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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY








    ENVIRONMENTAL HEALTH CRITERIA 175





    ANTICOAGULANT RODENTICIDES












    This report contains the collective views of an international group of
    experts and does not necessarily represent the decisions or the stated
    policy of the United Nations Environment Programme, the International
    Labour Organisation, or the World Health Organization.

    First draft prepared by Dr M. Tasheva, National Centre of Hygiene,
    Medical Ecology and Nutrition, Sofia, Bulgaria

    Published under the joint sponsorship of the United Nations
    Environment Programme, the International Labour Organisation, and the
    World Health Organization

    World Health Organization
    Geneva, 1995

         The International Programme on Chemical Safety (IPCS) is a joint
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    WHO Library Cataloguing in Publication Data

    Anticoagulant rodenticides.

    (Environmental health criteria ; 175)

    1.Rodenticides  2.Anticoagulants
    3.Occupational exposure  I.Series

    ISBN 92 4 157175 1                 (NLM Classification: WA 240)
    ISSN 0250-863X

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    CONTENTS

    ENVIRONMENTAL HEALTH CRITERIA FOR ANTICOAGULANT RODENTICIDES

    Preamble

    Introduction

    1. SUMMARY

         1.1. General
         1.2. Properties and analytical methods
         1.3. Sources of human and environmental exposure
         1.4. Environmental distribution, levels and exposures
         1.5. Mode of action and metabolism
         1.6. Effects on 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 and conclusion

    2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS

         2.1. Identity
         2.2. Physical and chemical properties
         2.3. Analytical methods

    3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

         3.1. Natural occurrence
         3.2. Anthropogenic sources

    4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION

         4.1. Transport and distribution between media
               4.1.1. Air, water and soil
               4.1.2. Vegetation and wildlife
         4.2. Transformation
               4.2.1. Biodegradation
               4.2.2. Abiotic degradation
                       4.2.2.1   Photolysis
                       4.2.2.2   Hydrolysis

    5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

         5.1. Environmental levels
         5.2. General population exposure
         5.3. Occupational exposure

    6. MODE OF ACTION AND METABOLISM

         6.1. Vitamin K and its antagonists
         6.2. Metabolism
               6.2.1. Absorption, distribution and elimination
               6.2.2. Metabolic transformation
               6.2.3. Retention and turnover

    7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS

         7.1. Acute effects
               7.1.1. Rodent species
               7.1.2. Non-target species
         7.2. Short-term exposure
               7.2.1. Rodent species
               7.2.2. Non-target species
         7.3. Long-term exposure
         7.4. Skin and eye irritation; sensitization
         7.5. Reproductive toxicity and teratogenicity
         7.6. Mutagenicity
         7.7. Factors modifying toxicity
         7.8. Adverse effects in domestic and farm animals
               7.8.1. Domestic animals
                       7.8.1.1   Poisoning incidents
                       7.8.1.2   Diagnosis and treatment of poisoning
               7.8.2. Farm animals

    8. EFFECTS ON HUMANS

         8.1. General population exposure
               8.1.1. Acute poisoning
               8.1.2. Poisoning incidents
               8.1.3. Controlled human studies
         8.2. Monitoring of biological effects
               8.2.1. Effects of short- and long-term exposure
               8.2.2. Epidemiological studies
         8.3. Developmental effects
         8.4. Other adverse effects
         8.5. Methods for assessing absorption and effects of
               anticoagulant rodenticides
         8.6. Treatment of anticoagulant rodenticide poisoning
               8.6.1. Minimizing the absorption
               8.6.2. Specific pharmacological treatment
                       8.6.2.1   Vitamin K1 (phytomenadione)
                       8.6.2.2   Blood components
                       8.6.2.3   Phenobarbital
               8.6.3. Response to therapy

    9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD

         9.1. Laboratory experiments
               9.1.1. Microorganisms
               9.1.2. Aquatic organisms
               9.1.3. Terrestrial organisms
                       9.1.3.1   Acute toxicity
                       9.1.3.2   Primary toxicity
                       9.1.3.3   Secondary toxicity
         9.2. Field observations
               9.2.1. Primary poisonings
               9.2.2. Secondary poisonings

    10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE
         ENVIRONMENT

         10.1. Evaluation of human health risks
         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

    RESUME

    RESUMEN
    

    NOTE TO READERS OF THE CRITERIA MONOGRAPHS

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    Criteria monographs, readers are requested to communicate any errors
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       A detailed data profile and a legal file can be obtained from the
    International Register of Potentially Toxic Chemicals, Case postale
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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

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    WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR ANTICOAGULANT
    RODENTICIDES

     Members

    Dr N. Gratz, Commugny, Switzerland

    Mr P. Howe, Institute of Terrestrial Ecology, Huntingdon,
       Cambridgeshire, United Kingdom

    Dr W. Jacobs, Office of Pesticide Programs, US Environmental   
       Protection Agency, Washington, USA

    Mrs M. Palmborg, Swedish Poison Information Centre, Stockholm, Sweden

    Dr A.F. Pelfrène, Technology Sciences Group (TSG) International Inc.,
       Brussels, Belgium  (Chairman)

    Mr D. Renshaw, Health Aspects of Environment and Food (Medical),
       Department of Health, London, United Kingdom

    Dr M. Tasheva, National Centre of Hygiene, Medical Ecology and
       Nutrition, Sofia, Bulgaria  (Rapporteur)

    Dr C. Vermeer, University of Limburg, Maastricht, Netherlands

     Observers

    Dr A. Buckle, ZENECA Public Health, Haslemere, Surrey, United Kingdom
        (Representative of GIFAP)

    Dr Y. Cohet, Lipha SA, Lyon, France  (Representative of GIFAP)

     Secretariat

    Dr R. Plestina, International Programme on Chemical Safety, World
       Health Organization, Geneva, Switzerland  (Secretary)

    ENVIRONMENTAL HEALTH CRITERIA FOR ANTICOAGULANT RODENTICIDES

         A WHO Task Group on Environmental Health Criteria for
    Anticoagulant Rodenticides met in Geneva from 14 to 18 November 1994.
    Dr R. Plestina, IPCS, welcomed the participants on behalf of
    Dr M. Mercier, Director of the IPCS, and the three IPCS cooperating
    organizations (UNEP/ILO/WHO).

       The first draft was prepared by Dr M. Tasheva of the National
    Centre of Hygiene, Medical Ecology and Nutrition, Sofia, Bulgaria. 
    The second draft was prepared by Dr R. Plestina, incorporating
    comments received following the circulation of the first draft to the
    IPCS contact points for Environmental Health Criteria monographs.  The
    Task Group reviewed and revised the draft document and made an
    evaluation of risks for human health and the environment from exposure
    to anticoagulant rodenticides.  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

    AAPCC    American Association of Poison Control Centers
    DT50     degradation time for 50% of a compound
    EC50     median effect concentration
    FD       fluorescence detection
    GC       gas chromatography
    HPLC     high-performance liquid chromatography
    I50      concentration of an inhibitor causing 50% inhibition of an
             enzyme under given conditions
    IUPAC    International Union of Pure and Applied Chemistry Kal
             adsorption coefficient
    LD50     median lethal dose
    MS       mass spectrometry
    MTD      maximum tolerated dose
    NOAEL    no-observed-adverse-effect level
    NOEL     no-observed-effect level
    PT       prothrombin time
    PTT      partial thromboplastin time
    WISN     warfarin-induced skin necrosis

    INTRODUCTION

         The anticoagulants included in this review are those that are
    used as rodenticides.  The development of coumarin anticoagulants
    occurred during the Second World War and they were introduced as
    effective antithrombotic agents for treatment of thromboembolic
    disease in humans.  Warfarin has been used both as a drug and a
    rodenticide, and has been extensively evaluated.  Several
    hydroxycoumarin and indandione derivatives have been synthesized and
    introduced as effective rodenticides.  They act by interfering with
    the blood coagulation mechanism.

         The appearance of rat strains resistant to warfarin and some
    other anticoagulants has stimulated the development of more potent,
    second-generation anticoagulants, some of which are also "single dose"
    anticoagulants or "superwarfarins".

         Many anticoagulant rodenticides are known, but it is not the aim
    of this monograph to include all available information on each
    compound.  The purpose is to describe the general characteristics of
    anticoagulants, using suitable illustrations to indicate their impact
    on humans and the environment.

         A distinction needs to be made between the characteristics of the
    technical compounds and those of their formulated products concerning
    the risks that their use poses to human health and the environment.

    1.  SUMMARY

    1.1  General

         The anticoagulants described in this monograph are those used
    mainly in agriculture and urban rodent control.  Warfarin, the first
    widely used anticoagulant rodenticide, was introduced as an effective
    agent for treatment of thromboembolic disease in humans.

         Based on their chemical structure, anticoagulant rodenticides may
    be grouped into two categories, hydroxycoumarins and indandiones,
    although their mechanisms of action are similar.

    1.2  Properties and analytical methods

         Anticoagulant rodenticides come in a solid crystalline or powder
    form, and are slightly soluble in water.  Most of them are stable
    under normal storage conditions.

         Most of the procedures for the determination of anticoagulant
    rodenticides are based on high-performance liquid chromatography.

    1.3  Sources of human and environmental exposure

         First-generation hydroxycoumarins were introduced as rodenticides
    in the late 1940s.  The appearance of resistance to warfarin and other
    first-generation anticoagulants led to the development of more potent,
    second-generation anticoagulants.  The concentrations of active
    ingredients in baits vary according to the efficacy of the
    rodenticides.

    1.4  Environmental distribution, levels and exposures

         Anticoagulant rodenticides are used mainly as bait formulations. 
    Since their volatility is low, concentrations in the air will be
    negligible.  As they are only slightly soluble in water, their use is
    unlikely to be a source of water contamination.

         Since anticoagulant rodenticides are not intended for direct
    application to growing crops, no residues in plant foodstuffs are
    expected.

         Non-target vertebrates are exposed to rodenticides primarily
    through consumption of bait and secondarily from consumption of
    poisoned rodents.  Small pellets and whole grain baits are highly
    attractive to birds.

         Warfarin is used as a therapeutic agent for thromboembolic
    disease.

         There is a potential for occupational exposure to anticoagulant
    rodenticides during manufacture, formulation and bait application, but
    data on the levels of exposure are not available.

    1.5  Mode of action and metabolism

         Anticoagulant rodenticides are vitamin K antagonists.  The main
    site of their action is the liver, where several of the blood
    coagulation precursors undergo vitamin-K-dependent posttranslation
    processing before they are converted into the respective procoagulant
    zymogens.  The point of action appears to be the inhibition of K1
    epoxide reductase.

         Anticoagulant rodenticides are easily absorbed from the
    gastrointestinal tract, and may also be absorbed through the skin and
    respiratory system.  After oral administration, the major route of
    elimination in various species is through the faeces.

         The metabolic degradation of warfarin and indandiones in rats
    mainly involves hydroxylation.  However, the second-generation
    anticoagulants are mainly eliminated as unchanged compounds.  The low
    urinary excretion precludes isolation of metabolites from the urine.

         The liver is the main organ for accumulation and storage of
    rodenticide anticoagulants.  Accumulation also occurs in the fat.

    1.6  Effects on mammals and in vitro test systems

         Signs of poisoning in rats and mice are those associated with
    increased bleeding tendency.

         There is wide variation in the LD50 of anticoagulant
    rodenticides, toxicity being greatest by the oral route.  Dermal and
    inhalation toxicities of anticoagulants are also high.

         Some anticoagulants show a similar range of acute toxicity for
    non-target mammals as for target rodents, but toxicity spectra for
    anticoagulants may vary between species.

         Following repeated oral administration in rats, the main effects
    seen are those associated with the anticoagulant action.

         There are few data available on repeated exposure of non-rodent
    species.

         One study on warfarin in rats has indicated developmental
    effects.  Otherwise, there is no convincing evidence that
    anticoagulants are teratogenic in experimental animals.

         There is no evidence to suggest that any anticoagulant
    rodenticides are mutagenic, but there are insufficient data available
    on individual compounds to demonstrate an absence of mutagenicity. 
    Strain, sex and diet are important factors modifying the toxicity of
    anticoagulants in rodents.

         Poisoning incidents in domestic animals after consumption of
    anticoagulant baits have been reported.  Fatalities and severe
    clinical syndromes are generally due to the second-generation
    anticoagulants.  The major difference between warfarin and the other
    anticoagulants (both indandiones and second-generation
    hydroxycoumarins) is that they have a longer retention time in the
    body and consequently a more prolonged effect than warfarin. 
    Therefore in cases of poisoning, antidote treatment with vitamin K1
    needs to be continued for a longer period.

    1.7  Effects on humans

         Many poisoning incidents (both intentional and unintentional)
    have been reported.  A few cases of intoxications from occupational
    exposure to anticoagulants have also occurred. Symptoms of acute
    intoxication by anticoagulant rodenticides range from increased
    bleeding tendency in minor or moderate poisoning to massive
    haemorrhage in more severe cases.  The signs of poisoning develop with
    a delay of one to several days after absorption.

