UNITED NATIONS ENVIRONMENT PROGRAMME INTERNATIONAL LABOUR ORGANISATION WORLD HEALTH ORGANIZATION INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY ENVIRONMENTAL HEALTH CRITERIA 199 Cholordimeform This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the United Nations Environment Programme, the International Labour Organisation, or the World Health Organization. Environmental Health Criteria 199 First draft prepared by Dr P.J. Abbott, Australia and New Zealand Food Authority, Canberra, Australia Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization World Health Organization Geneva, 1998 The International Programme on Chemical Safety (IPCS) is a joint venture of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization. The main objective of the IPCS is to carry out and disseminate evaluations of the effects of chemicals on human health and the quality of the environment. Supporting activities include the development of epidemiological, experimental laboratory, and risk-assessment methods that could produce internationally comparable results, and the development of manpower in the field of toxicology. Other activities carried out by the IPCS include the development of know-how for coping with chemical accidents, coordination of laboratory testing and epidemiological studies, and promotion of research on the mechanisms of the biological action of chemicals. WHO Library Cataloguing in Publication Data (Environmental health criteria ; 199) 1.Chlorphenamidine - toxicity 2.Chlorphenamidine - adverse effects 3.Environmental exposure 4.Occupational exposure I.International Programme on Chemical Safety II.Series ISBN 92 4 157199 3 (NLM Classification: QU 61) ISSN 0250-863X The World Health Organization welcomes requests for permission to reproduce or translate its publications, in part or in full. Applications and enquiries should be addressed to the Office of Publications, World Health Organization, Geneva, Switzerland, which will be glad to provide the latest information on any changes made to the text, plans for new editions, and reprints and translations already available. (c) World Health Organization 1998 Publications of the World Health Organization enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Convention. All rights reserved. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. CONTENTS ENVIRONMENTAL HEALTH CRITERIA FOR CHLORDIMEFORM PREAMBLE ABBREVIATIONS 1. SUMMARY 1.1. Identity, physical and chemical properties, and analytical methods 1.2. Sources of human and environmental exposure 1.3. Environmental transport, distribution and transformation 1.4. Environmental levels and human exposure 1.5. Kinetics and metabolism in laboratory animals and humans 1.6. Effects on laboratory mammals and in vitro test systems 1.7. Effects on humans 1.8. Effects on other organisms in the laboratory and field 1.9. Evaluation of human health risks and effects on the environment 1.10. Conclusions and recommendations 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS 2.1. Identity 2.2. Physical and chemical properties 2.3. Conversion factors 2.4. Analytical methods 2.4.1. Plants 2.4.2. Soil 2.4.3. Water 2.4.4. Formulations 2.4.5. Air 2.4.6. Urine 2.4.7. Tissues 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1. Natural occurrence 3.2. Anthropogenic sources 3.2.1. Production levels and processes 3.2.2. Uses 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION 4.1. Transport and distribution between media 4.1.1. Air 4.1.2. Water 4.1.3. Soil 4.1.4. Vegetation and wildlife 4.1.5. Entry into food chain 4.2. Biotransformation 4.2.1. Degradation in plants 4.2.2. Degradation in soils 4.2.3. Bioaccumulation 4.3. Ultimate fate following use 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1. Environmental levels 5.1.1. Air and water 5.1.2. Soil 5.2. General population exposure 5.2.1. Environmental sources 5.2.2. Residues in raw produce 5.2.3. Residues in processed food 5.3. Occupational exposure during manufacture, formulation or use 5.3.1. Exposure during manufacture and formulation 5.3.2. Exposure during use 6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS 6.1. Absorption, distribution and excretion 6.1.1. Mouse and rat 6.1.2. Other species 6.1.3. Human 6.2. Metabolic transformation 6.2.1. Mouse and rat 6.2.2. Other species 6.2.3. In vitro studies 7. EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO TEST SYSTEMS 7.1. Single exposure 7.1.1. Oral 7.1.2. Other routes 7.2. Short-term exposure 7.2.1. Dietary 188.8.131.52 Mouse 184.108.40.206 Rat 220.127.116.11 Dog 7.2.2. Intubation 18.104.22.168 Rat 7.3. Long-term dietary exposure 7.3.1. Mouse 7.3.2. Rat 7.4. Skin and eye irritation; skin sensitization 7.5. Reproductive toxicity, embryotoxicity and teratogenicity 7.5.1. Reproductive toxicity 22.214.171.124 Rat 126.96.36.199 Hamster 7.5.2. Embryotoxicity and teratology 188.8.131.52 Rat 184.108.40.206 Rabbit 7.6. Mutagenicity and related end-points 7.6.1. DNA damage and repair 7.6.2. Mutation 7.6.3. Chromosome damage 7.6.4. Cell transformation 7.7. Carcinogenicity 7.7.1. Mouse 7.7.2. Rat 7.8. Other special studies 7.8.1. Immunotoxicity 7.8.2. Behavioural effects 7.8.3. Pharmacological and biochemical effects 7.9. Factors modifying toxicity 7.10. Mechanisms of toxicity - mode of action 7.10.1. Mechanism of acute toxicity 7.10.2. Mechanism of carcinogenicity 8. EFFECTS ON HUMANS 8.1. General population exposure 8.1.1. Acute poisoning incidents 8.2. Occupational exposure 8.2.1. Acute poisoning incidents 8.2.2. Effects of long-term exposure 8.2.3. Epidemiological studies 220.127.116.11 4-Chloro- o-toluidine 18.104.22.168 Chlordimeform 9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD 9.1. Laboratory experiments 9.1.1. Microorganisms 9.1.2. Aquatic organisms 22.214.171.124 Plants 126.96.36.199 Invertebrates 188.8.131.52 Vertebrates 9.1.3. Terrestrial organisms 184.108.40.206 Plants 220.127.116.11 Invertebrates 18.104.22.168 Vertebrates 9.2. Field observations 9.2.1. Microorganisms 9.2.2. Aquatic organisms 9.2.3. Terrestrial organisms 22.214.171.124 Plants 126.96.36.199 Invertebrates 188.8.131.52 Vertebrates 10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT 10.1. Evaluation of human health risks 10.1.1. Exposure 10.1.2. Toxicity 10.1.3. Risk evaluation 10.2. Evaluation of effects on the environment 11. CONCLUSIONS AND RECOMMENDATIONS FOR PROTECTION OF HUMAN HEALTH AND THE ENVIRONMENT 11.1. Conclusions 11.2. Recommendations for protection of human health and the environment 12. FURTHER RESEARCH 13. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES REFERENCES RÉSUMÉ RESUMEN NOTE TO READERS OF THE CRITERIA MONOGRAPHS Every effort has been made to present information in the criteria monographs as accurately as possible without unduly delaying their publication. In the interest of all users of the Environmental Health Criteria monographs, readers are requested to communicate any errors that may have occurred to the Director of the International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland, in order that they may be included in corrigenda. * * * A detailed data profile and a legal file can be obtained from the International Register of Potentially Toxic Chemicals, Case postale 356, 1219 Châtelaine, Geneva, Switzerland (telephone no. + 41 22 - 9799111, fax no. + 41 22 - 7973460, E-mail firstname.lastname@example.org). * * * This publication was made possible by grant number 5 U01 ES02617-15 from the National Institute of Environmental Health Sciences, National Institutes of Health, USA, and by financial support from the European Commission. Environmental Health Criteria PREAMBLE Objectives In 1973 the WHO Environmental Health Criteria Programme was initiated with the following objectives: (i) to assess information on the relationship between exposure to environmental pollutants and human health, and to provide guidelines for setting exposure limits; (ii) to identify new or potential pollutants; (iii) to identify gaps in knowledge concerning the health effects of pollutants; (iv) to promote the harmonization of toxicological and epidemiological methods in order to have internationally comparable results. The first Environmental Health Criteria (EHC) monograph, on mercury, was published in 1976 and since that time an ever-increasing number of assessments of chemicals and of physical effects have been produced. In addition, many EHC monographs have been devoted to evaluating toxicological methodology, e.g., for genetic, neurotoxic, teratogenic and nephrotoxic effects. 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Content The layout of EHC monographs for chemicals is outlined below. * Summary - a review of the salient facts and the risk evaluation of the chemical * Identity - physical and chemical properties, analytical methods * Sources of exposure * Environmental transport, distribution and transformation * Environmental levels and human exposure * Kinetics and metabolism in laboratory animals and humans * Effects on laboratory mammals and in vitro test systems * Effects on humans * Effects on other organisms in the laboratory and field * Evaluation of human health risks and effects on the environment * Conclusions and recommendations for protection of human health and the environment * Further research * Previous evaluations by international bodies, e.g., IARC, JECFA, JMPR Selection of chemicals Since the inception of the EHC Programme, the IPCS has organized meetings of scientists to establish lists of priority chemicals for subsequent evaluation. 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It is accepted that the following criteria should initiate the updating of an EHC monograph: new data are available that would substantially change the evaluation; there is public concern for health or environmental effects of the agent because of greater exposure; an appreciable time period has elapsed since the last evaluation. All Participating Institutions are informed, through the EHC progress report, of the authors and institutions proposed for the drafting of the documents. A comprehensive file of all comments received on drafts of each EHC monograph is maintained and is available on request. The Chairpersons of Task Groups are briefed before each meeting on their role and responsibility in ensuring that these rules are followed. WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR CHLORDIMEFORM Members Dr P.J. Abbott, Australia and New Zealand Food Authority (ANZFA), Canberra, Australia Dr K. Barabás, Department of Public Health, Albert Szent-Gyorgyi, University Medical School, Szeged, Hungary Dr A.L. Black, Woden, ACT, Australia Professor J.F. Borzelleca, Pharmacology and Toxicology, Richmond, Virginia, USA Dr P.J. Campbell, Pesticides Safety Directorate, Ministry of Agriculture, Fisheries and Food, Kings Pool, York, United Kingdom Professor L.G. Costa, Department of Environmental Health, University of Washington, Seattle, USA Dr S. Dobson, Institute of Terrestrial Ecology, Monks Wood, Abbots Ripton, Huntingdon, Cambridgeshire, United Kingdom Dr I. Dewhurst, Mammalian Toxicology Branch, Pesticides Safety Directorate, Ministry of Agriculture, Fisheries and Food, Kings Pool, York, United Kingdom Dr V. Drevenkar, Institute for Medical Research and Occupational Health, Zagreb, Croatia Dr W. Erickson, Environmental Fate and Effects Division, US Environmental Protection Agency, Washington, D.C., USA Dr A. Finizio, Group of Ecotoxicology, Institute of Agricultural Entomology, University of Milan, Milan, Italy Mr K. Garvey, Office of Pesticide Programs (7501C), US Environmental Protection Agency, Washington, D.C., USA Dr A.B. Kocialski, Health Effects Division, Office of Pesticide Programs, US Environmental Protection Agency, Washington, D.C., USA Dr A. Moretto, Institute of Occupational Medicine, University of Padua, Padua, Italy Professor O. Pelkonen, Department of Pharmacology and Toxicology, University of Oulu, Oulu, Finland Dr D. Ray, Medical Research Council Toxicology Unit, University of Leicester, Leicester, United Kingdom Dr J.H.M. Temmink, Department of Toxicology, Wageningen Agricultural University, Wageningen, The Netherlands Observers Dr J.W. Adcock, AgrEvo UK Limited, Chesterford Park, Saffron, Waldon, Essex, United Kingdom Mr D. Arnold, Environmental Sciences, AgrEvo UK Ltd., Chesterford Park, Saffron Waldon, Essex, United Kingdom Dr E. Bellet, CCII, Overland Park, Kansas, USA Mr Jan Chart, AMVAC Chemical Corporation, Newport Beach, California, USA Dr H. Egli, Novartis Crop Protection AG, Basel, Switzerland Dr P. Harvey, AgrEvo UK Ltd., Chesterford Park, Saffron Walden, Essex, United Kingdom Dr G. Krinke, Novartis Crop Protection AG, Basel, Switzerland Dr A. McReath, DowElanco Limited, Letcombe Regis, Wantage, Oxford, United Kingdom Dr H. Scheffler, Novartis Crop Protection AG, Basel, Switzerland Dr A.E. Smith, Novartis Crop Protection AG, Basel, Switzerland Secretariat Dr L. Harrison, Health and Safety Executive, Bootle, Merseyside, United Kingdom Dr J.L. Herrman, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland Dr P.G. Jenkins, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland Dr D. McGregor, Unit of Carcinogen Identification and Evaluation, International Agency for Research on Cancer, Lyon, France Dr R. Plestina, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland Dr E. Smith, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland Dr P. Toft, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland IPCS TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR CHLORDIMEFORM The Core Assessment Group (CAG) of the Joint Meeting on Pesticides (JMP) met at the Institute for Environment and Health, Leicester, United Kingdom, from 3 to 8 March 1997. Dr L.L. Smith welcomed the participants on behalf of the Institute, and Dr R. Plestina on behalf of the three IPCS cooperating organizations (UNEP/ILO/WHO). The CAG reviewed and revised the draft monograph and made an evaluation of the risks for human health and the environment from exposure to chlordimeform. The first draft of the monograph was prepared by Dr P. Abbott, Canberra, Australia. Extensive scientific comments were received following circulation of the first draft to the IPCS contact points for Environmental Health Criteria monographs and these comments were incorporated into the second draft by the Secretariat. Dr R. Plestina and Dr P.G. Jenkins, both members of the IPCS Central Unit, were responsible for the overall scientific content and technical editing, respectively. The efforts of all who helped in the preparation and finalization of the monograph are gratefully acknowledged. ABBREVIATIONS ACTH adrenocorticotropic hormone ADI acceptable daily intake a.i. active ingredient BSP bromosulfophthalein CIMS chemical ionization mass spectrometry CNS central nervous system CORT corticosteroid DNA deoxyribonucleic acid EC emulsifiable concentrate ECG electrocardiography GC gas chromatography HPLC high performance liquid chromatography IgM immunoglobulin M JMPR Joint FAO/WHO Meeting on Pesticide Residues MRL maximum residue limit Mu Chinese measure of an area equivalent to 1/15 acre or 1/60 ha or 166 m2 MS mass spectroscopy NADPH reduced nicotinamide adenine dinucleotide NC cell activity natural cytotoxic cell activity NK cell activity natural killer cell activity NOEL no-observable-effect level PL prolactin SAP serum alkaline phosphatase SGOT serum glutamate-oxalate transaminase SGPT serum glutamate-pyruvate transaminase SIR standard incidence rate SMR standardized mortality ratio SPF specific pathogen free TLC thin layer chromatography TLm median tolerance limit UV ultraviolet 1. SUMMARY 1.1 Identity, physical and chemical properties, and analytical methods Chlordimeform is a base of medium strength and forms stable salts with strong acids. Both chlordimeform and its hydrochloride salt in the pure state are colourless crystalline solids. Chlordimeform base has a melting point of 32°C, while the hydrochloride salt has a melting point of 225-227°C. Chlordimeform base is sparingly soluble in water (250 mg/litre) and readily soluble in organic solvents, whereas the hydrochloride salt is readily soluble in water but less soluble in organic solvents. Chlordimeform base has a vapour pressure at 20°C of 48 mPa and a log Kow of 2.89. A wide range of analytical methods are available for detection and quantification of chlordimeform in plants, soil, water and urine. 1.2 Sources of human and environmental exposure Chlordimeform does not occur naturally. It is manufactured commercially by condensation of the Vilsmeier reagent (obtained by reaction of dimethylformamide with POCl3, SOCl2 or COCl2) either with 4-chloro- o-toluidine or with o-toluidine and subsequent chlorination of the resulting intermediate . It has been used as a broad spectrum acaricide and is active mainly against motile forms of mites and ticks and against eggs and early instars of some Lepidoptera insects. It is active in the vapour phase as well as by contact. In the early period of its use, it was used on a wide variety of crops such as pome fruits, stone fruits, cole crops, vegetables, grapes, hops, citrus fruits, apples, pears, cherries and strawberries. It was also used in cattle dips for the control of cattle ticks. In the latter years, its use was generally restricted to cotton, although in some countries, there was continued use on rice. Its registration was voluntarily withdrawn in 1988/1989 in most countries. In China, production stopped in 1992 and sales ceased in 1993. 1.3 Environmental transport, distribution and transformation Chlordimeform has a moderate vapour pressure but its evaporation from plant surfaces is less than would be expected. The hydrolytic stability of chlordimeform is strongly pH-dependent; it is stable in acid conditions but rapidly hydrolysed in alkaline conditions. Chlordimeform has the potential to adsorb to dissolved organic matter. In soils, chlordimeform is primarily dissipated by microbial action with some contribution by chemical hydrolysis. There is little evidence of leaching despite its water solubility, which may be due to its adsorption to clay minerals, soil organic matter and biodegradation. The principal metabolites are N-formyl-4-chloro- o-toluidine and 4-chloro- o-toluidine. There is a low but measurable uptake of chlordimeform into plants from soil, sufficient to affect plant-feeding pests. When applied to the leaves, chlordimeform has only limited capacity to penetrate the cuticular layers. Chlordimeform is degraded rapidly in plants. The principal metabolites are demethylchlordimeform, N-formyl-4- chloro- o-toluidine and 4-chloro- o-toluidine, though not all plants studied produced the 4-chloro- o-toluidine. In soils, chlordimeform and its metabolites are dissipated according to first-order kinetics with a half-life of 20-40 days. Bioaccumulation studies have demonstrated low uptake of chlordimeform by aquatic organisms and rapid depuration on transfer to clean water. 1.4 Environmental levels and human exposure Levels have not been measured in air and water. Following applications to paddy fields residues of up to 2900 µg/kg in the top 5 cm of soil and 150 µg/kg in the next 5 cm have been found. Maximum residue levels were set for a wide range of raw produce and, in some cases, the residues carried over into processed food. The Codex maximum residue limits for chlordimeform have been withdrawn. Occupational exposure to chlordimeform has taken place during manufacture, formulation and application. In recent years, total urinary levels of chlordimeform and its metabolites have been used as a monitor for exposure, and the urine level correlates well with the degree of skin contamination. Where agricultural workers in the cotton industry have undergone extensively surveillance for urinary excretion of chlordimeform, the highest exposure levels were in loaders, washers and mechanics, with lower levels in flagmen and pilots. 1.5 Kinetics and metabolism in laboratory animals and humans Chlordimeform is readily absorbed from the gastrointestinal tract and through the skin of mammals. Rapid excretion follows, with approximately 80% in the urine and 10-15% in faeces. Low residue levels are evident in all tissues after approximately 10 days, and there is no evidence of bioaccumulation. Following dermal administration in humans, similar rapid excretion through the urine is observed. Several oxidized and conjugated metabolites of chlordimeform are excreted in the urine, demethylchlordimeform, N-formyl-4-chloro- o-toluidine and 4-chloro- o-toluidine being the major metabolites. In in vitro studies, the same metabolites are formed, 4-chloro- o-toluidine being the major metabolite. 1.6 Effects on laboratory mammals and in vitro test systems Chlordimeform has moderate acute toxicity when tested in several species by oral and dermal routes of administration. The major metabolites have low oral toxicity when tested in rats. Chlordimeform causes only slight skin and eye irritation in rabbits. Following either short- or long-term exposure in both mice and rats with either chlordimeform or its metabolites, treatment-related changes can be observed in haematological parameters, and there is some evidence of hyperplasia of the epithelium of the bile duct and urinary bladder at the high dose levels. Chlordimeform does not cause an increase in tumour incidence in rats. In mice, following dietary administration of either chlordimeform, N-formyl-4-chloro- o-toluidine or 4-chloro- o-toluidine, there is a dose-related increase in haemorrhagic malignant tumours of vascular origin classified as malignant haemangioendotheliomas, which cause a dose-related increase in mortality. Chlordimeform does not affect reproductive parameters, nor does it have any teratogenic potential. Chlordimeform has been tested in a broad range of in vitro and in vivo genotoxicity assays. No positive responses have been reported with any of these tests in which unformulated chlordimeform was tested. In addition, there have been several sporadic and unconfirmed reports of mutagenic activity induced by N-formyl- 4-chloro- o-toluidine and 4-chloro- o-toluidine. A single report describes cell transformation induction by both chlordimeform and 4-chloro- o-toluidine. Binding to DNA occurs in the liver of dosed mice and rats. One major hydrophobic adduct is found at a much higher level in mice than in rats. Chlordimeform induces a variety of pharmacological and biochemical effects in animals, including cardiovascular changes, hypothermia, hyperexcitability, effects on central visual and auditory functions, and modulation of biogenic amines and drug-metabolizing enzymes. 1.7 Effects on humans Acute poisoning causes fatigue, nausea and loss of appetite, and, in more severe cases, somnolence, cyanosis, urgency in urination, cystitis, cardiovascular effects (tachycardia, bradycardia, ECG changes), coma and shock. Generally, there is complete recovery from acute intoxication. Following chronic exposure to chlordimeform, additional symptoms include abdominal pain, skin itching and rashes (dermal exposure), and gross and microscopic haematuria. A large number of cases with clinical symptoms of chronic exposure have been reported in both chlordimeform-manufacturing plants as well as in agricultural workers. Following occupational exposure, epidemiological evidence has provided a strong association between exposure to the metabolite 4-chloro- o-toluidine and the incidence of human urinary bladder cancer. There is currently only weak evidence for an association between exposure to chlordimeform and human bladder cancer. 1.8 Effects on other organisms in the laboratory and field There were no significant effects on populations of soil fungi, bacteria or actinomycetes following application of chlordimeform to soil. There are no laboratory toxicity data on freshwater invertebrates. Growth of larval oysters was inhibited by chlordimeform with an EC50 of 5.7 mg/litre. The 96-h LC50 for pink shrimp, the only crustacean studied, was 7.1 mg/litre and the 96-h LC50 values for fish ranged from 1 to 54 mg/litre. There are no chronic aquatic toxicity data available. A mixture of laboratory and field data shows that chlordimeform is toxic to a wide range of terrestrial non-target arthropods. The contact toxicity LD50 for bees has been reported to be 120 µg/g and that for oral toxicity 187 µg/g. There was no mortality in the field following exposure of species of bees to residues on alfalfa 3 h after spraying. The dietary LC50 for various birds species ranged from >1000 to >5000 mg/kg diet. 1.9 Evaluation of human health risks and effects on the environment Heavy exposure during manufacture or use, possibly resulting from inadequate safety precautions, has led to signs of acute poisoning in workers. Since both production and use are reported to have ceased worldwide, acute poisoning should no longer occur. The risk associated with chronic exposure, however, particularly the risk of bladder cancer, will continue to be of concern for many years. Health screening of significantly exposed individuals from manufacturing plants from those rural communities where chlordimeform was extensively used should be continued. Since chlordimeform is no longer used, no quantitative risk assessment for the environment has been performed. There are not expected to be any long-term detrimental effects on the environment as a result of past use of chlordimeform. 1.10 Conclusions and recommendations Chlordimeform has significant potential to cause both immediate and long-term toxicity in exposed individuals. Current information supports an association between an increased incidence of human bladder cancer and exposure to 4-chloro- o-toluidine and, to a lesser extent, chlordimeform. Chlordimeform does not persist in the environment, and therefore there are not expected to be any long-term detrimental effects on the environment as a result of past use. Future commercial production or use of chlordimeform is not recommended. Existing stocks should be disposed of safely. Those with occupational exposure to chlordimeform should participate in a health screening programme that includes urinary cytology and the detection of haematuria. 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS 2.1 Identity Common name: Chlordimeform Chemical structure: Chemical formula: C10H13ClN2 Relative molecular mass: 196.7 CAS name: N'(4-chloro-2-methylphenyl)- N, N-dimethyl-methanimidamide IUPAC name: N2-(4-Chloro- o-tolyl)- N1, N1-dimethylformamidine CAS registry number: 6164-98-3 (chlordimeform) 19750-95-9 (chlordimeform hydrochloride) RTECS number: LQ4375000 Common synonyms: Chlorphenamidine; chlorfenamidine; chlorophedine; chlorophenamide; chlorophenamidin; chlorophenamidine; N'-(4-chloro- o-tolyl)- N, N-dimethylformamidine; N, N-dimethyl- N'-(2-methyl-4- chlorophenyl)-formamidine; N, N-dimethyl- N'-(2-methyl-4- chlorophenyl)formadin; ENT 27335; ENT 27567; EP-333; N'-(2-methyl-4-chlorophenyl)- N, N-dimethylformamidine Trade names: Acaron; Bellotion Especial; Bermat; Bermatchlorfenamidine; C8514; Carzol; CDM; CDMS; CGS500; CGS800SP; Chlorfenamidine; Ciba 8514; Ciba C8514; COTIP 500EC; Fundal; Fundal 500; Fundex; Galecron; OMS-1209; Ovatoxion; OVINA; OVITIX; RS 141; Schering 36268; Sn 36268; Spanon; Spanone; SPIKE ULVAIR. Technical grade chlordimeform is greater than 95% pure and contains the following impurities: N-formyl-4-chloro-2-toluidine ( N-formyl-4-chloro- o-toluidine), 4-chloro-2-toluidine (4-chloro- o-toluidine hydrochloride) and sodium chloride. Chlordimeform free base has been formulated as a 500 g/litre emulsifiable concentrate. Chlordimeform hydrochloride has been formulated as a 300 or 800 g/kg water-soluble powder, a 20 g/kg dust or as 50 g/kg granules. 2.2 Physical and chemical properties Some of the physical and chemical properties of chlordimeform base and chlordimeform HCl are shown in Table 1. The molecular structure of chlordimeform has been investigated by Gifkins & Jacobson (1980) using single crystal X-ray diffraction. Table 1. Some physical and chemical properties of chlordimeform basea Physical state colourless crystalline solid Boiling point at 14 mmHg 163 - 165°C Melting point 32°C Log Kow 2.89 Vapour pressure at 20°C 48 mPa (3.5 × 10-4 mmHg) Density (d30) 1.10 Solubility in water at 20°C 250 mg/litre Solubility in acetone, benzene, chloroform, ethyl acetate, hexane, methanol at 20°C >200 g/litre Half-life at pH 7 (30°C in water, 5% methanol) 42 h Half-life at pH 9 (30°C in water, 5% methanol) 5 h Reactivity Forms salt with acids a From: Worthing (1979); IARC (1978) Chlordimeform has a solubility in water of 250 mg/litre but is readily soluble in organic solvents. It forms salts with acids and the hydrochloride salt is readily soluble in water. When pure, chlordimeform forms colourless crystals. Chlordimeform is a base of medium strength with pKa of 6.8 in 50% aqueous methanol (Voss et al., 1973) and forms stable salts with strong acids. Chlordimeform is sensitive to light, especially in alkali, and slowly decomposes in neutral and alkaline aqueous solution. The pH dependence of photodecomposition of chlordimeform was noted by Su & Zabik (1972), who observed that an aqueous solution of chlordimeform hydrochloride (pH 3.1) was unaffected by mercury lamp irradiation for up to 12 days at 25°C, while a solution of the free base at pH 7-8 decomposed in the same period to a mixture consisting of N-formyl-4- chloro- o-toluidine and a bis-formamidine. Photo-decomposition of chlordimeform has also been studied on silica gel chromatographic plates with irradiation by long- and short-wave ultraviolet light, fluorescent light and sunlight (under glass) for periods of 10 to 20 h (Knowles & Sen Gupta, 1969). The major degradation product was again N-formyl-4-chloro- o-toluidine with either sunlight or UV light. Fluorescent light caused little decomposition. Sunlight resulted in 12% decomposition in 10 h, while UV resulted in 25% decomposition in 20 h. When 4-chloro- o-toluidine was irradiated with UV light, numerous decomposition products were found but these were not characterized further. Chlordimeform has relatively high volatility and is thus capable of efficient fumigation action. The hydrochloride salt has negligible volatility. 2.3 Conversion factors 1 ppm = 8.04 mg/m3 1 mg/m3 = 0.12 ppm 2.4 Analytical methods 2.4.1 Plants Geissbühler et al. (1971) described in detail a method for the determination of total residues of chlordimeform and its metabolites, which can be used for routine analysis of plant and soil samples. In this method, chlordimeform and its metabolites are hydrolysed to 4-chloro- o-toluidine by successive treatments with acetic acid and sodium hydroxide, respectively. The hydrolysis product is then steam distilled, extracted with isooctane, diazotized and coupled with N-ethyl-1-naphthylamine yielding a purple dye, which, after column chromatography on cellulose, is determined by colorimetry. Interfering azo-dyes from aromatic plants or soil are removed by chromatography on a cellulose column. This colorimetric method has a limit of detection of 0.05 mg/kg. If required, the identity of the residues can be verified by thin-layer chromatography on a cellulose column. This procedure is sensitive to about 0.1 mg/kg. Alternatively, the hydrolysis product, 4-chloro- o-toluidine, is diazotized and iodinated, and the iodinated derivative is measured by electron- capture gas chromatography. This alternative method has a limit of detection of 0.05 mg/kg. Kossmann et al. (1971) refined the method of Geissbühler et al. (1971) to permit separate determination of residue quantities of the parent compound and its potential degradation products in plant materials. In this procedure, plant material is subject to a two-fold extraction, the first with methanol/hydrochloric acid and the second with the lipophilic mixture, methanol/methylene chloride. Separation of chlordimeform and its degradation products is accomplished by thin-layer chromatography. The separated eluants are converted to 4-chloro- o-toluidine and analysed as described by Geissbühler et al. (1971). The limits of detection for the separated compounds, chlordimeform, demethylchlordimeform and 4-chloro- o-toluidine are 0.02 to 0.03 mg/kg. Grübner (1977) described a thin-layer chromatographic method for the determination of chlordimeform residues alone or together with its metabolite, 4-chloro- o-toluidine, in cucumbers and apples. The limits of detection for chlordimeform and 4-chloro -o-toluidine were 0.1 and 0.05 mg/kg, respectively. The rates of recovery were 76-85 and 90-105%, respectively. Fan & Ge (1982) described an alkali flame ionization gas-chromatographic method for the determination of chlordimeform and three potential metabolites in cargo rice and husk. Residues of chlordimeform and its metabolites were extracted with absolute alcohol or hexane and cleaned up on neutral alumina columns, before being chromatographed in a column of 1% DEGS coated on 60-80 mesh 405 support (PEG 20M bonded phase). The detection limits for chlordimeform, 4-chloro- o-toluidine, 2,2'-dimethyl-4, 4'-dichloroazobenzene, and N-formyl-4-chloro- o-toluidine were 0.03, 0.028, 0.11 and 0.43 mg/kg, respectively, for cargo rice and 0.03, 0.028, 0.22 and 0.43 mg/kg, respectively, for husk. Recovery for chlordimeform was 81-93% for cargo rice and 103-104% for husk. Recovery for 4-chloro- o-toluidine was 71-73% for both cargo rice and husk. Recovery for 2,2'-dimethyl-4,4'-dichloroazobenzene was 81.8-112% for cargo rice and 109-118% for husk. Recovery for N-formyl-4-chloro- o-toluidine was 66% for husk. Mattern et al. (1991) described a rapid analytical procedure for 17 pesticides, including chlordimeform, using gas chromatography/chemical ionization mass spectrometry (GC/CIMS) for detection in various commodities including peppers, spinach, lettuce and snap beans. Percentage recoveries for chlordimeform were 87.8% (peppers), 72.6% (spinach), 99.7% (lettuce) and 94.7% (beans). The limits of detection for chlordimeform were 0.05 mg/kg (beans), 0.05 mg/kg (lettuce), 0.05 mg/kg (peppers) and 0.10 mg/kg (spinach). 2.4.2 Soil The method of Geissbühler et al. (1971) described in section 2.4.1 for plants can equally be applied to the determination of total residues of chlordimeform in soil. 2.4.3 Water Machin & Dingle (1977) described a UV spectrographic method for the determination of chlordimeform in cattle dipping baths and sprays. Preliminary clean-up removes UV-absorbing impurities and converts chlordimeform to its hydrochloride. Following silica gel chromatography, the absorbance of the non-eluted material is measured at 240 nm to determine chlordimeform content. Optimum results are obtained in the concentration range of 0.02-0.06% (w/v) chlordimeform. 