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    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
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    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

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    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
                       7.2.1.1   Mouse
                       7.2.1.2   Rat
                       7.2.1.3   Dog
               7.2.2. Intubation
                       7.2.2.1   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
                       7.5.1.1   Rat
                       7.5.1.2   Hamster
               7.5.2. Embryotoxicity and teratology
                       7.5.2.1   Rat
                       7.5.2.2   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
                       8.2.3.1   4-Chloro- o-toluidine
                       8.2.3.2   Chlordimeform

    9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD

         9.1. Laboratory experiments
               9.1.1. Microorganisms
               9.1.2. Aquatic organisms
                       9.1.2.1   Plants
                       9.1.2.2   Invertebrates
                       9.1.2.3   Vertebrates
               9.1.3. Terrestrial organisms
                       9.1.3.1   Plants
                       9.1.3.2   Invertebrates
                       9.1.3.3   Vertebrates
         9.2. Field observations
               9.2.1. Microorganisms
               9.2.2. Aquatic organisms

               9.2.3. Terrestrial organisms
                       9.2.3.1   Plants
                       9.2.3.2   Invertebrates
                       9.2.3.3   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

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         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

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    *    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
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         JMPR

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    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 STRUCTURE 1

    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 (œ-glucuronidase, œ-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).

    FIGURE 1

         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.

    7.2.1.1  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.

    7.2.1.2  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 œ-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 œ-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.

    7.2.1.3  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

    7.2.2.1  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

    7.5.1.1  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).

    7.5.1.2  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

    7.5.2.1  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).

    7.5.2.2  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 7.3.1.2. 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.

    FIGURE 2

    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

    8.2.3.1  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.

    8.2.3.2  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

    9.1.2.1  Plants

         There are no data on the effects of chlordimeform on aquatic
    plants.

    9.1.2.2  Invertebrates

         There are no laboratory data on the effects of chlordimeform on
    aquatic invertebrates.

    9.1.2.3  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

    9.1.3.1  Plants

         There are no data available for the effects of chlordimeform on
    plants.

    9.1.3.2  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.

    9.1.3.3  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

    9.2.3.1  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.

    9.2.3.2  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.

    9.2.3.3  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). In 1978, the temporary ADI was reduced to 0-0.0001 mg/kg body
    weight, the temporary MRLs for all crops except cotton and cottonseed
    were withdrawn, and the MRLs for meat and milk of cattle were set at
    the level of detection (FAO/WHO, 1979). In 1979, 1980 and 1985, the
    temporary ADI of 0-0.0001 mg/kg body weight was extended (FAO/WHO,
    1980, 1981, 1986). In 1987, the temporary ADI for chlordimeform was
    withdrawn (FAO/WHO, 1988).

    REFERENCES

    Abo-Khatwa N & Hollingworth RM (1972a) Chlordimeform: The relation of
    the mitochondrial uncoupling to toxicity in the German cockroach. Life
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    RÉSUMÉ

    1.  Identité, propriétés physiques et chimiques et méthodes d'analyse

         Le chlordiméform est une base de force moyenne qui forme des sels
    stables avec les acides forts. A l'état pur, le chlordiméform et son
    chlorhydrate sont des solides cristallins incolores. Le point de
    fusion du chlordiméform base est de 32°C, celui du chlorhydrate étant
    de 225-227°C. La base est légèrement soluble dans l'eau (250 mg/litre)
    et facilement soluble dans les solvants organiques, tandis que le
    chlorhydrate est facilement soluble dans l'eau mais plus difficilement
    dans les solvants organiques. 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:
       Toxicological Abbreviations
       Chlordimeform (ICSC)
       Chlordimeform (WHO Pesticide Residues Series 1)
       Chlordimeform (WHO Pesticide Residues Series 5)
       Chlordimeform (Pesticide residues in food: 1978 evaluations)
       Chlordimeform (Pesticide residues in food: 1979 evaluations)
       Chlordimeform (Pesticide residues in food: 1980 evaluations)
       Chlordimeform (Pesticide residues in food: 1985 evaluations Part II Toxicology)
       Chlordimeform (Pesticide residues in food: 1987 evaluations Part II Toxicology)
       Chlordimeform (IARC Summary & Evaluation, Volume 30, 1983)