         Warfarin is associated in humans with the induction of
    developmental malformations when taken as a therapeutic agent during
    pregnancy.  No cases of developmental defects following the use of
    anticoagulants as rodenticides have been reported.

         The plasma prothrombin concentration is one guide to the severity
    of intoxication.  This is a more sensitive indication than overall
    tests such as prothrombin time.  In repeated occupational exposure,
    direct measurement of either trace amounts of circulating
    descarboxyprothrombin or circulating vitamin K 2,3-epoxide may provide
    a more sensitive assessment.

         Treatment of anticoagulant poisoning is graded according to the
    severity of intoxication.  Specific pharmacological treatment consists
    of parenteral administration of vitamin K1 with, in serious cases,
    co-administration of blood components.  Measurement of prothrombin
    time helps to determine the effectiveness and required duration of
    treatment.

    1.8  Effects on other organisms in the laboratory and field

         The possible effects of anticoagulant rodenticides on non-target
    organisms can be considered to fall into two categories: primary
    (direct poisoning through consumption of bait) and secondary (through
    consumption of poisoned rodents).

         In the form of the technical product, anticoagulants are highly
    toxic to fish.  As bait formulations they are unlikely to present any
    hazard because of their low water solubility.  For this reason, they
    will not be available to fish unless misused.

         Bird species vary in their susceptibility to anticoagulant
    rodenticides.  It is difficult to assess the risks to birds resulting
    from direct consumption because most published studies consist of
    toxicity trials in laboratory conditions.  The attractiveness of whole
    grain bait to small birds increases the risk in field conditions.

         Secondary toxicity laboratory studies with wildlife have shown
    that captive predators can be intoxicated by no-choice feeding with
    anticoagulant-poisoned or -dosed prey.  Some deaths of predators in
    the field have been reported.

    1.9  Evaluation and conclusion

         Anticoagulant rodenticides disrupt the normal blood-clotting
    mechanisms, resulting in increased bleeding tendency and,
    eventually, profuse haemorrhage.

         Unintentional exposure of the general population to anticoagulant
    rodenticides is unlikely.

         Occupational contact is a potential source of significant
    exposure.  It may occur during manufacture and formulation as well as
    during bait preparation and application.

         Anticoagulant rodenticide compounds are readily absorbed from the
    gastrointestinal tract, and through the skin and respiratory system. 
    The liver is the major organ for accumulation and storage.  The plasma
    prothrombin concentration is a suitable guide to the severity of acute
    intoxication and to the effectiveness and required duration of the
    therapy.

         The specific antidote is vitamin K1.

         The major difference between first- and second-generation
    anticoagulant rodenticides is that the latter have longer body
    retention and therefore tend to lead to a longer period of bleeding.

         Most anticoagulants are stable under conditions of normal use. 
    Their low water solubility and low concentration in baits make them
    unlikely to be a source of water contamination.  They also appear to
    bind quickly to soil particles, with very slow desorption and no
    leaching properties.

         Non-target organisms are potentially at risk from direct
    consumption of baits (primary hazard) and from eating poisoned rodents
    (secondary hazard).

    2.  IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS

    2.1  Identity

         Based on their chemical structure, anticoagulant rodenticides may
    be grouped into two categories:

    *    hydroxycoumarins:

    FIGURE 2

    *    indandiones:

    FIGURE 3

         The common and chemical names of the rodenticides are given in
    Table 1.  Trade names, chemical structures, RTECS and CAS numbers,
    molecular formulae and relative molecular masses are listed in
    Table 2.

    2.2  Physical and chemical properties

         Anticoagulant rodenticides are solids (crystalline or powders),
    slightly soluble in water (Table 3) and readily soluble in acetone. 
    Most of them are stable under normal storage conditions.


        Table 1.  Identity of anticoagulant rodenticides
                                                                                                                                                

    Common name                         CAS name                                                                IUPAC name
                                                                                                                                                

    First generation hydroxycoumarins

    Coumachlor       3-[1-(4-chlorophenyl)-3 oxobutyl]-4-hydroxy-                   3-[1-(4-chlorophenyl)-3-oxobutyl]-4-hydroxycoumarin
                     2H-1-benzopyran-2-one

    Coumafuryl       3-[1-(2-furanyl)-3 oxobutyl]-4-hydroxy-                        3-[1-(2-furyl)-3-oxobutyl]-4-hydroxycoumarin
                     2H-1-benzopyran-2-one

    Coumatetralyl    4-hydroxy-3-(1,2,3,4-tetrahydro-1-naphthalenyl)-               4-hydroxy-3-(1,2,3,4-tetrahydro-1-naphthyl) coumarin
                     2H-1-benzopyran-2-one

    Warfarin         4-hydroxy-3-(3-oxo-1-phenylbutyl-2H-1-benzopyran-2-one         (RS)4-hydroxy-3-(3-oxo-1-phenylbutyl) coumarin

    Second generation hydroxycoumarins

    Brodifacoum      3-[3-(4'-bromo-[1,1'-biphenyl]-4-yl)-1,2,3,4-tetrahydro-       3-[3-(4'-bromobiphenyl-4-yl)-1,2,3,4-tetrahydro-
                     1-naphthalenyl]-4-hydroxy-2H-1-benzopyran-2-one                1-naphthyl]-4-hydroxycoumarin

    Bromadiolone     3-[3-(4'-bromo-[1,1'-biphenyl]-4-yl)-3-hydroxy-1-              3-[3-(4'-bromobiphenyl-4-yl)-3-hydroxy-1-phenylpropyl]-
                     phenylpropyl]-4-hydroxy-2H-1-benzopyran-2-one                  4-hydroxycoumarin

    Difenacoum       3-[3-(1,1'-biphenyl)-4-yl-1,2,3,4-tetrahydro-1-                3-(3-biphenyl-4-yl-1,2,3,4-tetrahydro-1-naphthyl)-
                     naphthalenyl]-4-hydroxy-2H-1-benzopyran-2-one                  4-hydroxycoumarin

    Difethialone     3-[3-(4-bromo-[1,1'-biphenyl]-4-yl)-1,2,3,4-tetrahydro-        3-[1RS,3RS;1RS,3SR)-3-(4'-bromobiphenyl-4-yl)-1,2,3,4-
                     1-naphthalenyl]-4-hydroxy-2H-1-benzothiopyran-2-one            tetrahydro-1-naphthyl]-4-hydroxy-1-benzothi-in-2-one

    Flocoumafen      4-hydroxy-3-[1,2,3,4-tetrahydro-3-[4-[-(trifluoromethyl)       4-hydroxy-3-[1,2,3,4-tetrahydro-3-[4-
                     phenyl]methoxy]phenyl-1-naphthalenyl]-2H-1-benzopyran-2-one    (4-trifluoromethylbenzyloxy)phenyl]-1-naphthyl] coumarin
                                                                                                                                                

    Table 1 (contd).
                                                                                                                                                

    Common name                         CAS name                                                                IUPAC name
                                                                                                                                                

    Indandione derivatives

    Chlorophacinone  2-[(4-chlorophenyl)phenylacetyl]-1H-indene-1,3 (2H)-dione      2-[2-(4-chlorophenyl)-2-phenylacetyl]indan-1,3-dione

    Diphacinone      2-(diphenylacetyl)-1H-indene-1,3 (2H)-dione                    2-(diphenylacetyl)indan-1,3-dione

    Pindone          2-(2,2-dimethyl-1-oxopropyl)-1H-indene-1,3 (2H)-dione          2-pivaloylindan-1,3-dione

    Valone           2-(3-methyl-1-oxopropyl)-1H-indene-1,3 (2H)-dione              2-isovaleryl-1,3-indandione
                                                                                                                                                

    Table 2.  Names, structures and identification details
                                                                                                                                                

    Common name         Trade/other              Chemical structure     RTECS          CAS             Molecular        Relative molecular
                        names                                           number         number          formula          mass
                                                                                                                                                

    Brodifacoum         Finale                                          GN4934750      56073-10-0      C31H23BrO3       523.4
                        Folgorat
                        Havoc
                        Klerat
                        Matikus
                        Mouser
                        Ratak +
                        Rodend
                        Talon
                        Volak
                        Volid

                                                 FIGURE 4
                                                                                                                                                

    Table 2 (cont'd)
                                                                                                                                                

    Common name         Trade/other              Chemical structure     RTECS          CAS             Molecular        Relative molecular
                        names                                           number         number          formula          mass
                                                                                                                                                

    Bromadiolone        Apobas, Bromard,                                GN4934700      28772-56-7      C30H23BrO4       527.4
                        Bromorat, Bromatrol,
                        Contrac, Deadline,
                        Hurex, Lanirat,
                        Maki, Morfaron,
                        Musal, Ramortal,
                        Ratimon, Rodine-c,
                        Slaymor, Super-caid,
                        Topidon

                                                 FIGURE 5
                                                                                                                                                

    Table 2 (cont'd)
                                                                                                                                                

    Common name         Trade/other              Chemical structure     RTECS          CAS             Molecular        Relative molecular
                        names                                           number         number          formula          mass
                                                                                                                                                

    Chlorophacinone     Caid                                            NK5335000      3691-35-8       C23H15ClO3       374.8
                        Delta
                        Drat
                        Lepit
                        Liphadione
                        Microzul
                        Muriol
                        Patrol
                        Quick
                        Raviac
                        Redentin OC
                        Rozol
                        Saviac

                                                 FIGURE 6
                                                                                                                                                

    Table 2 (cont'd)
                                                                                                                                                

    Common name         Trade/other              Chemical structure     RTECS          CAS             Molecular        Relative molecular
                        names                                           number         number          formula          mass
                                                                                                                                                

    Coumachlor          Ratilan                                         GN4830000      81-82-3         C19H15ClO4       342.8
                        Tomorin
                        (Discontinued by
                        Ciba-Geigy in 1984)

                                                 FIGURE 7
                                                                                                                                                

    Table 2 (cont'd)
                                                                                                                                                

    Common name         Trade/other              Chemical structure     RTECS          CAS             Molecular        Relative molecular
                        names                                           number         number          formula          mass
                                                                                                                                                

    Coumafuryl          Fumarin                                         GN4850000      117-52-2        C17H14O5         298.3
                        (Discontinued by
                        Rhône-Poulenc)
                        Fumasol
                        Kill-ko rat
                        Krumkil
                        Kumatox
                        Lurat
                        Mouse blues
                        Ratafin
                        Rat-a-way

                                                 FIGURE 8
                                                                                                                                                

    Table 2 (cont'd)
                                                                                                                                                

    Common name         Trade/other              Chemical structure     RTECS          CAS             Molecular        Relative molecular
                        names                                           number         number          formula          mass
                                                                                                                                                

    Coumatetralyl       Racumin                                         GN7630000      5836-29-3       C19H16O3         292.4
                        Raukumin 57
                        Rodentin

                                                 FIGURE 9
                                                                                                                                                

    Table 2 (cont'd)
                                                                                                                                                

    Common name         Trade/other              Chemical structure     RTECS          CAS             Molecular        Relative molecular
                        names                                           number         number          formula          mass
                                                                                                                                                

    Difenacoum          Compo                                           GN4934500      56073-07-5      C31H24O3         444.5
                        Diphenacoum
                        Matrak
                        Neosorexa
                        Rastop
                        Ratak
                        Ratrick
                        Silo

                                                 FIGURE 10
                                                                                                                                                

    Table 2 (cont'd)
                                                                                                                                                

    Common name         Trade/other              Chemical structure     RTECS          CAS             Molecular        Relative molecular
                        names                                           number         number          formula          mass
                                                                                                                                                

    Difethialone        Baraki                                          DM0013800      104653-34-1     C31H23BrO2S      539.5
                        Frap
                        Quell

                                                 FIGURE 11
                                                                                                                                                

    Table 2 (cont'd)
                                                                                                                                                

    Common name         Trade/other              Chemical structure     RTECS          CAS             Molecular        Relative molecular
                        names                                           number         number          formula          mass
                                                                                                                                                

    Diphacinone         Diphacine                                       NK5600000      82-66-6         C23H16O3         340.4
                        Gold Crest
                        Kill-ko rat killer
                        Pid
                        Promar
                        Ramik
                        Ratindan 1

                                                 FIGURE 12
                                                                                                                                                

    Table 2 (cont'd)
                                                                                                                                                

    Common name         Trade/other              Chemical structure     RTECS          CAS             Molecular        Relative molecular
                        names                                           number         number          formula          mass
                                                                                                                                                

    Flocoumafen         Stratagem                                       DJ3100300      90035-08-8      C33H25F3O4       542.6
                        Storm

                                                 FIGURE 13
                                                                                                                                                

    Table 2 (cont'd)
                                                                                                                                                

    Common name         Trade/other              Chemical structure     RTECS          CAS             Molecular        Relative molecular
                        names                                           number         number          formula          mass
                                                                                                                                                

    Pindone             Pivaldione                                      NK6300000      83-26-1         C14H14O3         230.3
                        Pival
                        Pivalyn
                        Tri-ban

                                                 FIGURE 14
                                                                                                                                                

    Table 2 (cont'd)
                                                                                                                                                