2.4.4 Formulations Voss et al. (1973) described two methods for the determination of chlordimeform in formulations. The first relies on acid titration of the free base with hydrochloric acid. The hydrochloride salt is converted into the free chlordimeform base, which is extracted into an organic solvent. After evaporation of the solvent, the active ingredient is determined potentiometrically. The second method makes use of gas chromatography, and in this case the chlordimeform hydrochloride preparations have to be converted into the base form prior to injection into the gas chromatograph. Gale & Hofberg (1985) described a gas chromatographic procedure for the determination of chlordimeform in emulsifiable concentrate formulations. Chlordimeform was extracted with methylene chloride, chromatographed on CBWX-20M and detected by flame ionization. 2.4.5 Air There are no published methods described for the determination of chlordimeform in air. 2.4.6 Urine Liu & Mao (1980) described a method for the gas chromatographic separation of chlordimeform, demethylchlordimeform, N-formyl-4- chloro- o-toluidine and 4-chloro- o-toluidine in urine. Optimum separation was achieved on a column with 1% polyvinylpyrolidone and 8% PEG 20M on 80-100 mesh white diatomeous support no. 101 (acid and base washed). The column was suitable for both qualitative and quantitative analysis. A method to analyse urinary residues of workers occupationally exposed to chlordimeform was developed by Ciba-Geigy in 1980 (Anonymous, 1980a). The method relies on the hydrolysis of chlordimeform and other residues to 4-chloro- o-toluidine with sodium hydroxide, followed by extraction with hexane and separation on reverse-phase liquid chromatography fitted with a UV detector. A published version of this method was prepared by Geyer & Fattal (1987) in which the alkaline hydrolysate of urine is extracted with hexane, the solvent is evaporated, and the hydrolysate is reconstituted with aqueous acetonitrile. Separation was performed on a reverse-phase Novo Pak 5 mm C18 column with a UV absorbance detector equipped with a 254 nm filter. A similar method was described by Cheung et al. (1989) for the analysis of chlordimeform from urine of field workers. Ross & Leisten (1989) have refined this method with the use of synchronous spectral data which provides a improved signal-to-noise ratio, which gives lower minimum detectable levels while still allowing a well-resolved spectrum. This system may allow detection of levels equivalent to 1 mg/litre in urine. 2.4.7 Tissues A gas chromatographic method for the determination of residues of chlordimeform in animal tissues was first described in the early 1970s (Anonymous, 1971a). The method involves hydrolysis of chlordimeform to 4-chloro- o-toluidine by successive treatments with acetic acid and sodium hydroxide. The hydrolysis product is steam distilled and extracted into isooctane. Following diazotization of the 4-chloro- o-toluidine, the diazo-moiety is exchanged for iodine by potassium iodide treatment. The iodinated derivative is gas chromatographed using electron-capture detection. The limit of detection using this method is 0.02 mg/kg. Rieger et al. (1985) have described a gas chromatography/flame ionization detection method for the determination of chlordimeform and its major metabolite, demethylchlordimeform, from human tissue samples, namely, human whole blood and human liver (1:1 aqueous homogenate). Tissues were first extracted with an organic solvent, transferred to an acid aqueous medium (0.1M hydrochloric acid), re-extracted into a small volume of organic solvent and separated on GC or GC/MS. Using extraction with either chloroform or n-butanol, recoveries of 81 and 75%, respectively, were obtained. 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1 Natural occurrence Chlordimeform does not occur naturally. 3.2 Anthropogenic sources 3.2.1 Production levels and processes Chlordimeform was first commercialized in 1966. It can be manufactured commercially by two methods (Voss et al., 1973), both starting with the conversion of dimethylformamide to the Vilsmeier reagent by reaction with POCl3, SOCl2 or COCl2. By the first method, condensation of the Vilsmeier reagent with 4-chloro-amino-toluene (or 5-chloro-2-aminotoluene, 5-CAT) leads directly to chlordimeform hydrochloride. Treatment with a strong base gives the free chlordimeform base. By the second method, the Vilsmeier reagent is reacted with o-toluidine to give phenamidine, which is chlorinated in a second step. The chlorination gives rise to a certain amount of isomers as unwanted side-products. The crude chlordimeform so obtained has to be purified either by recrystallization of its chlorohydrate or by rectification of the free base. Chlordimeform has been produced at various times in Switzerland, Germany, United Kingdom, USA, Italy, Argentina and China. Little information is available on the production levels of chlordimeform. Information from the US International Trade Commission (IARC, 1983) indicated that imports of chlordimeform to the USA through the principal US customs districts amounted to 745 tonnes in 1979 and 198 tonnes in 1980. In 1974, total usage of chlordimeform in the USA is estimated to have been 590 tonnes, 77% of which was used on cotton, 15% on deciduous fruits and nuts, and 8% on vegetables. In 1976, the US Department of Agriculture reported that 2000 tonnes of chlordimeform was used in the USA on major crops (IARC, 1983). In 1980, total usage in the USA was 227 tonnes, all of which was used on cotton to control budworm/bollworm. Chlordimeform has been used in China throughout the 1970s and the 1980s at the rate of approximately 10 000 to 15 000 tonnes per year (Xue, personal communication). In the Chinese province of Hu-bei, the average annual usage during the period 1984-1988 was 3276 tonnes (Huang et al., 1989). 3.2.2 Uses Chlordimeform is a broad spectrum acaricide and is active mainly against eggs and motile forms of mites and ticks and against eggs and early instars of some Lepidoptera insects. It kills eggs, larvae and adults not only by contact but also in the vapour phase. The major use initially was in the control of mites on deciduous fruit. In 1971, chlordimeform products were registered in many countries for use on a wide variety of crops such as pome fruits, stone fruits, cole crops, vegetables, grapes, hops, citrus, apples, pears, cherries and strawberries. Chlordimeform also had important veterinary uses as an acaricide. In Australia, chlordimeform was registered for use in cattle dips for the control of cattle ticks (Boophilis mictopus), in combination with organophosphorus acaricides (FAO/WHO, 1972). In 1975, it was reported that the use pattern of chlordimeform had been extended to include control of stemborers in irrigated rice, control of Lepidoptera larvae on cotton, and control of a wide range of Lepidoptera larvae on cabbage and tomatoes (FAO/WHO, 1976). At this time, the control of stemborers in irrigated rice proved to be one of the most important uses of chlordimeform. In the case of cotton, chlordimeform became one of the most important substitutes for DDT and other organochlorine pesticides. Chlordimeform has had no significant usage in non-crop situations other than on ornamentals. In 1976, the manufacturers temporarily suspended the sale of chlordimeform from all markets worldwide, on the basis of adverse carcinogenicity findings in chronic mouse studies. In 1978, having completed a number of toxicology, metabolism and residue studies, the manufacturers re-applied in a number of countries for registration to allow limited commercial use in cotton crops only. The proposal was to use chlordimeform by aerial application under supervised conditions that limited the uptake by operators and by-standers. Chlordimeform was re-introduced for insect control in cotton in USA, Central America, Columbia, Israel, Australia and China. Guidelines for the handling and use of chlordimeform were set in Australia, Columbia, Israel and USA (California). Application rates were set to minimize the occurrence of residues in cotton fibres and cotton seed oil. In China, extensive use of chlordimeform continued through the 1980s on rice and cotton. Use of chlordimeform ceased in most countries in the mid to late 1980. The Joint FAO/WHO Meeting on Pesticide Residues (JMPR) withdrew its temporary Acceptable Daily Intake (ADI) in 1987 and recommended that chlordimeform should not be used where its residues, or those of its metabolite, 4-chloro- o-toluidine, could arise in food. (FAO/WHO, 1988). In 1988-1989, Ciba-Geigy and Schering voluntarily and finally halted marketing of chlordimeform and decided to withdraw registration worldwide. In China, production stopped at the end of 1992, and sales ceased in June 1993. 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION 4.1 Transport and distribution between media 4.1.1 Air Chlordimeform has relatively high volatility, and thus when sprayed on crops considerable evaporation would be expected from plant surfaces as well as from the soil. Studies in plants, however, indicate a lower rate of evaporation than expected. In bean plants, disappearance from the surface in the first few hours was found to be of the order of only 30-40% of the original dose applied (FAO/WHO, 1972). This result was obtained when either chlordimeform or its hydrochloride salt was used and is considered to be due to the buffering capacity of plant exudates with a resulting equilibrium between the free base and salts. The low volatility from plant surfaces was confirmed by Sen Gupta & Knowles (1969) on apple seedlings and by Ehrhardt & Knowles (1970) on grapefruit seedlings. In cotton plants, approximately 55% of the dose applied to leaves was volatilized from the surface of the leaves within 2 h (Bull, 1973). No studies are available on the volatilization of chlordimeform from soil surfaces, but it is likely to be at least as high as from leaf surfaces. 4.1.2 Water While chlordimeform base has only low solubility in water, the solubility of the hydrochloride salt is relatively high. Its stability in water, however, is highly pH-dependent, and in the normally neutral to slightly alkaline conditions of rivers and lakes its half-life would be relatively short. It also has the potential to adsorb readily to dissolved organic matter resulting in precipitation (Maqueda et al., 1989). The hydrolytic stability of chlordimeform is highly pH-dependent. It slowly hydrolyses in neutral pH and is stable in strongly acid conditions. The half-life at 10°C is about 38 days at pH 7, compared to 8 days at pH 8. At 30°C, these values are reduced to about 3 and 0.5 days, respectively. A solution of the hydrochloride salt (pH 3-4) showed no appreciable hydrolysis over several days (Su & Zabik, 1972). The principal product of hydrolysis is N-formyl-4-chloro- o- toluidine, which at room temperature is very slowly converted to 4-chloro- o-toluidine by further hydrolysis. The second step may be accelerated by heating with strong acid or alkali. 4.1.3 Soil Hydrolysis of chlordimeform to N-formyl-4-chloro- o-toluidine would be expected to be significant under the slightly acid or slightly alkaline conditions that normally prevail in soils. Despite the reasonably high solubility of the hydrochloride salt of chlordimeform, there appears to be little leaching from the site of application in the soil (FAO/WHO, 1972). In the studies by Fischer & Cassidy on the uptake of chlordimeform from soil into cotton plants, the levels of chlordimeform in the soil were also analysed (FAO/WHO, 1979). Soil was treated when the cotton reached 10 weeks of maturity. Radioactivity in the top 75-mm layer of silt loam soil accounted for 1.23 mg/kg chlordimeform equivalents after treatment. At 7 weeks, this level had decreased to 0.33 mg/kg and at 13 weeks to 0.20 mg/kg. Extraction of this layer revealed partition of 32% into the organic layer and 20% into the polar fraction, and 44% was non-extractable, indicating rapid degradation. For all but one sample, the level of radioactivity as chlordimeform equivalents in the lower soil levels, 75-150 mm and 150-200 mm, was less than 0.01 mg/kg, indicating that leaching did not occur in silt loam. In later experiments with regular over-the-top spray treatment throughout the maturation of the cotton plants, the same rapid decrease in radioactivity (as chlordimeform equivalents) was seen in the top 75 mm of soil. Radioactivity in deeper layers was again equivalent to less than 0.01 mg/kg. At harvest of the cotton plants, up to 91% of the radioactivity in the soil could be converted to 4-chloro- o-toluidine. The nature of the non-extractable portion of chlordimeform in soil was investigated by Perez-Rodriguez & Hermosin (1979) and by Hermosin & Perez-Rodriguez (1981) in experiments examining the interaction of chlordimeform with clay minerals, montmorillonite, kaolinite, illite and vermiculite. The earlier work indicated that the adsorption of chlordimeform on clay is essentially a cation-exchange reaction and that chlordimeform ions lie between the silicate layers, thus being difficult to disperse with water or aqueous solutions of inorganic cations. In the later study, chlordimeform adsorption to the clay minerals montmorillonite, illite and vermiculite was found to be an irreversible process, whereas chlordimeform adsorbed on kaolinite is only weakly bonded and easily removed by washing with water. The role of soil organic matter in the adsorption and degradation of chlordimeform in soil was examined in experiments by Maqueda et al. (1983, 1989). In the first study, the interaction of chlordimeform with humic acid extracted from the top 20 cm of a clay soil classified as Typic Chromozerert soil was examined. Adsorption is essentially a cation-exchange process, although other mechanisms, such as charge transfer, H-bonding, and van der Waals forces may contribute to the high adsorption capacity. The variety of mechanism may make it difficult to ascertain the long-term fate in the environment. In the second study, the interaction of chlordimeform and other pesticides with fulvic acids extracted from a spodosol soil was examined. Fulvic acids are the fraction of humic substances that dissolves in both acid and alkaline media, and thus are readily found solubilized in lakes and rivers. The adsorption of chlordimeform was again shown to be a cation-exchange process, together with H-bonding and charge transfer mechanisms. Precipitation occurred upon interaction of chlordimeform with fulvic acids. The amount of precipitate increased in a dose-related manner up to levels of 100 mmol chlordimeform/litre. 4.1.4 Vegetation and wildlife Benezet & Knowles (1981) examined the degradation of chlordimeform by two algal types, Chlorella, the green alga, and Oscillatoria, a cyanobacterium. In the presence of either Chlorella or Oscillatoria, chlordimeform was hydrolysed to N-formyl-4-chloro- o-toluidine, probably by a largely non-enzymatic reaction. Further reaction formed 4-chloro- o-toluidine and some CO2. Oxidative N-demethylation was not a major path for chlordimeform degradation by algae. The solubility of chlordimeform was sufficient to allow uptake by the roots of bean and rice plants and to be transported to plant-feeding pests, as demonstrated by the efficacy experiments of Dittrich (1967) and Dittrich & Loncarevic (1971). The ability of plants to take up chlordimeform from soil was further demonstrated by the experiments of Fischer & Cassidy (FAO/WHO, 1979), where the soil of a cotton field was treated with [14C]-chlordimeform when the cotton was 10 weeks old. Uptake of the radioactivity by the cotton plant was noted to occur in small quantities, and the highest levels were found in the seeds and fibres. Biphasic extraction showed 42% in the organic fraction and 34% in the polar fraction, and 24% was not extractable. Thirteen weeks after treatment, the mature cotton contained 0.09 mg/kg in the leaves. The low level of translocation of chlordimeform in plants was demonstrated by Sen Gupta & Knowles (1969) in experiments where [14C]-chlordimeform was injected into the stem of apple seedlings followed by analysis of stem and leaf radioactivity for a period of 20 days. For the first 4 days after injection, 95% of the radioactivity was localized in the stems, predominantly as the parent compound. After 20 days, 71.6% of the radioactivity still remained in the stem, with 25.4% in the leaves, and only 17.9% remained as the parent compound. The major portion of the radioactivity in the stems after 20 days was unextractable with chloroform and acetone. In the experiments of Ehrhardt & Knowles (1970) with grapefruit seedlings, there was no detectable movement of radioactivity into adjacent stems and leaves 8 days after application of [14C]- chlordimeform to two upper leaves or two lower leaves. Considerable movement into stems and leaves was noted when [14C]-chlordimeform was injected into the main stem, and also to the periphery of grapefruit leaves when it is applied centrally. Thus, movement of chlordimeform occurred mainly in the direction of the xylematic transpiration stream. Application of chlordimeform directly to the leaves of apple seedlings (Sen Gupta & Knowles, 1969) or the leaves of grapefruit seedlings (Ehrhardt & Knowles, 1970) demonstrated the limited capacity of chlordimeform to penetrate the cuticular layers. Ercegovich et al. (1972) reported that chlordimeform appeared to adhere to the outer surface of fruit and did not appear to translocate readily to the fleshy parts. The chief factors which seem to account for the decrease of chlordimeform residues in fruit appear to be volatilization, weathering and growth dilution. Similarly, the application of [14C]-chlordimeform to cotton leaves resulted in little movement of radioactivity (and none of chlordimeform itself) into the untreated plant parts. The small amount of translocated radioactivity consisted exclusively of polar, mainly non-extractable substances (Gross, 1977). In a field experiment, Fischer & Cassidy treated a cotton field plot over-the-top with [14C]-chlordimeform at a rate of 1 kg/ha when plants were 12-14 weeks old (FAO/WHO, 1979). Radioactivity in the cotton plants immediately after treatment was the equivalent of 2.44 mg/kg chlordimeform. At harvest, the radioactivity calculated as [14C]-chlordimeform was 12.91 mg/kg in the leaves, 0.99 mg/kg in the stalks, 0.03 mg/kg in the fibre, and 0.26 mg/kg in the seed, with 0.07 mg/kg in the oil and 0.19 mg/kg in the meal. Parent chlordimeform accounted for 31% and 45.2% in the leaves and stalks, respectively. The data indicated that although leaf radioactivity is high, there is still little translocation of [14C]-chlordimeform metabolites to the seed or fibre. Supervised residue trials to determine the residue levels in cottonseed and cottonseed products have been conducted (FAO/WHO, 1979). In general, there is a correlation between the application rate and the residue level but the interval between the last application and the harvest also has a strong influence on the residue level. The decrease of residues with time was most pronounced during the first 10 days after treatment of the cotton plants. At the maximum application rate of 1 kg/ha, the residue level rarely exceeded 2 mg/kg in cottonseed, seed meal or crude oil. When used for the control of rice stem borer in Japan, chlordimeform resulted in low levels of residues in rice grains and straws. In rice grain after three treatments, the residue levels of chlordimeform, demethylchlordimeform, N-formyl-4-chloro- o- toluidine and 4-chloro- o-toluidine were 48, 0.4, 15 and 53 µg/kg, respectively. The results indicate a low level of penetration of chlordimeform into rice plants. The chlordimeform that entered the plant was gradually degraded to 4-chloro- o-toluidine (Iizuka & Masuda, 1979). There have been no studies conducted on the uptake of chlordimeform by wildlife. Studies with experimental animals suggest rapid metabolism and excretion, with negligible retention. 4.1.5 Entry into food chain Potential routes of entry of chlordimeform into the human diet include the direct consumption of crops containing chlordimeform residues, the consumption of processed food prepared from treated crops, or the consumption of animal products derived from animals treated topically with chlordimeform or raised on chlordimeform- containing feed such as cottonseed. Since the temporary withdrawal of the use of chlordimeform from the market in 1976 in most countries and the later restriction to use on cotton, dietary consumption of chlordimeform residues on crops in these countries has virtually ceased. However, dietary consumption of chlordimeform residues is likely to have continued at least until the late 1980s in some areas (see section 5.2.2). The maximum residue levels (MRLs) which were used for chlordimeform are discussed in section 5.2.2. 4.2 Biotransformation 4.2.1 Degradation in plants Data reviewed by JMPR (FAO/WHO, 1972) demonstrated that chlordimeform was quite rapidly degraded in plants with a high inherent metabolic activity (e.g., bean plants) but was only slowly degraded in ripe fruits. Green fruits (e.g., grapes) and stems have an intermediate rate of degradation of chlordimeform. Tentative identification of the observed metabolites indicated that in leaves both N'-(4-chloro- o-tolyl)- N-methylformamidine (demethylchlordimeform) and N-formyl-4-chloro- o-toluidine were major metabolites. In ripe apple and pear fruit, however, only N'-formyl-4-chloro- o-toluidine was detected. In all tissues, 4-chloro- o-toluidine was either not detected or present in small quantities, even when six-fold overdose treatment was used. In the experiments of Sen Gupta & Knowles (1969), [3H]- or [14C]-chlordimeform was applied to apple seedlings by either leaf treatment or stem injection. The half-life of degradation was about 12-16 days, and after 20 days 40% of the radioactivity was still unchanged chlordimeform. Organosoluble degradation products were identified as demethylchlordimeform, N-formyl-4-chloro- o-toluidine and 4-chloro- o-toluidine, with the last two representing less than 1% of the total radioactivity. Non-extractable radioactivity, possibly chlordimeform degradation products complexed with polymeric cell constituents, was observed only after stem application. In the experiments of Ehrhardt & Knowles (1970), both [14C]-chlordimeform and [14C]-chlordimeform hydrochloride were applied to the leaf surface of growing grapefruit seedlings. After 20 days, only 10-20% of total radioactivity was recovered, possibly due to evaporation from leaves, and only 1% of radioactivity was unchanged chlordimeform. The pattern of metabolites was essentially the same as in apple seedlings, but the levels were smaller. Witkonton & Ercegovich (1972) examined the metabolites found in six different fruits (apples, pears, cherries, plums, strawberries and peaches) following treatment at varying rates with chlordimeform. Samples of the fruit were collected at various intervals after the last application from orchards and plants that had been treated with aqueous sprays of chlordimeform. Of the three potential degradation products analysed for, only one, namely, N-formyl-4-chloro- o- toluidine, was detected, together with the parent compound. The other potential degradation products, namely, demethylchlordimeform and 4-chloro- o-toluidine, were not detected. There was no correlation between the amount of chlordimeform and 4-chloro- o-toluidine and the application rate or the sampling interval. The nature of the fruit and environmental factors were accredited as the major contributing factors governing the formation and retention of 4-chloro- o- toluidine. At harvest, the total residue in all crops was approximately 1 mg/kg, except in peaches, which had approximately 2 mg/kg of total residue. The chief factors which appeared to account for the decrease in chlordimeform residues were weathering and growth dilution, rather than chemical or enzymatic degradation. The potential formation of azo-derivatives of chlordimeform or its metabolite, 4-chloro- o-toluidine, in treated fruit and vegetables under field conditions was investigated by Geissbuhler et al. (1971) using a sensitive gas-chromatographic residue method that allowed the detection of 0.01 mg/kg of 2,2'-dimethyl-4,4'- dichloroazobenzene. At 20, 30 or 40 days after a 4-fold overdose treatment by chlordimeform to apple fruits and leaves, residues of the azobenzene compound were either not detectable or detected at very low levels (0.04 mg/kg) in leaves. At normal levels of treatment, residues of azobenzene compounds would be unlikely to be detected. This result is supported by the experiments of Witkonton (1973), who analysed the residues on apple surfaces 60 days after treatment with [14C]-chlordimeform. The results of these experiments do not support the in vitro studies of Rose (1969a,b), which indicate the potential formation of azobenzene derivatives in plants by plant peroxidases. The metabolism of chlordimeform in cotton plants was first examined by Bull (1973) following treatment of individual leaves with [14C]-chlordimeform by petiole injection or by foliar application. About 45% of the applied dose was absorbed by the leaves, and the balance volatilized from the leaf surface within 2 h. Tentative identification of metabolites included demethylchlordimeform, N-formyl-4-chloro- o-toluidine and 4-chloro- o-toluidine. After 1 h, only 2% of the applied dose could be recovered from leaf surfaces. The unextractable radioactivity was considered to represent decomposition products bound to insoluble plant material. Gross (1977) studied the metabolism of [14C]-chlordimeform in greenhouse-grown cotton plants following treatment of leaves at a rate equivalent to 0.6 kg a.i./ha. Metabolites were extracted into hexane, methylene chloride and water-soluble fractions at various times up to 11 weeks after treatment. The radioactivity in the organic fractions consisted of at least seven substances. Four were characterized by TLC as chlordimeform, N-demethylchlordimeform, 4-chloro- o-toluidine and N-formyl-4-chloro- o-toluidine. Fifty-six percent of the dose was found in the plant after one week, the balance being lost by volatilization. The main degradation pathway was hydrolysis, demethylation only being significant at later sampling times. The loss of chlordimeform from the surface of leaves was confirmed by Wolfenbarger et al. (1979) who noted that 24 h after cotton leaves were treated topically with chlordimeform, only 5% of the EC form was recovered, whereas 25% of the HCl salt was recovered. Fischer & Cassidy (FAO/WHO, 1979) identified the metabolites in leaves after [14C]-chlordimeform was sprayed over-the-top on cotton plants. At mature harvest, the radioactivity in the leaves consisted of chlordimeform (60.3%), demethylchlordimeform (4.1%), 4-chloro- o-toluidine ((7.6%) and N-formyl-4-chloro- o-toluidine (7%). The results indicate that the parent chlordimeform will be the major chemical residue in the mature cotton foliage. Honeycutt & Cassidy (1977) investigated the metabolism of chlordimeform in cottonseed following injection of [14C]- chlordimeform into the stem of a growing cotton plant. Forty percent of the radioactivity in the cottonseed was not extractable. Total hydrolysis of the radioactivity in the cottonseed showed that a total of 19.8% of the radioactivity could be converted to 4-chloro- o- toluidine. The data indicated that the metabolism of chlordimeform in cottonseed is extensive and results in conjugation to natural products. 4.2.2 Degradation in soils The potential for microbial degradation of chlordimeform in the soil was first identified by Johnson & Knowles (1970), who demonstrated the capability of several bacteria (Aerobacter aerogenes and Serratia marcesens), actinomycetes (Streptomyces griseus) and fungi (Fusarium moniliforme and Rhizopus nigricans) in culture media to degrade chlordimeform extensively. The principal metabolite of the bacterial and fungal species was N-formyl-4-chloro- o-toluidine, while for the actinomycete, Streptomyces griseus, the principal metabolite was 4-chloro- o- toluidine. 4-Chloro- o-toluidine was also formed by the bacteria and fungi. None of the microbes formed symmetrical azo-compounds. The metabolic fate of chlordimeform in sandy loam over a one-year period was examined by Iwan & Goller (1975). Soil samples containing 2 µCi of either [14C- ring]- or [14C- tolyl]-chlordimeform were prepared in an environmental chamber and methanol/benzene extracts examined at various intervals. Extractability decreased to 50% within 7 days and was less than 2% after 360 days. In sterilized soil samples, on the other hand, extractability decreased only slowly, and 70% was still extractable after 180 days. This result indicates that microbial activity plays a major role in soil degradation of chlordimeform to non-extractable components. Even though bound to soil, degradation of chlordimeform continued, as shown by the release of CO2 as a consequence of oxidative attack upon the tolyl group. Little CO2 was released under anaerobic conditions and no CO2 was released from sterile samples. The major pathway of metabolism was through hydrolysis to 4-chloro- o-toluidine but oxidative N-demethylation was also a significant pathway leading to 4-chloro- o-toluidine. Further hydrolysis steps followed. The azo compound, 2,2'-dimethyl-4,4'-dichloroazobenzene, was formed in small amounts only when the initial chlordimeform concentration was 200 mg/kg in the soil samples. Anaerobic conditions produced the same metabolic products with the exception of oxidative products such as demethylchlordimeform. The data suggests that even under sterile conditions, the degradation of chlordimeform is rapid and its half-life in non-sterile soils should not exceed one month. In a further study, Iwan et al. (1976) isolated from chlordimeform-treated soil four coupling products formed by one- electron oxidation of 4-chloro- o-toluidine by soil microorganisms. The four products, one of which is 2,2'-dimethyl-4,4'- dichloroazobenzene, are formed only from high concentrations of chlordimeform (70-100 mg/kg), which are at least 10 times higher than the levels occurring after field application. 4.2.3 Bioaccumulation There is no data to indicate that chlordimeform bioaccumulates in plant or animal tissues. However, with a low Kow of 2.89, this indicates a moderate potential to bioaccumulate. 4.3 Ultimate fate following use Chlordimeform in the air and in water would be expected to undergo photodecomposition. In water as well as in soil, chemical hydrolysis occurs together with adsorption to organic and clay materials. In plants, residues form complexes with polymeric cell constituents. Chlordimeform can be hydrolysed readily to 4-chloro- o-toluidine by heating with alkali. For the disposal of small quantities of unused pesticide, the following method is recommended: mix with excess lime (CaO) or sodium hydroxide (NaOH) and sand and bury at least 0.5 m below the surface in clay soils. Commercial formulations require 0.5-1.0 kg alkali per kg of pesticide. Alkali can be reduced by 50% for dilute formulations, e.g., 1% solution or dust. For very concentrated pesticides (> 50% a.i.), double the amount of alkali and mix the pesticide with soapy water, before reaction with alkali. Test reaction on small scale to discover whether or not it will be too vigorous. Larger quantities should be treated in small batches or burned in a high-temperature incinerator equipped with effluent gas scrubbing (IRPTC, 1992). 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1 Environmental levels 5.1.1 Air and water There are no specific data available on the monitoring of chlordimeform levels in air and water. In neutral and alkaline solutions, relatively rapid degradation would be expected owing to hydrolytic instability. Under acidic conditions, slower degradation would be expected (Su & Zabik, 1972). Adsorption to organic matter in water would also be expected under field conditions. In both media, there would be degradation due to photodecomposition (Knowles & Sen Gupta, 1969). 5.1.2 Soil Chlordimeform deposited inadvertently on soil surfaces after spray application may be expected to dissipate by the following processes: volatilization, chemical hydrolysis, photodecomposition and microbial degradation. Under field conditions, chlordimeform and its 4-chloro- o-toluidine-containing metabolites are dissipated according to first-order reactions with half-lives ranging from 20 to 40 days (Guth & Senn, 1969; FAO/WHO, 1972). The conclusion from these experiments is that accumulation of chlordimeform in the soil would not be expected. Following three applications to rice paddy fields for the control of rice stem borer, residues of chlordimeform, demethylchlordimeform, N-formyl-4-chloro- o-toluidine and 4-chloro- o-toluidine were 2900, 9, 190 and 68 µg/kg, respectively, in the top 5 cm of soil, and were 150, 1, 8 and 20 µg/kg, respectively, in the 5-10 cm level of soil. These results indicate the presence of chlordimeform and its degradation products mainly in the upper layer with minimal movement downward (Iizuka & Masuda, 1979). 5.2 General population exposure 5.2.1 Environmental sources There are no longer any environmental sources for exposure of the general population to chlordimeform. While chlordimeform was being used on cotton, there was potential for general population exposure to spray drift from aerial application. The persistence of residues of chlordimeform on the leaves of cotton also raised the possibility of exposure through contact with the leaves during the growing period or during harvesting. 