacillus subtilis TKJ5211 uvrA–. (1982) Dean et al. reverse mutation Salmonella typhimurium TA100. sex-linked recessive lethal mutation Unscheduled DNA synthesis. TA1537. gene conversion Drosophila melanogaster. (1986) Inoue et al. TA98. Genetic and related effects of triethanolamine Test system Resulta Without exogenous metabolic system Salmonella typhimurium TA100. reverse mutation Escherichia coli WP2 try– reverse mutation Escherichia coli WP2 and WP2 uvrA. TA1535. DNA damage assay Saccharomyces cerevisiae JD1. TA1538. TA1537. primary rat hepatocytes in vitro Sister chromatid exchange. TA1535. (1985) Hoshino & Tanooka (1978) Hoshino & Tanooka (1978) Hoshino & Tanooka (1978) Hoshino & Tanooka (1978) Inoue et al. mutation (plus sodium nitrite) Bacillus subtilis H17 rec+/M45 rec– . mutation (plus sodium nitrite) Bacillus subtilis TKJ5211 uvrA–. (1983) Galloway et al. mutation (plus sodium nitrite) Bacillus subtilis TKJ6321 uvrA– polA–. TA1535. mutation Bacillus subtilis WT. TA98. (1983) Inoue et al. (1985) Mortelmans et al. reverse mutation Salmonella typhimurium TA100. TA1538. TA98. reverse mutation Salmonella typhimurium TA100.394 Table 4. (1982) Dean et al. (1982) Dean et al. (1985) Beyer et al.

w. NT. lowest effective dose. –. mg/kg bw/day. NR. positive. negative.Table 4 (contd) Test system Resulta Without exogenous metabolic system Chromosomal aberrations. (+). (1985) Inoue et al. Chinese hamster ovary CHO cells in vitro Chromosomal aberrations. not reported. week c Feeding or injection GI50. (1982) Galloway et al. in-vivo tests. in-vitro tests. 8 d Reference TRIETHANOLAMINE – – – – Inoue et al. 24/48 h 10 100 0. μg/mL. Syrian hamster embryo cells in vitro a Doseb (LED or HID) With exogenous metabolic system NT – NT NT 100.5 × GI50 500. (1987) Dean et al. not tested LED. (1982) +. concentration causing 50% growth inhibition b 395 . rat liver RL cells in vitro Cell transformation. Chinese hamster lung CHL cells in vitro Chromosomal aberrations. highest ineffective dose. HID. weak positive.

1997). 1986.. as well as herbicide and algicide formulations. Occupational exposure may occur by inhalation and dermal ..1 Summary of Data Reported and Evaluation Exposure data Triethanolamine is a viscous liquid widely used as a corrosion inhibitor. 1985). interleukin-1α) that are known to be indicative of hyperproliferative and inflammatory events in human skin (Müller-Decker et al. It did not induce cell transformation in Syrian hamster embryo cells. In spite of the search for a possible mode of bioactivation of triethanolamine. N-nitrosodiethanolamine (Lijinsky et al. Chinese hamster lung cells or Chinese hamster ovary cells by in-vitro exposure to triethanolamine. 5. Biodegradation of triethanolamine to monoethanolamine or diethanolamine or to any other putative metabolite has not been shown in rodents. or that some other endogenous reactions convert triethanolamine to a putative carcinogen (Hoshino & Tanooka. unlikely since no treatment-related liver cancers have been observed in oral or dermal triethanolamine carcinogenicity studies in mice or rats (Hoshino & Tanooka. a surfaceactive agent and an intermediate in various products including metalworking fluids. in general. oils. Konishi et al. considered negligible in comparison with the nitrosation of secondary amines (Knaak et al. eicosanoids. 1978). 1980...5 Mechanistic considerations Triethanolamine is rapidly absorbed and excreted in urine (about 60%) and faeces (about 20%) mainly in the unchanged form. The potential reported for triethanolamine to undergo nitrosative dealkylation and form N-nitrosodiethanolamine under physiological conditions (including gastric pH) is.. nor its incorporation into natural products. however. 4. 1978. 1998). Maekawa et al. fuels. Lijinsky & Kovatch. It has been hypothesized that endogenous nitrosation of triethanolamine may produce a potent liver carcinogen. paints.396 IARC MONOGRAPHS VOLUME 77 Chromosomal aberrations were not induced in rat liver cells. In line with the in-vitro irritancy tests. The formation of N-nitrosodiethanolamine in amounts that would cause liver cancer in vivo appears. triethanolamine was found to be a non-irritant in a clinical patch testing study of human skin in 20 male volunteers (Müller-Decker et al. inks. 1994). cement. 1992). triethanolamine was categorized as a weak inducer of a delayed (≥ 4 h) stimulation of the release of key mediators (arachidonic acid. cosmetic and personal products. 5. Treatment of male and female mice with triethanolamine for 13 weeks by dermal application did not result in any change in the frequency of micronuclei in their blood cells. In h