    Common name         Trade/other              Chemical structure     RTECS          CAS             Molecular        Relative molecular
                        names                                           number         number          formula          mass
                                                                                                                                                

    Valone              Motomco trading                                 NK5775000      83-28-3         C14H14O3         230.3
                        powder

                                                 FIGURE 15
                                                                                                                                                

    Table 2 (cont'd)
                                                                                                                                                

    Common name         Trade/other              Chemical structure     RTECS          CAS             Molecular        Relative molecular
                        names                                           number         number          formula          mass
                                                                                                                                                

    Warfarin            Arthrombine-K                                   GN4550000      81-81-2         C19H16O4         308.4
                        Dethmore
                        Panwarfin
                        Warfarat
                        Warfarin +
                        Warficide
                        Zoocoumarin

                                                 FIGURE 16
                                                                                                                                                

    Table 3.  Water solubility and vapour pressure of various anticoagulant rodenticides
                                                                                                                                                

    Rodenticide                                         Solubility                                      Vapour pressure
                                                                                                                                  
                       in water (mg/litre)          at temperature (°C)    at pH               mPa              at temperature (°C)
                                                                                                                                                

    Brodifacoum           < 10                             20               7                 < 0.13                  25
    Bromadiolone          19                               20                                 0.002                   20
    Chlorophacinone       100                              20                                 negligible              20
    Coumachlor            0.5                              20               4.5               < 10                    20
    Coumatetralyl         4                                20               4.2               8.5 × 10-6              20
                          20                               20               5
                          425                              20               7
    Difenacoum            < 10                             20               7                 0.16                    45
    Difethialone          0.39                             25                                 0.074                   25
    Diphacinone           0.3                                                                 13.7 × 10-6             25
    Flocoumafen           1.1                              22                                 0.133 × 10-6            25
    Pindone               18                               25                                 very low                25
    Warfarin              practically insoluble
                                                                                                                                                
    

    2.3  Analytical methods

         Most of the procedures for the determination of anticoagulant
    rodenticides are based on high-performance liquid chromatography
    (Hunter, 1983; Hoogenboom & Rammell, 1983; Murphy et al., 1989;
    O'Bryan & Constable, 1991; Chalermchaikit et al., 1993; Kelly et al.,
    1993).

         Warfarin is an acid which, in its hydrogenated form, is
    practically insoluble in distilled water.  At neutral or higher pH,
    however, it is ionized and as such it readily dissolves in water.  In
    addition, compounds contaminating the water (such as proteins or
    detergents) may substantially increase the solubility of warfarin.

         Hunter (1983) developed a multi-residue method for the
    determination of warfarin, coumatetralyl, bromadiolone, difenacoum and
    brodifacoum in animal tissues by high-performance liquid
    chromatography with fluorescence detection.  A chloroformacetone (1:1)
    mixture was significantly better than chloroform for the extraction of
    residues of these rodenticides from liver tissues.  Detection limits
    in animal tissues of 2 µg/kg for coumatetralyl, difenacoum and
    brodifacoum, 10 µg/kg for bromadiolone, and 20 µg/kg for warfarin
    could be routinely achieved.

         Felice et al. (1991) developed a reversed-phase liquid
    chromatographic method with fluorescence detection for multicomponent
    determination of the above-mentioned five rodenticides in blood serum
    with detection limits of 10 to 20 ng/ml.  Acetonitrile was used for
    the extraction.

         Braselton et al. (1992) developed a special method for confirming
    the presence of indandione rodenticides (diphacinone and
    chlorophacinone) in intoxicated domestic animals by using mass
    spectrometry/mass spectrometry with collision-activated dissociation. 
    More details of analytical methods for individual rodenticides are
    given in Table 4.


        Table 4.  Methods for the determination of anticoagulant rodenticides
                                                                                                                                                

    Sample type          Extraction                     Analytical   Limit of            Rodenticide                       Reference
                                                        method       detection
                                                                                                                                                

    Animal tissues    Chloroform-acetone (1:1)          HPLC/FD      2 µg/kg        coumatetralyl, difenacoum,         Hunter (1983)
    brodifacoum
                                                                     10 µg/kg       bromadiolone                       Hunter (1983)
                                                                     20 µg/kg       warfarin                           Hunter (1983)

    Animal tissues    Chloroform-acetone (1:1)          HPLC/FDa     10 µg/kg       warfarin                           Hunter (1985)
                                                                     2 µg/kg        other rodenticides                 Hunter (1985)

    Serum             Acetonitrile and diethyl ether    HPLC         10 µg/litre    brodifacoum, coumatetralyl,        Felice et al. (1991)
                                                                                    difenacoum
                                                                     20 µg/litre    bromadiolone, warfarin             Felice et al. (1981)

    Serum             Acetonitrile and diethyl ether    HPLC         1 µg/litre     brodifacoum                        Felice & Murphy (1989)

    Serum             twice with diethyl ether and      HPLC/FD      3 µg/litre     brodifacoum                        Murphy et al. (1989)
                      twice with acetonitrile-ether
                      (1:1)
                                                                                                                                                

    Table 4 (contd).
                                                                                                                                                

    Sample type          Extraction                     Analytical   Limit of            Rodenticide                       Reference
                                                        method       detection
                                                                                                                                                

    Plasma            Acetonitrile-ethyl ether (9:1)    HPLC/FD      2 µg/litre     brodifacoum; no interference       O'Bryan & Constable
                                                                                    with bromadiolone,                 (1991)
    Liver tissue                                                     5 µg/kg        coumarin, difenacoum,
                                                                                    diphacinone, warfarin
                                                                                    and vitamin K1

    Liver tissue      Chloroform and acetone            GC/MS        60 µg/kg       protocol did not differentiate     Ray et al. (1989)
                                                                                    between brodifacoum and
                                                                                    bromadiolone
                                                                                                                                                

    a   Post-column pH-switching fluorescence detection
    

    3.  SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

    3.1  Natural occurrence

         Anticoagulant rodenticides do not occur naturally in the
    environment, although some plants do contain coumarinic derivatives. 
    Huebner & Link (1941), Overman et al. (1944) and Alstad et al. (1985)
    described the anticoagulant properties of dicumarol found in spoiled
    sweet clover and in connection with haemorrhagic disease in cattle.

    3.2  Anthropogenic sources

         Anticoagulant rodenticides are used worldwide, but figures for
    the total world production are not available.

         First-generation hydroxycoumarins were introduced as rodenticides
    in the late 1940s.  The appearance of resistance to warfarin and other
    early anticoagulant rodenticides stimulated the development of
    second-generation anticoagulants.  About 95% of all commensal rodent
    control in the USA is carried out with anticoagulants (Marsh, 1985a). 
    More than 50% of rodenticides used by professional pest controllers in
    the USA contain brodifacoum (Dubock, 1986).

         Depending on the toxicity of the rodenticide, the concentration
    of the active ingredient varies from 0.005 to 0.05% for indandiones
    and second-generation hydroxycoumarins and from 0.025 to 0.05% for
    first-generation anticoagulants.

         Anticoagulant rodenticides are available in a variety of
    different formulations, including paraffin wax blocks, whole grain
    baits, pelleted baits and tracking powder (FAO, 1979).  Baits are the
    most widely used formulations for rodent control.

         Some manufacturers have added bittering agents, such as Bitrex
    (denatonium benzoate), to anticoagulant baits.  According to Kaukeinen
    & Buckle (1992), adult humans found wax-block and pelleted placebo
    baits containing denatonium benzoate (10 mg/kg) to be unpalatable. 
    However, the concentration of Bitrex cannot be increased to levels
    that would make baits unpalatable to target rodents, and there is no
    evidence that concentrations of Bitrex that target rodents readily
    accept will deter bait-eating by non-target animals or by children
    under 14 months of age.

    4.  ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION

    4.1  Transport and distribution between media

    4.1.1  Air, water and soil

         Since anticoagulant rodenticides are generally used as bait
    formulations and have low volatility, increased levels in the air are
    unlikely.  As mentioned in section 2.2, most anticoagulants are
    slightly soluble in water and therefore their use is unlikely to be a
    source of water pollution.

         Newby & White (1978) studied the adsorption and desorption of
    14C-brodifacoum in soil under laboratory conditions. Adsorption
    coefficients (kd) for course sand (pH 6.6), sandy clay loam (pH 7.1)
    and calcareous sandy loam (pH 7.6) were 625, 1320 and 1180,
    respectively, indicating strong adsorption to soil particles. 
    Adsorption equilibria were established fairly rapidly with the large
    water:soil ratios used and despite very low brodifacoum water
    solubility.  Desorption was reported to be very slow and much less
    than that required for a reversible interaction.

         Lewis (1992b) applied 14C-difenacoum at 0.2 mg/kg (dry weight)
    to a sandy soil with low humous content.  After 142 days of incubation
    (the approximate half-life of difenacoum in this soil type), two soil
    samples were transferred to the top of soil columns.  The columns were
    eluted with deionized water at a rate and amount equivalent to
    approximately 200 mm of rain falling onto the soil surface area
    (91.6 cm2) for 50 h.  The percentages of applied radioactivity
    present in the leachates were 0.41 and 0.47%, representing only a very
    small amount of leaching under these test conditions.

         The leaching characteristics of aged soil residues of 14C-
    brodifacoum in four soil types were investigated.  14C-
    Brodifacoum was applied to soil at a nominal application rate of
    0.4 mg/kg and incubated under aerobic conditions for 30 days.  Samples
    were taken and transferred to soil columns.  After leaching, most of
    the radioactivity applied to the soil was recovered in the top segment
    of each column.  No detectable levels of 14C residues were found in
    the leachates.  The results indicated that 14C-brodifacoum was
    effectively immobile in all the soils tested (Jackson & Hall, 1992).

         A study was carried out with 14C-bromadiolone in four types of
    soil.  With a soil rich in clay and organic compounds, bromadiolone
    stayed in the superficial layer and scarcely moved.  However, in soil
    poor in clay and organic compounds, 67% of the added bromadiolone was
    eluted (Spare et al., 1980).

    4.1.2  Vegetation and wildlife

         Since anticoagulant rodenticides are not intended for direct
    application to growing crops, no residues in plant food stuffs are
    expected.  Unlike conventional crop protection products, which must be
    applied over relatively large crop areas, rodenticides are applied to
    discrete sites in the form of low concentration baits.  Even if the
    bait is spilled, it will not be taken up by plants.

         Small pellets and whole grain baits are highly attractive to
    birds and other non-target vertebrates.  The formulation in wax blocks
    consequently decreases the risk of primary poisoning of non-target
    species.

         Rodenticides may present a risk not only of primary poisoning
    (from direct consumption of the bait) but also of secondary poisoning
    (from consumption of poisoned rodents), in spite of the fact that many
    of the target rodents die below ground in their burrows (Gorenzel et
    al., 1982).  Commensal and wild rodents poisoned by anticoagulants may
    lead to the death of cats, pigs, foxes and birds of prey.  The risk of
    secondary poisoning depends mainly on the extent to which predators
    feed on the target animals (Dubock, 1986).

    4.2  Transformation

    4.2.1  Biodegradation

         Coveney & Forbes (1987) studied the degradation of flocoumafen in
    rat carcasses, rat faeces, loose grain and wax block baits placed on
    small soil plots.  Overall losses of flocoumafen ranged from 85% to
    95% over the 12-month study.  The majority of the rodenticide present
    in samples collected after 4 months was found in the upper 15 cm of
    the soil.  Only very small quantities were found in the lower soil
    layers.

         The degradation of 14C-difenacoum was studied in two standard
    soils under controlled conditions for a period of 108 days.
    Degradation time (DT50) values for the two soils were 146 and 439
    days, indicating that difenacoum is a relatively long-lived compound
    in soils (Lewis, 1992a).

         Hall & Priestley (1992) monitored the metabolism of 14C-
    brodifacoum in soil under aerobic conditions after applying it
    at a nominal rate of 0.4 mg/kg and incubating for up to 52 weeks.  A
    mean total of 35.8% of the applied radioactivity was recovered as
    14CO2 within the test period.  14C-Brodifacoum was the major
    radiolabelled component in the soil extracts throughout the 52 weeks. 
    Under the conditions of the study the half-life of brodifacoum was
    calculated to be 157 days.

         A study was carried out with 14C-bromadiolone in four types of
    soil.  The rodenticide was degraded significantly with half-lives
    ranging from 1.8 to 7.4 days (Wölkl & Galicia, 1992).

    4.2.2  Abiotic degradation

    4.2.2.1  Photolysis

         A photolysis study was carried out with 14C-bromadiolone
    (1 mg/litre) in a solution at pH 7.3 (Spare, 1982).  The rodenticide
    was very quickly degraded by exposure to artificial sunlight with a
    half-life of 2.1 h.

         The photolytic stability of 14C-difenacoum was investigated in
    sterile buffered aqueous solutions of pH 5, 7 and 9 over a 24-h
    irradiation period.  The photolytic half-lives for total difenacoum
    were calculated to be 3.26, 8.05 and 7.32 h at pH 5, 7 and 9,
    respectively (Hall et al., 1992).