5.2.2 Residues in raw produce Prior to the temporary suspension of its use in 1976, chlordimeform was used on a wide variety of crops and on livestock. The temporary maximum residue levels (MRLs) shown in Table 2 were established at the 1971 meeting of the Joint Meeting on Pesticide Residues (JMPR) (FAO/WHO, 1972) as a result of numerous residue trials in various countries. Residue trials indicated that whilst there was a sharp drop in the residue level between the day of application and the second or third day post-treatment, thereafter the rate of decline was remarkably slow with a half-life on apples, grapes, pears and tomatoes exceeding 21 days. Table 2. Temporary tolerances for chlordimeform established in 1971 (FAO/WHO, 1972) Temporary tolerance mg/kg Pears, peaches, prunes 5 Apples, grapes, plums, strawberries 3 Brassicas, cherries, citrus fruit, cotton seed oil (crude and refined), cotton seed 2 Beans 0.5 Fat, meat and meat products of cattle 0.5 Milk (whole) 0.05 Butter 0.5 In 1975, the temporary MRL for pears was raised to 10 mg/kg, and new temporary MRLs were established for tomatoes (1 mg/kg) and hulled rice (0.1 mg/kg) (FAO/WHO, 1976). In 1978, the JMPR meeting retained only the MRLs for cottonseed and recommended that for cottonseed oil (edible), meat of cattle, pigs, poultry and sheep, and milk and milk products no residues should occur at the current limit of detection (0.05 mg/kg) (FAO/WHO, 1979). The proportion of metabolites and parent compound in the residues remaining on fruits at various times after application have been determined in numerous trials. In general, the parent compound represents the major residue (>80%), followed by N-formyl-4-chloro- o-toluidine, N'-(4-chloro- o-tolyl)- N-methylformamidine (demethylchlordimeform) and 4-chloro- o-toluidine. In Chinese residue trials, chlordimeform residues on green cabbage after application by direct spraying of a 800-fold dilution of 25% chlor-dimeform formulation were 20.9 mg/kg after 4 h, 11.5 mg/kg after 2 days, 4.2 mg/kg after 7 days and 0.02 mg/kg after 14 days (Anonymous, 1980b). In a paper by the Chinese Special Task Group on the residues of chlordimeform (Anonymous, 1981), the residues of chlordimeform in rice plants during the period 1974-1980 were examined. In the period 1974-1975, after a single application of 25% aqueous chlordimeform (9-11 litre/ha) the residue levels on rice harvested after 33-40 days were 0.25-0.28 mg/kg. When applied at half this rate, residue levels on rice harvested after 20-74 days were 0.17-0.71 mg/kg. In field studies in Beijing in 1977, with the same single rate of application, residue levels on rice harvested after 19-42 days were 0.37-0.51 mg/kg. If 2-3 applications were used, the residue levels on rice harvested after 19-31 days were 1.3-1.8 mg/kg. The authors noted the difficulty in meeting the requirement for a residue level of 0.1 mg/kg regardless of the pattern of application. In field studies in Hu-bei Province in 1978 with the same application rate, the residue levels in rice harvested after 25-42 days were 0.19-1.20 mg/kg. In field studies in Zhe-jiang Province in 1978, residue levels in rice when harvested after 30 days were 0.080-0.112 mg/kg, while residues in rice harvested after 80 days were 0.039-0.100 mg/kg. In field studies in Guang-dong Province in 1978, residues in rice harvested after 30 days were 0.042-0.149 mg/kg. In other field studies in the Guang-dong Province in 1980, residue levels on rice harvested after 56 days were 0.185 mg/kg, but when the harvest was performed at 72 days, the residue level was less than 0.10 mg/kg (Anonymous, 1981). Huang et al. (1989) reported the residues of chlordimeform on both rice and cotton plants in the Hu-bei Province of China between 1984 and 1988. With 1-3 applications to rice plants, followed by harvest after 25-55 days, the chlordimeform residues were generally in the range of 0.066-0.820 mg/kg for the rice, 7.70-22.30 mg/kg for the husk, and 16.5-21.2 mg/kg for the stem. The authors noted that the residue levels seldom met the 1975 JMPR recommended MRL of 0.10 mg/kg for hulled rice (FAO/WHO, 1976). In further work on rice plants, it was noted that the residue levels for late rice were generally higher (approximately 2-fold) in late rice compared to early rice, and that the residue levels in both the rice and the husk reduced by more than 90% when the time to harvest was increased from 26 to 72 days. With a 72-day harvest, the residue level in the rice was 0.065 mg/kg. The residue levels in the stem (18-41 mg/kg), on the other hand, remained relatively unchanged over the 72-day period. With 1-3 applications to cotton plants, followed by harvest after 40 days, the chlordimeform residues were 0.053-0.151 mg/kg in the kernel and 0.118 mg/kg in the bracket. Chlordimeform residues were also found in 8/15 honey samples (Huang et al., 1989). The highest residue found was 32.2 mg/kg, and the majority of the samples contained less than half this level. In 1994 the US FDA collected and analysed samples of honey imported from the People's Republic of China. Of 60 samples analysed, 39 had detectable residues, the highest being 0.058 mg/kg (Krick, 1994). Moore (1971) summarized the results of residue trials on the use of chlordimeform as an acaricide in cattle dips in Australia. The residues were examined in cattle muscle, fat and liver as well as in milk and butter from the first milking. Chlordimeform was used at concentrations of 0.0125-0.1% in buffered cattle dips. Residues in muscle, fat and liver did not increase greatly with increasing dose of chlordimeform, and showed significant reductions between day 1 and day 3 post-treatment. The maximum residue levels found at day 3 post-treatment in muscle, fat and liver were 0.33, 0.51 and 0.69 mg/kg, respectively. At the first milking, the residues levels showed a closer relationship with the concentration of chlordimeform in the dip. The residue levels in milk and butter at a concentration of 0.0125% were 0.01 and 0.30 mg/kg, respectively. The maximum residue levels in milk and butter, which were found at the highest concentration used (0.2 %), were 0.31 and 1.6 mg/kg, respectively. In the study by Burkhard (1971), cows washed with a 0.5% solution of chlordimeform to the hindquarters (3 treatments at 7-day intervals), had total residue levels in milk, meat and fat below the level of detection (0.03 mg/litre), except in milk on the day after treatment when the levels rose to 1 mg/kg. In a further study by Voss & Burkhard (1971), when cows were fed a concentrate containing 40-240 mg/kg chlordimeform for periods up to 42 days, the total residues of chlordimeform and its metabolites in all milk, meat and fat samples were below the limit of detection (0.03 mg/litre or mg/kg). In liver and kidney samples, residues rose to a peak between 14 and 21 days (0.58 mg/kg in liver and 0.13 mg/kg in kidney), which was followed by a slow decline. In a study by Palmer et al. (1977), residues of chlordimeform were determined in tissues and milk of cattle after spray application to control cattle tick. In subcutaneous fat from animals sprayed with 0.45, 0.15 or 0.05% chlordimeform, the residue levels were 2.88, 0.46 and 0.15 mg/kg, respectively. The half-life of disappearance in all cases was 2.46 days. Lower residue levels were found in six other tissues, including kidney, muscle and liver. Residue levels in whole milk of lactating cows at the three treatment levels were 1.42, 0.28 and 0.03 mg/litre, respectively. The half-life of disappearance from milk was 0.45 days. White Leghorn hens fed a laying mash containing chlordimeform at levels of 0.25, 0.75 or 1.0 mg/kg were examined for residues in eggs and tissues (breast, fat and liver) for periods of up to 28 days. No residues were detected in breast meat. Residues were detected in fat (0.22 mg/kg) at the 21 days only. Residues in the liver were highest between 7 and 14 days (0.20 mg/kg) and reduced rapidly upon withdrawal from the chlordimeform-containing feed. There were no detectable residues of chlordimeform in eggs (FAO/WHO, 1972). Residue trails on cotton were conducted between the years 1969 and 1978 (FAO/WHO, 1979). The application rates ranged from 0.125 to 3.6 kg/ha and resulted in mean residue levels of 0.1 to 13.1 mg/kg in cottonseed when it was harvested immediately after application. The final residue level was dependent on a number of factors including application rate, number of applications, and length of waiting period before harvest. The application rate had the largest influence. 5.2.3 Residues in processed food Total residues of chlordimeform and its metabolites do not reduce substantially during cooking processes, since while the proportion of parent compound is reduced, there is an increase in the hydrolysis product, N-formyl-4-chloro- o-toluidine. Residues of chlordimeform itself in crops decrease through hydrolysis, but volatilization in steam during cooking is not an important factor. The rate of hydrolysis of chlordimeform is a function of pH and occurs much more rapidly in weakly acid or neutral crops such as cauliflower (pH 6) or green beans (pH 5) than in strongly acid crops such as apples (pH 2.5) or tomatoes (pH 3). These results have been derived from studies in different crops such as apples, grapes, tomatoes, cauliflower, beans and sugar beet. These studies have also shown that residues of chlordimeform and its metabolites are located in the outer parts of crops, such as fruit peel. Excessive residues might therefore be removed by peeling fruit (apples, citrus) or trimming the outer leaves of leaf crops. In general, washing will remove only a small part of the total residue (FAO/WHO, 1972). Chlordimeform residues in whole apples reduced to approximately 40% of this level in pressed apple juice, while the level in the wet pomace doubled (FAO/WHO, 1972) This is consistent with studies that have shown that the residue level in the skin and outer layer is approximately 50-fold higher than that found in the flesh (FAO/WHO, 1972). Chlordimeform residues in tea leaves were found to be extractable into tea prepared from these leaves to the extent of approximately 50% of the total residues (Blass, 1972a). Chlordimeform residues in grapes reduced to approximately 60% of this level in grape juice (Blass, 1972b). This is consistent with studies that have shown that the residue level in the grape skin was between 60 and 76% of total residues (FAO/WHO, 1972). Fermentation of the grape juice over a period of 72 days yielded a wine that contained residue levels similar to those in grape juice (Blass, 1972c), indicating that the fermentation process does not significantly lower the total chlordimeform residue level. Chlordimeform residues in green hop cones, when used to prepare beer, were found to be reduced to levels below the level of detection (0.03 mg/kg) (Voss, 1971). Residues associated with the processing of cottonseed have been reported (FAO/WHO, 1979). Separation of the cottonseed oil leaves the majority of the residues in the hulk and meal, although a significant residue still remains in the crude oil. Additional refining processes including bleaching, hydrogenating and deodorizing reduce the residue level to below the level of detection. Cottonseed oil for human consumption is subject to the bleaching and deodorizing processes and thus residues of chlordimeform will be virtually zero. 5.3 Occupational exposure during manufacture, formulation or use 5.3.1 Exposure during manufacture and formulation In the cases described by Folland et al. (1978) of hospitalization of three factory workers in the USA who were exposed to chlordimeform, the urinary levels of chlordimeform plus 4-chloro- o-toluidine were 1.29, 6.32 and 4.85 mg/litre, respectively, three days after exposure. This report is described in more detail in section 8.2.2. In a study on workers in the USA engaged in chlordimeform production and packaging in 1976, urine was monitored in more than 100 workers. In more than 800 individual urine samples, total urinary levels ranged from 0.05 to 50 mg/litre (personal communication by J.W. Barnett, Ciba-Geigy Agricultural Division, Greenborough, North Carolina, USA, to California Department of Food and Agriculture). In China, there have been several studies in which the level of exposure of workers to chlordimeform in chemical factories has been examined together with a medical examination to detect any evidence of toxicity in these workers. These are described in section 8.2. In the study by Lu et al. (1981), the air concentrations in 1974 in a chlordimeform-producing factory were generally below 0.036 mg/m3, with shorter periods at higher levels (0.108- 0.33 mg/m3), during specific tasks. Skin contamination on hands and forearms was 9.1 mg/h for chemical operators and 964.2 mg/h for packers. The urinary excretion levels for chlordimeform and 4-chloro- o-toluidine in controls were 0.015 and 0.042 mg/litre, respectively, in chemical operators were 0.065 and 0.108 mg/litre, respectively, and in packers were 0.263 and 0.398 mg/litre, respectively. In the study by Li et al. (1985b), 24 packers (9 male, 15 female) in a chlordimeform manufacturing plant in Jiang-su Province of China, were exposed to chlordimeform air concentrations (9 samples over 3 consecutive days) of 0.066 mg/m3 (range 0.017-0.121 mg/m3). Skin contamination of the hands and forearms was 110 µg/100 cm2 (S.D. 39 µg/100 cm2). Urinary chlordimeform levels were 0.20 ± 0.13 mg/litre, and urinary 4-chloro- o-toluidine levels were 0.48 ± 0.29 mg/litre. In a further study (Anonymous, 1985a) in a chlordimeform manufacturing factory in China, packers had the highest urinary chlordimeform and 4-chloro- o-toluidine levels at 0.39 mg/litre which significantly correlated with skin contamination but not with air concentration. In the study by Tao et al. (1985), 61 employees (25 chemical operators, 36 packers) of a pesticide factory in China were exposed to air levels in the range 0.074 to 0.160 mg/m3. Skin contamination of packers (2.99 mg/day) was higher than for chemical operators (0.784 mg/day). The urinary excretion rate of chlordimeform and 4-chloro- o-toluidine in packers was also higher (0.513 mg/litre) than for chemical operators (0.206 mg/litre) or controls (0.055 mg/litre). 5.3.2 Exposure during use In a company report by Kossmann (1980), summary data was provided on the results of occupational exposure surveillance programmes on agricultural workers associated with chlordimeform in nine countries. Surveys of aerial pesticide applications to cotton entailed the monitoring of about 600 airstrips in 1979 in the nine countries. Over 28 000 urine samples were analysed from workers in all phases of the application situation. The urine was monitored and residue data expressed as chlordimeform equivalents. In 1% of the assays, substantial chlordimeform urinary residues indicated a significant occupational exposure. Over 75% of the samples were at or below the lowest level of analytical detection. This report states that, in general, the conditions in two countries, the USA and Australia, were indicative of favourable working conditions where only about 1% of the samples contained a residue level indicating a higher-than-desired level of exposure. In a subsequent report by Kenyon et al. (1993), however, it is stated that at least 20% of the urine samples in agricultural workers associated with chlordimeform in New South Wales, Australia, exceeded the maximum permissible exposure level for chlordimeform equivalents in urine, which was set at 0.2 mg/litre. Operators who exceeded this level were required to be withdrawn from the site until the urinary level fell below 0.1 mg/litre. The mean sample assays for both ground rig operators and workers involved in aerial application exceeded the set level in 1984-1985. Furthermore, a number of workers experienced exposures that exceeded the limit on multiple occasions. The urine monitoring programme in operation in New South Wales, Australia, also grossly underestimated the worker exposure levels since its protocol did not allow urine sample collection in the first 24 h following potential exposure (Kenyon et al., 1993). In the report by Kossman (1980), it is stated that working conditions in some other countries (i.e., Colombia, El Salvador, Guatemala and Honduras) were less favourable and thus exposure was higher. However, in some areas where flagmen were unavoidably exposed, the urinary residue levels were low, indicating that with precautions exposure can be controlled. In New South Wales and Israel, urine monitoring for agricultural workers was mandatory, while in the USA, urine monitoring was conducted on a voluntary basis. In a report by Henderson (1985), monitoring studies on operator exposure during the 1984-1985 cotton season in NSW, Australia, were summarized. Urine samples were examined in operators involved in application of chlordimeform by both ground-rig (Strong & Bull, 1985a) and aerial (Strong & Bull, 1985b) methods. Chlordimeform application by ground-rig to 26 444 hectares involved 48 people. A total of 85 urine samples were examined; in 78.8% of samples the chlordimeform level was below 0.20 mg/litre, and in 90.5% of samples it was below 0.50 mg/litre. The mean sample assay was 0.21 mg/litre. Chlordimeform application by aerial spraying to 315 694 hectares involved 222 people. A total of 919 urine samples were examined and in 80.3% of samples, the chlordimeform level was below 0.20 mg/litre, and in 89.8% of samples was below 0.50 mg/litre. The mean sample assay was 0.24 mg/litre. The exposure data for chlordimeform used on cotton in seven countries (Australia, Columbia, El Salvador, Guatemala, Mexico, Nicaragua, USA) for the period 1980-1984 has been compiled in a company report by Limmer (1985). Urine samples indicated that in all countries, the chlordimeform level was less than 0.3 mg/litre for between 70 and 92% of the exposed workers, and was >5 mg/litre in less than 2% of workers. The highest levels were recorded in the loaders, washers and mechanics, while the lower levels were found in the pilots and flagmen. In a study by Jiang et al. (1985), exposure of workers engaged in spraying chlordimeform with fine mist sprayers in both rice fields and cotton fields was examined. The air concentration of chlordimeform surrounding the workers during spraying was 0.80 mg/m3. Skin contamination from spraying in a rice field was 0.777 mg/100 cm2/h (16 samples), and from spraying in a cotton field was 0.445 mg/100 cm2/h for one group (40 samples) and 1.216 mg/100cm2/h for a second group (40 samples). Urinary excretion of chlordimeform and 4-chloro- o-toluidine together was 0.756 mg/litre for rice workers, and 0.490, 0.465 and 1.125 mg/litre in three separate groups (40 each) for cotton workers. Good correlation was noted between skin contamination and urinary excretion. It was noted that contamination of the lower extremities of the body was significantly different between workers with protection (0.490 mg/100 cm2 per h) and those without (1.179 mg/100 cm2 per h). In a study by Ling et al. (1986) and Zhang et al. (1986a), excretion of chlordimeform and 4-chloro- o-toluidine was examined as a measure of occupational exposure. Chlordimeform applicators (7 male, 6 female; 20-41 years) were examined during spraying of cotton for three consecutive days for 4.7, 3.0 and 4.4 h respectively in July 1985. Protective measures included gauze mask, plastic gloves and plastic apron, although it was noted that extensive contamination occurred. Air levels in the breathing zone on each of the three days were 0.011, 0.014, 0.011 mg/m3, respectively. Skin contamination on each of the three days was estimated by the method of Zhang et al. (1986b) to be 10.99, 4.32, and 4.45 mg/day, respectively. Urinary chlordimeform and 4-chloro- o-toluidine together were measured over the 3 days of exposure and for 7 days after cessation of exposure. Urinary levels ranged from a peak of 2.408 mg/litre during exposure to 0.036 mg /litre after 7 days. Excretion of chlordimeform occurred very rapidly and the highest level was detected in the sample collected at the end of each shift. There was a close correlation between skin contamination and urinary excretion. Metabolism occurred very rapidly since 4-chloro- o-toluidine usually accounted for 70-93 % of the total amount in the urine. The authors concluded that the level of urinary chlordimeform plus 4-chloro- o-toluidine is an accurate index of chlordimeform exposure. Maddy et al. (1986) reported the results between 1982 and 1985 of a programme of monitoring the urine of more than 200 workers who had received training in the use of chlordimeform on cotton in California. Protective clothing was required for all employees who handled containers, prepared mixtures, loaded application vehicles, applied chemical, flagged or did repair work on equipment exposed to chlordimeform. This included cloth overalls, washable hat, waterproof boots, waterproof gloves, and a full-face shield. Chlordimeform was detectable in urine as early as 4 h after dermal exposure, but did not increase during the work season. The chlordimeform concentrations averaged about 90 µg/litre, with the highest levels found in mixer-loaders and somewhat less in equipment washers, and the lowest levels in pilots and flaggers. Urinary levels in the 8-10 h following a work shift gave a good indication of exposure for the shift just completed. Kurtz et al. (1987) reported the results of a monitoring programme of agricultural workers exposed to chlordimeform when used on cotton in Imperial Valley, California, during the 1982 season. More than 1000 urine samples were taken from 132 workers, including pilots, mixers/loaders, flaggers and equipment maintenance workers. Chlordimeform metabolites were detected in all workers at some time during the study despite the use of protective clothing. The level of urinary metabolites was positively correlated with the length of exposure and the nature of job activity as shown in Table 3. Mixer/loaders and maintenance workers had the highest levels. Metabolites appeared in urine within 4 h and approximately 75% of urinary excretion occurred within the first 24 h. Table 3. Chlordimeform metabolite concentrations in urine (mg/litre) of agricultural workers during an 11-week application period (Kurtz et al., 1987) Work group Immediately post-work Following morning No. Mean SD No. Mean SD All groups 535 0.12 0.41 572 0.10 0.23 Pilots 145 0.08 0.10 163 0.08 0.10 Mixers/Loaders 156 0.19a 0.71 162 0.15b 0.36 Flaggers 202 0.07 0.08 213 0.07 0.09 Others 32 0.25 0.45 34 0.21c 0.36 a Significantly greater versus flagger group (P<0.01) b Significantly greater versus pilots (P<0.01) and flaggers (P<0.001) c Significantly greater versus pilots (P<0.001) and flaggers (P<0.001) Lemesch et al. (1987) provided the results of monitoring for chlordimeform exposure in agricultural workers in Israel during 1980-1985. Chlordimeform was used only on cotton by aerial application and all workers were monitored for urinary chlordimeform and its metabolites on a weekly basis. The results indicated 86.8% of the urine samples contained less 0.05 mg/litre, and 1.4% contained more than 0.30 mg/litre. Overall, the loaders had the highest exposure followed by the mechanic and then the pilots (see Table 4). Table 4. Chlordimeform metabolite concentrations in urine (mg/litre) of agricultural workers in Israel during 1980-1985 according to occupation (Lemesch et al., 1987) Occupation < 0.05 0.05 - 0.30 > 0.30 Total No. % No. % No. % Loaders 666 79.0 157 18.6 20 2.4 843 Mechanics 383 94.8 19 4.7 2 0.5 404 Pilots 287 98.2 5 1.7 - - 292 Total 1336 86.8 181 11.8 22 1.4 1539 Balu (1989) has provided the results of monitoring field worker exposure to chlordimeform from aerial application on cotton. During the years 1978-1984, urine samples using a grab sample technique from approximately 4600 field workers were examined. For mixer/loaders, between 0.5 and 1.9% had levels >5 mg/litre, and between 2.1 and 18% had levels of 1.0-5.0 mg/litre. The majority (46-78%) had levels in the range <0.05-0.10 mg/litre. There was no apparent change in the proportion of workers in the various exposure levels over the course of the study. For the pilots, between 0.3 and 0.7% had levels >5.0 mg/litre, while 63-90% had levels between <0.05 and 0.10 mg/litre. The clinical signs associated with chlordimeform exposure in these studies are described in section 8.2.2. 6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS 6.1 Absorption, distribution and excretion 6.1.1 Mouse and rat The earliest investigations on the kinetics and distribution of chlordimeform were performed in a series of studies on rats (FAO/WHO, 1972). Four male and four female rats were treated orally with 270 µg [3H-phenyl]-chlordimeform. Over a 24 h period, 52.8% (range 41.8-59.6%) of the radioactivity was eliminated in urine and 2.5% (range 0.13-5.30%) in faeces, while 19-23% of the dose was excreted into the bile. Following intravenous injection of 270 µg [3H-phenyl]- chlordimeform in rat, elimination of radioactivity over 24 h consisted of 53.7% (range 52.0-55.6%) in urine and 1.42% (range 1.19-1.84%) in faeces. Oral dosing of male rats with 270 µg [3H-phenyl]-chlordimeform resulted in residues in liver (0.78 mg/kg), kidney (0.59 mg/kg) and lymph nodes (0.35 mg/kg) after 8 h. After 24 h, residues in gastrointestinal tract (and contents) and liver were 0.95 and 0.35 mg/kg, respectively. All other tissues contained residue levels of <0.16 mg/kg at 8 h, and <0.27 mg/kg at 24 h (FAO/WHO, 1972). Oral dosing of male rats with 270 µg [3H-phenyl]-chlordimeform for seven consecutive days resulted in excretion of 59% of the administered label in urine and 10% in faeces during the dosing period. Tissue residues at the termination of dosing were less than 0.03% of the administered dose (FAO/WHO, 1972). Knowles & Sen Gupta (1970) further studied the toxicokinetics in rats. A group of two male and two female rats was given [14C-tolyl]-chlordimeform (3 µCi) orally (dose unspecified). Over a 72-h period, 88% of the administered radioactivity was eliminated in the urine, with the highest concentration occurring at 12 h, and 7.5% was eliminated in the faeces. At sacrifice (72 h), tissue levels based upon [14C]-label levels were 0.21 mg/kg in liver, 0.15 mg/kg in muscle, 0.11 mg/kg in fat and less than 0.1 mg/kg in other tissues. As part of the same study (Knowles & Sen Gupta, 1970), a similar group of male and female rats received an oral dose of [14C-methyl]-4-chloro- o-toluidine. Tissue levels based upon [14C]-label levels at 72 h after dosing were 0.33 mg/kg in fat, 0.26 mg/kg in liver, 0.2 mg/kg in kidney and oviduct, 0.1 mg/kg in brain, and less than 0.1 mg/kg in other tissues. In a more recent study by Watanabe & Matsumura (1987) concerning the comparative metabolism of chlordimeform and sulfamidine in rats, it was found that after administration of [14C]-chlordimeform as a single oral dose (130 mg/kg), radioactivity was eliminated in the urine (87%) and faeces (8%) within 3 days. Most of the radioactivity was excreted within 2 days. After 5 daily doses of [14C]- chlordimeform (26 mg/kg), 78% of the radioactivity was excreted in the urine and 15% in the faeces. After 10 days, the residue level in all tissues, except blood and liver, was below 1 mg/kg. In a study by Ifflaender (1977a), groups of mice (8/sex; strain Tif:MAG f) and rats (3/sex; strain TIF:RAI f) were administered [14C- ring]-chlordimeform orally at a dose of 25 mg/kg body weight. The general excretion pattern was similar for both mice and rats with more than 70% of the [14C]-label being excreted within 24 h. Of the excreted dose, 80-95% was excreted through the urine, while 10-15% was excreted through the faeces. After 144 h, 95-113% of the administered dose was recovered. Over the period of the experiment (144 h), the levels of radioactivity in the urine were found to range from 82-97% of the administered dose. Residues of chlordimeform were found in liver, kidney and blood, with the highest level found to be 1 mg/kg. Slightly higher residue levels were found to be present in females than in males. In a subsequent study by Ifflaender (1977b) to determine the quantitative differences between mice and rats, animals were administered 25 mg/kg body weight [14C- ring]-chlordimeform. Rapid urinary excretion of chlordimeform was again observed in both mice (85%) and rats (75%) within 24 h. In a more detailed toxicokinetic study by Kopp et al. (1977), chlordimeform was administered orally to female mice at two dose levels (1.2 or 120 mg/kg body weight) using either a single acute or multiple daily administration for up to 21 days. The results again indicated rapid excretion of chlordimeform and/or its metabolites through the urine and did not provide any indication of bioaccumulation at either dose level. At the high dose level, a slightly reduced 24-h excretion pattern of the radioactivity was observed following a single administration. This pattern returned to normal within two to three doses in the multiple dosing regime. The percentage of excretion was the same after a period of 21 days, irrespective of the dose level. The authors concluded that chlordimeform excretion was largely complete within 24 h of discontinuation of administration. No accumulation of residues was evident. Knowles & Benezet (1977) studied the kinetics of chlordimeform in mice following intraperitoneal injection of 0.6 µCi [14C-tolyl]- chlordimeform. Over the 96-h period, 95.5% of the administered dose was eliminated, with 42.5% in the urine and 53% in the faeces. In the first 3 h, 43.7% was eliminated, with 27.3% in urine and 16.4% in faeces. In a study in mice by Crowder & Whitson (1980), the excretion and retention of [14C]-chlordimeform in mice was found not to be affected by oral co-administration of toxaphene or methyl parathion. Low residue levels of chlordimeform were evident at 196 h in all tissues following oral administration. 6.1.2 Other species In a study by Sen Gupta & Knowles (1970), two female dogs (18 and 20 kg) were given 10 µCi [14C]-chlordimeform as a single oral dose (0.3 mg/kg), and one male dog (12 kg), which had undergone cannulation of the gall-bladder and ligation of the bile duct, was given 20 µCi [14C]-chlordimeform orally. Urine was collected (by catheterization) at 1, 3, 6, 12, 24, 48 and 72 h. Faeces were collected at similar time intervals. Of the administered [14C] label, 85% was recovered in urine, 0.6% in faeces, and 5% in the bile within 72 h. In the same study, two brush goats, a male (36 kg) and a lactating female (39 kg) were administered 10 µCi [14C]-chlordimeform orally. The male goat eliminated 87% of the administered dose through the urine within 48 h, while the lactating female eliminated only 67% during the same period. Only about 0.3% of the applied dose was eliminated in the milk within 96 h. In a review by Knowles (1970), the metabolites found in three species, namely, rat, dog and goat, were compared. In all three species, oral treatment with radioactive chlordimeform resulted primarily in elimination through the urine. Cumulative percentages of the dose excreted in the urine 24 h after treatment were 85% for rats, 70% and 80% for the two dogs, 65% for a lactating goat, and 80% for a male goat. Rats eliminated 7.5% of the dose in the faeces by 72 h, and only 0.6% and 1.8% of the administered radioactivity was accounted for in dog and goat faeces, respectively. The rate of degradation of chlordimeform was also different in the three species. By 24 h after treatment, 25% of the radioactive material in rat urine was organosoluble and partitioned into chloroform, but in the dog and goat urine less than 10% was organosoluble. Levels of chlordimeform expressed as percentages of organosoluble urinary radioactivity at 24 and 72 h post-treatment were 9.9 and 2.1% for the rat, 1.3 and 0.2% for the dog, and 0.1 and <0.1% for the goat. 6.1.3 Human In a study by Nixon & Neal (1983), the excretion of chlordimeform residues was examined in eight volunteers following dermal application. A dose of 24.75 mg chlordimeform was applied to the forearm via a patch which was removed after 4 h and the application site washed in propanol followed by detergent. The average absorbed dose was calculated to be 7.95 mg. Urine was collected for 72 h following treatment. During this period, an average of 38.3% of the absorbed dose was recovered from the urine. The half-life for excretion was between 5.9 and 12.1 h, with an average of 8.8 h. A number of studies have been conducted that monitored the urine of workers exposed to chlordimeform during use (see section 5.3.2). The data indicate rapid metabolism of chlordimeform to 4-chloro- o- toluidine, followed by urinary excretion. Detection in the urine was as early as 4 h after exposure, and approximately 75% was excreted within 24 h. 6.2 Metabolic transformation 6.2.1 Mouse and rat In an early study (FAO/WHO, 1972), the urine from a male rat collected over 72 h subsequent to oral administration of 1.1 mg [3H-phenyl]-chlordimeform contained free extractables representing 22% of the [3H] label, of which 10% was in the water phase and 17% was extractable glucuronides. The free extractable [3H]-label comprised chlordimeform, 4-chloro- o-toluidine, N-formyl-4-chloro- o-toluidine, and N'-(4-chloro- o-tolyl)- N-methylformamidine (demethylchlordimeform). Glucuronides were based on the same compounds found as free extractables. In a study by Knowles & Sen Gupta (1970), pairs of male and female rats were treated orally with 3 µCi [14C-tolyl]-chlordimeform. Urine and faeces were collected at 3, 12, 24, 48 and 72 h after dosing. Urinary and faecal elimination of [14C] label after 72 h comprised 88% and 7.5% of the administered dose of [14C]- chlordimeform, and 71 and 24.5% of the administered [14C]-4- chloro- o-toluidine. Chloroform extraction removed 30% of the radioactivity from the urine of [14C]-chlordimeform-treated rats, the extract containing chlordimeform, N'-(4-chloro- o-tolyl)- N-methylformamidine (demethylchlordimeform), N-formyl-4-chloro- o-toluidine, and 4-chloro- o-toluidine, in addition to three unidentified metabolites. A considerable amount of radioactivity remained at the point of origin of the chromatograph, with the amount remaining increasing with time, (30% at 3 h and 75% at 72 h). At 3 h, the four identified compounds were present in approximately equal amounts. By 12 h, the level of N'-(4-chloro- o-tolyl)- N- methylform-amidine had decreased to approximately 25% of the level of any of the other three compounds. By 48 h, chlordimeform levels were half those of the other two compounds, and, by 72 h, N-formyl- 4-chloro- o-toluidine was present in the greatest proportion. As part of the same study (Knowles & Sen Gupta, 1970), a similar group of male and female rats received an oral dose of [14C-methyl]- 4-chloro- o-toluidine. The metabolites found in ethyl acetate- extracted urine comprised 5-chloroanthranilic acid, and N-formyl- 5-chloroanthranilic acid increased. The level of 5-chloroanthranilic acid remained constant. A large amount (20-50%) of the radioactivity remained at the origin of the chromatograph. Five unidentified compounds were noted. The metabolic transformation of the metabolite demethyl-chlordimeform ( N'-(4-chloro- o-tolyl)- N- methylformamidine) in the rat was investigated by Benezet & Knowles (1976). Eight Sprague-Dawley rats were each administered 1.5 µCi [14C]-demethylchlordimeform by oral intubation. Urine and faeces were analysed over a 72-h period. The majority of the radioactivity was eliminated through the faeces (64%) but significant amounts were also eliminated in the urine (35%). The peak level of radioactivity occurred in the urine between 12 and 24 h, and in the faeces between 18 and 48 h. Of the urinary radioactivity, 16-26% could be extracted with ethyl acetate. Compounds present included demethylchlordimeform, N'-(4-chloro- o-tolyl)formamidine, N-formyl-4-chloro- o- toluidine, 4-chloro- o-toluidine and several unidentified compounds. The aqueous fraction remaining after ethyl acetate extraction (74-85% of the total radioactivity) was largely acid-labile and probably consisted of conjugates, possible glucuronides and ethereal sulfates. Approximately 25% of the total radioactivity of the faeces was extractable with ethyl acetate, and similar metabolites were present. Ifflaender (1977b) examined the quantitative differences in urinary metabolites between mice and rats following oral administration of [14C]-chlordimeform at a dose level of 25 mg/kg body weight. Little quantitative difference in individual metabolites was observed between the species. Of the total metabolites, N'-(4-chloro- o-tolyl)- N-methyl formamidine (demethylchlordimeform) represented 11.3% in rats and 2.4% in mice, while 4-chloro-2-methyl-phenylurea represented 6.3% in rats and 1.2% in mice. Sulfuric acid conjugates represented 20.8% in mice compared to 14.0% in rats. Glucuronic acid conjugates (representing 28% of metabolites) and all other minor metabolites were in similar amounts in the urine of rats and mice. Acid hydrolysis of the urine released degradation products in similar amounts in the urine of rats and mice. Knowles & Benezet (1977) reassessed the metabolism of chlordimeform in rat and also assessed the metabolism in mice. Ten male rats were treated orally with 2 µCi [14C]-chlordimeform and urine samples collected at 12 and 24 h. Twelve male mice were injected intraperitoneally with 0.6 µCi [14C]-chlordimeform, and urine and faeces samples were collected at various times up to 96 h. In rat urine, the major organosoluble metabolites (>10%) included 3-(4-chloro- o-tolyl)urea, N-formyl-4-chloro- o-toluidine, 4-chloro- o-toluidine, and N-formyl-5-chloroanthranilic. Demethylchlordimeform, didemethylchlordimeform, 1,1-dimethyl-3- (4-chloro- o-tolyl)urea and 5-chloroanthranilic acid were minor metabolites. In mouse urine, the majority of the radioactive material was water soluble, probably consisting mainly of conjugates such as glucuronides and ethereal sulfates (based on analogy with metabolism in rats). The major organosoluble metabolites (>10%) were N-formyl-4-chloro- o-toluidine, 4-chloro- o-toluidine and N-formyl-5-chloroanthranilic acid. The minor metabolites identified in rat urine were also present in mouse urine. The identity of the major metabolites in rat urine were confirmed in the study of Watanabe & Matsumura (1987). Knowles & Benezet (1977) proposed the metabolic pathway for chlordimeform metabolism in rats and mice shown in Fig. 1. 6.2.2 Other species In the study of Sen Gupta & Knowles (1970) in dogs described in section 6.1.2, chloroform extraction of the urine removed 10% of the radioactivity. Thin-layer chromatography of the extract revealed chlordimeform, N'-(4-chloro- o-tolyl)- N-methylformamidine (demethyl-chlordimeform) and 4-chloro- o-toluidine in about equal quantities, but about four times as much N-formyl-4-chloro- o- toluidine at 1 h after treatment. The level of unchanged chlordimeform and N'-(4-chloro- o-tolyl)- N-methylformamidine decreased steadily with time, whereas 4-chloro- o-toluidine and N-formyl-4-chloro- o- toluidine rose to maximum levels between 6 and 12 h prior to tapering off. Three unidentified metabolites were present. In addition, a lot of the radioactivity remained at the origin of the chromatograph. Re-runs of this material in polar solvents showed 5-chloroanthranilic acid, N-formyl-5-chloroanthranilic acid and three unidentified compounds were present. Some radioactivity still remained at the origin. The urinary [14C] label not extracted by chloroform was treated with enzymes (-glucuronidase, -glucu-ronidase-aryl sulfatase) to form "aglycones". About 75% of the remaining [14C] label was extracted in this manner (hydrochloric acid released 62%), and thin-layer chromatography showed the same compounds as found in the chloroform extract, the major metabolite being N-formyl- 4-chloro- o-toluidine. In addition, more of one of the unidentified metabolites was present. Again re-chromatography of the 45% of the radioactivity remaining at the origin with more polar solvents revealed 5-chloroanthranilic acid to be the major product. In the bile, peak concentration of radioactivity occurred at 8 h. About 10% of this activity could be partitioned into ether, and thin-layer chromato-graphy of the extract indicated the same four compounds seen in urine chloroform extract. N'-(4-chloro- o-tolyl)- N- methylformamidine (demethylchlordimeform), N-formyl-4-chloro- o-toluidine and an unidentified compound accounted for most of the activity at 2 h. By 6 h, 75% of the activity was due to N-formyl-4- chloro- o-toluidine. Incubation of extracted bile with enzymes or acid gave the same "aglycone" compounds as found in urine. Tissue residues of [14C] label at 72 h ranged from 72 µg/kg in liver to 30 µg/kg (kidney), 13.5 µg/kg (lung), 11.9 µg/kg (spleen and brain) and 5 µg/kg (heart and fat and pancreas). In the same study, metabolites extracted from goat urine were analysed by thin-layer chromatography. The major urinary metabolite was N-formyl-4-chloro- o-toluidine. The metabolites in goat urine showed a similar pattern to those in rats, with a similar proportion of conjugated material. The comparative metabolic fate of chlordimeform in rats, goats and dogs is considered in a review by Knowles (1970), which emphasizes the similarity between these species. 6.2.3 In vitro studies Initial studies on the in vitro metabolism of chlordimeform were conducted with [3H-phenyl]-chlordimeform (FAO/WHO, 1972). Incubation of [3H-phenyl]-chlordimeform for 120 min with rat liver homogenate resulted in 24% unchanged chlordimeform, 45% 4-chloro- o- toluidine, and 11% unidentified metabolites being formed. Rabbit liver homogenate yielded 53, 40 and 7% of the same metabolites, respectively. Incubation of 60 µg [3H-phenyl]-chlordimeform (30 µCi) with 5 ml human plasma yielded N-formyl-4-chloro- o-toluidine only. Conversion was 25% in 5 h, and 50% in 20 h. Rose (1969a) confirmed the rat liver homogenate studies using [14C]-chlordimeform. Three unidentified metabolites were also observed and, in addition, chlordimeform degradation was shown to require the presence of nicotinamide. Spleen homogenates were inactive with regard to chlordimeform degradation. The metabolism of chlordimeform in vitro was first reported by Ahmad & Knowles (1971). Incubation of [14C]-chlordimeform with various rat liver enzyme preparations identified demethylchlordimeform as the major metabolite, which was formed by microsomal N-demethylase in the presence of exogenous nicotinamide. This reaction was inhibited by mixed function oxidase inhibitor, SKF-525A. The chlordimeform metabolites formed in vitro were qualitatively similar to those detected in urine from chlordimeform-treated mammals. This has been confirmed by others (Hill et al., 1979; Ghali & Hollingworth, 1985; Kimmel et al., 1986; Watanabe & Matsumura, 1987). Knowles & Benezet (1977) confirmed that the major in vitro metabolite was demethylchlordimeform, but also found that N-formyl-4-chloro- o-toluidine and 4-chloro- o-toluidine were present in appreciable amounts. Ahmad & Knowles (1971) also investigated the metabolism of [14C]- N-formyl-4-chloro- o-toluidine) in the presence of rat liver enzyme preparations. Eighty percent of this metabolite was metabolized by an enzyme, probably a hydrolase, in the soluble fraction, with major metabolites being 4-chloro- o-toluidine (52%) and an unknown substance (26%). The question of the possible formation of azo-derivatives in animal tissues was investigated by Rose (1969a). A number of experiments were conducted to investigate the presence or absence of azobenzene formation from chlordimeform or 4-chloro- o-toluidine. In the first experiment, it was demonstrated that peroxidase activity was negligible in rat liver and spleen. Furthermore, catalase, which was abundant in the same tissues, and which, like peroxidase, catalyses reactions between hydroxyperoxides and many oxidizable compounds, was shown to be unable to form symmetrical azo-derivatives from 4-chloro- o-toluidine. In the second experiment, it was demonstrated that rat liver and spleen homogenates, which were fortified with nicotinamide, and which degrade chlordimeform to demethylchlordimeform and small quantities of N-formyl-4-chloro- o-toluidine and 4-chloro- o-toluidine, respectively, did not form any azobenzene derivatives. These compounds therefore do not represent metabolites of chlordimeform or its aromatic amine degradation products in animal tissues. Lin et al. (1975) have investigated the metabolism of chlordimeform in primary embryonic lung cell cultures. In 2 h of incubation, 97% of chlordimeform was metabolized to N-formyl-4- chloro- o-toluidine (81.9%) and 4-chloro- o-toluidine (2.3%). The route of metabolism, which was different to that seen in mammals, appeared to be first demethylation followed by cleavage at the carbon-nitrogen double bond to form N-formyl-4-chloro- o-toluidine. The formation of the demethylchlordimeform was minute compared to that of the N-formyl derivative. The minor metabolites observed were demethylchlordimeform and two unknown metabolites. When incubated in culture media without cells, chlordimeform decomposed to N-formyl-4-chloro- o-toluidine. 7. EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO TEST SYSTEMS 7.1 Single exposure 7.1.1 Oral The acute oral toxicity data for chlordimeform is presented in Table 5 and for chlordimeform hydrochloride in Table 6. The general signs of toxicity in rats are hyperactivity, dyspnoea, muscular weakness, tremors, "Straub's tail", spasms, convulsions and respiratory arrest. No pathological changes were noted in the rat following oral treatment. In mice, signs of toxicity were similar, but some differences were noted. Symptoms included restlessness, hyperreflexia and tremors, particularly of the head and forelimbs, that developed to one or more episodes of clonic convulsions. Death usually occurred within one hour during one of the convulsive episodes. If the animal survives this hyperexcitation and tremor, it becomes sedated, locomotion is suppressed, and it usually recovers within 24 h. The acute oral toxicity data for metabolites of chlordimeform is presented in Table 7. 7.1.2 Other routes The acute dermal toxicity data for chlordimeform in rats is presented in Table 5 and for chlordimeform hydrochloride in Table 6. The base, but not the hydrochloride, is readily absorbed by the skin (Knowles, 1991). The general signs of toxicity are dyspnoea, exophthalmos, prostration, spasms and convulsions. Pathological changes in the rat included pale or blotchy liver, pale kidneys, and haemorrhagic intestinal contents. No local skin irritation occurred. In the dog, a lethal intravenous dose of chlordimeform (50 mg/kg body weight) caused rapid and irreversible hypotension, and respiratory arrest followed cardiovascular collapse within a few seconds. Artificial respiration did not protect the animals against hypotension and death, suggesting cardiovascular collapse is probably the primary cause of death in dogs. Pathological examination following oral administration in dogs revealed congestion of liver, kidneys and lungs. The acute inhalation LC50 of chlordimeform base in rats (male and female) was 17 400 mg/m3 and for chlordimeform HCl was >5800 mg/m3 (FAO/WHO, 1972). The acute dermal toxicity data for metabolites of chlordimeform is presented in Table 7. Table 5. Acute toxicity of chlordimeform in experimental animals Species Sex Route LD50 References (mg/kg body weight) Rat male/female oral 250 FAO/WHO (1972) male/female oral 340 Worthing & Walker (1983) male/female oral 123 Robinson et al. (1975) male/female oral 301 Gaines & Linder (1986) male/female oral 178-220 FAO/WHO (1972) male/female oral 178 FAO/WHO (1972) female oral 170-460 FAO/WHO (1972) female oral 265 Gaines & Linder (1986) female oral 460 FAO/WHO (1972) male/female dermal 640 FAO/WHO (1972) male dermal 337 Gaines & Linder (1986) female dermal 263 Gaines & Linder (1986) Mouse male/female oral 290 Haddow & Shankland (1969) male oral 267 Ghali & Hollingworth (1985) male/female ip 110 FAO/WHO (1972) Rabbit - oral 625 FAO/WHO (1972) - oral 625 Worthing & Walker (1983) Dog male oral approx.150 Hurni & Sachsse (1969) female oral approx.100 Hurni & Sachsse (1969) Table 6. Acute toxicity of chlordimeform hydrochloride in experimental animals (FAO/WHO, 1972) Species Sex Route LD50 (mg/kg body weight) Rat male oral 305 male oral 325 female oral 330 male iv 95 - dermal approx. 4000 Mouse male/female oral 220 Rabbit - dermal >4000 Table 7. Acute toxicity of chlordimeform metabolites in the rat (FAO/WHO, 1972) Metabolite Sex Route LD50 (mg/kg body weight) N-formyl-4-chloro-o-toluidine male/female oral 2900 4-chloro-o-toluidine (base) male/female oral approx.1000 4-chloro-o-toluidine-HCl male/female oral 860 N-formyl-4-chloro-o-toluidine male/female dermal (24 h) >2150 4-chloro-o-toluidine (base) male/female dermal (24 h) approx.1800 4-chloro-o-toluidine-HCl male/female dermal (24 h) >2150 7.2 Short-term exposure 7.2.1 Dietary Dietary studies of 60 days duration have been conducted in the mouse and rat with each of chlordimeform, N-formyl-4-chloro -o- toluidine, and 4-chloro- o-toluidine. 184.108.40.206 Mouse In a study with chlordimeform by Sachsse et al. (1979a), groups of Tif:MAGf mice (30/sex/group), housed under SPF conditions, were fed a diet containing chlordimeform at concentrations of 0, 750, 1500, 3000 or 6000 mg/kg for 60 days. This corresponded to dietary intakes of 0, 107, 194, 717 or 1525 mg/kg body weight per day for females and 0, 110, 200, 669 or 1519 mg/kg body weight per day for males. At the end of the 60-day period, all animals were examined for haematology, blood chemistry and urinalysis parameters, and groups of 10 male and 10 female animals from the control and the lower three dose groups were subjected to gross and microscopic examination of tissues and organs. Mortality was observed in the two highest dose groups over the course of the study. The highest dose group was terminated after two weeks because of a poor general condition of the animals. Growth, as shown by body weight gain, was reduced in all dietary groups. Food consumption was reduced at all dietary levels in females only. No clinical signs of toxicity were noted. Ophthalmological and auditory examinations were normal. Haematological investigations showed haemolytic anaemia in both sexes of all treated groups, which was characterized as a reduction in haemoglobin concentration, red blood cell count, and packed cell volume. The anaemia was associated in a dose-related manner with an increased methaemoglobin concentration and an increase in Heinz body formation. At 3000 mg/kg diet, there was a slight reticulocytosis noted in both sexes. This was accompanied in females by a shift in the differential leucocyte count noted as an increase in the percentage of polymorphonuclear neutrophile and a decrease in the percentage of lymphocytes. Small changes were observed in alkaline phosphatase activity, which was slightly increased in male mice at the highest dose level. Total protein concentration was also slightly reduced in female mice at the highest dose level. Urinalysis was unremarkable. In the animals that died or were killed within the first 2-week period, all were found to be emaciated and in poor general condition. In all treated animals dying during the test period, congestion of the organs, especially of the liver, was observed. At the highest dose level, atrophy of thymic tissue was observed. There was an increased haemosiderosis at the two highest dose levels. There were no other pathological findings associated with the presence of chlordimeform in the diet. In a study with N-formyl-4-chloro- o-toluidine by Sachsse et al. (1980a), groups of Tif:MAGf mice (30/sex/group), housed under SPF conditions, were fed a diet containing N-formyl-4-chloro- o- toluidine at concentrations of 0, 750, 1500, 3000 or 6000 mg/kg for 60 days. This corresponded to dietary intakes of 0, 138, 379, 1203, or 3153 mg/kg body weight per day for females and 0, 140, 349, 1023, 2549 mg/kg body weight per day for males. At the end of the 60-day period, all animals were examined for haematology, clinical chemistry and urinalysis parameters. A group of 10 males and 10 females from each dose level was examined for gross and microscopic pathological changes at the conclusion of the study. Mortality was observed predominantly at the high-dose level over the course of the study. There were no clinical signs of toxicity, although food consumption and growth were depressed at 1500 mg/kg and above in both sexes over the course of the study. Ophthalmological and auditory examinations were unremarkable. Significant haematological abnormalities were observed at all dose levels at the conclusion of the study. Haemolytic anaemia was observed in both males and females and was characterized as a reduction in haemoglobin concentration, erythrocyte count and packed cell volume. There was a dose-related increase in methaemoglobin concentration and an increase in Heinz body formation. Additionally, both males and females in all treated groups showed a significant reticulocytosis, thrombocytaemia, and leucocytosis. At higher dose levels in both males and females, the leucocytosis was accompanied by a shift in the differential leucocyte count. There was a slight increase in the activity of SGOT, SGPT and SAP. Urinalysis revealed somewhat lower specific gravity and the presence of bile pigment in animals at the two highest dietary concentrations. Microscopic examination of tissues and organs revealed cytomegaly and hyperplasia of the bile duct epithelium and Kupffer cells in some animals at 750 mg/kg and in most animals at higher dose levels. Nuclear inclusion bodies were also evident in all treated animals and, at the highest dose level, moderate centrilobular fatty changes were observed. Additionally, at the higher dose levels, atrophy of thymic lymphoid tissue and of splenic white pulp was observed. Substantial hyperplasia of the epithelium of the urinary bladder was observed in most animals at the highest dose level and sporadically throughout the treated groups. In a study with 4-chloro- o-toluidine by Suter et al. (1976a), groups of mice (30/sex/group, TIF:NMRI strain) were bred and maintained under SPF conditions and fed a diet containing 4-chloro- o-toluidine at concentrations of 0, 750, 1500, 3000 and 6000 mg/kg for 60 days. Mortality of 50% was observed in the 6000 mg/kg group. There were no clinical signs of toxicity, although food intake and growth were retarded at the two highest dose levels. Eye examinations did not indicate adverse ocular changes. Haemolytic anaemia occurred in both sexes of all treated groups and was characterized by reticulocytosis and Heinz body formation. In the male mice of all treated groups, haemoglobin concentration, packed cell volume and erythrocyte counts were slightly below that of controls. In addition, leucocytosis was observed in all animals of all dosage groups with the exception of females at the 750 mg/kg level. In both sexes at 6000 mg/kg and in the females at 3000 mg/kg total protein concentration was reduced and blood glucose and urea nitrogen values were increased. Plasma GPT was increased in male mice at 3000 mg/kg and above and in females at 1500 mg/kg. Microscopic examination of tissues and organs at the conclusion of the studies showed slight to moderate vacuolar changes in hepatocytes, which were pronounced in animals at the 3000 mg/kg level and above. There was also a marked congestion of the spleen at these high dose levels. In addition, the urinary bladder revealed hyperaemia and dilation of the capillaries in the mucosal layer. These changes were accompanied by oedema, multiple intra-epithelial haemorrhage and focal proliferation of the transitional cell epithelium. On occasion, these changes in the urinary bladder were noted at the lowest concentration. 220.127.116.11 Rat In a study with chlordimeform by Sachsse et al. (1979b), groups of Tif:RAIf rats (20/sex/group) were fed a diet containing chlordimeform at concentrations of 0, 750, 1500, 3000 or 6000 mg/kg for 60 days. This corresponded to dietary intakes of 0, 84, 137, 222 or 462 mg/kg body weight per day for males and 0, 71, 121, 231 or 464 mg/kg body weight per day for females. Groups of 10 males and 10 females were killed at 60 days and had complete haematology, clinical chemistry and urinalysis parameters examined. At the end of the study, 10 males and 10 females from each group were subjected to gross and microscopic pathological examination. Animals that died during the course of the study were similarly examined. Food intake and growth were reduced over the course of the study at all dose levels. Slight mortality was observed at the highest concentration. There were no clinical signs of toxicity or adverse behaviour at any dose level. Slight changes in several haematological parameters were noted at the two highest levels. Methaemoglobin levels were increased in a dose- related manner at all treatment levels. Heinz bodies were noted in haematological examination at 1500 mg/kg and above. Slight changes were noted in several clinical chemistry para-meters including decreased glucose concentration, increased alkaline phosphatase activity and increased -glutamyl transpeptidase activity, predominantly at the three highest dose levels. Urinalyses showed slight changes at the two highest dose levels including a reduced urine volume, reduced protein concentration, and reduced electrolyte (potassium) level, predominantly at the highest dietary levels. Terminal body weights of all animals administered chlordimeform were significantly reduced in a dose-related fashion. Substantial changes in growth and relative organ weights were noted in both males and females at all dietary levels. Reductions in the weight of such organs as the brain, heart, liver, kidneys, adrenals and thymus were reported for both males and females. In males, reduced kidney and testes weights were noted only at the highest dose level while reduced ovarian weights were noted at all dose levels. Other than excessive emaciation at the highest dose level, no gross anatomical changes were noted in the animals killed for pathological examination. In most rats of the highest-dose groups, haemosiderosis in the spleen was observed. Reduced spermatogenesis was noted at the highest concentration. Focal hyperplasia of small biliary ducts and of the transitional epithelium, and increased vascularization in the mucous membrane of the bladder were observed in the highest-dose group. In addition, the highest-dose group showed thymic atrophy in several of the animals examined. No compound-related histopathological changes were noted in rats fed 1500 mg/kg or below in the diet. In a study with N-formyl-4-chloro- o-toluidine by Sachsse et al. (1980b), groups of Tif:RAI rats (30/sex/group) were fed a diet containing N-formyl-4-chloro- o-toluidine at concentrations of 0, 750, 1500, 3000 or 6000 mg/kg for 60 days. This corresponded to dietary intakes of 0, 91, 176, 347 or 875 mg/kg body weight per day for males and 0, 87, 165, 329 and 719 mg/kg body weight per day for females. Groups of 10 males and 10 females were killed at the conclusion of the study for complete haematological, clinical chemistry and urinalysis examinations, and gross and microscopic pathological examinations of tissues and organs. Extensive mortality was observed at the high-dose level within the first few weeks of the experiment. At the end of the third week of treatment, the highest- dose group was terminated. There was no substantial mortality at 3000 or lower. Food intake and growth were reduced over the course of the study in a dose-dependent fashion in all dose groups. Apart from the mortality noted at the high dose level, no clinical signs of toxicity or adverse behaviour were observed. Auditory and ophthalmological examinations showed no evidence of loss of these functions in any of the animals examined. Haematological examination indicated haemolytic anaemia in both sexes of all treatment groups; characterized by a reduction in haemoglobin concentration, erythrocyte count and packed cell volume, and an increase in methaemoglobin level. Heinz bodies were observed at 3000 mg/kg only. In addition, at 1500 mg/kg and above there was a slight reticulocytosis and reduced partial thromboplastin time in these dose groups. Changes in the clinical chemistry parameters were noted at both 1500 and 3000 mg/kg. Gross examination of certain tissues and organs showed changes in absolute weights and relative weight ratios at all dosage levels. These reductions appeared to follow a dose-dependent relationship. Animals administered 6000 mg/kg showed atrophy of the thymus and spleen within the first three weeks of the test. Liver changes were noted in all dose groups characterized as hyperplasia of the bile duct epithelium and changes in the distribution of lipid. At the highest dose level, hyperplasia of the urinary bladder epithelium and testes was noted. About half the animals of both sexes in the 6000 mg/kg group showed an increase in the mitotic index in hepatocytes. In a study of 4-chloro- o-toluidine by Suter et al. (1976b), groups of rats (30/sex/group; Tif/RAI strain) were fed a diet containing 4-chloro- o-toluidine at concentrations of 0, 750, 1500, 3000 and 6000 mg/kg for 60 days. There was no mortality over the course of the study and clinical signs of toxicity were not observed. Ophthalmological examinations did not suggest changes related to the presence of 4-chloro- o-toluidine in the diet. Growth was reduced at dietary levels of 1500 mg/kg and above. Haemolytic anaemia in both sexes of all treated groups was characterized by a variety of haematological changes, including reduced haemoglobin content, reduced haematocrit content, reduced blood cell count, increased methaemoglobin content, Heinz body formation, reticulocytosis and polychromatophilia. In the highest-dose group, an increased number of immature red blood cells (normoblasts) were observed. An increased leucocyte count and prothrombin time was recorded at 3000 and 6000 mg/kg. Total protein was slightly reduced at 3000 and 6000 mg/kg and there was a shift in the globulin content as observed by electrophoresis. Plasma -glutamyl transpeptidase of males and alkaline phosphatase of females was increased at 6000 mg/kg. Urinalysis was not significantly affected. In all treated animals, the liver showed an increase in size accompanied by hypertrophy of the hepatocytes. In the two highest-dose groups, the spleen was enlarged and microscopic examination showed pronounced congestion and haemorrhage. In the highest-dose group, slight or moderate proliferation of the transitional cell epithelium was noted in the urinary bladder. 18.104.22.168 Dog In a study with chlordimeform by Blackmore (1969a), four groups of beagle dogs were fed a dry diet containing either 0 mg/kg (10/sex), 250 mg/kg (8/sex), 500 mg/kg (8/sex) or 1000 mg/kg (10/sex) of chlordimeform for 2 years. Two male and two female dogs were sacrificed from each group at 26 and 52 weeks. Body weight was reduced at 1000 mg/kg, the effect being slightly more pronounced in the females. Total leucocyte counts were sporadically elevated in both sexes at 1000 mg/kg and in females at 500 mg/kg. Haematocrit, haemoglobin and erythrocyte counts tended to be depressed after 2 years in both sexes at 1000 mg/kg. Sporadic slight decreases in serum albumin were observed, more frequently in males, at 1000 mg/kg. Terminal spleen-to-body weight ratio was elevated in males at 500 and 1000 mg/kg, and in females at 1000 mg/kg. Histopathological examinations revealed bile duct hyperplasia, pericholangitis and nodular hepatocytic hyperplasia at 500 and 1000 mg/kg in both sexes, and nodular hepatocytic hypertrophy at 1000 mg/kg in both sexes in the liver. Kidneys showed an increased amount of pigmentation at 500 and 1000 mg/kg in both sexes. 7.2.2 Intubation 22.214.171.124 Rat Four groups of 10 male and 10 female rats were intubated six times weekly for one month with 5 ml/kg body weight of a 2% solution of carboxymethylcellulose containing chlordimeform base at concentrations such as to give dose levels of 0, 25, 50 or 100 mg/kg (FAO/WHO, 1972). Body weight was markedly reduced in both sexes at 100 mg/kg. Hyperexcitability was observed in all test animals. At 100 mg/kg, this was apparent 20-30 min after dosing, and was followed 2 to 3 h after dosing by decreased activity and apathy. Recovery was complete at 4 h. Similar but reduced effects were observed at 50 and 25 mg/kg, and with inconsistent frequency. 7.3 Long-term dietary exposure 7.3.1 Mouse While there have been a number of long-term studies in mice with chlordimeform and its metabolites, these were specifically designed to study carcinogenic potential and are described in section 7.7.1. 7.3.2 Rat In a study with chlordimeform by Blackmore (1969b), groups of rats (35/sex/group) were fed a diet containing 0, 100, 250, 500 or 1000 mg/kg chlordimeform for 2 years. The 100 mg/kg group commenced treatment 7 weeks after the other groups. This group was originally part of the control group. Animals at that time were of similar weight to those that had already been on test. The 1000 mg/kg group was discontinued at 3 months due to severe growth inhibition. Growth inhibition was observed in the males at 500 and 1000 mg/kg. In the females, weight gain was reduced at 250 mg/kg and above. In addition, female body weight gain was reduced at 100 mg/kg between weeks 20 and 48. Food intake was significantly reduced at 500 and 1000 mg/kg in both sexes. Dose-related decreases in haematocrit, haemoglobin, and erythrocyte counts, and a dose-related increase in the leucocyte count occurred in females at 250 and 500 mg/kg up to one year. During the second year, haematocrit only was consistently depressed in females at 500 mg/kg. Histopathological changes in the liver (nodules, and foci of hyperplasia of hepatocytes) occurred in all groups, but the incidence was greater at 250 and 500 mg/kg and was more severe at 500 mg/kg. Some females at 500 mg/kg showed slight hypertrophy and vacuolation of focal groups of cells in the adrenal cortex. Terminally organ to body weight ratios were increased in the liver (females at 250 and 500 mg/kg and males at 100 and 250 mg/kg), kidney (females at 250 and 500 mg/kg), thyroid (females at 250 and 500 mg/kg), heart (males at 250 mg/kg and females at 500 mg/kg), adrenals (males at 100 and 250 mg/kg) and testes (100 and 500 mg/kg). In a study with chlordimeform by Zak et al. (1973), groups of rats (25/sex/group) were fed a diet containing chlordimeform at concentrations of 0, 50, 75, 100, 250 and 500 mg/kg for one year. Food consumption and weight gain data were recorded through the study. Terminal organ weights and gross and microscopic examinations of tissues and organs were the only other parameters reported. The 500 mg/kg group was terminated at 37 weeks after 10 males and 8 females had died. At the conclusion of the study, there was considerable mortality noted in all groups. Food consumption was decreased at 500 mg/kg in both sexes and was slightly reduced at 100 mg/kg and above in males only. This reduced food consumption was not significantly reflected in the growth curves of males and females. Gross examination did not show any compound-related abnormalities. Organ weights and organ/body weight or organ/brain weight ratios did not differ from control values. Histological examinations of liver and spleen were performed on all animals. There were no significant differences from control values with respect to fatty changes and inflammatory changes in the liver. Slight proliferation of the bile duct was more frequent in female rats treated with 500 mg/kg than in the rats of other treated groups or the control group. Results of examinations of the spleen for haemosiderosis suggested that, while this condition was more pronounced in females, there were no significant differences from control values. In a study by Sachsse et al. (1980c), rats (90/sex/group; Tif: RAIf strain) were fed a diet containing chlordimeform at concentrations of 0, 2, 20, 100 or 500 mg/kg for 24 months. This was equivalent to dosage levels of 0, 0.1, 1.0, 5.0 and 24 mg/kg body weight per day for males and 0, 0.1, 1.2, 6.0, and 28 mg/kg body weight per day for females. At the conclusion of the dietary feeding study, all remaining rats were fed control diets for a period of time until a survival rate of 20% per sex (10 rats) per group was attained, at which time the animals were killed and examined. Groups of 20 male and 20 female rats per group were examined periodically (4, 13, 26, 52, 78 and 104 weeks) for clinical laboratory investigations including haematology, blood chemistry and urinalysis. Groups of 10 animals/sex/ group were sacrificed at 27 and 52 weeks for gross and microscopic examination of tissues and organs. At the conclusion of the study, all animals sacrificed (also those that died prior to the termination) were examined for gross and microscopic pathology. There was no mortality in the study attributable to the presence of chlordimeform. Growth and body weight were maintained in all groups with the exception of the 500 mg/kg group, where growth in both sexes was slightly retarded. There were no clinical signs of toxicity or abnormal behaviour. Ophthalmological and auditory examinations, performed at periodic intervals, revealed no adverse effects attributable to chlordimeform. Methaemoglobinaemia was observed at dose levels of 20 mg/kg diet and above. At week 4, both males and females showed a slight, but statistically significant, increase in methaemoglobin content. At weeks 13 and 26, this condition abated but returned at the end of one year and was significant in both sexes at the highest dose level for the remainder of the study. Changes in several other blood chemistry parameters were observed at the highest dose level. Heinz body formation generally associated with methaemoglobinaemia was not observed at week 4, but at the end of year one and thereafter Heinz bodies were observed at the highest dose level. A slight but significant reduction in blood glucose concentration was noted at the higher dose levels throughout a major part of the study. Slight changes in urinalysis parameters were observed in the highest dose group, including a slightly reduced urinary volume and a slightly higher specific gravity. Ketonuria and proteinuria were observed at the high dose level at the earliest examination periods only and were not observed at 13 weeks and thereafter. Gross pathology and organ weight measurements (provided for 27, 52 and 106-week sacrifice intervals) did not show any significant dose-related responses. Microscopic histopathological analyses of tissues and organs (performed at weeks 27 and 52 and at the termination of the study) indicated no significant changes attributable to chlordimeform in the diet. Although numerous benign and malignant tumours were observed in both treated and control animals, the frequency and type of neoplasms, reported