    4.2.2.2  Hydrolysis

         Lewis (1992c) studied the stability of 14C-difenacoum in
    sterile buffered aqueous solutions of pH 5, 7 and 9.  No hydrolysis
    was observed at pH 5, at pH 7 there was very slow hydrolysis
    (half-life estimated to be 847-1332 days), and at pH 9 the half-life
    was estimated to be 77-85 days.

         Jackson et al. (1991) studied the hydrolytic stability of
    14C-brodifacoum (0.04 mg/kg) in sterile buffered aqueous solutions
    at pH 5, 7 and 9 over a 30-day period.  The hydrolytic half-life of
    brodifacoum at pH 7 and 9 was found to be much greater than 30 days,
    but precise calculation was not possible because the degradation seen
    after one day did not continue.

         Spare (1992) demonstrated that 14C-bromadiolone was slowly
    hydrolysed in pH 5 buffer, with an estimated half-life of 392 days. 
    No degradation was observed at pH 7 and 9.

         In the absence of a co-solvent, bromadiolone has a half-life of
    67 days at pH 7 and 20°C (Morin, 1988).  Degradation is more
    significant in the presence of H3O+ ions, in saline water and at
    increased temperatures.

    5.  ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

    5.1  Environmental levels

         There is no information available on concentrations of
    anticoagulant rodenticides in air, water and soil.

         Since anticoagulant rodenticides are not intended for direct
    application to growing crops, no residues in plants are expected.

         Residues of difenacoum and brodifacoum were detected in the
    bodies of 15 out of a total of 145 dead barn owls  (Tylo alba) received
    from various parts of the United Kingdom during the period 1983-1989. 
    Levels of difenacoum were in the range of 0.005-0.106 mg/kg body
    weight, whilst levels of brodifacoum were in the range
    0.019-0.515 mg/kg body weight (Newton et al., 1990).

         Merson & Byers (1984) analysed eastern screech owl  (Otus asio)
    pellets following the application of 0.001% brodifacoum to an orchard
    for rodent control.  The brodifacoum residues in pellet samples ranged
    from 0.06 to 0.09 mg/kg, indicating some exposure of the birds. 
    Hegdal & Colvin (1988) analysed screech owl tissues up to 52 days
    after application of brodifacoum in an orchard.  Brodifacoum was
    detected in livers (detection limit = 0.3 mg/kg) from 9 out of 16
    birds, the concentrations ranging from 0.3 to 0.8 mg/kg.  No
    detectable residues were found in the remainder of the carcasses
    (detection limit = 0.1 mg/kg).

         Hegdal & Blaskiewicz (1984) sampled six barn owls of different
    ages in the vicinity of farm buildings treated with brodifacoum. 
    Analysis of carcasses revealed only one with trace (< 0.05 mg/kg)
    levels of brodifacoum; the other carcasses did not contain detectable
    concentrations.

         Brodifacoum residues in the liver, muscle and fatty tissue of
    rabbits poisoned during field trials with bait containing 0.005%
    active ingredient were 4.4, 0.26 and 0.86 mg/kg, respectively.  During
    the same field trials, brodifacoum residues in seven poisoned birds of
    various species ranged from 0.12 to 8.1 mg/kg in the liver, < 0.05 to
    0.14 mg/kg in muscle and < 0.05 to 0.25 mg/kg in fatty tissue (Rammel
    et al., 1984).

    5.2  General population exposure

         As mentioned in the previous section, residues are unlikely to be
    found in plant foods.  The use of dry baits to protect grain stores
    can result in contamination of the stored food.  Although on average
    the concentration of residues would be expected to be low, occasional
    areas of high concentration can occur.

         With respect to residues in animals used for human food (pigs,
    sheep and birds), there are no residue data concerning animals that
    have survived anticoagulant poisoning.  It should be emphasized,
    however, that in some countries rodents are used as food.

         Warfarin is widely used as a therapeutic agent.

    5.3  Occupational exposure

         Exposure may occur during manufacture, formulation and bait
    application.  The available information is discussed in section 8.2.

    6.  MODE OF ACTION AND METABOLISM

    6.1  Vitamin K and its antagonists

         Vitamin K is a collective name for a number of related compounds,
    which all may function as co-enzymes for the enzyme gamma-glutamate
    carboxylase.  They all contain the functional naphthoquinone ring
    structure, but differ in their aliphatic side chains.  Vitamin K1
    (phytomenadione) contains a side chain composed of four isoprenoid
    residues, one of which is unsaturated.  The vitamin K2 compounds
    (menaquinones) have side chains which vary from 1 to 13 isoprenoid
    residues, all of which are unsaturated.  They are generally referred
    to as MK-n, where n is the number of isoprenoid residues.  Vitamin
    K3 (menadione) has no side chain, but upon ingestion it is converted
    into MK-4 by a liver enzyme.  The two products commercially available
    for human use are K1 and MK-4. Both are equally active, but for some
    reason K1 is almost exclusively used in Europe and North America,
    whereas MK-4 (also known as menatetrenone) is used in Asia, notably
    Japan.  K3 is not used any more for humans because of its adverse
    side effect, haemolysis, but is frequently added to animal food.

         Both 4-hydroxycoumarin derivatives and indandiones (also known as
    oral anticoagulants) are antagonists of vitamin K.  Their use as
    rodenticides is based on the inhibition of the vitamin K-dependent
    step in the synthesis of a number of blood coagulation factors.  The
    vitamin K-dependent proteins involved in the coagulation cascade
    (Fig. 1) are the procoagulant factors II (prothrombin), VII
    (proconvertin), IX (Christmas factor) and X (Stuart-Prower factor),
    and the coagulation-inhibiting proteins C and S.  All these proteins
    are synthesized in the liver.  Before they are released into the
    circulation the various precursor proteins undergo substantial
    (intracellular) post-translational modification.  Vitamin K functions
    as a co-enzyme in one of these modifications, namely the carboxylation
    at well-defined positions of 10-12 glutamate residues into
    gamma-carboxyglutamate (Gla). The presence of these Gla residues is
    essential for the procoagulant activity of the various coagulations
    factors. Vitamin K hydroquinone (KH2) is the active co-enzyme, and its
    oxidation to vitamin K 2,3-epoxide (KO) provides the energy required
    for the carboxylation reaction.  The epoxide is than recycled in two
    reduction steps mediated by the enzyme KO reductase (Fig. 2).  The
    latter enzyme is the target enzyme for coumarin anticoagulants.  Their
    blocking of the KO reductase leads to a rapid exhaustion of the supply
    of KH2, and thus to an effective prevention of the formation of Gla
    residues.  This leads to an accumulation of non-carboxylated
    coagulation factor precursors in the liver.  In some cases these
    precursors are processed further without being carboxylated, and
    (depending on the species) may appear in the circulation.  At that

    stage the under-carboxylated proteins are designated as descarboxy
    coagulation factors (Stenflo et al., 1974; Nelsestuen et al., 1974). 
    Normal coagulation factors circulate in the form of zymogens, which
    can only participate in the coagulation cascade after being activated
    by limited proteolytic degradation (see Fig. 1).  Descarboxy
    coagulation factors have no procoagulant activity (i.e. they cannot be
    activated) and neither they can be converted into the active zymogens
    by vitamin K action. Whereas in anticoagulated humans high levels of
    circulating descarboxy coagulation factors are detectable, these
    levels are negligible in warfarin-treated rats and mice.  Reviews by
    Vermeer (1990) and Furie & Furie (1990) give further details.

         Leck & Park (1981) compared the effects of warfarin and
    brodifacoum on vitamin K metabolism and blood-clotting factor activity
    in warfarin-susceptible and warfarin-resistant rats.  In
    warfarin-susceptible rats both brodifacoum and warfarin induced a
    significant increase in the circulating KO (measured as the KO/K ratio
    using 3H-vitamin K1), indicating that KO reductase is the target
    enzyme for both drugs.  However, whereas warfarin (1 mg/kg) only
    inhibited the KO reductase in the susceptible strain, brodifacoum
    (1 mg/kg) produced the same decrease of plasma prothrombin
    concentration in both warfarin-susceptible and warfarin-resistant
    animals.

         The KO/K ratio in warfarin-resistant rats is five times higher
    than in warfarin-susceptible animals.  This is explained by the fact
    that the hepatic KO reductase in the resistant animals has not only a
    reduced affinity for warfarin, but also for KO.  Hence the vitamin K
    requirement of warfarin-resistant animals is 5-10 times higher than
    that of warfarin-susceptible ones.  Second-generation anticoagulants,
    if given in doses which cause anticoagulation, further increase the
    KO/K ratio (Leck & Park, 1981).

         The much stronger potency of difenacoum and brodifacoum, as
    vitamin K-antagonists, was reported by Park & Leck (1982), who
    concluded that in the case of poisoning with these second-generation
    anticoagulants it will be necessary to give repeated and frequent
    doses of vitamin K to maintain clotting factor synthesis.  The potency
    of second-generation anticoagulants can be partly explained by their
    highly lipophilic nature, which enables them to bind strongly to
    membranes.  Their target enzyme KO reductase is an integral membrane
    protein with, in addition, a highly lipophilic nature.  It is to be
    expected that the dissociation of enzyme/inhibitor complexes will be
    extremely slow.  Moreover, their effectiveness in warfarin-resistant
    rats demonstrates that the mutation leading to warfarin resistance
    does not significantly affect their interaction with the KO reductase.

    FIGURE 17

    FIGURE 18

         Vermeer & Soute (1992) compared the inhibition of each of the
    three enzymes from the vitamin K cycle by four anticoagulants
    (warfarin, flocoumafen, difenacoum and brodifacoum).  The studies were
    performed using  in vitro enzyme systems prepared from rat, cow and
    human liver.  It was shown that in all three species the inhibitor
    concentration required for 50% inhibition (I50) was comparable for
    the KO reductase and K reductase activity, but that the I50 for
    gamma-glutamylcarboxylase was 2-3 orders of magnitude higher. It was
    concluded that for all four anticoagulants the reductions of KO and K
    are the target reactions for inhibition.  Moreover, it was found that
    there is no species specificity of the inhibitors, which means that
    they are equally active in cell-free systems derived from rat, cow and
    human liver.  Any species-dependent differences which might be found
     in vivo will presumably be brought about by a different
    pharmacokinetic or pharmacodynamic behaviour in these species.

    6.2  Metabolism

    6.2.1  Absorption, distribution and elimination

         Anticoagulant rodenticides are easily absorbed through the
    gastrointestinal tract, skin and respiratory system.

         After a single oral dose of 14C-flocoumafen (0.14 mg/kg body
    weight) to rats, the absorption into blood was rapid, reaching maximum
    concentrations (0.03-0.05 µg/ml) in plasma within 4 h (Huckle et al.,
    1989).

         The major route of elimination in rats and sheep after oral
    administration of anticoagulants is through the faeces.  The
    intestinal levels of brodifacoum in rats began increasing 24 to 72 h
    after an oral dose of 0.2 mg/kg body weight (Bachmann & Sullivan,
    1983).  Faecal elimination of radiolabelled flocoumafen following an
    oral dose of 0.14 mg/kg body weight accounted for 23-26% of the dose
    over the 7-day period; approximately half of this was recovered within
    the first 24 h.  Less than 0.5% of the dose appeared in the urine
    within 7 days (Huckle et al., 1989).

         After single oral administration of brodifacoum (0.2 and 2 mg/kg
    body weight) to sheep, about 20% and 30%, respectively, was excreted
    in the faeces within 8 days (Laas et al., 1985).

         A larger proportion of a percutaneous dose of 14C-flocoumafen
    (0.17 mg/kg body weight) dissolved in acetone was found in the urine
    of rats (10%) than in the case of an equivalent oral dose (less than
    0.5%) over a 7-day period.  Faecal elimination accounted for 31% of
    the percutaneous dose (Huckle & Warburton, 1986b).

         After oral 14C-flocoumafen doses of 0.02 mg/kg body weight or
    0.1 mg/kg body weight were given to rats, once weekly for up to 14
    weeks, approximately one-third of each weekly low dose was eliminated
    through the faeces within 3 days, mostly within the first 24 h.  At
    the higher dose the elimination ranged from 18% after the first dose
    to 59% after the tenth dose (Huckle et al., 1988).

         Following repeated oral administration of 14C-flocoumafen to
    rats at 0.02 mg/kg body weight per week for 14 weeks or 0.1 mg/kg body
    weight per week for 10 weeks, appreciable accumulation was seen in the
    liver.  At both dose levels tissue concentrations were highest in the
    liver, followed by the kidney > skin > muscle > fat > blood.  The
    hepatic residue in the low-dose group ranged from 0.1 mg/kg tissue
    after one week to 2.1 mg/kg by week 14 (Huckle & Warburton, 1986a).

         Brodifacoum could not be detected in the omental fat of sheep 8
    days after the oral administration of 0.2 and 2 mg/kg body weight
    (Laas et al., 1985).