at 12 and 24 months with pathology analyses, were not dose-related nor were they attributable to chlordimeform in the diet. Several inherent, degenerative or inflammatory changes were attributed to disease, common in older animals. There was no indication of carcinogenic potential to rats as a result of the presence of chlordimeform in the diet. Based on the haematological occurrence of methaemoglobinaemia, the no-observed-effect level of chlordimeform for rats was 2 mg/kg diet, corresponding to an intake of 0.1 mg/kg body weight per day. In a study with N-formyl-4-chloro- o-toluidine by Sachsse et al. (1980d), groups of Tif:RAIf rats (90/sex/group) were fed a diet containing N-formyl-4-chloro- o-toluidine at concentrations of 0, 2, 20, 100, or 500 mg/kg for 2 years. This corresponded to dietary intakes of 0, 0.1, 1.0, 5 or 30 mg/kg body weight per day for females and 0, 0.1, 1.0, 4.0 or 24 mg/kg body weight per day for males. Groups of 10 males and 10 females were killed at periodic intervals (26 and 52 weeks) for examination of gross and microscopic pathology. Complete haemato-logical, clinical chemistry, and urinalysis examinations were performed at 4, 13, 26, 52 and 78 weeks on 20 males and 20 females of each group. At 24 months, 20 males and 20 females were killed and examined for clinical laboratory parameters and gross pathology. The remaining animals were fed control diets for additional periods of time until a survival rate of 20% per sex per group was attained. At that time the remaining animals were killed and examined microscopically for patho-logical changes, especially neoplastic and non-neoplastic lesions. In the high-dose group, food intake and growth were affected over the course of the study and slight growth retardation was observed. Clinical signs of toxicity or adverse behaviour were not observed. There was no mortality in the study attributable to the presence of N-formyl-4-chloro- o-toluidine. Ophthalmological examinations and auditory tests were normal. The results of the haematological investigation showed haemoglobin concentration to be slightly, but significantly, below that of the controls in both male and female rats at the two highest dose levels. In addition, slight but significant decreases in the erythrocyte count and packed cell volume, a slight increase in reticulocytes and somewhat higher methaemoglobin values were also seen in both male and female rats at 500 mg/kg. With the exception of lower body weights of the animals at the highest concentration, the most obvious change was a significant increase in absolute and relative liver weights in both sexes, but more pronounced in females, in the 500 mg/kg group. A significantly increased incidence of hyperplasia of small biliary ducts was seen in the liver of rats of the 500 mg/kg dose group. In rats of the 500 mg/kg group that were killed after 2 years or died after 12 months, a marked increase in the frequency of multioculated cholangiogenic biliary cysts in the liver was noted. Both of these finding were more pronounced and more frequent in female than in male animals. Numerous benign and malignant tumours were observed in both control and treated rats, but the frequency and types of neoplasms was not treatment-related. All gross and histopathological lesions and changes seen in both control and test animals were described as inherent, degenerative or inflammatory in origin and were attributed to naturally occurring diseases common in aged rats. There was no indication of oncogenic potential in rats as a result of the presence of N-formyl-4-chloro- o-toluidine. On the basis of the minor haematological changes, the no-observed-effect level in this study was 20 mg/kg diet, corresponding to an intake of 1 mg/kg body weight per day. In a study with 4-chloro- o-toluidine by Sachsse et al. (1980e), groups of Tif:RAIf rats (90/sex/dose level) were fed a diet containing 4-chloro- o-toluidine at concentrations of 0, 2, 20, 100 or 500 mg/kg for two years. This corresponded to dietary levels of 0, 0.1, 1.0, 5.0 or 28 mg/kg body weight per day for females, and 0, 0.1, 1.0, 4.6 or 24.6 mg/kg body weight per day for males. Groups of 10 males and 10 females were killed at periodic intervals (27 and 54 weeks) for gross and microscopic pathological examinations. Complete haematological, clinical chemistry and urinalysis examinations were performed at 4, 13, 26, 52, and 78 weeks on 20 females and 20 males of each group. At 24 months, 20 males and 20 females were killed and examined for clinical laboratory parameters. Several animals were examined for gross pathology. The remaining animals were fed control diets for additional periods of time until a survival rate of 20% per group was attained. At that time, the remaining animals were killed and examined for microscopic pathology and oncogenic response. A complete microscopic analysis was made on at least 10 rats of each sex of each group at the termination of the experiment. All rats dying during the course of the study were examined for tumours or neoplasms. In the high-dose group of female rats, food intake and growth were affected over the course of the study and slight growth retardation was observed. There was no effect on growth in male rats at any dose level. Clinical signs of toxicity were not observed. There was no mortality in the study attributable to the presence of 4-chloro- o- toluidine in the diet. Ophthalmological examinations and auditory tests did not reveal changes that were related to the administration of 4-chloro- o-toluidine. The results of the haematological investigation, blood chemistry data and the urinalysis were similar for both treated and control rats. Periodically, the haemoglobin concentration was slightly but significantly below that of the controls in the female rats at 100 mg/kg diet and above. Slight but significant decreases were observed in the erythrocyte count and packed cell volume in the female rats at 500 mg/kg. Marginal reticulocytosis was also found to occur at 500 mg/kg in the female rats at week 13 and in both sexes at week 26. In both male and female rats at 500 mg/kg, the methaemoglobin level was found to be slightly though significantly increased when compared to controls. Periodically, this change was observed in the females of the 100 mg/kg dose group, and, occasionally, Heinz bodies were also observed in female rats. There were some changes to organ weights, organ-to-body weight ratios and organ-to-brain ratios that were statistically significant, but only the increase in absolute and relative liver weights were dose-related. In rats from the 500 mg/kg dose group only, a slightly but significantly increased incidence of multilobular cholangiogenic cysts was observed in the liver. These biliary cysts were found in 10/89 female and 3/90 male rats from the 500 mg/kg group, compared to 4/89 female and 0/90 male rats in the control group. Numerous benign and malignant tumours were observed in both control and treated rats, but the frequency and types of the neoplasms occurring in these animals was not treatment-related. Gross and histopathological lesions and changes seen in both control and treated animals were described as inherent, degenerative or inflammatory in origin, and were attributed to naturally occurring diseases, common in aged rats. There was no indication of oncogenic potential in rats as a result of the presence of 4-chloro- o-toluidine in the diet. On the basis of minor haematological changes, the no-observed-effect level in this study was 20 mg/kg diet, corresponding to a dietary intake of 1 mg/kg body weight per day. 7.4 Skin and eye irritation; skin sensitization Potential skin irritation was assessed by the application of 0.5 g chlordimeform or its hydrochloride salt to the shaved skin of six male rabbits. When evaluated at 24 and 72 h, both compounds produced a very slight irritation (FAO/WHO, 1972). Potential eye irritation was assessed by application of 0.1 ml of chlordimeform to one eye of each of nine rabbits, followed by assessment over 7 days. All animals exhibited slight conjunctival redness, while one showed slight chemosis. All effects had reversed within 7 days. There was no evidence of corneal damage. Chlordimeform may be considered a slight eye irritant (FAO/WHO, 1972). There were no studies performed to assess the potential for chlordimeform to cause skin sensitization. 7.5 Reproductive toxicity, embryotoxicity and teratogenicity 7.5.1 Reproductive toxicity 126.96.36.199 Rat Four groups of 10 male and 20 female rats were fed a diet containing 0, 100, 250 and 500 mg/kg chlordimeform in corn oil during three parental and three two-litter filial generations. Parental body weight prior to mating tended to be reduced in all treatment groups, especially at the highest dose level. The same tendency was apparent with regard to food consumption. The fertility index, gestation index, live birth index, sex ratio, mean litter size and birth weight of pups were comparable between treatment and control groups in all generations. At the 500 mg/kg dose level, the lactation index was reduced in Fla, Flb and F3a litters. Weaning weight of offspring was depressed in all high-dose litters. Gross pathological examinations were performed on parents and pups dying during the study, and on 10 male and 10 female weanlings of the F3b generation. No compound- related effects were noted in the pathological examination (Blackmore, 1969c). In a study by Goldman et al. (1991), treatment of ovariectomized Long-Evans rats with a single intraperitoneal injection of chlordimeform at dose levels of 25 or 50 mg/kg caused a complete suppression of luteinizing hormone surge. The observed suppression did not persist beyond the day of treatment. In a more recent study by Cooper et al. (1994), the effect of this delay in hormone surge on pregnancy outcome in females was examined. Chlordimeform at a dose level of 50 mg/kg resulted in a delay in breeding as well as a significant reduction in litter size. Adult male Sprague-Dawley rats were administered chlordimeform by gavage at 200 mg/kg body weight on one day or 50 mg/kg body weight per day for 5 days. Rats were killed on either 3 or 13 days after the last dose. Body weights were reduced at the earlier time points only. There were no changes in the weights of the testes or associated organs. Caudal sperm counts were reduced after the single dose only. No other changes were observed, including sperm motility, velocity or morphology (Linder et al., 1992). 188.8.131.52 Hamster Goldman et al. (1993) reported that a single intraperitoneal dose of chlordimeform (75 mg/kg and above) is capable of delaying the luteinizing hormone surge and altering the timing of oocyte release in the hamster. The reproduction consequences of this effect were not investigated. 7.5.2 Embryotoxicity and teratology 184.108.40.206 Rat Groups of pregnant rats (25/treatment group, 30 controls) were administered chlordimeform in carboxymethylcellulose at dose levels of 0, 10, 25 or 50 mg/kg body weight per day from days 6 to 15 of pregnancy. Only a slight reduction in feed intake was noted at the intermediate dose level. At the high dose level, dams showed somnolence through the first 3 days of treatment. There was also a reduced body weight gain and decrease in feed consumption at this dose level. Examination of fetuses removed by caesarean section on day 21 showed there was a slight delay in growth of the fetuses at the two highest dose levels. This effect was probably a direct result of the toxic response in the dams. No teratogenic events were observed in the offspring, although an increased incidence of sternal ossification defects occurred at 25 mg/kg body weight (Fritz, 1975). 220.127.116.11 Rabbit Three groups of 10 impregnated female New Zealand white rabbits were administered chlordimeform by gavage on days 8 to 16 of gestation at dose levels of 0, 7.5 or 30 mg/kg body weight per day. Five rabbits per group were killed on day 28 of gestation. Parental mortality, abortion rate, corpora lutea to implantation ratio, litter size, incidence of resorption, stillbirths, fetal weight, fetal length, and incidence of skeletal and tissue abnormalities were unaffected by the test compound. In the remaining rabbits, which were allowed to litter normally, gestation length, litter size and litter weights were similar in both treated and control groups (Blackmore, 1969d). Groups of rabbits (group size ranged from 17 to 38 dams per group) were given chlordimeform orally from days 6 to 18 of pregnancy at dose levels of 0, 10, 30 and 100 mg/kg body weight per day. Fetuses were removed by caesarean section on day 28 of pregnancy. The administration of chlordimeform at 100 mg/kg body weight produced a distinct adverse effect on dams for 2-3 h for the first 4 days of treatment. Examination of dams and fetuses at 28 days suggested that the low dose had no teratogenic or embryotoxic effect. In the intermediate and high dose groups, the implantation to corpora lutea ratio was found to be reduced compared to controls. In the high dose group, the number of incompletely ossified sternebrae showed a slight increase over that observed in the controls and in the other groups. In addition, the number of fetuses with malformations was slightly increased at 100 mg/kg. These malformations included a median cleft palate and exencephaly and an omphalocele. Further examination of spontaneous malformations observed in a cumulative control of 2495 rabbit fetuses suggested that these abnormalities may be spontaneous and not a consequence of the administration of chlordimeform (Fritz, 1971). 7.6 Mutagenicity and related endpoints Referenced summaries of the test results with chlordimeform, N-formyl-4-chloro- o-toluidine, and 4-chloro- o-toluidine are given in Tables 8, 9 and 10, respectively. The important features of these data are given below. 7.6.1 DNA damage and repair Chlordimeform gave no evidence of unscheduled DNA synthesis in rat hepatocytes (dose levels: 5-625 µg/ml) or in human fibroblasts (dose levels: 2-250 µg/ml). 4-Chloro- o-toluidine, on the other hand, gave a slight to moderate dose-related increase in the mean number of silver grains per nucleus in rat hepatocytes over a dose range of 0.625-78.15 µg/ml, but not in human fibroblasts over the dose range of 1.25-156.25 µg/ml. Table 8. Summary of mutagenicity and related end-point studies on chlordimeform HC1 Organism Test Test system Strain +/- References Microorganisms Point mutation Salmonella typhimurium TA98 +/-S9 - Arni & Müller (1976a); TA100 +/-S9 - Konopka & Heymann (1977); TA1535 +/-S9 - Muecke et al. (1979); TA1537 +/-S9 - Rashid et al. (1984) TA1538 +/-S9 - Salmonella typhimurium TA98 - Arni & Müller (1983a) Intrasanguine host- TA100 - mediated assay TA1535 - Saccharomyces cerevisiae D7 +/-S9 - Arni & Müller (1983c) Escherichia coli WP2 +/-S9 - Rashid et al. (1984) WP2uvrA +/-S9 - WP67 +/-S9 - CM611 +/-S9 - CM571 +/-S9 - Insects Sex-linked recessive lethals Drosophila +a,b Kale et al. (1995) Mammalian cells Gene mutation Mouse lymphoma L5178Y- TK+/-/ - Beilstein & Müller (1984a) in vitro +/-S9 Unscheduled DNA synthesis Rat hepatocytes - Puri & Müller (1983a) Unscheduled DNA synthesis Human fibroblasts - Puri & Müller (1983c) Mammalian cells Cell transformation Mouse BALB/3T3 cells +a,b Beilstein & Müller (1983) in vitro Table 8. (con't) Organism Test Test system Strain +/- References Mammals Testicular cell chromosome Mouse spermatocytes - Hool et al. (1983) damage Mouse spermatocytes - Arni et al. (1983a) Micronucleus assay Chinese Hamster bone - Langauer & Müller (1977) marrow interphase cells Chromosome aberrations Chinese hamster bone - Hool & Müller (1978) marrow metaphase cells Sister chromatid exchange Chinese hamster bone - Hool & Arni (1983a) marrow cells Heritable translocation Mouse - Lang & Adler (1982) Mammalian spot test Mouse - Lang (1984) Dominant lethal Mouse - Fritz (1978a) a Chlordimeform formulation b Not dose-related Table 9. Summary of mutagenicity and related end-point studies on N-formyl-4-chloro-o-toluidine Organism Test Test Systems Strain +/- References Microorganisms Point mutation Salmonella typhimurium TA98 +/-S9 - Arni & Müller (1976c); TA100 +S9 + Konopka & Heymann (1977); TA100 -S9 - Muecke et al. (1979); TA1535 +/-S9 - Rashid et al. (1984) TA1537 +/-S9 - TA1538 +/-S9 - Escherichia coli WP2 +/-S9 - Rashid et al. (1984) WP2uvrA +/-S9 - Wp67 +/-S9 - CM611 +/-S9 - CM571 +/-S9 - Mammalian cells in Gene mutation Mouse lymphoma L5178Y +a Strasser & Müller (1984b) vitro Mouse lymphoma L5178Y / -a Strasser & Müller (1983b) host-mediated assay Mammals Testicular cell chromosome Mouse spermatogonia - Arni (1983b) damage Mouse spermatocytes +b Arni & Müller (1983e) Micronucleus assay Chinese hamster bone - Langauer & Müller (1978a) marrow interphase cells Chromosome aberrations Chinese hamster bone - Hool & Arni (1983f) marrow metaphase cells Table 9. (con't) Organism Test Test Systems Strain +/- References Mammals Heritable translocation Mouse - Lang & Adler (1982) Mammalian spot test Mouse - Lang (1984) Dominant lethal Mouse - Fritz et al. (1978b) a No positive control b Chromosome aberrations; not dose-related Table 10. Summary of mutagenicity and related end-point studies on 4-chloro-o-toluidine Organisms Test Test system Strain +/- References Microorganisms Point mutation Salmonella typhimurium TA98 +S9 + Arni & Müller (1976b); TA98 +S9 - Haworth et al. (1983); TA98 -S9 + Konopka & Heymann (1977); Haworth et al. (1983); TA100 +S9 - Meuke et al. (1979); TA100 +S9 - Haworth et al. (1983) TA100 -S9 - Rashid et al. (1984); TA1535 +/-S9 - Haworth et al. (1983) TA1537 +/-S9 - TA1538 +/-S9 - S. typhimurium TA98 - Arni & Müller (1983b) Intrasanguine host- TA100 - mediated assay TA1535 - Saccharomyces cerevisiae D7 +/-S9 - Arni & Müller (1983d) Escherichia coli WP2 +/-S9 - Rashid et al. (1984) WP2uvrA +/-S9 - Wp67 +/-S9 - CM611 +/-S9 - CM571 +/-S9 - Mammalian cells in Gene mutation Mouse lymphoma L5178Y/TK+/- -S9 - Beilstein & Müller (1984b) vitro +S9 + Mouse lymphoma L5178Y + Strasser & Müller (1984a) Mouse lymphoma L5178Y - Strasser & Müller (1983a) /host-mediated Table 10. (con't) Organisms Test Test system Strain +/- References Mammalian cells in Unscheduled DNA synthesis Rat hepatocytes + Puri & Müller (1983b) vitro Human fibroblasts - Puri & Müller (1983d) DNA strand breakage V79 cells + Zimmer et al. (1980) Cell transformation Mouse BALB/3T3 cells + Beilstein & Müller (1984c) Mammals Testicular cell chromosome Mouse spermatogonia - Hool & Arni (1983b) damage Mouse spermatocytes - Hool & Arni (1983c) Micronucleus assay Chinese hamster bone - Langauer & Müller (1978b) marrow interphase cells Chromosome aberrations Chinese hamster bone - Hool & Arni (1983d) marrow metaphase cells Sister chromatid exchange Chinese hamster bone - Hool & Arni (1983e) marrow Sister chromatid exchange Chinese hamster ovary + Galloway et al. (1987) cells Heritable translocation Mouse - Lang & Adler (1982) Mammalian spot test Mouse + Lang (1984) Dominant lethal Mouse - Fritz et al. (1978) N-Formyl-4-chloro- o-toluidine was not directly tested for its ability to induce DNA damage and repair. The macromolecular binding of 4-chloro- o-toluidine to macro- molecules of rat and mouse liver has been investigated by several authors. In a report by Hill et al. (1979), the binding of 4-chloro-2-[methyl-14C]-methylaniline (4-chloro- o-toluidine) in vivo and in vitro was investigated. The major binding in vivo was in the liver. In vitro binding was dependent on the presence for microsomal preparations and NADPH. Two soluble products of microsomal enzymes were identified as 5-chloro-2- hydroxylaminotoluene and 4,4'-dichloro-2,2'-dimethylazobenzene. The hydroxylamino compound appeared to be the more activated form of 4-chloro- o-toluidine. 4-Chloro- o-toluidine caused DNA strand breaks in Chinese hamster V79 cells (Zimmer et al., 1980). In studies by Bentley et al. (1986a,b), the covalent binding of [14C- ring]-4-chloro- o-toluidine to mouse and rat liver macromolecules was compared. After a single administration to either species, the extent of binding decreased in the order: protein>RNA> DNA. The level of binding to mouse liver DNA was approximately twice as high as the binding to rat liver DNA after either single or repeated administration, while the binding to liver RNA and protein was greater in the rat. There was no evidence of an increased level of incorporation of [3H]-thymidine into DNA as a result of 4-chloro- o-toluidine binding. Two major hydrophobic DNA adducts were formed in both species, and one of these was formed to a much greater extent (6-30 fold) in mice. 7.6.2 Mutation The ability of chlordimeform and its metabolites to induce mutations has been investigated in both microbial and mammalian systems. Chlordimeform itself gave uniformly negative results in Salmonella typhimurium (0.1-2000 mg/ml), Saccharomyces cerevisiae (15-400 mg/ml), and Escherichia coli (250-2000 mg/ml), with or without S9 microsomal activation, as well as in a thymidine kinase mutation assay in mouse lymphoma L5178Y/TK+/- cells with (75-3000 mg/ml) or without microsomal activation (42.5-1700 mg/ml). Kale et al. (1995) reported that a chlordimeform formulation diluted to provide a dose level of 10 000 µg/ml is a potent sex-linked recessive mutagen in male pre-meiotic and meiotic cells of Drosophila. N-Formyl-4-chloro- o-toluidine was also negative in all Salmonella typhimurium assays (0.1-1000 µg/ml) except for TA100 with microsomal activation, in which there was a dose-related increase in revertants. All Escherichia coli assays (250-2000 µg/ml) were negative. In a forward mutation assay in mouse lymphoma L5178Y cells (213 & 640 µg/ml), N-formyl-4-chloro- o-toluidine gave a positive result in two out of three 18-h incubation experiments. In a host- mediated experiment with mouse lymphoma L5178Y cells (300 mg/kg), the result was negative. 4-Chloro- o-toluidine was negative in all assays with Salmonella typhimurium (10-2000 µg/ml) except for TA100 with S9 microsomal activation and TA98 with S9 microsomal activation. Assays with Saccharomyces cerevisiae (3.2-90 µg/ml) and with Escherichia coli (250-2000 µg/ml) were negative. In a thymidine kinase forward mutation assay in mouse lymphoma L5178Y/TK+/- cells, 4-chloro- o- toluidine was negative without S9 microsomal activation (31.25- 500 µg/ml) and positive with microsomal activation (37.5-600 µg/ml). In a separate forward mutation assay in mouse lymphoma L5178Y cells (111 & 255 µg/ml), a positive result was obtained in one out of three 18-h incubation experiments. In a host-mediated experiment with mouse lymphoma L5178Y cells (330 mg/kg), the result was negative. 7.6.3 Chromosome damage Sister chromatid exchange assays in Chinese hamster bone marrow cells were conducted following oral treatment with chlordimeform (31-324 mg/kg) and 4-chloro- o-toluidine (100-400 mg/kg). In both cases, the result was negative. Similarly, in an assay for chromosome aberrations in Chinese hamster bone marrow metaphase cells, a negative result was obtained following oral treatment with chlordimeform (2 × 60-240 mg/kg), N-formyl-4-chloro- o-toluidine (2 × 300- 1200 mg/kg) and 4-chloro- o-toluidine (2 × 100-800 mg/kg), although the results were somewhat erratic. A micronucleus test in Chinese hamster bone marrow interphase cells following oral treatment with chlordimeform (2 × 60-240 mg/kg), N-formyl-4-chloro- o-toluidine (2 × 300-1200 mg/kg) and 4-chloro- o-toluidine (2 × 100-400 mg/kg) was also negative. Testicular cell chromosomal damage was assessed in mouse spermatocytes and spermatogonia. To investigate the potential cytogenetic effects on mouse spermatogonia, chlordimeform (9-66 mg/kg), N-formyl-4-chloro- o-toluidine (80-320 mg/kg) or 4-chloro- o-toluidine (85-500 mg/kg) was administered orally on 5 consecutive days. The results were negative in each case. To investigate the potential cytogenetic effects on mouse spermatocytes, chlordimeform (18-72 mg/kg), N-formyl-4-chloro- o-toluidine (80-320 mg/kg), or 4-chloro- o-toluidine (85-500 mg/kg) was administered orally over 10 days on days 0, 2, 3, 5 and 9. The results were negative in the case of both chlordimeform and 4-chloro- o- toluidine, but non-dose-related evidence of chromosome damage was indicated from the results with N-formyl-4-chloro- o-toluidine. The heritable translocation assay, dominant lethal test, and mammalian spot test, each of which might indicate minor or major genomic changes, were conducted on all three compounds. In the heritable translocation assay, chlordimeform (120 mg/kg/day), N-formyl-4-chloro- o-toluidine (100 mg/kg/day) or 4-chloro- o- toluidine (200 mg/kg/day) was administered orally for 49 days. No induction of translocation heterozygosity was found. In the dominant lethal assay, chlordimeform (22 or 66 mg/kg), N-formyl-4-chloro- o-toluidine (105 or 315 mg/kg) or 4-chloro- o- toluidine (110 or 330 mg/kg) was administered orally as a single dose. There was no evidence of any dominant lethal effects in the progeny of male mice. In the mammalian spot test, chlordimeform (160 mg/kg), N-formyl-4-chloro- o-toluidine (100 mg/kg) or 4-chloro- o- toluidine (100 mg/kg) was administered orally on days 8-10 of embryonic development. The results were negative except in the case of 4-chloro- o-toluidine, which induced a 3.2% incidence of spots of genetic relevance compared to an incidence of 0.9% in controls. 7.6.4 Cell transformation Cell transformation assays conducted with both chlordimeform and 4-chloro-toluidine in mouse BALB/3T3 cells produced an increased incidence of transformed cell colonies with both compounds. With chlordimeform, the experiment was conducted at dose levels up to 1 µg/ml, and increased transformation frequency was observed only at 0.0625 and 0.125 µg/ml. The transformative properties of chlordimeform were considered weak. With 4-chloro- o-toluidine, three experiments were conducted at dose levels up to 36 µg/ml, and a significant increase in transformation frequency was observed. The transformative properties of 4-chloro- o-toluidine were considered definite. 7.7 Carcinogenicity A number of carcinogenicity studies have been conducted in mice. With chlordimeform, there are two dietary studies and one dermal study. With N-formyl-4-chloro- o-toluidine, there is one dietary study. With 4-chloro- o-toluidine, there are four dietary studies. In rats, the carcinogenic potential of chlordimeform and its metabolites was generally investigated as part of more detailed long-term studies, and details are provided in section 18.104.22.168. Three studies on 4-chloro- o-toluidine that primarily investigated carcinogenicity are reported below. 7.7.1 Mouse The carcinogenicity of chlordimeform has been examined in two dietary studies and in one dermal study. In a study by Suter et al. (1978), groups of mice (50/sex/group; Tif: MAG strain, SPF derived) were fed a diet containing chlordimeform at concentrations of 0, 20, 100 and 500 mg/kg for 24 months. At the conclusion of the dietary feeding interval, animals were maintained on control diet until 90% of a group had died, at which time the remaining animals of the group were sacrificed. There were no signs of acute toxicity related to chlordimeform in the diet over the course of the feeding trial. Growth and food consumption were similarly unaffected by the presence of chlordimeform in the diet. Mortality was significantly increased in females after 60 weeks at 500 mg/kg, and after 90 weeks at both 100 and 500 mg/kg. In males, significantly increased mortality was observed after 70 weeks at 500 mg/kg, and after 110 weeks at both 100 and 500 mg/kg. However, lifespan was not significantly affected in males at 100 mg/kg. The animals fed dietary levels of 100 mg/kg and above displayed an increased incidence of haemorrhagic tissue masses in subcutaneous tissues, retroperitoneum and some internal organs (kidney, liver and spleen), which upon examination were classified as malignant haemangioendotheliomas. These malignancies which were reported to occur rarely in control populations were found predominantly in the 100 and 500 mg/kg dietary groups (see Table 11). In some animals the tumours were of multiple origin and metastases were observed in the lungs. There were no other types of neoplasm observed in the study that were attributable to chlordimeform in the diet. Under the conditions of this study, 20 mg/kg in the diet appeared to be a no-observed-effect level. In a study by Li et al. (1985a), groups of Swiss mice (50/group, sex not stated) were fed a diet containing chlordimeform at concentrations of 0, 20, 100, or 300 mg/kg for a period of 18 months. A positive control group was administered 300 mg/kg of 4-chloro- o- toluidine in the diet for 18 months. All animals were killed at the end of the study and assessed for tumour formation. The main results of the study are presented in Table 12. The author described the majority of the neoplasms as angiomas, and the malignant neoplasms as angiosarcomas. These neoplastic changes were considered to be similar to those observed in the study by Suter et al. (1978). In a paper by Jiang et al. (1988), the dermal carcinogenicity of chlordimeform was investigated in mice. Groups of Swiss mice (50 per dose level, sex not stated) were treated dermally with chlordimeform twice per week at dose levels of 0, 100, 500, 2000 or 4000 mg/kg body weight for a total of 17-20 treatments, together with croton oil (0.5% in acetone). Positive controls received coal tar pitch (20 treatments) plus croton oil (30 treatments). All animals were sacrificed after 18 months and analysed for tumour formation. Chlordimeform induced both skin and liver tumours in this assay (see Table 13). The changes observed in the skin consisted of epidermal hyperplasia, papillomas and squamous cell carcinomas. The effect of croton oil application was Table 11. Incidence of haemangioendotheliomas in mice following dietary administration of chlordimeform, N-formyl-4-chloro-o-toluidine or 4-chloro-o-toluidine (Suter et al., 1978; Sachsse et al., 1978a,b) Control Dietary concentration (mg/kg) 2 20 100 500 Chlordimeform HCl Male 1/44 (2%) - 0/44 (0%) 15/49 (30%) 39/48 (83%) Female 1/43 (2%) - 2/46 (4%) 22/46 (50%) 35/49 (71%) Total 2/87 (2%) - 2/90 (2%) 37/95 (41%) 74/97 (80%) N-formyl-4-chloro-o-toluidine Male 0/46 (2%) - 0/49 (0%) 15/48 (38%) 40/47 (89%) Female 0/47 (0%) - 0/47 (0%) 23/43 (56%) 38/48 (79%) Total 1/93 (1%) - 0/96 (0%) 38/91 (47%) 78/95 (84%) 4-Chloro-o-toluidine Male 0/50 (0%) 0/47 (0%) 4/48 (8%) 23/47 (48%) 40/48 (83%) Female 1/45 (7%) 1/45 (2%) 3/48 (6%) 30/47 (62%) 34/49 (72%) Total 1/95 (1%) 1/92 (1%) 7/96 (19%) 53/94 (66%) 74/97 (78%) to shorten the latent period for tumour formation and also to hasten the malignant progression of existing neoplasms in the skin. At 500 mg/kg body weight, the time of first appearance of tumours was 483 days without croton oil and 154 days with croton oil. The latency period also decreased with increasing dose levels of chlordimeform. In the liver, changes consisted of enlargement, hepatocytic hyperplasia, and hepatocytic carcinomas. In a carcinogenicity study by Sachsse et al. (1978a), groups of mice (50/sex/group; Tif: MAG strain) were fed N-formyl-4-chloro- o- toluidine in the diet at concentrations of 0, 20, 100 and 500 mg/kg for 24 months. After this time, all animals were fed a control diet until the study was concluded when 90% of the animals in a group had Table 12. Incidence of tumours in mice after dietary administration of chlordimeform or 4-chloro-o-toluidine (Li et al., 1985a) Dietary Number of animals Number of animals Incidence Number of animals Incidence Days to concentration necropsied bearing haemangiomas (%) bearing (%) appearance of (mg/kg diet) or haemangiosarcomas haemangiosarcomas neoplasm Chlordimeform 0 50 0 0 0 0 - 20 50 8 16 0 0 494 100 50 22 44 5 10 469 300 50 36 72 15 30 448 4-Chloro-o-toluidine 300 50 31 62 13 26 283 Table 13. Incidence of tumours in mice following dermal application of chlordimeform (Jiang et al., 1988) Group/treatment Skin Liver Number Carcinomas Papillomas Hyperplasia Number Carcinomas Papillomas Hyperplasia of animals (%) (%) (%) of animals (%) (%) (%) Water 18 0.0 0.0 6.1 17 0.0 0.0 0.0 Croton oil alone 17 0.0 0.0 17.6 24 0.0 0.0 8.3 100 mg/kg + croton oil 19 0.0 5.3 21.1 21 23.8 0.0 9.5 500 mg/kg 22 4.6 4.6 18.2 20 25.0 0.0 0.0 chlordimeform alone 500 mg/kg + croton oil 23 4.4 4.4 52.2 25 8.0 0.0 4.0 2000 mg/kg + croton oil 15 20.0 20.0 26.7 14 14.3 0.0 0.0 4000 mg/kg + croton oil 15 60.0 13.3 13.3 16 18.8 6.2 0.0 Coal tar pitch 18 88.9 11.1 0.0 19 15.8 5.3 0.0 died. There was no sign of adverse behaviour, and acute mortality was not noted. Growth and food consumption were unaffected. There were significant differences noted in survival after one year of age. Both males and females showed an increased mortality at 100 and 500 mg/kg after approximately one year of feeding. The onset of increased mortality occurred earlier in females. The females at the 20 mg/kg dietary level showed a slightly higher, non-significant, mortality during the same period. Detailed gross and microscopic examination of a variety of tissues and organs showed the presence of numerous gross anatomical lesions. There was an increased number of haemorrhagic masses in the subcutaneous tissues in the retroperitoneum and in some internal organs of mice at all treatment levels. Detailed microscopic examination confirmed that the increased incidence of haemorrhagic masses were malignant tumours of vascular origin. These tumours were histologically classified as malignant haemangioendotheliomas (see Table 11). In addition to the occurrence of tumours, the time to tumour relationship was decreased as the dietary concentration was increased. Other neoplasms occurring in the study were not considered to be treatment-related. A no-observed-effect-level was not demonstrated under the condition of this experiment. The carcinogenicity of 4-chloro -o-toluidine has been examined in four dietary studies. An early study by Ezumi and Nakao conducted in 1974 was reviewed by the JMPR in 1978 and considered inadequate (FAO/WHO, 1979). In a large study on the carcinogenicity of 21 aromatic amines and their derivatives described by Homburger et al. (1972) and Weisburger et al. (1978), groups of CD-1 mice (25/sex/dose level) were administered 4-chloro- o-toluidine in the diet at dose levels of 0, 750 or 1500 mg/kg for males, and 0, 2000 or 4000 mg/kg for females for a period of 18 months. All mice were placed on a control diet for an additional 3 months before sacrifice and complete necropsy and histopathological examination of tissues. The incidence of haemangioendotheliomas was increased in males at both low (12/20) and high (13/20) dose levels compared to concurrent controls (0/14) and historical controls (5/99), and in females at both low (18/19) and high (12/16) dose levels compared to concurrent controls (0/15) and