    6.2.2  Metabolic transformation

         Warfarin is readily hydroxylated  in vitro and  in vivo by rat
    liver microsomal enzymes to form 6-, 8- and, especially
    7-hydroxy-warfarin (Ullrich & Staundinger, 1968; Ikeda et al.,
    1986a,b).  These inactive metabolites are to some extent conjugated
    with glucuronic acid, undergo enterohepatic recirculation, and are
    excreted in the urine and faeces (Ellenhorn & Barceloux, 1988).

         The metabolic pattern of indandiones in rats also mainly involves
    hydroxylation (Yu et al., 1982).

         The second-generation anticoagulants have mainly been found as
    unchanged compounds (Bachmann & Sullivan, 1983; Huckle et al., 1988). 
    The low urinary elimination following oral dosing has precluded
    accurate isolation of metabolites in urine (Warburton & Hutson, 1985;
    Waburton & Huckle, 1986; Huckle & Warburton, 1986a).

         Following administration of flocoumafen, liver residues in rats
    consisted mainly of unchanged flocoumafen, although in a repeat dose
    study a polar metabolite was detected.  Eight urinary metabolites were
    detected after percutaneous exposure to 14C-flocoumafen (Huckle &
    Warburton, 1986b).

         Studies in male Japanese quail have shown more rapid metabolism
    and elimination than in the rat following an oral dose of
    14C-flocoumafen.  Up to 12 radioactive components were detected in
    the excreta (Huckle & Warburton, 1986c).

         Bromadiolone, brodifacoum and coumatetralyl were also found in
    rats as unchanged parent compounds, whereas in the case of difenacoum
    metabolites predominated (Parmar et al., 1987).  The metabolism and
    elimination of the difenacoum trans isomer was more rapid than for the
    cis isomer (Bratt, 1987).

         The suggestion that the anticoagulant effect in rats is mediated
    by the unchanged compound itself rather than by its metabolites has
    been confirmed by the effects of phenobarbital and SKF525A
    pretreatments on the general pattern of responses to warfarin and
    brodifacoum (Bachmann & Sullivan, 1983).

    6.2.3  Retention and turnover

         Metabolic studies of anticoagulant rodenticides show that the
    liver is the main organ of accumulation and storage. Liver
    concentrations of brodifacoum after a single oral dose of 0.2 mg/kg
    body weight to rats remained high and relatively constant for 96 h,
    with a maximum of 5.0 mg/kg after 50 h (Bachmann & Sullivan, 1983).

         A high degree of body retention was found 7 days after a single
    oral dose of 0.14 mg/kg body weight 14C-flocoumafen (74-76% of the
    administered dose); approximately half the dose was found in the liver
    (Huckle et al., 1989).

         Brodifacoum was detected in the liver of sheep 128 days after
    oral administration (0.2 and 2 mg/kg body weight) in concentrations of
    0.64 and 1.07 mg/kg dry weight (equivalent to 0.22 and 0.36 mg/kg wet
    weight), respectively.  The peak levels occurred at 2 days in the
    high-dose group and at 8 days in the low-dose group, being 6.50 and
    1.87 mg/kg dry weight (2.21 and 0.64 mg/kg wet weight), respectively
    (Laas et al., 1985).  Woody et al. (1992) observed an elimination
    half-life for brodifacoum in serum of 6 ± 4 days in four dogs.

         The largest proportion of a percutaneous flocoumafen dose of
    0.17 mg/kg body weight was located in the liver (25% of the dose at a
    concentration of 0.8 mg/kg), although this was 10 times lower than
    that following an oral dose (Huckle & Warburton, 1986b).

         Parmar et al. (1987) found that elimination of radiolabelled
    brodifacoum, bromadiolone and difenacoum from the liver was biphasic,
    consisting of an rapid initial phase lasting from days 2 to 8 after
    dosing and a slower terminal phase when the elimination half-lives
    were 130, 170 and 120 days, respectively.  Elimination of
    coumatetralyl was more rapid, with a half-life of 55 days.

         Similar results for difenacoum were found by Bratt (1987).  After
    a single oral 14C-difenacoum dose of 1.2 mg/kg body weight, the
    highest concentration of radioactivity (41.5% of the dose) was found
    in the rat liver 24 h after dosing.  The elimination from the liver
    was biphasic.  The half-life of elimination of the radioactivity
    during the first rapid phase was three days, and for the slower phase
    was 118 days.  A similar biphasic elimination was also apparent in the
    kidney.  In the pancreas the concentration declined more slowly than
    in any of the other tissues (182 days).  The parent compound was the
    major component in the liver 24 h after dosing (42%).

         Unchanged flocoumafen comprised the major proportion of the
    hepatic radioactivity in rats and was eliminated with a half-life of
    220 days (Huckle et al., 1989).  Veenstra et al. (1991) found
    retention of about 8% of an administered flocoumafen dose of 0.4 mg/kg
    in the liver of beagle dogs 43 weeks after dosing.

         Despite the more rapid metabolism of flocoumafen in Japanese
    quail, a proportion of the administered dose is retained in the liver,
    with an elimination half-life of 115 days after oral dosing (Huckle &
    Warburton, 1986c).

         Six Hereford heifers weighing approximately 230 kg each were
    dosed with diphacinone (1 mg/kg body weight) by injecting it into the
    rumen.  The highest residue level of parent compound found in the
    liver was 0.15 mg/kg at days 30 and 90 after treatment.  No detectable
    levels (> 0.01 mg/kg) could be found in any of the other tissues
    analysed (kidney, plasma, brain, heart, muscle and fat).  The residues
    in the liver were almost constant from 30 to 90 days post-treatment
    (Bullard et al., 1976).

         The plasma half-life of brodifacoum determined in three patients
    with severe bleeding disorders was found to be approximately 16 to 36
    days (Weitzel et al., 1990).

         The half-life for disappearance from the plasma of human
    volunteers given a single oral or intravenous warfarin dose of
    1.5 mg/kg body weight varied from 15 to 58 h, with a mean of 42 h
    (O'Reilly et al., 1963).

    7.  EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS

    7.1  Acute effects

    7.1.1  Rodent species

         Wide variations exist in the literature for LD50 values of
    anticoagulant rodenticides.  A particular reason for these variables
    is the use of single or repeated (5-day) doses.  LD50 values also
    vary according to the animal strain and sex, but both have not always
    been indicated in reported data (Ashton et al., 1987)
    (Tables 5 and 6).

    Table 5.  Acute oral LD50 values for various rodenticides in albino
              Norway rats
                                                                        

    Rodenticide            LD50 (mg/kg)          Reference
                                                                        

    Brodifacoum                0.26         Redfern et al. (1976)
    Bromadiolone               1.125        Grand (1976)
    Chlorophacinone           20.5          Thomson (1988)
    Coumachlor               900.0          Thomson (1988)
    Coumafuril                 0.4          Wiswesser (1976)
    Coumatetralyl             16.5          Thomson (1988)
    Difenacoum                 1.8          Bull (1976)
    Difethialone               0.56         Lechevin & Grand (1987)
    Diphacinone                3.0          Thomson (1988)
    Flocoumafen                0.46         Sharples (1983a)
    Pindone                   50.0          Tomlin (1994)
    Warfarin                  58.0          Thomson (1988)
                                                                        

         Reported oral LD50 values for warfarin in rats vary by a
    considerable magnitude.  Values of 11 mg/kg body weight (Lund, 1982),
    58 mg/kg body weight (Thomson, 1988) and 58 mg/kg body weight and
    323 mg/kg body weight for female and male, respectively (Hagan &
    Radomski, 1953), have been reported.

         The second-generation anticoagulants are more toxic than the
    first-generation ones in the sense that a single feeding may be
    lethal.  Apparent discrepancies between the single oral LD50 values
    for Norway rats, as shown in Table 5, and the multiple dose oral
    LD50 values, shown in Table 6, can be explained by the cumulative
    effects resulting from multiple dose (5 day) administration (Table 6)
    and characteristics of this family of compounds.


        Table 6.  Five-day oral LD50 (mg/kg body weight per day) of various anticoagulants
              for the Norway rat (Rattus norvegicus)a
                                                                                                                                                

    Anticoagulant     Strainb             Male                     Female             Both sexes              Number of
                                                                                                              animalsc
                                                                                                                                                

    Pindone             SD            1.21 (0.70-2.11)        1.60 (0.83-3.08)      1.34 (0.87-2.06)             40
                        wild          7.60 (2.61-22.2)        25.60 (5.34-123)      12.80 (1.73-84.8)            40

    Warfarin            SD            0.29 (0.14-0.57)        0.38 (0.22-0.66)      0.33 (0.22-0.50)             40
                        wild          0.39 (0.16-0.91)        0.60 (0.23-1.09)      0.44 (0.25-0.76)             40

    Diphacinone         SD            0.19 (0.11-0.33)        0.23 (0.12-0.43)      0.21 (0.14-0.31)             40
                        wild          0.39 (0.15-0.84)        0.60 (0.22-0.57)      0.44 (0.23-0.54)             40

    Chlorophacinone     SD            0.18 (0.18-0.18)        0.20 (0.15-0.27)      0.19 (0.16-0.22)             40
                        wild          0.13 (0.10-0.19)        0.23 (0.14-0.36)      0.16 (0.12-0.22)             40

    Bromadiolone        SD            0.13 (0.10-0.19)        0.10 (0.08-0.13)      0.12 (0.10-0.15)             40
                        wild          0.06 (0.03-0.12)        0.09 (0.09-0.09)      0.07 (0.05-0.10)             16
                                                                                                                                                

    a   Modified from: Ashton et al. (1987); figures in parentheses represent 95% confidence limits
    b   SD = Sprague-Dawley
    c   50% males and 50% females in all tests
    

         Reported oral LD50 values for mice show similar variations,
    from less than 1 mg/kg body weight for second-generation rodenticides
    to 374 mg/kg body weight for warfarin (Hagan & Radomski, 1953; Redfern
    et al., 1976; Bull, 1976; Sharples, 1983a).

         Signs of anticoagulant poisoning in rats and mice include
    lethargy, hunched posture and vein clearing in the ears. Blood around
    the eyes, mouth and anus, indicating internal haemorrhaging, appears
    prior to death (Sharples, 1984).

         The percutaneous toxicity of anticoagulants to rats varied for
    different compounds from an LD50 of 0.54 mg/kg body weight for
    flocoumafen (master mix in corn oil) to > 50 mg/kg body weight for
    brodifacoum and difenacoum (Price, 1985b; Tomlin, 1994).  The signs of
    intoxication were identical to those observed after an oral dose.

         Second-generation anticoagulants appeared to be highly toxic by
    inhalation.  An acute inhalation study in Wistar rats exposed (nose
    only) for 4 h was conducted using the 0.5% manufacturing master mix
    specifically prepared to give a mass median aerodynamic diameter of
    less than 5 µm.  The acute LC50 values were between 0.16 and
    1.4 mg/litre.  Signs of intoxication, characteristic of an
    anticoagulant action, were observed within 3 days after exposure with
    deaths occurring between 4 and 9 days (Blair, 1984).

    7.1.2  Non-target species

         The acute toxicity of various anticoagulant rodenticides to
    non-target mammalian species is presented in Table 7.

         From the data presented it appears that some anticoagulants show
    a similar range of acute toxicity for both non-target mammals and
    target rodents.

    7.2  Short-term exposure

    7.2.1  Rodent species

         In a study by Hadler (1974), groups of Wistar rats (male and
    female) were given brodifacoum by gavage (0.01, 0.02, 0.05, 0.1 and
    0.2 mg/kg body weight) for 5 consecutive days.  All rats receiving the
    two lowest doses survived the 21-day experimental period, but all rats
    given the two highest doses died within 11 days of cessation of
    dosing.  No abnormalities were detected in surviving animals
    sacrificed at the end of the observation period.  In animals which
    died during the study, only massive internal haemorrhages, mainly in
    the peritoneum, were observed.  The no-observed-effect level of
    brodifacoum for Wistar rats was 0.02 mg/kg body weight per day.


        Table 7.  Acute oral toxicity (LD50, mg/kg) of various anticoagulant rodenticides for non-target mammalian speciesa
                                                                                                                                                

    Rodenticide       Guinea-pig   Rabbit       Dog            Cat               Sheep       Pig                     References
                                                                                                                                                

    Brodifacoum       2.78 (F)     0.29 (M)     0.25-1 (F)     approx. 25 (F)    > 25 (M)    0.5-2                Hadler (1975a,b); Parkinson
                                                3.56                             11-33                            (1975, 1976); Godfrey (1984);
                                                                                                                  Godfrey et al. (1985)

    Bromadiolone      2.8          1.0          10 (F) MTD     > 25 MTD                      3                    Grand (1976)

    Chlorophacinone                50                                                                             Pelfrène (1991)

    Difenacoum        50 (F)       2 (M)        approx. 50     100               100         80-100               Bull (1976); Tomlin (1994)

    Diphacinone                    35           3-7.5          14.7                          150                  Kosmin & Barlow (1976)

    Difethialone                   0.75         5 MTD          > 16 MTD                      2-3                  Lechevin & Grand (1987)

    Flocoumafen       > 10 (M)     0.7 (M/F)    0.075-0.25     > 10 (M/F)        > 5         approx. 60 (M,F)     Sharples (1983b); Price (1985a);
                                                (M/F)                                                             Chesterman et al. (1984);
                                                                                                                  Roberts et al. (1985a, 1986)

    Warfarin                       800          20-50          6-40                          1-5                  Anonymous (1976)
                                                                                                                                                

    a   F = female; M = male; MTD = maximum tolerated dose
    

         Wistar rats (male and female) were continuously given a diet
    containing brodifacoum at 0.1 mg/kg body weight, with no choice of
    other feed, for 12 weeks.  Prothrombin time was increased and
    mortality occurred in 9 out of 20 males and 5 out of 20 females. 
    Macroscopic examination of the major organs of surviving rats revealed
    no abnormalities other than those expected of anticoagulant action
    (Hadler, 1976).

         Feeding Fisher-344 rats with diets containing flocoumafen at
    concentrations of 0.2 or 0.4 mg/kg feed for 5 days produced no
    toxicologically significant effects during the 15-day observation
    period (Price, 1985c).

         In a 28-day feeding study in rats, fed on a diet containing 0,
    0.01, 0.05, 0.1 or 0.2 mg-flocoumafen/kg, there were no overt signs of
    toxicity.  Highest-dose females showed a slight but significant
    increase in mean prothrombin time (PT) and activated partial
    thromboplastin time (PTT), and a slight decrease in mean plasma total
    protein.  No toxicological or pathological changes were observed in
    rats fed diets containing 0.01 or 0.05 mg/kg of diet (Price, 1985d).

         No effect on prothrombin time was observed in a 12-week study
    with Wistar rats administered oral flocoumafen doses (by gavage) of
    0.0125, 0.0625 and 0.125 mg/kg body weight once a week (Forsey, 1985).

         Groups of six male and six female Sprague-Dawley rats were fed
    diphacinone in their diets at concentrations of 0 (control), 0.0313,
    0.0625, 0.125, 0.25 and 0.5 mg/kg diet (equivalent to 0, 1.7, 3.3,
    6.4, 13 and 27 µg/kg body weight per day) for 90 days (Elias & Johns,
    1981).  Additional satellite groups of one rat of each sex and each
    dose group were killed for gross pathological examination at 30 and
    60 days.  Mortality was unaffected by the treatment.  In the survivors
    of the main groups and satellite groups, there were no gross
    pathological changes, but in the two males that died prematurely (in
    the 0.0625 and 0.25 mg/kg groups) there was subdural haemorrhage.
    Prothrombin time was unaffected by treatment.  Routine haematological
    and clinical chemistry tests were performed on only two rats of each
    sex from each group, and no effects were observed apart from a reduced
    blood fibrinogen concentration in both sexes at the highest dose
    level.

         As no clear NOAEL was indicated by the results of the 90-day
    study, a 21-day study was performed using two rats of each sex at
    diphacinone levels of 0 (control), 0.125, 0.25, 0.5, 1, 2 and 4 mg/kg
    diet.  All the rats in the 2 and 4 mg/kg groups died within the first
    14 days, but all others survived until the end of the study.  The mean

    dosages received by the survivors were equivalent to 0, 9, 17, 34 and
    67 µg/kg body weight per day.  At 21 days, there was no effect on
    prothrombin time.  Gross necropsy revealed haemorrhage in the thymus
    of one rat in the 0.5 mg/kg group, but no effects were seen in any
    other survivors.  All the animals in the 2 and 4 mg/kg groups had
    massive and extensive internal haemorrhage (Elias & Johns, 1981).

    7.2.2  Non-target species

         There are few data available on the repeated exposure of
    non-rodent species to anticoagulant rodenticides.

         Death followed five daily warfarin doses of 3 mg/kg body weight
    in cats and 1 mg/kg body weight in pigs (Tomlin, 1994).  The route of
    administration was not specified.

         The dermal administration of warfarin (188 mg/kg per day) to
    female baboons caused profuse bleeding in 5 days followed by death
    (Dreyfus et al., 1983).

         When it became known that vitamin K was not an antagonist for
    warfarin action in bone, it became possible to study the long-term
    effects of this anticoagulant in bones without the risk of inducing
    haemorrhages and bleeding. By feeding lambs with high doses of
    warfarin (up to 150 mg/kg body weight per day) under the protection of
    4 mg/kg body weight per day of vitamin K, Pastoureau et al. (1993)
    showed that within 3 months the lambs had developed a marked
    osteopenia that resulted from a decrease in resorption and a much more
    pronounced decrease in bone formation. As a result the bone density in
    the warfarin-treated animals was substantially lower than that in
    control animals. This is in agreement with earlier studies in rats
    (Price et al., 1982) where it was found that, under the protection of
    vitamin K, warfarin induced excessive mineralization and growth plate
    closure, due to which bone growth came to a halt.

         The maximum tolerated 5-day oral dose of bromadiolone was
    considered to be 25 mg in pigs (Large White strain) weighing 25 kg.
    After 45 oral daily doses of 0.5 mg no change in the prothrombin time
    was observed (Grand, 1976).

         Woody et al. (1992) studied the effect of a cumulative dose of
    1.1 mg brodifacoum/kg body weight administered orally to dogs over a
    3-day period.  Signs of coagulopathic effects appeared within 24 h
    (greater PT and PTT) and increased over a 10-day period.  Treatment
    with Vitamin K1 at 10 days post-exposure reduced the effects.

         Six horses were treated by gavage with brodifacoum containing
    bait at a dosage of 0.125 mg/kg body weight (Boermans et al., 1991). 
    Four of the horses became anorexic and depressed, one requiring K1
    therapy.  Peak plasma concentration occurred 2-3 h after
    administration.  Pharmacokinetic evaluation indicated that brodifacoum
    has a plasma half-life of 1.22 days.  An increase in clotting time was
    observed as early as 24 h after dosing, returning to the pre-treatment
    level by day 12.

         Male pigs (five per group) were fed difenacoum in the diet for 14
    days at dose levels of 0.01, 0.05, 0.1, 0.5 and 1.0 mg/kg diet.  With
    the exception of the lowest-dose group, all groups showed a marked
    increase of prothrombin time values.  Extensive subcutaneous, inter-
    and intra-muscular haemorrhage and oedema was observed in animals
    dosed at levels of 0.5 mg/kg or more (Ross et al., 1979).

    7.3  Long-term exposure

         No data on long-term exposure are available.

         Studies with second-generation anticoagulants are difficult to
    carry out for more than a few weeks due to the rapid acute effects,
    and NOEL values for second-generation rodenticides and longer exposure
    periods have not been established.  In any chronic study approaching
    two years in length, the dose level would have to be less than the
    analytical limit of detection.

    7.4  Skin and eye irritation; sensitization

         Brodifacoum is a slight skin irritant and a mild eye irritant in
    the rabbit (Hadler, 1975c).  No skin or eye irritation was observed in
    New Zealand white rabbits treated with flocoumafen (Forsey, 1983a,b). 
    Neither of these rodenticides was a skin sensitizer when tested in the
    guinea-pig maximization test (Parkinson, 1979; Price, 1986).

    7.5  Reproductive toxicity and teratogenicity

         Brodifacoum was given by oral gavage to female rats at daily dose
    levels of 0.001, 0.01 or 0.02 mg/kg body weight during days 6-15 of
    pregnancy.  There was no evidence of adverse effects on the fetus at
    termination.  Higher daily doses (above 0.05 mg/kg) caused an
    anticoagulant effect in the dams which resulted in a high incidence of
    abortion (Hodge et al., 1980a).

         Pregnant female rabbits were given oral gavage doses of 0.001,
    0.002 or 0.005 mg brodifacoum/kg body weight per day from days 6-18 of
    pregnancy.  A the highest dose level a high proportion of maternal
    deaths occurred as a result of haemorrhage.  Although the survivors
    showed signs of haemorrhage, there were no effects on the developing
    fetus.  No effects were observed at either of the other dose levels
    used (Hodge et al., 1980b,c).

         Bromadiolone was given orally to four groups of 25 female rats
    from day 6 to 15 of pregnancy at doses of 0, 17.5, 35 and 70 µg/kg
    body weight per day.  Maternal toxicity occurred at the higher dose
    levels.  There was no evidence of embryotoxicity or teratogenic
    effects at any dose level (Monnot et al., 1981).  A similar absence of
    effects was reported in a study on rabbits treated orally with daily
    doses of either 2, 4 or 8 µg/kg body weight per day on days 6-18 of
    pregnancy, although there was maternal toxicity at the highest dose
    level (Virat, 1981).

         Groups of 18 pregnant F-344 rats were given daily oral doses of
    0, 0.01 or 0.04 mg flocoumafen/kg body weight from day 8 to 17 of
    gestation.  Eight animals in the 0.04 mg/kg body weight group either
    died or were killed with signs of anticoagulant poisoning.

         In contrast, Mirkova & Antov (1983) found warfarin to be
    embryotoxic and teratogenic to Wistar rats when administered by gavage
    in single doses or repeatedly throughout the periods of
    pre-implantation (1-7 days of gestation) and organogenesis (8-16
    days), and also throughout the whole gestation (1-21 days) at a wide
    range of dose levels (0.04-8 mg/kg body weight).  At these dose levels
    and treatment regimens, warfarin induced substantially increased rates
    of embryolethality, subcutaneous and internal haemorrhage and gross
    structural malformations (pes varus, internal hydrocephalus and
    anomalies of skeletal ossification).

    7.6  Mutagenicity

         Various  in vitro and  in vivo studies have been undertaken to
    assess the genotoxic potential of brodifacoum. No mutagenic activity
    was detected in the Salmonella reverse mutation assay in any of the
    five tester strains employed (TA98, TA100., TA1535, TA1537 and TA1538)
    either in the presence or absence of Arochlor 1254-induced rat liver
    S9 fraction at brodifacoum concentrations ranging from 1.6 to
    5000 µg/plate (Callander, 1984).  Brodifacoum showed no activity in a
    forward mutation assay using L5178 mouse lymphoma cells, either with
    or without metabolic activation, at concentrations of 47.5, 63.3 and
    84.4 mg/litre (Cross & Clay, 1984).

         Brodifacoum caused no significant chromosomal aberrations in
    cultured human lymphocytes (concentrations 1, 10, 100 and
    1000 mg/litre), either with or without metabolic activation, and did
    not induce unscheduled DNA synthesis in cultured HeLa cells at the
    same range of concentrations (Mellano, 1984a,b).  Difenacoum did not
    induce unscheduled DNA synthesis in rat hepatocytes  in vivo at
    either dose level or time-point (Kennelly, 1990).

         An  in vivo micronucleus test, in which mice were given single
    brodifacoum intraperitoneal doses of 0.187 or 0.30 mg/kg body weight,
    showed no induction of micronuclei in bone marrow polychromatic
    erythrocytes (Sheldon et al., 1984).

         Bromadiolone was tested in the Salmonella reverse mutation assay
    at concentrations ranging from 10 to 3330 µg per plate on strains
    TA1535, TA1537 and TA1538.  No evidence of mutagenic effect was found
    either with or without Aroclor metabolic activation (Lawlor, 1992).

         Bromadiolone did not induce forward mutations in Chinese hamster
    ovary cells either with or without metabolic activation
    (Cifone, 1993).

         In a mouse micronucleus test at four dose levels from 50 to
    400 mg/kg, bromadiolone did not induce micronuclei in bone marrow
    polychromatic erythrocytes (Murli, 1993).

         Flocoumafen did not induce reverse gene mutation in  Salmonella
     typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 nor in
     Escherichia coli WP2uvrA pkm 101 either with or without metabolic
    activation.  Flocoumafen was tested at concentrations ranging from 31
    to 2000 µg/plate, beyond which precipitation from suspension occurred
    (Brooks et al., 1984).

         Flocoumafen did not increase the frequency of mutation to
    6-thioguanine resistance in Chinese hamster V79 cells either in the
    presence or absence of an Arochlor-induced rat liver S9 fraction. 
    Doses ranging from 5 to 150 mg/litre were used, beyond which
    cytotoxicity occurred (Clare & Wiggins, 1986).

         Flocoumafen did not induce  in vitro cell transformation in
    C3H1OT´ mouse fibroblasts, either in the presence or absence of a rat
    S9 metabolizing system, at concentrations ranging from 12.5 to
    100 µg/litre (Meyer & Wiggins, 1986).

         Flocoumafen did not induce mitotic gene conversion in liquid
    suspension cultures of  Saccharomyces cerevisiae JD1, either in the
    presence or absence of a rat liver S9 fraction, at concentrations
    ranging from 0.01 to 2 g/litre (Brooks et al., 1984).

         When incubated at concentrations ranging from 5 to 25 mg/litre
    for 24 h in monolayer cultures of rat liver RL4 cells, flocoumafen did
    not induce  in vitro chromosomal damage (Brooks et al., 1984).

         Oral administration of flocoumafen to rats at doses of 0.25 mg/kg
    or 1000 mg/kg body weight (a dose 4000 times the acute oral LD50 for
    rats) did not produce chromosomal damage (Allen et al., 1986).