historical controls (9/102). In a study by Sachsse et al. (1978b), groups of mice (50/sex/group; Tif: MAGf strain) were fed a diet containing 4-chloro- o-toluidine at concentrations of 0, 2, 20, 100 and 500 mg/kg for 24 months. After 24 months, all animals were fed control diets until the study was concluded when 90% of the animals in a group had died. There were no overt signs of toxicity. Growth and food consumption were unaffected by treatment. An adverse effect on longevity (lifespan) was noted in both males and females at the two highest dietary levels. At the conclusion of the study upon gross examination there was a marked increase number of haemorrhagic masses in subcutaneous tissue, in the retroperitoneum, and in some internal organs. Microscopic examination revealed an increased incidence of haemorrhagic malignant tumours of vascular origin at dose levels of 20 mg/kg and above (see Table 11). The tumour incidence in control exceeded the incidence observed at 2 mg/kg. The tumours were histologically classified as malignant haemangioendotheliomas and, on occasion, metastases were observed. There was not only a significant dose-dependent increase in the total incidence of malignant tumours but the time to appearance of tumours occurred at a markedly earlier date in animals at the higher concentrations than in those at the lower concentrations. A benign variant of the haemangioma was observed in all groups, and although without the characteristics of malignancy, did cause local invasion. Thus, the benign and malignant tumours have been grouped together. The vascular tumours (haemangiomas and haemangioendotheliomas) of the type that occurred in the mice appeared to be peculiar to this rodent species. The occurrence of other types of neoplasms in the study was not influenced by the presence of 4-chloro- o-toluidine in the diet. Under the conditions of this experiment, 2 mg/kg in the diet appeared to be a no-observed-effect level. In a study with 4-chloro- o-toluidine conducted by the National Cancer Institute (NCI, 1979), groups of B6C3F1 mice (50/sex/dose level) were administered 4-chloro -o-toluidine in the diet at dose levels of 3750 or 15 000 mg/kg for males and 1250 or 5000 mg/kg for females for 99 weeks. Control groups consisted of 20 males and 20 females. There was a dose-related increase in mortality in both sexes. There was also a dose-related increase in the incidence of haemangiosarcomas as shown in Table 14. The haemangiosarcomas apparently originated in fatty tissue adjacent to the genital organs and not in a particular organ. In some instances, they were observed to infiltrate the abdominal muscles, uterus, ovary, prostate or urinary bladder. The haemangiosarcomas were lethal to 89 (75%) of the affected mice, owing to haemorrhage in the peritoneal cavity and to the space-consuming character of the lesions. Pulmonary metastasis was observed in only 5 (4%) of the 119 dosed animals bearing haemangiosarcomas. Associated pathological alterations that were recorded at necropsy were haemorrhage in the peritoneal cavity and variable enlargement of the spleen. It was concluded that 4-chloro- o-toluidine was carcinogenic in B6C3F1 mice. In the study of Li et al. (1985a), a single dietary dose of 4-chloro- o-toluidine (300 mg/kg) was given to mice for 18 months as a positive control. The incidence of tumours was similar to that seen in mice receiving 300 mg/kg of chlordimeform, but the latency period was considerably reduced (Table 12). Table 14. Incidence of tumours in mice following dietary administration of 4-chloro-o-toluidine (NCI, 1979) Male Female Control 3750 15 000 Control 1250 5000 mg/kg mg/kg mg/kg mg/kg Number of animals 20 50 50 18 49 50 Haemangiosarcomas 0 3 37 0 40 39 (0%) (6%) (74%) (0%) (82%) (78%) Haemangioma 0 3 5 1 6 0 (0%) (6%) (10%) (6%) (12%) (0%) 7.7.2 Rat The carcinogenicity of chlordimeform and its metabolites has generally been considered as part of more detailed long-term studies (see Section 7.3). In the studies below, carcinogenicity was the primary consideration. In a study conducted by the National Cancer Institute (NCI, 1979), groups of F344 rats (50/sex/dose level) were fed a diet containing 4-chloro- o-toluidine at concentrations of 1250 mg/kg or 5000 mg/kg for 107 weeks. Control groups contained 20 animals of each sex. There was no significant dose-related trend in mortality in either sex. There was a variety of neoplastic and non-neoplastic changes in control and treated rats. There was a small increase in adenomas of chromophobe cells of the pituitary gland in both male and female rats compared to controls (see Table 15). All of these tumours were benign, are also common in this strain of rat and have occurred in 21% of control female rats in the NCI laboratories. The authors concluded that on the basis of histopathological examination, 4-chloro- o-toluidine was not carcinogenic in F344 rats. In a large study on the carcinogenicity of 21 aromatic amines and their derivatives by Weisburger et al. (1978), groups of male Charles River CD rats were administered 4-chloro- o-toluidine in the diet at dose levels of 0, 2000 or 4000 mg/kg diet for the first 3 months, which was then reduced to 0, 500 or 1000 mg/kg diet for the following 15 months. There was no statistically significant increase in tumours in either of the treated groups. Table 15. Incidence of tumours in rats following dietary administration of 4-chloro-o-toluidine (NCI, 1979) Male Female Control 1250 5000 Control 1250 5000 mg/kg mg/kg mg/kg mg/kg Number of animals 19 48 47 19 48 48 Chromophobe 2 6 15 1 13 15 adenoma (11%) (13%) (32%) (5%) (27%) (31%) Chromophobe 0 0 2 0 3 1 hyperplasia (0%) (0%) (4%) (0%) (6%) (2%) 7.8 Other special studies 7.8.1 Immunotoxicity In a study by Wiltrout et al. (1978), the potential of various pesticides to influence the primary humoral immune response in the mouse with respect to both dose and time of exposure was examined. Mice receiving a single oral dose of chlordimeform at approximately the LD50 level (148 mg/kg body weight) experienced a significant suppression of humoral response when the dose was administered on the day of immunization or two days after immunization. No response was observed at one tenth of the LD50 dose, even when administered for 8 or 28 days. Further studies by Shopp et al. (1985) investigated the effect of chlordimeform on both humoral and cell-mediated immunity in the mouse following both acute and 14-day exposures by the intraperitoneal route. There was a decrease in IgM antibody-forming (plaque-forming) cells when measured 4 days after intraperitoneal administration at 20 or 40 mg/kg body weight per day. These dose levels did not result in any alteration of cell-mediated immunity. When administered orally, chlordimeform at doses as high as 120 mg/kg body weight per day did not have any effect on the 4- or 5-day antibody response. Immunological parameters that may be related to the carcinogenic activity of chlordimeform in rats were investigated by Thomas et al. (1990). These included spleen/body weight ratio, splenocyte viability, T and B cell mitogenesis, natural killer (NK) cell and natural cytotoxic (NC) cell activity. Chlordimeform was administered intraperitoneally on three consecutive days at 0, 1, 10 or 75 mg/kg body weight per day. 4-Chloro- o-toluidine was administered intraperitoneally on three consecutive days at 0, 10, 50 or 100 mg/kg body weight per day. Spleen/body weight changes were observed only at the highest dose of chlordimeform. No changes were observed with either chemical on splenocyte viability or T and B cell mitogenesis. An inhibition of NC activity at all chlordimeform doses was observed, and an inhibition of NK activity was observed at 10 mg/kg body weight per day and above. The relevance of this result to the carcinogenic activity of these chemicals is doubtful. 7.8.2 Behavioural effects Behavioural studies of the effects of chlordimeform in rats were first investigated by Olson et al. (1978). The effects of exposure prenatally and post-natally were examined following a dietary intake of 0.1 mg/kg body weight per day. Early development testing (swimming and righting reflex) was conducted on rat pups from post-natal days 7 to 17, while motivational, learning and retention tests were conducted on days 70 to 90. The most significant differences between control and treated groups was in the swimming task, retarded maturation being observed in the chlordimeform-fed group. There was no treatment- related effect with regard to maze tests or with regard to the tests of motivation. Moser et al. (1988) examined the behaviour of rats using a functional observation battery following a single oral administration of chlordimeform at dose levels of 0, 1, 25 or 56 mg/kg body weight. Rats were examined at 1, 5 or 24 h. Chlordimeform produced a decrease in body weight as well as a decrease in body temperature. There was a dose-related increase in general activity, CNS excitability and sensory responsiveness, coupled with a decrease in rearing, gait and arousal. Chlordimeform also produced an increase in grip strength. Other behavioural effects observed with chlordimeform have included appetite stimulation in rats (Pfister et al., 1978b), flavour aversion in both rats (MacPhail & Leander, (1980) and mice (Leander et al., 1984) and alteration in schedule-controlled performance in rats (MacPhail & Leander, 1981), mice (Glowa, 1986) and pigeons (Leander & MacPhail, 1980). Witkin & Leander (1982) also demonstrated that, while causing appetite stimulation in rats, chlordimeform produced a dose- related decrease in water consumption, in contrast to other appetite stimulants. 7.8.3 Pharmacological and biochemical effects The pharmacological and biochemical effects of chlordimeform in animals have been reviewed by Knowles (1991). The cardiovascular effects of chlordimeform treatment were recognized from an early stage with the observation that chlordimeform administered intraperitoneally to rabbits caused a marked decrease in arterial blood pressure of almost 50% within 30 min of treatment (Matsumura & Beeman, 1976). Cardiovascular changes were also noted in the dog (Lund et al., 1979a,b; Rieger et al., 1981) but in this case the effect was biphasic, consisting of an initial depressor response associated with decreased cardiac contractility and vascular resistance, and a secondary pressor response associated with increased cardiac contractility and vascular resistance. These actions of chlordimeform were noted to be similar to those of local anaesthetics such as procaine and lidocaine (Pfister et al., 1978a; Lund et al., 1979a,b,c). In studies by Watkinson (1985, 1986a,b), the effects of chlordimeform on cardiovascular functional parameters were examined in post-weaning and geriatric rats following intravenous treatment at dose levels up to 60 and 120 mg/kg body weight, respectively, or intraperitoneal treatment of post-weaning rats at dose levels up to 60 mg/kg body weight. Chlordimeform produced profound and abrupt decreases in heart rate and blood pressure within 3 min, together with multiple arrhythmias and alterations in electrocardiogram waveforms and intervals. The effects observed in post-weaning rats were less severe than those observed in geriatric rats. The inhibition of monoamine oxidase in rats in vivo and in vitro by chlordimeform and/or its metabolites has been extensively studied (Beeman & Matsumura, 1973; Maitre et al., 1978; Benezet et al., 1978; Hollingworth et al., 1979; Kadir & Knowles, 1981; Kaloyanova et al., 1981; Bailey et al., 1982). The lack of correlation of toxicity of chlordimeform metabolites to monoamine oxidase inhibition and the fact that chlordimeform is a relatively weak monoamine oxidase inhibitor suggest that monoamine oxidase inhibition is not the primary factor involved in the acute toxicity of chlordimeform (Neumann & Voss, 1977; Robinson & Smith, 1977; Hollingworth et al., 1979). Chlordimeform also has an effect on the level of biogenic amines in brain and plasma of rats, which may in part at least be due to the inhibition of monoamine oxidase levels. Administration of chlordimeform to rats was found to produce an increase of 25-70% in 5-hydroxytryptamine, norepinephrine or dopamine levels in brain (Maitre et al., 1978; Benezet et al., 1978; Bailey et al., 1982). However, Johnson & Knowles (1983) treated rats subcutaneously with chlordimeform (200 mg/kg body weight) and found no change in any of the amines. Chlordimeform and some of its metabolites have been shown to affect platelet function, as measured by the uptake of radioactive 5-hydroxytryptamine (Knowles, 1991). Chlordimeform also has antipyretic and anti-inflammatory actions, as shown by its ability to reduce yeast-induced fever in rats. It also antagonizes both early (5-hydroxytryptamine- and histamine-mediated) and late (prostaglandin-mediated) phases of carrageenan-induced hind-paw oedema, albumin-induced oedema, and oedema induced by direct injection of 5-hydroxytryptamine and histamine (Yim et al., 1978). Chlordimeform also induced mild gastric ulceration in rats after intraperitoneal injection (20-80 mg/kg body weight) but not after oral treatment (80-240 mg/kg body weight). The above actions may be related to the ability of chlordimeform to inhibit prostaglandin biosynthesis (Yim et al., 1978; Holsapple & Yim, 1981). Chlordimeform induces hypothermia in rats (Watkinson & Gordon, 1987) and mice (Gordon et al., 1985). Watkinson et al. (1989) examined the effect of core body temperature on both the survival and cardiovascular functions of rats following treatment with chlordimeform. The results indicated that at a given dose of chlordimeform, the magnitude and duration of the observed toxic effects are primarily a function of core body temperature. The authors concluded that moderate hypothermia, but not extreme hypothermia, may have a beneficial effect with respect to survival after exposure to chlordimeform. Chlordimeform has been shown to have an effect on both visual and auditory functions in mammals. Intraperitoneal treatment of male rats with acute dosages of chlordimeform (5-40 mg/kg body weight) before testing revealed a temporary increase in both the amplitude and latency of pattern reversal-evoked potentials and an increase only in the latency of pattern flash-evoked potentials (Dyer & Boyes, 1983; Boyes & Dyer, 1984). Boyes & Moser (1988) provided evidence to support the hypothesis that these effects are evoked through actions as a central nervous system alpha-adrenegic agonist. Janssen et al. (1983) demonstrated effects on the brain stem auditory-evoked response after injection of chlordimeform at a dose levels of 40 mg/kg body weight. It has been suggested that these effects may by secondary to the hypothermic effects induced by chlordimeform (Gordon et al., 1985). Chlordimeform has been shown to affect the activity of hepatic drug-metabolizing enzymes in both rats and mice. Studies have been conducted following gastric intubation at dose levels up to 150 mg/kg body weight per day for 7 days, and also following intraperitoneal injections either singly (100 mg/kg body weight) or daily (75 mg/kg body weight per day) for 4 days. Chlordimeform treatment induced several of these hepatic drug-metabolizing enzymes with significant species and/or sex specificity. Cytochrome P-450 content was increased in all cases. 7.9 Factors modifying toxicity The factors modifying the acute toxicity of chlordimeform have been reviewed by Knowles (1991). 7.10 Mechanisms of toxicity - mode of action 7.10.1 Mechanism of acute toxicity A large number of studies that investigated the mechanism of action following acute poisoning with chlordimeform have been reported. Based on the early in vitro and in vivo studies of Aziz & Knowles (1973) and Beeman & Matsumura (1973), it was suggested that the increase in biogenic amines resulting from inhibition of monoamine oxidase by chlordimeform could account for the variety of toxic signs following acute poisoning. However, Maitre & Gfeller (1975) and Robinson et al. (1975) demonstrated that this mechanism does not play a significant role in the acute toxicity in rats. A number of other studies have attempted to define the mode of action of chlordimeform. Studies in insects have shown that chlordimeform has little activity on cholinergic transmission although it is an uncoupler of oxidative phosphorylation and an inhibitor of electron transport (Abo-Khatwa & Hollingworth, 1972a). A number of biochemical mechanisms have been postulated to explain the effects of chlordimeform in insects, including uncoupling of respiration (Abo-Khatwa & Hollingworth, 1972a,b), inhibition of monoamine oxidase (Knowles & Roulston, 1972) and blockage of neuromuscular transmission (Wang et al., 1975; Watanabe et al., 1975), and motor stimulation through actions on central non-cholinergic synapses (Lund et al., 1979a; Lund et al., 1979c). The latter effect is thought to be mediated through the neurotransmitter, octopamine (Lund et al., 1979b). Both chlordimeform and particularly demethylchlordimeform have been shown to interact with the octopamine receptor and partially mimic the pharmacological effects of octopamine (Evans & Gee, 1980; Nathanson & Hunnicutt, 1981; Bokisch et al., 1985). In mammalian systems, oxidative phosphorylation is uncoupled (Abo-Khatwa & Hollingworth, 1972b) and RNA synthesis is inhibited by chlordimeform, but only at very high concentrations (Murakami & Fukami, 1974). The effects of chlordimeform on hepatic drug- metabolizing enzymes was dependent on both sex and species and did not show any particular pattern that would indicate a consistent mechanism of action (Budris et al., 1983; Bentley et al., 1985; Leslie et al., 1988). Chlordimeform, acting as a direct depressant on cardiac and vascular muscle, induced a hypotensive state in dogs. Chlordimeform did not interfere with the autonomic nervous system. The mechanism of cardiovascular depression may be related to that noted with frog nerve preparations treated with procaine, a local anaesthetic. The metabolite, 4-chloro- o-toluidine has been shown to interfere with rat cardiac receptors (Wang et al., 1975; Watanabe et al., 1975; Matsumura & Beeman, 1976; Knowles, 1976; Hollingworth, 1976; Lund et al., 1978a). More recent research has shown that formamidine pesticides may exert their effects on the central nervous system by interacting directly with adrenergic receptors, particularly the alpha-2 subtype (Costa & Murphy, 1987; Costa et al., 1988, 1989). This interaction appears to mediate several of the observed effects of formamidines, such as changes in heart rate (Hsu & Kakuk, 1984, Watkinson, 1985; 1986a,b), pupil diameter (Hsu & Kakuk, 1984), visual evoked potential (Boyes & Moser, 1988) and hormonal secretion (Goldman et al., 1990; 1991). Costa et al. (1991) demonstrated that chlordimeform decreases the hepatic glutathione content by up to 40% in a time- and dose-dependent manner, through an interaction with alpha2-adrenoreceptors. Wu et al. (1990) have demonstrated that chlordimeform inhibits the binding of the known alpha2-adrenoreceptor blockers, clonidine and yohimbine, in rat forebrain tissue in vitro. Furthermore, Stoker et al. (1991), in a further study on the effects of chlordimeform on hormone release, have demonstrated in rats, treated intraperitoneally with chlordimeform (20 or 50 mg/kg body weight), that there is an increase in adrenocorticotropic hormone (ACTH), circulating corticosteroid (CORT) and prolactin (PL) in a dose-dependent manner. alpha-Adrenergic agonists specifically inhibited these effects indicating the interference with a regulatory signal mediated by alpha-adrenergic receptor-associated activity. Candura et al. (1992) demonstrated that the inhibition induced by chlordimeform in the intestinal tract is mediated by calcium channel blockade rather than by alpha2-adrenoceptor activation. In a study by Robinson et al. (1975), it was found that using drugs to block the serotonergic or alpha-adrenergic receptors did not reduce the chlordimeform-induced lethality in male rats. 7.10.2 Mechanism of carcinogenicity Chlordimeform and its metabolites, N-formyl-4-chloro- o- toluidine and 4-chloro -o-toluidine, have been shown to induce mouse tumours of a vascular origin characterized histologically as haemangioendotheliomas and haemangiosarcomas. 4-Chloro- o-toluidine has been shown to be a more potent carcinogen than chlordimeform, both with respect to dose-response and to a reduced latency period. Haemangioendotheliomas and haemangiosarcomas were not induced in rats. Cases of bladder cancer in humans associated with occupational exposure to high levels of chlordimeform or 4-chloro- o-toluidine have been seen in groups with high urinary levels of chlordimeform and 4-chloro- o-toluidine. The exact mechanism of induction of these tumours is unknown but there is evidence that a genetic mechanism involving mutations induced by 4-chloro- o-toluidine is involved. Metabolic studies in mice and rats indicate a similar metabolic pathway for chlordimeform in both species. The kinetics of absorption and elimination in mice and rats also seem to be similar. However, the overall DNA binding was higher in mice than rats, and one DNA adduct was formed to a 6- to 30-fold higher extent in mice. There is considerable evidence that 4-chloro- o-toluidine causes severe toxic effects in the human bladder leading to haemorrhagic cystitis (see section 8). Monitoring of urinary metabolites in humans also indicates that chlordimeform is rapidly metabolized to 4-chloro- o-toluidine in vivo. 4-Chloro- o-toluidine also has a close structural similarity to aromatic amines for which there is established evidence of carcinogenicity by animal experimentation and also by human epidemiological data (Parkes, 1984). Taken together, the evidence strongly implicates 4-chloro- o- toluidine as the causative agent in the induction of tumours in both mice and humans. A proposed route of activation that may be associated with carcinogenicity is shown in Fig. 2. However, the mechanism of the carcinogenicity remains unclear. 8. EFFECTS ON HUMANS 8.1 General population exposure 8.1.1 Acute poisoning incidents The most comprehensive data on acute poisoning cases associated with exposure to chlordimeform has come from China. Details of these published poisoning cases are shown in Table 16. While many were due to intentional ingestion, there were also cases of unintentional poisoning as a result of consumption of contaminated food, as well as occupational exposure to the spray. In a brief report prepared by Deng et al. (1984) of a 1983 symposium in Hu-bei Province on chlordimeform poisoning, which featured some 29 papers and 859 case studies, it was stressed that the main cause of death was suppression of cardiac contracture and dilation of blood vessels resulting in circulatory failure. Arima et al. (1976) described an unsuccessful suicide attempt involving a 76-year-old male who ingested 100 g chlordimeform. He vomited several times before arriving at hospital 50 min after ingestion. He was lethargic with a weak pulse and cyanosis associated with his lips, nails and skin. Methaemoglobin levels represented 17% of total haemoglobin at 5 h but returned to normal levels by 2 days. He regained consciousness by 50 h, although complained of headache and blurred vision. The only treatment received was gastric lavage, which was performed shortly after his arrival at the hospital. 8.2 Occupational exposure 8.2.1 Acute poisoning incidents Currie (1933) reported nine cases of haematuria in workers exposed to 4-chloro- o-toluidine (erroneously called 5-chloro- o- toluidine) by inhalation or possibly by absorption through the skin. All patients had difficulty urinating and had suprapubic pain. Most of the workers were exposed to the material for only 1-2 days. Despite efforts to control exposure to the chemical in the factory, further cases of poisoning occurred, and manufacture was ceased. In a follow-up study of three of the nine cases after 3 years, one patient had no bladder trouble, one had a slight cystitis and urethritis, and one had carcinoma of the bladder. Jurincic et al. (1991) reported cases of acute haemorrhagic cystitis in two men (aged 19 and 50) following involvement in cleaning of a water-tank that had likely been used to transport chlordimeform. Both developed abdominal pain, dysuria and haematuria in the evening following exposure. Cystoscopy revealed haemorrhagic cystitis, which was confirmed by bladder mucosa biopsy. Serum levels of 4-chloro- o-toluidine (referred to as 4-chloro-2-methylaniline) were >1 mg/litre in both patients and urine levels were 16 mg/litre in the Table 16. Case studies of acute chlordimeform poisoning in China Study Number of patients Route of exposure Number Clinical features Reference number (sex and/or age) of deaths 1 71 4 dermal absorption 5 ECG: 26 tachycardia; 6 bradycardia; 11 ectopic Wang & Tong (1992) (28 male, 67 ingestion rhythm; 6 premature beat; 2 atrial fibrillation; 43 female) ventricular fibrillation; 1"Torsade de Pointes"; 2 high pike P, 6 A-V block, 17 S-T depression, 3 inverse T, 1 S-T elevation ,7 Q-T elongation. In 33 severe cases, 28 has ECG abnormalities; 38 moderate cases, 14 had abnormal ECG. Changes in heart were found in 32 cases. Deaths were from respiratory failure (3); ventricular fibrillation (1) and supraventicular tachycardia (1). 2 4 ingestion(?) 0 Mild cyanosis, cystitis (2 cases occupational, Nui et al. (1990) 2 cases non-occupational; OPs also in formulation. 3 1 female ingestion 0 Jaundice on 3rd day which progressively deepened. Liu et al. (1990) (30 years old) (150 ml) Hb 40 g/litre (70 g/litre on admission); complete recovery, discharged on day 20. 4 52 ingestion 0 Loss of appetite (86.5%), urgency in urination He (1989) (19 male, (20-350 ml) (84.6%), cyanosis (81.1%), coma (67.3%), miosis 33 female) (34.6%), mydriasis (15.4%),hypotension (38.5%), tachycardia (32.7%), bradycardia (3.8%). Impairment of liver and renal functions. 15 ECGs: 7 tachycardia, 2 bradycardia, Q-T elongation, 8 T-wave changes. Treated with methylene blue, vitamin C, fresh blood transfusion and sopolamine. Table 16. (con't) Study Number of patients Route of exposure Number Clinical features Reference number (sex and/or age) of deaths 5 35 ingestion(?) 0 18 severe cases. Suggested use of 5-36 mg He et al. (1987) atropine for chlordimeform poisoning and 50-128 mg for mixed pesticide poisoning. 6 1 female ingestion 0 Cyanosis, pin-point myosis. Given atropine Zhou (1987) (30 years old) (80 ml 25% (15 mg/min) after lavage until total of 530 mg. chlordimeform) Symptoms indicated overdose of atropine. Methylene blue given, recovery and discharge at day 7. 7 23 4 contaminated food; 3 Mild case: nausea, vomiting, light cyanosis, (6 male, 19 ingestion no somnolence. Moderate case: somnolence and Xu (1987) 17 female) (10-350 ml) light consciousness. Severe case: Marked cyanosis, coma, shock. 5 ECG examined: 2 bradycardia, 1 tachycardia, A-V block, S-T change. Mild impairment of liver renal functions. Treated with methylene blue, 19 recovered. 8 1 female ingestion 0 Lavage and treatment led to recovery from danger. Liu & Li (1987) (52 years old) (30 ml conc. Black stool, tachycardia occurred on 3rd day. formulation) Complete recovery. 9 187 27 occupational spray; 13 Cyanosis (63.6%), nausea (49.2%), vomiting Ding & Huang (1987) (66 male, 16 ingestion (20-250 ml (44.9%), mydriasis (32.1%), somnolence (33.7%), 121 female) 25% chlordimeform coma (32.1%), irritation in urination (30.5%), formulation) hypotension. 27 ECGs: 4 tachycardia, 6 bradycardia, 4 S-T & T wave change, 2 pre-mature beat, 2 conductive blockage. 158 cases received methylene blue and 174 recovered within 1-5 days. Table 16. (con't) Study Number of patients Route of exposure Number Clinical features Reference number (sex and/or age) of deaths 10 1 male occupational spray 0 Sprayed incorrect dilution spray. Complained of Gu et al. (1987) (28 years old) fatigue, somnolence, loss of appetite, nausea, vomiting, but no cyanosis, or signs of cystitis, pulse 68, BP 128/94 (normally 120/80), MAO 25.12 U (normally 38.87 U). Total chlordimeform in urine on admission, 6.4 mg/ml. Recovered quickly. 11 6 (?) ? Main clinical features: drowsy, cyanosis, Chan (1985) loss of consciousness, mydriasis, cystitis, hypotension, bradycardia, myocarditis, shock, methaemaglobinaemia. 12 47 ingestion 4 Symptoms: drowsy, cyanosis, cystitis, Ke (1985) (11 male, 20-1900 ml 2 hypotension (severe case), 8 hypertension, 36 female) 10 ECG: 1 tachycardia and T-wave change. 13 25 ingestion(?) 1 Cyanosis, cystitis, hypotension, arrhythmia, Wang & Dong (1985) S-T and T changes, Q-T elongation. Treatment with gastric lavage, methylene blue, vitamin C 14 682 340 occupational spray; 25 279 cyanosis, 147 cystitis, 197 somnolence, Liu & Zhang (1985) (331 male, 342 ingestion 211 coma, 81 shock, 109 tachycardia, 351 female) 64 bradycardia, 54 hypertension, 22 hypotension. 59 ECG: 8 premature beat, 4 Q-T elongation, 16 S-T and T changes. 15 358 283 ingestion 37 Somnolence, cyanosis, loss of appetite, Ding & Ru (1985) haemorrhagic cystitis, often myocardium damage, A-V block, cardiac failure. Table 16. (con't) Study Number of patients Route of exposure Number Clinical features Reference number (sex and/or age) of deaths 16 49 3 occupational 4 13 cases were severe. Clinical features: cyanosis Liu & Ke (1985) 46 non-occupational and cystitis with haematuria in all cases, most with severe somnolence and a few with coma. Two severe cases had hypothermia. Hypertension was more common than hypotension. 10 ECGs: only one case of T-wave change and tachycardia. Treatment with methylene blue and lavage. 17 1 ingestion (300 ml 25% 0 Coma and cyanosis. Sudden cardiac arrest during Yang (1984) (female, form.) lavage, rescued with mechanical respiration. 25 years old) Recovered after 14 days. 18 24 ingestion (15-150 ml 2 16 cyanosis, 14 drowsiness, 8 haematuria, Wu et al. (1983) (11 male, 25% form.) 6 methaemoglobin, 1 cardiac arrest, which 13 female) recovered after resuscitation. 19 101 35 occupational spray, 2 89 chlordimeform alone cases: 66% cyanosis, Xie (1983) (49 male, 66 ingestion; 32 comas, 14 cystitis, 14 hypotension, 3 cardiac 52 female) chlordimeform +Ops) failure. 8 ECGs: 6 myocardium damage (changes in Q-T, S-T, and T waves). Treatment with methylene blue, vitamin. C. All recovered. 12 cases with mixed pesticides (OPs and Ocs). 20 1 ingestion 1 Loss of consciousness, cyanosis, mydriasis, Wu (1982) (female, 85 years) (30 ml) arrhythmia. ECG: bradycardia, T-wave changes. Died on day 6. Table 16. (con't) Study Number of patients Route of exposure Number Clinical features Reference number (sex and/or age) of deaths 21 20 occupational spray 0 Farmers applied wrong dilution chlordimeform to Li et al. (1982) (18 male, 2 female) cotton for one day. 7 drowsy, 10 loss of appetite, 4 cystitis. Symptomatic treatment. All recovered in 2-4 days. 22 2 male ingestion (100 & 200ml) 0 Cyanosis, coma, respiratory-circulation failure, Zhang et al. (1976) cystitis during 2nd day. Treatment with methylene blue and atropine. 23 1 male 100 ml 0 Deep cyanosis, pulse 166. Xia & Gao (1977) 24 6 male occupational spray 0 Contamination of body surface and clothing. Su (1977) Symptoms from day 1-4: cyanosis, haemorrhagic cystitis, fatigue. Recovery after 18 days. 25 2 male occupational spray 0 Clothing contaminated. Haemorrhagic cystitis, Anonymous (1977) no cyanosis, ECG normal. Symptomatically treated. 26 4 male occupational spray 0 Clothing contaminated. Haemorrhagic cystitis, Ming (1977) cyanosis, somnolence, loss of appetite, haematuria, RBC in urine for 20 days. Treatment: vitamin C, antibiotics, coagulators. 51-year-old patient. Case studies of chlordimeform poisoning in China due to occupational exposure are given in Table 17, together with a brief account of the clinical features observed. Table 17. Levels of urinary chlordimeform and its metabolites in hospitalized workers (3 days following exposure) (Folland et al., 1978) Worker Total aminesa Chlordimeform 4-Chloro-o-toluidine Conjugate (mg/litre) (mg/litre) (mg/litre) (mg/litre) 1 11.0 1.10 3.75 6.25 2 15.2 2.16 4.16 8.67 3 2.6 0.04 1.25 1.17 a Measured following hydrolysis with 10N NaOH and 2 h at 80°C. A brief account of the signs and symptoms of chlordimeform poisoning and suggested interventions has been provided by Xue & Loosli (1994). 