         The cytogenic effect of chlorophacinone was investigated  in vivo
    in metaphase bone marrow cells taken at 48 and 96 h after oral dosing
    of male CFLP mice with 20 mg/kg body weight.  No induction of
    chromosomal aberrations was observed (Nehéz et al., 1985).

         In the same study, chlorophacinone was investigated in
    spermatocytes taken from male CFLP mice at 1, 2, 3 and 4 weeks after a
    single oral dose of 20 mg/kg body weight.  The spermatocytes were
    analysed in the diakinesis phase of meiosis.  There was no increased
    incidence of chromosomal aberrations (Nehéz et al., 1985).

         Rabbits were treated orally with 20 mg chlorophacinone/kg body
    weight and chromosomal analysis was performed in bone marrow and
    spermatocytes taken at 48 h post-dosing.  No increase in the incidence
    of chromosomal aberrations was seen (Selypes et al., 1984).

    7.7  Factors modifying toxicity

         Phenobarbital pretreatment of rats followed by a single
    administration of brodifacoum or warfarin decreased the anticoagulant
    effects of both compounds, more markedly in the case of warfarin
    (Bachmann & Sullivan, 1983).

         The non-steroid anti-inflammatory drugs ibuprofen and
    phenylbutazone potentiated the anticoagulant effects of brodifacoum
    and bromadiolone in rats (Sridhara & Krishnamurthy, 1992).

         Strain and sex are important factors modifying the toxicity of
    anticoagulants in rodents (see Table 5, section 7.1.1).  Winn et al.
    (1987) observed greater sensitivity of male rats and mice to
    difenacoum, compared with female rats and mice.  The possible
    explanation of different responses to difenacoum was the greater
    turnover of plasma proteins in male rats or the marked
    inter-individual variation of vitamin K1 level in male rat liver.

         Large amounts (relative to farm animals' dietary requirements) of
    vitamin K3 (menadione and its salts) are sometimes added to animal
    feedstuffs.  This gives rodent pests ready access to a substance which
    acts as an antidote to anticoagulant rodenticides, and thus can reduce
    the efficacy of these rodenticides.

         Differences in metabolism to inhibitors of vitamin K synthesis
    among strains of mice and rats has been attributed to a number of
    different factors (Misenheimer et al., 1994).  Among these are: 1)
    reduced sensitivity of vitamin K epoxide hydrolase to inhibition; 2)
    greater reversibility of inhibition of epoxide hydrolase; and 3)
    faster clearance of the rodenticide.  The mechanism of resistance
    differs between strains.

         A single flocoumafen dose of 0.5 mg/kg body weight resulted in
    clear signs of anticoagulation in five out of eight beagle dogs
    (Veenstra et al., 1991).  Administration of a second dose 5 weeks
    later resulted in clinical evidence of anticoagulation in two out of
    the three remaining dogs.  Vitamin K treatment (2 to 5 mg/kg
    subcutaneously) reversed the effects in all cases.

    7.8  Adverse effects in domestic and farm animals

    7.8.1  Domestic animals

    7.8.1.1  Poisoning incidents

         The main cause for accidental poisoning of domestic animals is
    direct consumption of anticoagulant baits.  Secondary poisoning
    through the consumption of rats and mice killed with anticoagulants
    may occur in dogs and cats in urban situations, but is more likely in
    farm situations (Marsh, 1985b).  The majority of fatalities and severe
    clinical syndromes are connected with the second-generation
    anticoagulants (Dodds & Frantz, 1984). Du Vall et al. (1989) studied
    10 cases of second-generation anticoagulant rodenticide poisoning in
    dogs and cats.  The presence of anticoagulants (brodifacoum,
    bromadiolone or diphacinone) in serum or liver was confirmed by HPLC
    or GC/MS.  Several other cases of brodifacoum poisonings in dogs have
    been reported and some of them were fatal (Mc Sporran & Phillips,
    1983; Stowe et al., 1983; Dodds & Frantz, 1984).

         Until 1983, only first-generation anticoagulants were available
    in New Zealand and, between 1977 and 1983, warfarin, coumatetralyl and
    diphacinone were involved in 45 reported incidents of accidental
    poisoning of domestic and farm animals.  Brodifacoum and flocoumafen
    were introduced after 1983.  From 1983 to 1994, 40 cases of poisoning
    of domestic and farm animals resulted from the use of
    second-generation compounds and 17 cases were due to the ingestion of
    first-generation anticoagulants (Hoogenbroom, 1994).

         Schulman et al. (1986) reported five cases of coagulopathy in
    dogs caused by consumption of diphacinone-containing baits.  Lethargy
    and respiratory distress, associated with pulmonary interstitial
    haemorrhage and pleural and/or pericardial effusion, were the most
    consistent signs.  The prothrombin time and the activated partial
    thromboplastin time were moderately to markedly prolonged.

    7.8.1.2  Diagnosis and treatment of poisoning

         The major difference between warfarin and the other anticoagulant
    rodenticides is that the latter have longer body retention and a
    tendency to induce bleeding for a longer period of time.  It is
    therefore necessary to continue the treatment for weeks rather than
    days.

         Signs of poisoning occur after a latent period of 12 h to several
    days and may include:

    *  bruising easily with occasional nose or gum bleeds
    *  blood in stools or urine
    *  excessive bleeding from minor cuts or abrasions
    *  laboured breathing
    *  pale mouth and cold gums
    *  anorexia and general weakness

         A reliable indication of an anticoagulant effect is the
    determination of prothrombin time.

         Vitamin K1 (phytomenadione) is the antidote of choice.  The
    recommended dosage is 2-5 mg/kg body weight in dogs. Intravenous
    injection is the quickest route but it is not recommended because of
    reported anaphylaxis.  Vitamin K1 is much safer if given
    subcutaneously for the initial 2-3 treatments.  A dose of 0.25 to
    2.5 mg/kg body weight is recommended for use in small animals (Mount
    et al., 1982).  The recommended period of treatment with vitamin K1
    is 3-6 weeks (Braithwaite, 1982; Mackintosh et al., 1988).

         Mount & Feldman (1983) observed that therapy effective for
    warfarin poisoning was ineffective against diphacinone toxicosis due
    to inadequate vitamin K1 dosage and duration of therapy.  Oral
    therapy was ineffective.

         In a study on dogs, Mount & Kas (1989) found that the serum
    concentration of vitamin K epoxide was significantly higher in
    diphacinone-treated dogs than in controls, the difference being
    significant as early as 1-4 h after vitamin K application.  The
    authors suggested that this phenomenon could be used as diagnostic
    information.

         In cases of severe blood loss, fresh plasma should be infused
    every 6 h to the extent of 5-10% of total blood volume, assuming this
    to be 90 ml/kg in the dog and 70 ml/kg in the cat (Mount et al.,
    1982).

         Vitamin K1 (phytomenadione) is the antidote for treating dogs
    exposed to anticoagulant rodenticides. The therapeutic dosages and
    duration of treatment needed vary with the compound involved.  Mount &
    Feldman (1983) reported that dogs poisoned with diphacinone require
    treatments for at least three weeks, while treatment for 4-6 days is
    sufficient for treating warfarin poisonings.  Initial treatments are
    by subcutaneous injection, subsequent therapy being given orally.

         For animals exposed to the second-generation rodenticides
    brodifacoum, bromadiolone, difenacoum and flocoumafen, the treatment
    regimen that has been recommended includes one or more injections of
    vitamin K1 at 2-5 mg/kg body weight until prothrombin times return
    to normal levels.  Once this has been achieved, daily oral doses of
    vitamin K1 are recommended for a period of 3-4 weeks.  Oral doses of
    2-5 mg/kg body weight are recommended initially, with some reduction
    being possible over time if the animal is observed closely for signs
    of recurrence of symptoms.  If prothrombin times increase following
    withdrawal of vitamin K1, the oral dosing should be resumed for 2-3
    weeks.  If blood loss was severe, transfusions (10-15 ml/kg body
    weight) with fresh whole blood may be needed (Anonymous, 1988).

    7.8.2  Farm animals

         Feinsod et al. (1986) reported an outbreak of abortions and
    haemorrhages in sheep and goats in Egypt caused by brodifacoum
    intoxication.  Clinical signs appeared within 3-7 days after exposure
    to the rodenticide and included epistaxis, haematochezia, recumbency,
    subcutaneous haemorrhage, flank ecchymosis, lameness, abdominal
    bloating and abortion of various stages of gestation.  The signs
    resembled epidemic Rift valley fever, but there was no fever, icterus
    or loss of appetite in affected animals.

    8.  EFFECTS ON HUMANS

    8.1  General population exposure

         Incidents of human exposures to rodenticides are reported to
    poison control centres in countries where such facilities exist.  In
    1988, for example, the American Association of Poison Control Centers
    (AAPCC) received accounts of 10 626 cases of human exposures to
    rodenticides.  These incidents represented 17% of reported exposures
    involving pesticides and 0.8% of the total number of cases reported in
    the AAPCC system.  The rodenticide incidents included 4190 cases
    involving "anticoagulants" (principally warfarin) and 5133 involving
    "long-acting anticoagulants" (second-generation anticoagulants plus
    the indandione compounds).  More than 95% of the rodenticide cases
    were classified as "accidental".  Most of the remainder were
    classified as "intentional" and included attempted suicides.  Of the
    10 540 rodenticide incidents for which the ages of victims were
    reported, 9406 (89%) involved children under 6 years of age (Litovitz
    et al., 1994).

         Victims in nearly 32% of the rodenticide exposure incidents
    reported to the AAPCC in 1988 were treated in health care facilities. 
    However, the medical outcome "none" was reported in more than 93% of
    the 5708 incidents for which information regarding outcomes was
    reported.  The remaining 380 cases included 333 with "minor" medical
    effects, 41 with "moderate" effects, 4 with "major" effects, and two
    deaths (Litovitz et al., 1994).

         In 1993, the Swedish Poison Information Centre received 338
    enquiries concerning exposures to anticoagulant rodenticides.  This
    number represented 0.6% of all enquiries to the centre and 37% of the
    enquiries concerning pesticides.  Of the anticoagulant rodenticide
    enquiries, 202 pertained to warfarin and 136 to "superwarfarin"
    compounds (Persson, 1994).

         Human exposure to second-generation and indandione anticoagulants
    produces symptoms consistent with anticoagulation effects (e.g.,
    haematomas, haematemesis, haematuria, easy bruisability).  Treatment
    of cases of exposure, particularly of substantial and repeated
    exposure, may require vitamin K1 therapy and monitoring of
    prothrombin times for periods of many months (Rauch et al., 1994).

         Suicide and/or unintentional poisonings with anticoagulant
    rodenticides have occurred in many countries.  Thus, Ungvary (1994)
    reported 70 cases, mostly involving children, that occurred in Hungary
    between 1988 and 1993.

         Warfarin is widely used as a therapeutic and preventive agent in
    the treatment of thromboembolic disease.  Patients have been
    maintained for years on this treatment with control of the prothrombin
    level, which should be kept between 10 and 30% of normal.

         Diphacinone has also been used as a drug because of its
    long-lasting action (the half-life in humans is 15-20 days).  It
    ceased to be listed in the American Medical Association Drug
    Evaluations, (AMA, 1980) because of its structural relation to
    phenindion, which had been reported to have adverse effects.

    8.1.1  Acute poisoning

         Typical features of poisoning result from increased bleeding
    tendency and include:

    *    minor poisoning: coagulation disturbance detected only by   
         laboratory analyses;

    *    moderate poisoning: coagulation disturbance resulting in   
         haematomata, haematuria, blood in faeces or excessive bleeding   
         from minor cuts or abrasions, gum bleeding;

    *    severe poisoning: retroperitoneal haemorrhage, severe 
         gastrointestinal bleeding, cerebrovascular accidents, massive
         haemorrhage (internal bleeding) resulting in shock.

         If anaemia or liver disease is present then the above features
    may be more severe and persistent and the poisoning may be more
    difficult to control (Anonymous, 1988).

         The onset of the signs of poisoning may not be evident until a
    few days after ingestion.

    8.1.2  Poisoning incidents

         Cases of human poisoning with "superwarfarins" were reviewed by
    Katona & Wason (1989).

         Fourteen members of a family in the Republic of Korea were
    poisoned by eating warfarin-containing maize meal.  The first symptoms
    appeared 7-10 days after the beginning of exposure and were followed
    by massive bruises or haematomata on the buttocks in all cases (Lange
    & Terveer, 1954).

         Pribilla (1966) reported a total dose of about 1000 mg of
    warfarin to be fatal after 13 days of consumption.

         Out of a total of 741 infants, 177 died after the use of
    warfarin-contaminated talc in Viet Nam.  The concentrations of
    warfarin in the powder varied from 1.7 to 6.5% (Martin-Bouyer et al.,
    1983).

         A 73-year-old woman suffered from recurrent episodes of
    hypoprothrombinaemia.  Clotting tests and further investigation showed
    that this was due to a warfarin rodenticide intentionally mixed in the
    woman's cough syrup by her daughter-in-law.  As the patient had as
    many as seven relapses, it was possible to compare different types of
    therapy.  Menadione had no effect (Nilsson, 1957).