8.2.2 Effects of long-term exposure A report of an outbreak of haematuria in employees of a chemical packaging plant in the USA over a 4-day period in 1975 was first reported by Armstrong et al. (1975). Further details were described by Folland et al. (1978). Nine of 22 workers who packaged chlordimeform became severely ill with abdominal pain, dysuria, urgency to void, or haematuria. In the previous year, four workers who had packaged the chemical had similar symptoms. While six workers recovered within 7 to 18 days, three were hospitalized with symptoms which lasted from one to two months. In these three workers, abnormalities noted were microscopic haematuria and pyuria, proteinuria, low creatinine clearance, elevated SGOT, prolonged BSP retention, elevated serum amylase level, small bladder capacity, ureteral reflux and an intense inflammatory reaction in three bladder biopsy specimens. The highest concentrations of total amines were found in the urine of workers who had become ill and were hospitalized. Low but measurable levels were also found in workers who had not become ill. The major part of the urinary amines was present as 4-chloro- o-toluidine or as conjugates. Urinary total amines (following hydrolysis with 10 N sodium hydroxide and 2 h at 80°C), as well as chlordimeform and 4-chloro- o-toluidine, were measured in the hospitalized cases and are shown in Table 17. The results of a monitoring programme on packaging workers in a chlordimeform plant in the USA during 1976 have been described (personal communication by J.W. Barnett, Ciba-Geigy Agricultural Division, Greenborough, North Carolina, USA, to the California Department of Food and Agricultural). The programme involved more than 100 workers and over 800 urine samples, monitoring for the presence of red blood cells, for residues of chlordimeform metabolites, and for clinical signs of toxicity in workers. Residues in urine samples were reported to range from <0.05 to 50 mg/litre. There was no evidence of microscopic haematuria found in the samples analysed nor of any clinical signs of toxicity. Four separate incidents resulting in 7 cases of frank haematuria following industrial exposure were reported in the USA during the period 1980-1984 (personal communication by J.W. Barnett, Ciba-Geigy Agricultural Division, Greenborough, North Carolina, USA, to Ciba-Geigy Ltd., Switzerland). Chemical cystitis, confirmed by cystoscopy and biopsy, was diagnosed in one case while non-specific bladder mucosal lesions were found in another. Six cases required hospitalization, but all resolved after cessation of exposure. In a study by Maddy et al. (1986), the results of a programme of monitoring (1982-1985) the urine of more than 200 workers, who had received training in the use of chlordimeform on cotton in California, were described. Although urinalysis was unremarkable and no significant cytological changes were found, a single case of bladder cancer was detected in a pilot who had seven seasons of exposure to chlordimeform. By contrast, in the same period (1980-1984), no cases of chlordimeform-induced haematuria occurred at manufacturing plants in Switzerland and West Germany or formulation plants in Australia, Columbia, Central America, Mexico and the USA. No cases of haematuria reportedly resulted from application or use of chlordimeform in the field (Anon., 1985b; personal communications by F.E. Pfister and P. Duback (Ciba-Geigy Ltd., Agricultural Division, Switzerland) and by N. Reckefus and K. Kossmann (Schering Aktiengesellschaft Agrochemical Division, Berlin, Germany), 1985). In a study by Lu et al. (1981), data on the effects of chlordimeform exposure of factory workers in China was examined. In this study, conducted in 1974, the air concentrations in the factory were generally below 0.036 mg/m3, with shorter periods at higher levels (0.108-0.33 mg/m3), during specific tasks. Skin contamination on hands and forearms was 9.1 mg/h for chemical operators and 964.2 mg/h for packers. The urinary excretion levels of chlordimeform and 4-chloro- o-toluidine in controls were 0.015 and 0.042 mg/litre, respectively; in chemical operators they were 0.065 and 0.108 mg/litre, respectively; and in packers were 0.263 and 0.398 mg/litre, respectively. The health of the workers was examined during the following 3 years (1974-1976). In 44-56 workers (equal number of each sex) at an average age of 32 years and working period of 2 years, the main finding were neurosis, sore throat and disorders of the nervous system. There were no treatment-related effects on ECG, liver function, clinical chemistry or urinalysis parameters. In the same report (Lu et al., 1981), the effect of chlordimeform exposure on rice field workers during 1974 was also examined. The air concentration in the breathing space in all cases was below 0.02 mg/m3. Skin contamination was examined at the front of the thorax, on the right forearm and on the right thigh. The applicators applied chlordimeform for 4-5 h per day for 1-3 consecutive days, wearing shirts and shorts with no other protection. Skin contamination was from splash or from spray. The levels found from splash on thorax, forearm and thigh were 0.0436, 0.0303 and 0.131 mg/100 cm2 per h, respectively. The levels found from spray on thorax, forearm and thigh were 0.235, 0.299 and 0.804 mg/100 cm2 per h, respectively. Medical examination during 1974/1975 revealed complaints of light-headedness, headache, fatigue, nausea, abdominal pain, skin itching and burning sensation, and hypotension. There were no changes in ECG or blood chemistry, and no reported cases of acute intoxication. In a study by Li et al. (1985b), the health of 24 packers (9 male, 15 female) in a chlordimeform manufacturing plant in Jiang-su Province of China, was examined. The chlordimeform division of the factory started manufacturing in 1975 and continued to do so at the time of the study. The employees were working in the factory for between 3 months and 4 years (average 1.5 years). Another 24 employees from the kitchen and kindergarten served as controls. The air concentration of chlordimeform (9 samples over 3 consecutive days) was 0.066 mg/m3 (range 0.017-0.121 mg/m3). Skin contamination of the hands and forearms was 110 µg/100cm2 (S.D. 39 µg/100 cm2). Urinary chlordimeform levels were 0.20 ± 0.13 mg/litre, and urinary 4-chloro-o-toluidine levels were 0.48 ± 0.29 mg/litre. Medical examination revealed no difference between packers and controls with regard to symptoms, laboratory examinations including liver enzymes and urinalysis parameters, chest X-rays, ECG, or other parameters of cardiac function. The only symptom associated with exposure was skin rashes and itching in 21% of exposed individuals. There was no difference in the micronucleus counting in cultured peripheral lymphocytes between exposed and control groups, nor were there any positive mutagenicity results from urine samples with or without glucuronidase or sulfatase in the medium. In a further study in a Chinese chlordimeform manufacturing factory, the health of employees involved in chlordimeform production was studied for the 5-year period, 1977 to 1981 (Anon., 1985a). The urinary chlordimeform plus 4-chloro- o-toluidine levels of packers was the highest at 0.39 mg/litre, which significantly correlated with skin contamination but not with air concentration. The major medical findings were complaints of lightheadedness, disorders in sleep, memory impairment, fatigue, loss of appetite, skin rashes and itching, and skin spot pigmentation. There were no features of cystitis. ECG findings in 36 employees indicated premature beats, partial A-V block, tachycardia and bradycardia. There was no evidence of chromosome aberrations in metaphase chromosomes of cultured peripheral lymphocytes. In a study by Tao et al. (1985), the health of 61 employees (25 chemical operators, 36 packers) of a pesticide factory in China was examined. Chlordimeform was produced in the factory for 5 months per year. Air levels ranged from 0.074 to 0.160 mg/m3. Skin contamination of packers (2.99 mg/day) was higher than for hemical operators (0.784 mg/day). The urinary excretion rate of chlordimeform plus 4-chloro- o-toluidine in packers was also higher (0.513 mg/litre) than for chemical operators (0.206 mg/litre) or controls (0.055 mg/litre). Symptoms of exposure noted in packers included loss of appetite, fatigue, somnolence and skin rashes. Hepatomegaly was observed. There was no difference in blood pressure or heart rate. Abnormalities in ECG were noted in 10/61 exposed employees compared to 6/76 controls. In a study by Wang et al. (1987), the health of 16 applicators (8 males, 8 females) spraying chlordimeform in cotton fields in Xin-yang Farm in the Jiang-su Province of China over a 3-day period (July 1986) was examined. Air levels in the breathing zone were 0.031 mg/m3 and the skin contamination was 4.17 mg per shift. Urinary levels of chlordimeform plus 4-chloro- o-toluidine ranged between 1 and 3 mg/litre over the exposure period. A close correlation was noted between the level of chlordimeform on the skin and the levels of chlordimeform plus 4-chloro- o-toluidine in the urine. Rapid excretion of chlordimeform plus 4-chloro- o-toluidine was noted following exposure. There was no change noted in heart rate, blood pressure, monoamine oxidase activity or urinalysis between exposed individuals and controls. Mild chlordimeform exposure, however, appeared to be related to loss of appetite and drowsiness. In a study by Zhang et al. (1986a), conducted at the same farm over the same period, 13 applicators (7 male, 6 female, 20-41 years of age) were examined during spraying chlordimeform on cotton over three consecutive days. Protective measures included gauze mask, plastic gloves and plastic apron, although it was noted that extensive contamination occurred. Air levels in the breath zone on each of the three days were 0.011, 0.014 and 0.011 mg/m3, respectively. Skin contamination on each of the three days was estimated by the method of Zhang et al. (1986b) to be 10.99, 4.32 and 4.45 mg/person per day, respectively. Urinary chlordimeform plus 4-chloro- o-toluidine levels were measured over the 3 days of exposure and for 7 days after cessation of exposure. Urinary levels ranged from a peak of 2.408 mg/litre during exposure to 0.036 mg/litre after 7 days. Excretion of chlordimeform occurred very rapidly with the highest level being detected in the sample collected at the end of each shift. There was a close correlation between skin contamination and urinary excretion. Metabolism occurred very rapidly since 4-chloro- o- toluidine usually accounted for 70-93 % of the total amount in the urine. Serum monoamine oxidase activity varied from 26.18 U to 19.26 U. Clinical symptoms were somnolence, headache, dizziness and fatigue. Heart rate and blood pressure dropped on the 2nd and 3rd days. Analysis of ECG indicated elongation of P-R, Q-T intervals. One person complained of urgency and pain in urination, gross haematuria, and the urinary chlordimeform plus 4-chloro- o-toluidine level was more than 6 mg/litre. Another four subjects were found to have microscopic haematuria. Liver function tests were normal. In a study by Xue et al. (personal communication by S.-Z. Xue, M. Wang, C.-M. Chu and X.-W. Zhou entitled "Effects of chlordimeform on cardiovascular function in humans with occupational exposure", 1993), the effect of chlordimeform on cardiovascular function was studied in exposed farm workers and in manufacturing workers in China. Four separate exposure groups were studied. The first (short-term) exposure group consisted of 16 farmers engaged in spraying chlordimeform (0.125% solution) in a cotton field. Exposure was for a 3- to 4-h period for 3 consecutive days. The second (long-term) exposure group consisted of 21 chlordimeform packers in a factory who had worked for 6 months on this task. The third exposure group consisted of 19 factory plant operators who had minimal exposure to chlordimeform. The fourth group consisted of 9 control (non-exposed) factory workers. Exposure was measured in the breathing zone air (personal sampler for the working shift, usually 6 h), by dermal contact (pooled aliquot of 10 swabs from various body sites), and by urine measurements. In each case, chlordimeform and its major metabolite, 4-chloro- o-toluidine, were measured. The cardiovascular system function was determined by measurement of blood pressure, heart rate and electrocardiography (ECG). Exposure data indicated the packer group had a higher inhalation exposure (0.107 mg/m3) than the sprayers (0.031 mg/m3). Dermal exposure, on the other hand, was higher in the sprayers group (4.251 mg/m2) than in the packers group (2.713 mg/m2). Urinary levels collected at the end of the working shift indicated the highest level in sprayers (1.950 mg/litre) compared to packers (1.267 mg/litre) and operators (0.097 mg/litre). In the farmer group, analysis of cardiovascular activity indicated a significant decrease in heart rate, and an increase in P-wave duration, Q-T interval and amplitude of T-wave compared to the control group. In the factory workers, the packers had significantly lower diastolic and systolic blood pressure, and an increase in T-wave amplitude compared to the plant operators. The heart beat of packers was also higher than controls, but not significantly. The cardiovascular function parameters of the plant operators were slightly but not significantly different to those of controls. Examination of the cardiovascular function parameters of the packers during a month of continuous exposure indicated a relationship between length of exposure, total urinary chlordimeform, and cardiac function parameters (see Table 18). Analysis of the exposure-effect correlation indicated the drop in blood pressure was the most sensitive parameter, with the change in amplitude of the T-wave the next most sensitive parameter. The changes of P-R interval were the least sensitive. Table 18: Cardiovascular function and urinary chlordimeform in factory workers (personal communication by S.-Z. Xue, M. Wang, C.-M. Chu and X.-W. Zhou entitled "Effects of chlordimeform on cardiovascular function in humans with occupational exposure", 1993) Parameter measured Duration of continuous exposure (days) 0 1 7 15 30 Total urinary 0.000 0.311 0.627 0.642 0.773 chlordimeform (mg/litre) Systolic BP (mmHg) 111/8.6a 105/7.7b 105/12c 102/10d 102/9.6c Diastolic BP (mmHg) 71/7.1 69/9.3 63/10c 65/8.2d 64/9.8d Heart rate (beat/min) 64.3/9.9 69.6/8.6d 67.2/6.5 70.0/9.4 71.4/12d Q-T interval (msec) 398/18.2 404/23.1 412/16.7d 418/22.3d 412/23.6d P-R interval (msec) 131/215 140/178d 140/212d 141/200a 143/317d a Figures are mean/standard deviation b P < 0.001 c P < 0.01 d P < 0.05 The authors attributed major importance to the alteration in cardiovascular function in relation to chlordimeform intoxication, and in most cases considered cardiac failure to be the cause of death. Recognition of the effects on cardiac function may have been overlooked previously, firstly, because of the diversity of mild changes induced by chlordimeform and, secondly, because of the tendency to concentrate on the effects of the aniline-containing metabolites, such as methaema-globinaemia, haematuria, and haemorrhagic cystitis. A no-observed-effect-level (NOEL) of 0.1 mg/litre of urinary chlordimeform plus 4-chloro- o-toluidine excretion is proposed as the threshold for effects on cardiovascular function following long-term, exposure while 0.3 mg/litre is proposed as the threshold for effects on cardiovascular function following short term exposure, even as short as one day. While the cardiovascular function parameters are unlikely to be useful as indicators of exposure, an understanding of the mechanism of action should assist in designing appropriate treatment. In a post-exposure surveillance programme, the chlordimeform- exposed group showed an increased prevalence of malignancy-associated surface markers on exfoliated urine cells, compared to geographical controls, but no tumours were found (Kenyon et al., 1993). 8.2.3 Epidemiological studies 22.214.171.124 4-Chloro- o-toluidine In a retrospective epidemiological study by Ott & Langner (1983) the mortality experience of 342 employees assigned to three aromatic amine-based dye production areas between 1914 and 1958 was examined in relation to duration of employment (<1 to 5 years) and interval since entry into these areas. 4-Chloro- o-toluidine represented one of a number of chemicals to which the workers were potentially exposed. 4-Chloro- o-toluidine and two other aromatic amines ( o-toluidine and 4-chloro-acetyl- o-toluidine) to which the workers were exposed have been shown to be carcinogenic in animal studies. No deaths due to bladder cancer were observed, and no statistically significant increases in mortality by work area or duration of exposure within work area were found. In a retrospective study by Stasik (1988; 1991) of 116 workers occupationally exposed in Germany to 4-chloro- o-toluidine during manufacture prior to 1970, eight cases of bladder cancer, diagnosed between 1967 and 1985, were identified. This represents an incidence more than 70-fold higher than expected. Although occupational exposure to two other aromatic amines, o-toluidine and 6-chloro- o- toluidine, may have occurred, analysis of the production process indicated that exposure to 4-chloro- o-toluidine in the plant was considerably higher than exposure to these other two chemicals. The workers were exposed to relatively high levels (before 1970) for a median of 14 years. In two cases, however, the exposure period was only 1.5 and 4.0 years. No quantitative measurements of exposure were available. Two of the patients had suffered from haemorrhagic cystitis as a consequence of massive acute exposure to 4-chloro -o-toluidine at 4 and 14 years, respectively, before the tumour was diagnosed. The latency periods for these eight cases ranged from 17 to 38 years. The significantly increased incidence of bladder cancer in this study is remarkable. 126.96.36.199 Chlordimeform An epidemiological study has been conducted on the incidence of cancer deaths of employees and their relatives on Xin-Yang Farm in Jiang-su Province of China (Gu et al., 1991). In this area, chlordimeform has been applied aerially in large amounts since 1974, in a relatively indiscriminate manner, with contamination of land, ponds, creeks, and gardens of adjacent houses. The study involved 7321 people (3911 male, 3410 female and 1265 retired agricultural workers) over the period 1 January 1971 to 30 June 1987. During this period, there were 706 registered deaths (510 males, 196 females), of which 198 were cancer deaths (160 males, 38 females). The standardized mortality ratio (SMR) was calculated on the basis of the specific mortality due to cancer in the adjacent Hai-men County. Many of the SMRs were significantly exceeded on the Xin-Yang Farm, as shown in Table 19. The incidences of bladder cancer adjusted to the national level were 2.65 (males) and 1.47 (females) per 100 000. The SMRs were 260 (males) and 420 (females). During the period 1 July 1987 to 30 June 1990, there were three more cases of bladder cancer (with one death) among the cohort members. The authors concluded there is evidence for an association between bladder cancer and exposure to chlordimeform, but that further data is needed to strengthen this association. It is noted that there was a high incidence of other tumour types in this study which makes the association between bladder cancer and exposure to chlordimeform more difficult to establish. Table 19. Standardized mortality ratio (SMR) for workers on the Xin-Yang farm (Gu et al., 1991) Cause of death Adjusted mortality Standardized mortality ratio (per 100 000) (95% C.I.) Male Female Male Female All deaths 785.1 610.0 134 (124-145) 139 (128-151) All cancers 214.0 130 113 (107-120) 128 (117-139) Oesophageal cancer 35.6 32.5 228 (208-249) 388 (352-428) Stomach cancer 61.4 24.5 175 (161-190) 120 (110-130) Liver cancer 31.6 6.9 40 (37-44) 27 (24-29) Colon cancer 8.8 6.5 133 (123-145) 79 (72-86) Lung cancer 34.2 16.9 135 (124-146) 147 (135-169) Leukemia 3.9 5.6 144 (133-157) 260 (235-285) Bladder cancer 4.1 3.0 197 (180-214) 750 (671-839) Breast cancer - 16.0 - 380 (345-419) Cervical cancer - 30.7 - 216 (198-234) Further epidemiological data on the association between cancer incidence and exposure to chlordimeform has been provided in papers by Xue et al. (1990; 1991). A summary of the findings of epidemio-logical studies between 1984 and 1988 is given in Table 20. Data from three counties and one farm are shown. The counties are located close to one another, with comparable environmental and socio-economic situations. The agricultural products are mainly rice and cotton. County A acted as a control, with little or no use of chlordimeform; County B was the largest user of chlordimeform; and County C started using chlordimeform at the earliest time. The results from the Xin-Yang farm are included for comparison. A comparison between the mortality rate in recent years (1984-1988) and the mortality rate in the years prior to the use of chlordimeform in these counties and Xin-Yang farm is shown in Table 21. There were excesses in the incidence of all deaths, deaths from cancer, and urinary bladder cancer for both sexes, although the data may not yet have reached the level of statistical significance. Table 21. Comparison of adjusted mortalities of urinary bladder cancer between 1984-1988 (county) and 1973-1975 (prefecture) (Xue et al., 1990, 1991) Item County A County B County C Xin-Yang Farm / / Prefecture / Prefecture Prefecturea Male 1.52 / 1.10 0.77 / 0.77 1.12 2.65 / 1.02 SRR 1.38 1.04 1.10 2.65b Female 0.41 / 0.35 0.46 / 0.17 0.55 1.47 / 0.35 SRR 1.17 2.71b 1.57b 4.20b a The duration of observation was 1971-1987 (June 30) b p < 0.05 In a retrospective study by Popp & Norpoth (1991) and Popp et al. (1992), the exposure and incidence of bladder cancer in a German chemical plant was examined. Chlordimeform was manufactured from 4-chloro- o-toluidine and production commenced in December 1965. Production was not continuous, but rather was in response to orders, so workers were subject to different periods of exposure (generally 8-12 weeks per year). Between 1965 and 1976, the exact levels of exposure were not available because measurement of the concentration in the air or monitoring of urine excretion was not carried out at that time. In 1976, production was ceased in order to improve working conditions and minimize human exposure. Production recommenced in 1980 with improved containment and monitoring of urinary excretion of Table 20. Data on Epidemiological Studies with Chlordimeform during 1984-1988 (Xue et al., 1990, 1991) Items County A County B County C Xin-Yang Farm (control) (largest amount) (earliest in using) Year started using chlordimeform 1979 1977 1973 1973 Population (annual average) 993 549 1 076 456 736 037 8732 Average amount of chlordimeform 1.1 65.0b 29.8 89.2 used (g/Mu/year)a All causes of mortality Male 584.5 675.7 761.0 785.1 RR 1.2 (1.1-1.3) 1.3 (1.2-1.4) 1.3 (1.2-1.5) Female 438.1 891.7 668.5 625.9 RR 2.0 (1.4-2.3) 1.5 (1.1-1.7) 1.4 (1.3-1.5) Cardiovascular mortalityc Male 143.2 167.7 221.6 - RR 1.2 (1.1-1.3) 1.6 (1.4-1.7) Female 138.2 280.2 234.8 - RR 2.0 (1.9-2.2) 1.7 (1.6-1.8) Respiratory mortalityc Male 99.6 100.4 127.2 RR 1.0 (0.9-1.1) 1.3 (1.2-1.4) Female 82.1 145.1 124.0 RR 1.8 (1.7-1.9) 1.5 (1.4-1.6) Table 20. (con't) Items County A County B County C Xin-Yang Farm (control) (largest amount) (earliest in using) All cancer mortalityc Male 188.5 246.6 232.2 214.9 RR 1.3 (1.2-1.4) 1.2 (1.1-1.3) 1.1 (1.1-1.2) Female 101.7 227.1 145.5 130.0 RR 2.3 (2.0-2.5) 1.4 (1.3-1.6) 1.3 (1.2-1.4) Bladder cancer mortalityc Male 2.08 (95)d 0.90 (26) 2.10 (32) 4.10 (4) RR 0.4 (0.39-0.47) 1.0 (0.9-1.2) 2.0 (1.8-2.2) Female 0.40 (15) 0.20 (9) 0.90 (14) 3.00 (2) RR 0.5 (0.46-0.55) 2.3 (2.1-2.5) 7.5 (6.7-8.4) a The Mu is a measure of area equivalent to 1/15th acre b Considered over the last 5 years c All mortality figures were age-adjusted d Figure in parentheses is the actual number of cases of bladder cancer workers. Production finally ceased in 1986. The company identified 170 individuals who had come into contact with chlordimeform but many had minimal exposure. The number of workers involved in the production of chlordimeform was 49, and these comprised the study group. The period under investigation was from the year of employment to the end of 1990. The expected incidence of bladder cancer (age- and sex-specific) was extracted from the cancer registers of Saarland (1988), the former German Democratic Republic (GDR) (1978-1982) and Denmark (1978-1982). The standard incidence rate (SIR) was the ratio of the number of cases observed to the expected number (see Table 22). Table 22. Standard incidence rates (SIRs) of bladder carcinoma in a group of 49 workers engaged in chlordimeform synthesis (Popp et al., 1992) Observed cases Expected number SIR 95% CI p value 7 0.078 (GDR) 89.7 35.6 - 168.6 0.000002 7 0.200 (Denmark) 35.0 13.9 - 65.7 0.00001 7 0.130 (Saarland) 53.8 21.3 - 101.1 0.000005 The average age for workers starting work was 30 (range 18-51), and the exposure ranged from 3 to 956 days. By the end of 1990, an average of 18 (10-25) years had passed since the start of exposure. Bladder cancer was detected in 7 of the 49 subjects by the end of 1990. Of the seven cases, six were diagnosed as transitional cell carcinoma and one as papillary carcinoma. In five cases, the exposure period could be determined, with an average of 575 days (range 291-766). The latency period was an average of 19 years (range 15-23), with an average age at diagnosis of 54 years (range 42-62). This study provides strong evidence of an association between exposure to 4-chloro- o-toluidine and human bladder cancer. All of the cases involved workers who were exposed to 4-chloro- o-toluidine while synthesizing chlordimeform before 1976. None of those workers who were handling the final product, chlordimeform, had developed bladder cancer by the end of 1990. In a historical cohort study (personal communication by P. Boyle & G.J. Macfarlane to the IPCS, 1997), the bladder cancer incidence of 847 men involved in the manufacture of chlordimeform in Australia, Switzerland, the United Kingdom and the USA was compared with that expected on the basis of population-based cancer registry rates. Subjects eligible to be included in the cohort were those who had been employed in the production or formulation of chlordimeform or who had otherwise been an integral part of a chlordimeform unit in a plant where it had been produced or formulated for at least 6 months. The results presented in Table 23 show an incidence rate of bladder cancer which was significantly higher than expected. Overall, ten cases were observed while 2.6 were expected. When the cohort was divided according to whether members had been exposed to chlordimeform and 4-chloro- o-toluidine, or to chlordimeform alone, it was found that a significant excess of risk of bladder cancer also occurred in those workers thought not to have been exposed to 4-chloro- o-toluidine. In this group of 592 men, 5 cases of bladder cancer were observed, while 1.4 cases were expected (SIR = 3.5, 95% CI (1.1, 8.3)). The authors concluded that despite the lack of information on potentially confounding factors in this study, the data indicated an association between excess risk of bladder cancer in this cohort and one or more aspects of the manufacture of chlordimeform. Table 23. Bladder cancer risk in a cohort of men exposed to chlordimeform (Boyle & Macfarlane, 1997) Plant location Cohort numbers Bladder cancer cases Observed Expected SIRa Switzerland 273 4 0.72 5.6 USA (A)b 182 1 0.32 3.1 United Kingdom 174 3 1.06 2.8 USA (B)b 163 1 0.26 3.8 Australia 55 1 0.27 3.7 All plants 847 10 2.63 3.8 95% CIc (1.8, 7.1) a Standardized Incidence Ratio b Different production sites c Confidence Interval 9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD 9.1 Laboratory experiments 9.1.1 Microorganisms There are no data on the effects of chlordimeform on microorganisms. 9.1.2 Aquatic organisms 188.8.131.52 Plants There are no data on the effects of chlordimeform on aquatic plants. 184.108.40.206 Invertebrates There are no laboratory data on the effects of chlordimeform on aquatic invertebrates. 220.127.116.11 Vertebrates The toxicity of chlordimeform to some species of fish has been determined (FAO/WHO, 1972; Mayer & Ellersieck, 1986), and is shown in Table 24. 9.1.3 Terrestrial organisms 18.104.22.168 Plants There are no data available for the effects of chlordimeform on plants. 22.214.171.124 Invertebrates Dittrich (1966, 1967) first reported studies on the efficacy of chlordimeform as an acaricide with both ovicidal activity against insect eggs and adulticidal activity. It killed adult spider mites when applied as a vapour and as a spray, and penetrated plant tissues where it was released in ovicidal quantities. Since then, its efficacy as an insecticide has been studied in a wide range of species. Chlordimeform not only has a direct lethal action, particularly against eggs and early instar larvae of insects and acarines, but also has important sublethal effects, including sterilization of eggs, induction of hyperactivity, detachment of feeding ticks from hosts, Table 24. Toxicity of chlordimeform to fish Species Duration LC50 Reference (h) (mg/litre) Bluegill sunfish 24 1.0 FAO/WHO (1972) 48 1.0 96 1.0 Trout 24 11.7 (8.73-15.8) FAO/WHO (1972) 48 10.6 (7.80-14.50) 96 7.14 (4.70-10.80) Cat fish 24 11.9 (8.98-15.9) FAO/WHO (1972) 48 8.72 (6.26-21.1) 96 4.54 (3.08-6.68) Rainbow trout 24 29 Mayer & Ellersiek 96 13.2 (1986) Channel catfish 24 20.7 Mayer & Ellersiek 96 20.2 (1986) Carp 24 65a FAO/WHO (1972) 48 60a 96 50a a Values are for TLm colony dispersal behaviour in ticks and mites, anti-feeding effects and disruption of mating and oviposition in Lepidoptera (Hollingworth, 1976). Knowles & Shrivastava (1973) investigated its toxicity in house flies. The LD50 was 25 µg/fly, a dose which was not at a practical level for house-fly control, possibly due to the high rate of metabolism in this species. Pimley (1986) investigated the toxicity of chlordimeform to tsetse fly (Glossina morsitans). The median lethal dose was approximately 0.4 µg/fly for unfed tsetse, and 100% mortality was achieved with 2 µg/fly. Sublethal doses also caused a significant depression of feeding activity. The specificity of chlordimeform with regard to both eggs and larval stages was examined by Streibert & Dittrich (1977). Eggs of the three noctuid cotton pest moths, Heliothis armigera, Heliothis virescens, and Spodoptera littoralis, when exposed to a saturated atmosphere of 4 mg/m3, have very similar sensitivity to vaporized chlordimeform. Agrotis ipsilon, also a noctuid, on the other hand, is definitely less sensitive, and the coccinillid Epilachna varivestis was the most tolerant. The larval stages of all of these species were considerably less sensitive to chlordimeform vapour than the egg stage, but chlordimeform does seem to cause a decrease in the number of larvae in the field, possibly due to a repellent effect or a behavioural change rather than a direct toxic effect. These results with Spodoptera littoralis on the relative sensitivity of eggs and larvae were confirmed in the studies of Salvisberg et al. (1980). Davenport & Wright (1985) have also demonstrated the differential susceptibility of adult and larvae of the noctuid moths, Spodoptera littoralis and Heliothis virescens, and also highlighted the significantly higher toxicity of the hydrochloride salt, compared to the base, to the adults of both species. Sparks et al. (1993) studied the effects of several insecticides on ovicidal activity and alteration of octopamine titres in eggs of the tobacco budworm (Heliothis virescens). Chlordimeform was highly toxic to eggs of H. virescens. The authors reported that the alteration in the biogenic amine octopamine titres by chlordimeform might lead to a disruption in the ability of larvae to hatch from the egg. Crecelius & Knowles (1976) studied the sensitivity of the larvae of the cabbage looper, Trichoplusia ni, to the toxic effects of chlordimeform. Third instar larvae were more sensitive to the toxic effects of chlordimeform than the fifth instar larvae, possible due to slower penetration and slower metabolism of chlordimeform in the latter instar larvae. Bailey & Cathey (1985) demonstrated the effectiveness of chlordimeform in reducing the percentage egg hatch of Lygus lineolaris (Palisot de Beauvois) on pole bean ( Phaseolus vulgaris L.) pods and cotton ( Gossypium hirsutum L.). A solution of 0.09% chlordimeform, while not significantly reducing nymph emergence from eggs deposited on bean pole pods, did significantly reduce nymph emergence from eggs deposited on cotton plants. Salvisberg et al. (1980) also demonstrated that Spodoptera littoralis moths, when treated at doses as low as 10% of the LD50, showed symptoms of hyperexcitation, which resulted in abnormal patterns of egg-laying, a reduced number of eggs and lower fertility. Further studies by Davenport & Wright (1987) on Spodoptera littoralis have shown that chlordimeform hydrochloride significantly reduces food consumption in fifth-instar larvae when incorporated into the diet at a level of 0.1-10 mg/g or when topically applied. No mortality occurred during feeding, but mortality was increased during subsequent pupation and during emergence of the adult from the pupae. In adult moths, egg laying was significantly decreased when chlordimeform hydrochloride was applied topically (1 or 10 µg/moth). Further evidence that behavioural changes may be more important in reducing both the larval and insect populations following chlordimeform treatment has been provided by Shimizu & Fukami (1983) in studies of the larvae of the cabbage armyworm, Mamestra brassicae, which showed a prolonged period of wandering behaviour in the presence of chlordimeform. This may have caused a failure to find or prepare a suitable site for pupation. The behaviour-modifying effects of chlordimeform have also been studied by Blackwell (1988a,b; 1889) in the larvae of the large cabbage white butterfly, Pieris brassicae L. When placed on chlordimeform-dipped leaves, the larvae become excited, in contrast to their normal communal feeding behaviour. Locomotion was increased and feeding was significantly reduced as a result of disaggregation (Blackwell, 1988a). When applied directly to the larvae, chlordimeform caused excitation and inhibition of feeding (Blackwell, 1989). Direct application also caused developmental delays and mortality was increased at later developmental stages (Blackwell, 1988b). O'Brian et al. (1985) have studied the effect of insecticides on beneficial insects, and in particular, the effect of chlordimeform on the ecoparasitoid, Bracon mellitor, an important parasitoid of the boll weevil (Anthonomus grandis grandis). Chlordimeform was found to be more toxic to Bracon mellitor than to the boll weevil, and also reduced the number of egg deposited. The toxicity of chlordimeform hydrochloride to bees has been examined after both ingestion and contact. Ingestion of a 0.3% solution killed 18%, while ingestion of 0.15% killed approximately 7%. Contact with the same solutions did not increase the mortality rate (FAO/WHO, 1972). In a study by Johansen (1972), bees were exposed to field-weathered residues of a range of insecticides, including chlordimeform, on alfalfa foliage. Over a 24-h period, zero mortality was obtained with alfalfa leafcutter bees, alkali bees and honey bees exposed to 3-h-old residues. 126.96.36.199 Vertebrates Fleming et al. (1985) examined the toxic and behavioural effects of chlordimeform on the game bird, the bobwhite quail (Colinus virginianus). When added to the diet of newborn chicks over a 7-day period, the lethal concentration to chicks was 2835 mg/kg diet (2169-3705 mg/kg diet). When chicks were fed a diet containing chlordime-form at a concentration of 1000 mg/kg diet for 7 days, they ate less, weighed less, travelled further from a fright stimulus in an avoidance test, and had a high locomotor activity in an open-field test than at lower dose levels. Chicks fed 100 or 1000 mg chlordimeform/kg diet scored more highly than controls in a visual cliff performance test. After a further 8 days on control diet, the chicks fed 1000 mg/kg diet still scored higher than controls on the avoidance test, but the open-field and cliff performance scores were similar to those of controls. In studies conducted on bobwhite quails and ducks, groups of animals (10 per treatment group, 30 per control group) were fed chlordimeform technical or chlordimeform 48% EC formulation in their diets for 5 consecutive days. The dose levels were 0, 10, 31.6, 100, 316 or 1000 mg/kg diet. Both quails and ducks were tolerant of the presence of chlordimeform in the diet. With the technical material, one quail in each of the groups fed 100 and 316 mg/kg diet died, while, with the formulation, one quail in each of the groups fed 316 and 1000 mg/kg diet died. All ducks survived treatment, even at the highest dose level (FAO/WHO, 1972). Hill et al. (1975) exposed three bird species, Japanese quail (Coturnix japonica), ring-necked pheasant (Phasianus colchicus) and mallard (Anas platyrhynchos), to chlordimeform. LC50 values for Japanese quail and ring-necked pheasant were determined to be 1749 and 2608 mg/kg diet, respectively. The LC50 for mallard was determined to be >5000 mg/kg diet; only 20% mortality was reported at the highest exposure group, 5000 mg/kg diet. Hill & Camardese (1986) reported an LC50 of 5079 mg/kg diet for Japanese quail exposed to chlordimeform. 9.2 Field Observations 9.2.1 Microorganisms There are no field data on the effects of chlordimeform on microorganisms. 9.2.2 Aquatic organisms There are no field data on the effects of chlordimeform on aquatic organisms. 9.2.3 Terrestrial organisms 188.8.131.52 Plants The possibility that some insecticides might enhance the growth of cotton plants has been suggested for some time. However, in the case of chlordimeform, debate has continued as to whether this effect is due to early season insect suppression (Bailey & Cathey, 1985) or to a physiological effect (Phillips et al., 1977). Cathey & Bailey (1987) have conducted controlled studies to examine the effects of multiple applications of chlordimeform on the growth and development of cotton ( Gossypium hirsutum L.) in both greenhouse and field studies. Plants were sprayed six times with chlordimeform either alone or in combination with fenvalerate at 5- to 7-day intervals, beginning at the six-leaf stage of plant development. In the absence of early season insects and when insect populations were maintained at a relatively low level, no increases in lint yield occurred on the chlordimeform-treated plants. However, yield increases did occur and insect populations became lower in these treated plots when early season insect populations in the test area were relatively high. None of the treatments influenced the boll components, boll size, seed index and lint percentage, or the first fibre properties, length, strength and micronaire. Field studies by Youngman et al. (1990) to determine the effects of several insecticides on growth, fruiting patterns and yield of the cotton plant, Gossypium hirsutum L., supported the conclusion that chlordimeform does not significantly increase any plant growth parameter when compared with the control. 184.108.40.206 Invertebrates In a small field study conducted by Bull & House (1978), tests were conducted in 0.05-ha plots of cotton to compare lower and more frequent applications of chlordimeform with commercial mixtures of insecticides against natural populations of Heliothis species. The results indicated that the protection afforded was as good as with commercial mixtures, probably as a result of careful observation of the cotton to pinpoint the onset of significant egg production. In a another small field study by Wilson (1981), the potential of chlordimeform to control Heliothis species in cotton was tested separately or in combination with amitraz and the microbial insecticide, Bacillus thuringiensis. Chlordimeform was the most efficient of the three materials and controlled Heliothis species reasonably efficiently, but no control of the rough bollworm, Earias hueglei was obtained. There was also good control of the cotton looper, Anomis flava, and some indication of suppression of mites and aphids was obtained. The behaviour-modifying effects of chlordimeform have been demonstrated in field studies by Uk & Dittrich (1986) on the adult whitefly, Bemisia tabaci (Genn.), which attacks cotton in the Sudan. At dose levels of 500-2500 g chlordimeform/ha together with 960 g endosulfan/ha, there was evidence of irritation and mass emigration of adults from treated cotton foliage without detectable direct mortality. 220.127.116.11 Vertebrates There are no field data on the effects of chlordimeform on vertebrates. 10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT 10.1 Evaluation of human health risks 10.1.1 Exposure Production and use of chlordimeform has now ceased worldwide and no further human exposure should occur. During the years of chlordimeform production and use, dietary and incidental exposure to chlordimeform occurred. Occupational exposure to chlordimeform and 4-chloro- o-toluidine (used as a starting material for synthesis) occurred during manufacture and formulation, as well as during application. The major route of exposure was through dermal contamination. Application of chlordimeform occurred extensively by aerial spraying, but it was also applied by ground-rigs as well as by backpack spray equipment. Thus, agricultural workers were exposed during mixing, loading, washing, and flagging operations. General population exposure occurred through the consumption of food containing residues of chlordimeform, and to a lesser extent through by-stander exposure. In some cases, there was intentional ingestion of the formulation. Monitoring of urinary 4-chloro- o-toluidine has been found to be a useful indicator of exposure. 10.1.2 Toxicity In both experimental animals and humans, there is extensive metabolism of chlordimeform, followed by rapid excretion via the urine. A major urinary metabolite is 4-chloro- o-toluidine. In experimental animals, symptoms of acute toxicity included neurotoxic as well as cardiovascular effects. There was no evidence of teratogenicity or reproductive effects. Following chronic administration, there was a dose-related increase in haemangioendotheliomas in mice. There was no treatment-related increase in tumour incidence in rats. Most of the mutagenicity studies with chlordimeform itself were negative, but there were sporadic reports of genotoxicity with 4-chloro- o-toluidine and to a more limited extent with N-formyl-4-chloro- o-toluidine. In humans, chlordimeform has been shown to have both acute and chronic effects. Acute poisoning caused fatigue, nausea and loss of appetite, and, in more severe cases, somnolence, cyanosis, urgency in urination, cystitis, cardiovascular effects (tachycardia, bradycardia, ECG changes), coma and shock. The significance of the cardiovascular effects in chlordimeform-induced mortality has only recently been recognized. While there have been fatalities as a result of acute chlordimeform exposure, in the majority of cases complete recovery occurs. Symptoms of chronic exposure include those of acute exposure as well as abdominal pain, skin itching and rashes, and gross or microscopic haematuria. With regard to carcinogenicity, the International Agency for Research on Cancer (IARC) has concluded that there is limited evidence in humans and sufficient evidence in experimental animals for the carcinogenicity of 4-chloro- o-toluidine. The available epidemiological data indicate an association between excess risk of bladder cancer and exposures entailed in the manufacture of chlordimeform. There is currently preliminary epidemiological evidence of an association between chlordimeform use and excess risk of bladder cancer. 10.1.3 Risk evaluation With the withdrawal of the use of chlordimeform in agriculture and a cessation of production worldwide, there is no longer any risk associated with acute exposure except during the disposal of existing stocks. The risk associated with chronic exposure, however, particularly the risk of bladder cancer, will continue to be of concern for many years. Human bladder cancer has a long latency period, and establishing whether or not there is a link between chlordimeform exposure and bladder cancer will require continued health screening of significantly exposed individuals both from manufacturing plants and from those rural communities where chlordimeform was extensively used. 10.2 Evaluation of effects on the environment Since chlordimeform is no longer used, no quantitative risk assessment for the environment has been performed. There are not expected to be any long-term detrimental effects on the environment as a result of past use of chlordimeform. 11. CONCLUSIONS AND RECOMMENDATIONS FOR PROTECTION OF HUMAN HEALTH AND THE ENVIRONMENT 11.1 Conclusions Chlordimeform has significant potential to cause both immediate and long-term toxicity in exposed individuals. Current information supports an association between an increased incidence of human bladder cancer and exposure to 4-chloro- o-toluidine, and, to a lesser extent, chlordimeform. Chlordimeform does not persist in the environment and therefore there are not expected to be any long-term detrimental effects on the environment as a result of past use. 11.2 Recommendations for protection of human health and the environment Future commercial production or use of chlordimeform is not recommended. Existing stocks should be disposed of safely. Those with occupational exposure to chlordimeform should participate in a health screening programme that includes urinary cytology and the detection of haematuria. 12. FURTHER RESEARCH The following studies are needed: 1. epidemiological investigations on exposed populations. 2. studies on the dose-response relationship between exposure to 4-chloro- o-toluidine or chlordimeform and the induction of urinary bladder cancer in humans. 13. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES Chlordimeform was considered by the International Agency for Research on Cancer (IARC) in 1983. IARC noted that no published study on the carcinogenicity of chlordimeform was available. However, it considered data on the carcinogenicity of 4-chloro- o-toluidine and concluded that the results of experiments on mice provided sufficient evidence that 4-chloro- o-toluidine, a metabolite of chlordimeform, is carcinogenic to experimental animals. No relevant data on humans were available. IARC concluded the available data were inadequate to evaluate the carcinogenicity of chlordimeform to humans (IARC, 1983). The carcinogenicity of 4-chloro- o-toluidine, the breakdown product and major metabolite of chlordimeform, was evaluated by IARC in 1990 (IARC, 1990). On the basis of the available published data, it was concluded that there is limited evidence in humans and sufficient evidence in experimental animals for the carcinogenicity of 4-chloro- o-toluidine. 4-chloro- o-toluidine and its strong acid salts were classified as probably carcinogenic to humans (Group 2A). Chlordimeform was considered at the 1971, 1975, 1978, 1979, 1980, 1985 and 1987 FAO/WHO Joint Meeting on Pesticide Residues (JMPR). In 1971, a temporary acceptable daily intake (ADI) for chlordimeform of 0-0.01 mg/kg body weight was established, and temporary maximum residue levels (MRLs) were set for a number of crops and for the meat and milk of cattle (FAO/WHO, 1972). In 1975, the temporary ADI was maintained and some new temporary MRLs were established (FAO/WHO, 1976). 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La tension de vapeur du chlordiméform est de 48 mPa à 20°C et son coefficient de partage entre l'eau et l'octanol (log Kow) est égal à 2,89. On peut faire appel à de nombreuses méthodes d'analyse pour la recherche et le dosage du chlordiméform dans les végétaux, le sol, l'eau et l'urine. 2. Sources d'exposition humaine et environnementale Le chlordiméform n'existe pas à l'état naturel. On le prépare industriellement par condensation du réactif de Vilsmeier (obtenu par réaction du diméthylformamide sur POCl3, SOCl2 ou COCl2) soit avec la 4-chloro- o-toluidine, soit avec la l' o-toluidine, suivie d'une chloration du dérivé intermédiaire obtenu. On l'utilise comme acaricide à large spectre et il est principalement actif contre les formes mobiles des acariens et des tiques ainsi que contre les oeufs et les premiers stades de certains lépidoptères. Il agit en phase gazeuse aussi bien que par contact. Les premiers temps de son utilisation, on l'employait pour traiter des cultures très variées comme les fruits à pépins, les drupes, les choux et autres légumes, les raisins, le houblon, les agrumes, les cerises et les fraises. On l'utilise aussi en bains contre les tiques des bovins. Ces dernières années son usage s'est généralement limité au coton, mais on continue tout de même à l'utiliser sur le riz dans certains pays. Depuis 1988/89 il n'est plus homologué dans la plupart des pays. En Chine, la production a cessé en 1992, de même que la vente en 1993. 3. Transport, distribution et transformation dans l'environnement Bien que sa tension de vapeur ait une valeur moyenne, le chlordiméform ne s'évapore pas autant qu'on le penserait des surfaces végétales. Sa stabilité vis-à-vis de l'hydrolyse dépend fortement du pH; il est stable en milieu acide mais s'hydrolyse rapidement en milieu alcalin. Le chlordiméform est capable de s'adsorber sur les matières organiques dissoutes. Dans le sol, la disparition du chlordiméform est essentiellement imputable à l'action des microorganismes et, pour une moindre part, à l'hydrolyse chimique. Malgré la solubilité du composé dans l'eau, on ne trouve guère de traces de lessivage, ce qui peut s'expliquer par une adsorption aux matériaux argileux ou aux matières organiques du sol ainsi que par la biodégradation. Les principaux métabolites sont la N-formyl-4-chloro- o-toluidine et la 4-chloro- o-toluidine. Les plantes fixent le chlordiméform présent dans le sol en proportion faible mais mesurable et la concentration est suffisante pour affecter les ravageurs qui se nourrissent à leurs dépens. En traitement foliaire, la pénétration du chlordiméform dans la cuticule est limitée. Le chlordiméform est rapidement décomposé par les végétaux. Les principaux métabolites sont le déméthylchlordiméform, la N-formyl-4-chloro -o-toluidine et la 4-chloro -o-toluidine, cette dernière n'étant pas produite par toutes les plantes. Dans le sol, le chlordiméform et ses métabolites disparaissent selon une cinétique du premier ordre avec une demi-vie de 20 à 40 jours. Les études de bioaccumulation montrent que les organismes aquatiques ne fixent qu'une petite quantité de chlordiméform et que celui-ci s'élimine rapidement une fois ces organismes replacés en eau pure. 4. Concentrations dans l'environnement et exposition humaine On n'a pas procédé à des mesures de concentration dans l'air ou l'eau. Après traitement de rizières, on a retrouvé des résidus allant jusqu'à 2900 µg/kg dans les 5 premiers centimètres du sol et jusqu'à 150 µg/kg dans les 5 centimètres suivants. On a fixé des teneurs limites en résidus pour un grand nombre de produits crus et dans certains cas, pour des préparations contenant ces produits. Les limites maximales de résidus fixées par le Codex ont été supprimées. Il y a eu des cas d'exposition au chlordiméform au cours de la préparation, de la formulation et de l'épandage de ce produit. Depuis quelques années, on utilise la concentration urinaire totale du chlordiméform et de ses métabolites pour surveiller l'exposition et il y a d'ailleurs une bonne corrélation entre cette concentration et le degré de contamination cutanée. Dans les industries cotonnières où l'on a soumis les ouvriers agricoles à une surveillance générale de la concentration urinaire en chlordiméform, on a constaté que les plus exposés étaient les chargeurs, les laveurs et les mécaniciens et les moins exposés les signaleurs et les pilotes. 5. Cinétique et métabolisme chez les animaux de laboratoire et l'Homme Chez les mammifères, le chlordiméform est facilement résorbé au niveau des voies digestives ainsi que par la voie transcutanée. Il est ensuite rapidement excrété à raison de 80% environ dans l'urine et de 10-15% dans les matières fécales. De petites quantités de résidus sont présentes au bout de 10 jours dans tous les tissus mais rien n'indique qu'il y ait bioaccumulation. Après application cutanée chez l'Homme, on constate également une excrétion urinaire rapide. On retrouve dans l'urine plusieurs métabolites du chlordiméform sous forme oxydée et conjuguée, à savoir principalement la N-formyl-4-chloro- o-toluidine, et la 4-chloro- o-toluidine. In vitro, on retrouve les mêmes métabolites, mais avec prédominance de la 4-chloro- o-toluidine. 6. Effets sur les mammifères de laboratoire et les systèmes d'épreuve in vitro Les épreuves pratiquées sur un certain nombre d'espèces montrent que la chlordiméform présente une toxicité aiguë modérée par la voie orale et la voie transcutanée. Chez le rat, les principaux métabolites sont peu toxiques par voie orale. Chez le lapin, le chlordiméform ne provoque qu'une légère irritation oculaire et cutanée. Après exposition de courte ou de brève durée au chlordiméform ou à ses métabolites, on peut observer, au niveau des constantes hématologiques, des modifications qui sont imputables au traitement et on constate, à dose élevée, certains signes qui dénotent une hyperplasie de l'épithélium des canaux biliaires et de la vessie. Il n'y pas d'accroissement de la fréquence des tumeurs chez le rat. Chez la souris, on observe, après administration par voie alimentaire de chlordiméform, de N-formyl-4-chloro -o-toluidine ou de 4-chloro- o-toluidine, une augmentation, liée à la dose, des tumeurs malignes hémorragiques d'origine vasculaire appartenant à la classe des hémangio-endothéliomes, dont la présence entraîne un accroissement de la mortalité parallèle à la dose. Le chlordiméform n'a pas d'effet indésirable sur les différents aspects de la fonction de reproduction et il n'a aucun pouvoir tératogène. Le chlordiméform a fait l'objet d'un grand nombre d'épreuves de génotoxicité in vitro et in vivo. Aucune d'elles n'a donné de résultat positif, étant entendu qu'il s'agissait de la matière active et non de formulations. Par ailleurs, un certain nombre d'observations sporadiques non confirmées font état d'une activité mutagène induite par la N-formyl-4-chloro- o-toluidine et par la 4-chloro- o- toluidine. Il n'existe qu'une seule description de transformations cellulaires provoquées par le chlordiméform et par la 4-chloro -o- toluidine. Chez des souris et des rats traités par le chlordiméform, on a constaté une que le composé se liait à l'ADN des cellules hépatiques. A dose beaucoup plus élevée, il se forme chez les mêmes animaux un important adduit hydrophobe. Le chlordiméform provoque des effets pharmacologiques et biochimiques divers chez l'animal, et notamment des effets cardiovasculaires, une hypothermie, une hyperexcitabilité, une modification des fonctions visuelle et auditive ainsi que la modulation des amines biogenèse et des enzymes pharmacométabolisantes. 7. Effets sur l'Homme Les intoxications aiguës se traduisent par une fatigue, des nausées et une perte d'appétit, avec, dans les cas graves, somnolence, cyanose, besoin impérieux d'uriner, cystite, effets cardiovasculaires (tachy-cardie, bradycardie, anomalies de l'ECG), coma et état de choc. En général, la récupération est totale. Après une exposition de longue durée au chlordiméform, on peut observer encore d'autres symptômes tels que des douleurs abdominales, des démangeaisons et des éruptions (en cas d'exposition cutanée) accompagnés d'une hématurie macroscopique ou micro-scopique. On a signalé de nombreux cas d'intoxication présentant des symptômes d'exposition de longue durée parmi les ouvriers d'unités de production de chlordiméform et des ouvriers agricoles. Les données épidémiologiques obtenues à la suite de cas d'exposition professionnelle montrent qu'il existe une forte corrélation entre l'exposition à la 4-chloro -o-toluidine et le cancer de la vessie. En revanche, on n'a guère obtenu d'éléments qui militeraient en faveur d'une association entre ce type de cancer et l'exposition au chlordiméform. 8. Effets sur les autres êtres vivants au laboratoire et dans leur milieu naturel Après épandage de chlordiméform sur le sol, on n'a pas observé d'effets sensibles sur les populations de champignons, de bactéries ou d'actinomycètes terricoles. Il n'existe pas de données toxicologiques de laboratoire concernant les invertébrés dulçaquicoles. En présence de chlordiméform, il y a inhibition de la croissance des larves d'huîtres, avec une CE50 de 5,7 mg/litre. Pour la crevette rose, le seul crustacé étudié, la CL50 à 96 h a été trouvée égale à 7,1 mg/litre et des valeurs allant de 1 à 54 mg/litre ont été obtenues pour le même paramètre chez les poissons. On ne possède aucune donnée relative à la toxicité chronique pour les espèces aquatiques. L'ensemble des résultats de laboratoire et des données recueillies sur le terrain indique que le composé est toxique pour de nombreux arthropodes terrestres non visés. Chez l'abeille, la toxicité de contact se traduit par une DL50 de 120 µg/g, la toxicité par voie orale correspondant à une valeur de 187 µg/g. Trois heures après l'épandage de chlordiméform sur de la luzerne, l'exposition de certaines espèces d'abeilles aux résidus encore présents sur les plantes, n'a provoqué aucune mortalité. La CL50 par voie alimentaire varie de >1000 à > 5000/kg de nourriture pour diverses espèces d'oiseaux. 9. Evaluation des risques pour la santé humaine et des effets sur l'environnement On a observé des signes d'intoxication aiguë chez des travailleurs qui, peut-être par suite de l'inobservation des mesures de sécurité, avaient été fortement exposés à du chlordiméform au cours de la préparation ou de l'utilisation de ce produit. Comme, à ce qu'il semble, il n'est plus produit ni utilisé nulle part dans le monde, il ne devrait plus y avoir de cas d'intoxication aiguë. Le risque lié à une exposition chronique et en particulier, le risque de cancer de la vessie, subsistera cependant pendant de nombreuses années. Il faut continuer à effectuer des contrôles sanitaires chez les sujets qui ont subi une exposition notable pour avoir travaillé dans des ateliers de production de chlordiméform ou avoir vécu dans des zones rurales où le produit était largement utilisé. Comme il s'agit d'un produit qui n'est plus en usage, on n'a pas procédé à une évaluation quantitative du risque qu'il représente pour l'environnement. On ne pense pas que celui-ci puisse subir des effets nocifs à long terme qui soient attribuables à l'utilisation antérieure du produit. 10. Conclusions et recommandations Il existe un risque non négligeable que le chlordiméform produise des effets toxiques immédiats ou à plus long terme chez les individus exposés. Les données disponibles accréditent l'idée d'une association entre l'augmentation de l'incidence du cancer de la vessie chez l'homme et l'exposition à la 4-chloro- o-toluidine et, dans une moindre mesure, au chlordiméform. Le chlordiméform ne persiste pas dans l'environnement et il ne devrait donc pas y avoir d'effets nocifs à long terme sur celui-ci qui résulteraient de l'usage antérieur du composé. Il n'est pas recommandé de reprendre la production ou l'usage du chlordiméform dans un but commercial. Les stocks existants doivent être éliminés selon les règles de sécurité. Les personnes exposées au chlordiméform de par leur profession doivent être soumises à des examens cytologiques vésicaux et à une recherche systématique de l'hématurie dans le cadre d'un programme général de dépistage. RESUMEN 1. Identidad, propiedades físicas y químicas y métodos analíticos El clordimeformo es una base de fuerza media que forma sales estables con ácidos fuertes. Tanto el clordimeformo como su sal hidroclorada en estado puro son sólidos cristalinos incoloros. El punto de fusión del clordimeformo (base) es de 32°C, mientras que el de la sal hidroclorada es de 225-227°C. El clordimeformo (base) es poco soluble en agua (250 mg/litro) y fácilmente soluble en disolventes orgánicos, mientras que la sal hidroclorada es fácilmente soluble en agua pero menos soluble en disolventes orgánicos. El clordimeformo (base) tiene una presión de vapor de 48 mPa a 20°C y un log Kow de 2,89. Se dispone de una amplia gama de métodos analíticos para detectar y cuantificar la presencia de clordimeformo en las plantas, el suelo, el agua y la orina. 2. Fuentes de exposición humana y ambiental El clordimeformo no existe en la naturaleza. Se produce comercialmente mediante condensación del reactivo de Vilsmeier (obtenido por reacción de la dimetilformamida con POCl3, SOCl2 o COCl2) con 4-cloro- o-toluidina o bien con o-toluidina y cloración ulterior del producto intermedio resultante. Se ha utilizado como acaricida de amplio espectro y actúa principalmente contra las formas móviles de ácaros y garrapatas, así como contra los huevos y las crisálidas en estado inicial de algunos insectos del orden Lepidóptera. Es activo en la fase de vapor, así como por contacto. Cuando comenzó a utilizarse, se aplicaba a productos de una amplia variedad de cultivos, tales como frutas de pipas, frutas de hueso, berzas, hortalizas, uvas, lúpulo, cítricos, manzanas, peras, cerezas y fresas. También se utilizaba en baños antiparasitarios para combatir las garrapatas del ganado. En los últimos años, su uso se limitaba por lo general al algodón, aunque en algunos países se seguía aplicando al arroz. En la mayoría de los países, su registro se abandonó voluntariamente en 1988/1989. En China dejó de producirse en 1992 y de venderse en 1993. 3. Transporte, distribución y transformación en el medio ambiente El clordimeformo tiene una presión de vapor moderada pero su evaporación de la superficie de las plantas es inferior a la que cabría prever. La estabilidad hidrolítica del clordimeformo depende mucho del pH; es estable en condiciones ácidas pero se hidroliza rápidamente en condiciones alcalinas. El clordimeformo tiene un potencial de adsorción a la materia orgánica disuelta. Hay dispersión del clordimeformo en el suelo, principalmente por acción microbiana y, en menor medida, por hidrólisis química. Pese a la solubilidad del clordimeformo en agua, hay pocos indicios de lixiviación, lo que puede deberse a su adsorción a minerales arcillosos y a la materia orgánica del suelo, así como a su biodegradación. Los principales metabolitos son la N-formil- 4-cloro- o-toluidina y la 4-cloro- o-toluidina. La absorción del clordimeformo por las plantas a partir del suelo es escasa pero detectable, y suficiente para afectar a las plagas que se alimentan de ellas. El clordimeformo aplicado a las hojas sólo tiene una capacidad limitada de penetrar en las capas cuticulares. El clordimeformo se degrada rápidamente en las plantas. Sus principales metabolitos son el demetilclordimeformo, la N-formil-4-cloro- o-toluidina y la 4-cloro- o-toluidina, aunque no todas las plantas estudiadas produjeron 4-cloro- o-toluidina. El clordimeformo y sus metabolitos se dispersan en el suelo conforme a una cinética de primer orden, con una semivida de 20-40 días. Los estudios sobre bioacumulación indican una escasa absorción del clordimeformo por los organismos acuáticos y una rápida depuración de éstos después de haber sido transferidos a un agua limpia. 4. Niveles medioambientales y exposición humana No se han medido los niveles de clordimeformo en el aire ni en el agua. Tras la aplicación de clordimeformo a unos arrozales, en el suelo se hallaron residuos en concentraciones de hasta 2900 µg/kg en los 5 cm primeros de profundidad, y de 150 µg/kg en los 5 cm siguientes. Se establecieron niveles máximos de residuos aplicables a una amplia variedad de productos sin elaborar y, en algunos casos, de residuos trasladados a los alimentos elaborados. Los límites máximos aplicables a los residuos de clordimeformo se han retirado del Codex Alimentarius. Había exposición ocupacional al clordimeformo durante la fabricación, la formulación y la aplicación del producto. En los últimos años la exposición se ha vigilado mediante la determinación de los niveles totales de clordimeformo y de sus metabolitos presentes en la orina, y hay una correlación positiva entre el nivel en la orina y el grado de contaminación cutánea. Entre los trabajadores agrícolas de los algodonales sometidos a una amplia vigilancia de la excreción urinaria de clordimeformo, los niveles más altos de exposición se hallaban en los cargadores, lavadores y mecánicos, y los niveles más bajos en los obreros señalizadores y pilotos. 5. Cinética y metabolismo en animales de laboratorio y en el ser humano Los mamíferos absorben fácilmente el clordimeformo por el tracto gastrointestinal y a través de la piel. Lo excretan rápidamente, alrededor del 80% por la orina y del 10-15% por las heces. Al cabo de unos 10 días se observan niveles bajos de residuos en todos los tejidos y no hay indicios de bioacumulación. Tras la administración cutánea a seres humanos, se observa una excreción rápida semejante por la orina. Varios metabolitos oxidados y conjugados del clordimeformo se excretan por la orina; los principales son el demetilclordimeformo, la N-formil-4-cloro- o-toluidina y la 4-cloro- o-toluidina. En estudios in vitro se han observado los mismos metabolitos, siendo el principal la 4-cloro- o-toluidina. 6. Efectos en mamíferos de laboratorio y en sistemas de pruebas in vitro En ensayos realizados en varias especies, el clordimeformo administrado por vía oral y cutánea ha mostrado tener una toxicidad aguda moderada. Los principales metabolitos han mostrado tener una toxicidad oral baja en ensayos realizados en ratas. El clordimeformo provoca solamente una ligera irritación cutánea y ocular en el conejo. Tras una exposición breve o prolongada de ratones y ratas al clordimeformo o a sus metabolitos pueden observarse cambios asociados al tratamiento en los parámetros hematológicos y, con dosis elevadas, indicios de hiperplasia del epitelio de las vías biliares y de la vejiga. El clordimeformo no aumenta la incidencia de tumores en las ratas. En los ratones, después de administrar a través de la dieta clordimeformo N-formil-4-cloro- o-toluidina o 4-cloro- o- toluidina, se observa, de forma relacionada con la dosis, un aumento de los tumores malignos hemorrágicos de origen vascular clasificados como hemangioendoteliomas malignos, que producen un aumento de la mortalidad asociado con la dosis. El clordimeformo no afecta a los parámetros reproductivos ni tiene potencial teratogénico. Se ha ensayado el clordimeformo en una amplia variedad de pruebas de genotoxicidad in vitro e in vivo. No se han comunicado reacciones positivas a ninguna de esas pruebas, en las que se ensayó clordimeformo en estado puro. Además, se han comunicado varios informes esporádicos y no confirmados de actividad mutagénica inducida por la N-formil-4-cloro- o-toluidina y la 4-cloro- o-toluidina. Un informe describe una inducción de la transformación celular por efecto tanto del clordimeformo como de la 4-cloro- o-toluidina. En el hígado de los ratones y las ratas expuestos se producen enlaces con el ADN. Se ha observado un importante aducto hidrofóbico, en los ratones en niveles mucho mayores que en las ratas. El clordimeformo induce diversos efectos farmacológicos y bioquímicos en los animales, tales como cambios cardiovasculares, hipotermia, hiperexcitabilidad, efectos sobre las funciones visual central y auditiva y modulación de las aminas biogénicas y de las enzimas que metabolizan fármacos. 7. Efectos en el ser humano La intoxicación aguda causa fatiga, náuseas, pérdida del apetito y, en casos más graves, somnolencia, cianosis, micción imperiosa, cistitis, efectos cardiovasculares (taquicardia, bradicardia, alteraciones del ECG), coma y choque. En general se produce una recuperación completa de la intoxicación aguda. Otros síntomas asociados a la exposición crónica al clordime-formo son dolores abdominales, prurito y exantemas (exposición cutánea), así como hematuria macroscópica y microscópica. Se ha comunicado un gran número de casos con síntomas clínicos de exposición crónica tanto entre los obreros de las plantas de producción de clordimeformo como entre los trabajadores agrícolas. Los indicios epidemiológicos relacionados con la exposición ocupacional muestran una fuerte asociación entre la exposición al metabolito 4-cloro- o-toluidina y la incidencia de cáncer de vejiga en el ser humano. Actualmente se dispone de pocos indicios de asocia-ción entre la exposición al clordimeformo y el cáncer de vejiga en el ser humano. 8. Efectos en otros organismos en el laboratorio y en el medio ambiente No se observaron efectos significativos en poblaciones de hongos de la tierra, bacterias o actinomicetos tras la aplicación de clordime-formo al suelo. No existen datos de laboratorio sobre la toxicidad en los invertebrados de agua dulce. El clordimeformo inhibió el crecimiento de larvas de ostras, con una CE50 de 5,7 mg/litro. La CL50 a las 96-h para los camarones rosados, único crustáceo estudiado, fue de 7,1 mg/litro y los valores de la CL50 a las 96-h para los peces oscilaron entre 1 y 54 mg/litro. No se dispone de datos sobre toxicidad acuática crónica. La combinación de datos obtenidos en el laboratorio y sobre el terreno revela que el clordimeformo es tóxico para una amplia gama de artrópodos terrestres no combatidos. Con respecto a las abejas, se ha comunicado una DL50 de toxicidad por contacto de 120 µg/g y una DL50 de toxicidad oral de 187 µg/g. No se produjo mortalidad sobre el terreno tras la exposición de especies de abejas a los residuos presentes en la alfalfa tres horas después del rociado. La CL50 en la dieta de varias especies de pájaros osciló entre >1000 y >5000 mg/kg de dieta. 9. Evaluación de los riesgos para la salud humana y efectos en el medio ambiente La exposición intensa durante la producción o la utilización, debida posiblemente a la insuficiencia de las medidas de seguridad, dio lugar a síntomas de intoxicación aguda en los trabajadores. Como se ha notificado que se ha suspendido la producción y la utilización de clordimeformo en todo el mundo, no deberían producirse nuevos casos de intoxicación aguda. Sin embargo, el riesgo asociado a la exposición crónica, en particular el riesgo de cáncer de vejiga, seguirá siendo preocupante durante muchos años. Debería proseguir el reconoci-miento médico de las personas que han estado muy expuestas en las plantas de producción y en las comunidades rurales donde se haya aplicado extensamente el clordimeformo. Dado que el clordimeformo ha dejado de utilizarse, no se ha realizado ninguna evaluación cuantitativa de los riesgos para el medio ambiente. A largo plazo no se prevén efectos perjudiciales para el medio ambiente como consecuencia de la utilización de clordime-formo en el pasado. 10. Conclusiones y recomendaciones El clordimeformo tiene un potencial significativo para causar tanto toxicidad inmediata como a largo plazo en las personas expuestas. La información de que se dispone actualmente apunta a una asociación entre una mayor incidencia de cáncer de vejiga en el ser humano y la exposición a la 4-cloro- o-toluidina y, en menor medida, al clordimeformo. El clordimeformo no persiste en el medio ambiente, por lo que a largo plazo no se prevén efectos perjudiciales como consecuencia de su utilización en el pasado. Se recomienda que el clordimeformo no se produzca comercialmente ni se utilice en el futuro. Las reservas existentes deberían eliminarse sin correr riesgos. Las personas expuestas profesionalmente al clordimeformo deberían participar en un programa de reconocimiento médico que comprenda citología urinaria y detección de hematuria.
See Also: Chlordimeform (IARC Summary & Evaluation, Volume 30, 1983) Chlordimeform (ICSC)