         Several suicidal attempts with chlorophacinone have been
    reported.  Murdoch (1983) reported a case of ingestion of 625 mg
    chlorophacinone (250 ml of a 0.25% concentrate formulation) by a
    37-year-old woman.  The prolonged anticoagulant action of
    chlorophacinone persisted for at least 45 days even though treatment
    was given.  It was found that menadiol, the synthetic analogue of
    vitamin K1, was ineffective.  The natural form, phytomenadione, was
    effective only when given at high dosage (20 mg daily) 30 days after
    the ingestion of chlorophacinone.

         In a case reported by Dusein et al. (1984), the amount of
    ingested chlorophacinone was unknown.  After adequate therapy, the
    prothrombin level became normal within 4 weeks.

         Vogel et al. (1988) reported the case of an 18-year-old woman
    hospitalized 3 days after ingesting approximately 100 mg
    chlorophacinone.  Under high-dose vitamin K1 therapy (160 mg) the
    prothrombin time was normalized, but it increased again following
    withdrawal of vitamin K1.  After prolonged vitamin K1 administration,
    the prothrombin time finally became normal after 7 weeks.

         Brodifacoum poisoning has occurred in South Sumatra, Indonesia. 
    Some of the villagers used a 0.005% brodifacoum rice grain bait as a
    food source even though they knew it was poisonous and unfit for human
    consumption.  They attempted to remove the rodenticide by repeated
    washing, rinsing and cooking before eating the rice.  Because of the
    delay in the appearance of poisoning symptoms it appeared that they
    had been successful, thus encouraging further attempts to purify the
    rice baits.  As a result, deaths occurred before appropriate remedial
    treatment could be initiated (Anonymous, 1985).

         Jones et al. (1984) reported the first case of human brodifacoum
    poisoning in a 17-year-old boy who attempted suicide by ingesting
    approximately 7.5 mg (0.12 mg/kg) of brodifacoum in Canada.  He was
    initially seen with gross haematuria, followed by epistaxis and gum
    bleeding.  The prothrombin time and the activated partial
    thromboplastin time were notably prolonged.  He was treated for 56
    days with either parenteral or oral vitamin K1 and either fresh or
    stored plasma until coagulation values remained normal and stable.

         Lipton & Klass (1984) reported a similar case in a 31-year-old
    mentally disturbed woman who ingested over a 2-day period
    approximately thirty 50-g packages of Talon-G (approximately 75 mg of
    brodifacoum).  Prothrombin time and activated partial thromboplastin
    time were considerably prolonged (respectively 6-fold and 4-fold above
    normal values).  After 4 days of therapy with high doses of vitamin
    K1 (up to 125 mg/day), partial correction in the prothrombin time
    occurred.  Vitamin K1 therapy continued with interruptions for 8
    months until normal prothrombin time levels were found.

         Chong et al. (1986) reported a case of suicidal poisoning after
    ingestion of 10 mg brodifacoum (as 0.05% Klerat). The coagulation test
    became normal after large doses and prolonged use of vitamin K1 over
    6 months.

         A case of intentional ingestion of brodifacoum (200 g of Talon G,
    0.005% brodifacoum) was reported by Hoffman et al. (1988).  A profound
    decrease in the levels of factors II, VII, IX and X, lasting 43 days
    after ingestion, was observed.  Treatment with subcutaneous vitamin
    K1 in doses up to 100 mg per day was effective.

         Weitzel et al. (1990) described three patients with severe
    bleeding disorders due to deficiency of the vitamin K-dependent blood
    clotting proteins after ingestion of an anticoagulant.  Although the
    patients denied any ingestion, brodifacoum was detected in their serum
    at concentrations of 7.6 nmol/litre, 270.7 nmol/litre and
    2759 nmol/litre, respectively.  The anticoagulant effect was found to
    persist long after brodifacoum was no longer detectable in the serum. 
    A half-life of approximately 16-36 days was determined for brodifacoum
    in the plasma.

         Kruse & Carlson (1992) reported the case of a 25-year-old man who
    attempted suicide by consuming a brodifacoum rodenticide.  He
    developed a severe coagulopathy that was treated with vitamin K1 and
    fresh frozen plasma and he was discharged from hospital with oral
    phytomenadione.  Fifteen weeks later the man presented again with a
    history of further brodifacoum ingestion.  He suddenly became comatose
    and computer tomography revealed a subarachnoid haemorrhage that led
    to brain death 24 h later.

         Wallace et al. (1990) described the clinical course of a patient
    poisoned with brodifacoum in a suicide attempt.  He developed
    microhaematuria and melaena.  His clotting factors were depressed and
    were poorly responsive to vitamin K treatment.

         Barlow et al. (1982) reported a case of attempted suicide with
    25 mg of difenacoum (500 g of rat bait) followed several months later
    by 1800 g of rat bait.  The patient was treated with vitamin K1
    (phytomenadione) for 48 and 42 days, respectively, until the
    pharmacological effect of difenacoum ceased.

         Nighoghossian et al. (1990) reported an unusual coagulopathy
    after accidental exposure to a diphenacoum rodenticide.  A 59-year-old
    man developed subacute tetraparesis following severe sudden neck pain,
    which on clinical examination was shown to be due to a subdural
    cervical haematoma.  Prothrombin complex activity was low and
    diphenacoum was present in the plasma.  Specific medical management
    led to a complete recovery.

         Greeff et al. (1987) reported accidental bromadiolone poisoning
    in two children, resulting in prolonged anticoagulation.
    Descarboxyprothrombin levels were increased in both cases by 27% and
    29.9%, respectively (normal, non-detectable level).  The first child
    rapidly recovered after treatment with high-dose intravenous factor
    IX-prothrombin complex and vitamin K1.  The clotting profile became
    normal on the third day after admission.  The second child gave a poor
    response to 10 mg intravenous vitamin K1 and the dose was increased
    to 20 mg.

    8.1.3  Controlled human studies

         Single oral doses of 60, 70, 80 or 120 mg warfarin  decreased the
    prothrombin concentrations in volunteers to zero by the third day. 
    After the administration of 50 mg vitamin K1, the prothrombin
    concentrations returned by the sixth day to 60, 70, 55 and 63%,
    respectively, of the normal value (Anonymous, 1965).

         When a single oral dose of 20 mg chlorophacinone was given to
    three volunteers, the lowest prothrombin times were 35, 34 and 38% of
    the pretreatment value on days 2, 4 and 2, respectively.  Eight days
    after administration without any treatment the values were 80, 100 and
    90%, respectively (Anonymous, 1965).

    8.2  Monitoring of biological effects

    8.2.1  Effects of short- and long-term exposure

         Two cases of occupational exposure to brodifacoum and difenacoum
    were reported by Park et al. (1986).  The exposure was of a chronic
    nature (2 and 4 years, respectively).  Plasma analysis in the first
    patient revealed the presence of both difenacoum and brodifacoum in
    the  range of 30-50 µg/litre.  In both patients unexpectedly high
    concentrations of vitamin K1 2,3-epoxide were found in the presence
    of normal clotting factor activities and antigen levels suggesting the
    presence of coumarin anticoagulants in the liver.

         A case of poisoning in a 23-year-old man resulting from prolonged
    skin contact during the process of preparing and distributing warfarin
    baits has been reported (Fristedt & Sterner, 1965).

    8.2.2  Epidemiological studies

         During a production run preparing ready-to-use flocoumafen bait
    (0.005% in baits) in a formulation plant, the effect of the
    rodenticide on blood coagulation factors was monitored in 12 subjects,
    using the classical prothrombin time test, a modified prothrombin time
    technique and measurement of prothrombin (factor II) concentration in
    blood.  No adverse health effects were observed in any subject
    involved in formulation operations.  No changes were observed in any
    of the three tests that could be ascribed to absorption of flocoumafen
    into the body (Tuinman & Van Sittert, 1986).

    8.3  Developmental effects

         Developmental effects have been reported when anticoagulants,
    particularly warfarin, have been administered as therapeutic agent
    during pregnancy.  Hall et al. (1980) reviewed 418 reported
    pregnancies in which coumarin and indan-1,3-dione derivatives were
    used, and found that one-sixth resulted in abnormal liveborn infants,
    one-sixth in abortion or stillbirth, and two-thirds in apparently
    normal infants.

         According to Hall (1976), there are two types of defects
    associated with anticoagulants, dependent upon the time of
    administration during pregnancy.  The first, a characteristic
    embryopathy described by the terms "warfarin embryopathy" or "fetal
    warfarin syndrome", occurs from early, first-trimester use.  Fetal
    wastage and other abnormalities, especially central nervous system
    anomalies, result from treatment later during gestation (usually the
    second or third trimesters).  Clotting factors are not present in
    first-trimester embryos and this may explain the differences in fetal
    abnormalities.

         The most consistent feature of warfarin embryopathy is nasal
    hypoplasia.  Choanal stenosis has been observed, and respiratory
    difficulty is typical because of the narrowed nasal passages (Smith &
    Cameron, 1979; Pauli & Hall, 1979).

         The other common feature is bone abnormalities of the axial and
    appendicular skeleton.  Laryngeal and tracheal-thyroid calcification
    has also been noted (Schardein, 1985).

         Other non-skeletal abnormalities reported include
    ophthalmological malformations of several types, including defects
    leading to blindness, developmental delay, low birth weight (premature
    birth), mental retardation, hypotonia and ear anomalies (Carson &
    Reid, 1976; Schardein, 1985).

         No cases of embryopathy from anticoagulants in their use as
    rodenticides have been reported.

    8.4  Other adverse effects

         One of the more common side-effects of coumarin therapy is skin
    necrosis (Brooks & Blais, 1991; Eby, 1993; Locht & Lindström, 1993). 
    Although this effect has been associated with a number of different
    agents, it is commonly referred to as warfarin-induced skin necrosis
    (WISN).  The frequency of this effect has been estimated to be between
    1 in 100 and 1 in 10 000, with the majority of cases occurring in
    women.  Symptoms of WISN typically appear 3-6 days after the
    initiation of warfarin therapy, and the areas of skin involved are
    most frequently areas with subcutaneous fat, particularly the breasts,
    thighs, and buttocks.  There is some evidence that deficiencies in
    vitamin K-dependent anticoagulant proteins (protein C and protein S)
    may underlie the susceptibilities of at least some individuals to WISN
    (Anderson et al., 1992). The occurrence of WISN may be avoided in at
    least some cases by the co-administration of heparin.

         Warfarin therapy has been noted to result in increases in the
    incidence of haematomas, including intraspinal epidural haematoma
    (Murphy & Nye, 1992), liver haematoma (Erichsen et al., 1993), and
    intramural haematoma of the small intestine (Avent et al., 1992).

         Bone contains three vitamin-K-dependent proteins, i.e.
    osteocalcin, matrix Gla-protein and protein S. It has been shown that
    oral anticoagulants (phenprocoumon, acenocoumarol) reduce both the
    plasma antigen level as well as the Gla content of osteocalcin (Van
    Haarlem et al., 1988). Furthermore it was found that a poor vitamin K
    status was associated with a high urinary calcium loss (Knapen et al.,
    1993).  These data are consistent with the observation that the bone
    mass in patients on long-term anticoagulant treatment was
    significantly lower than in a group of age- and sex-matched controls
    (Fiore et al., 1990; Resch et al., 1991). Vitamin-K-dependent proteins
    have also been identified in calcified atherosclerotic plaques, where
    they were suggested to contribute to the prevention of vascular
    calcification (Gijsbers et al., 1990).  In this respect it is
    remarkable that warfarin treatment has been shown to increase rather
    than to reduce atherosclerosis in an animal model (Antov et al.,
    1985).  Vitamin-K-dependent proteins not related to blood coagulation
    are still discovered regularly (Manfioletti et al., 1993), and it is
    important to remain alert for unexpected side-effects of vitamin K
    antagonists, notably after long-term exposure.

    8.5  Methods for assessing absorption and effects of anticoagulant
         rodenticides

         The laboratory control of orally administered coumarin
    derivatives has been carried out using the classical one-stage
    prothrombin time test (Quick, 1935) or modified techniques such as
    "Thrombotest" (Owren, 1959).  However, these tests have been designed

    for clinical monitoring of circulating clotting factors during
    anticoagulant therapy.  The monitoring of occupational exposure to
    rodenticides requires the prothrombin time test to be of sufficient
    sensitivity to measure changes in the normal range (Tuinman & Van
    Sittert, 1985).

         Repeated occupational exposure to low levels of anticoagulant
    rodenticides could gradually deplete vitamin-K-dependent coagulation
    factors in the blood.  To detect unwanted exposure of humans in an
    early state, a careful screening of those at risk is recommended.  The
    question is which screening method is the most suitable to monitor low
    levels of rodenticide ingestion. Prothrombin time and related tests
    are "overall" clotting tests, which were developed for monitoring
    patients under deep anticoagulation.  These tests are easy to perform
    and do not require complicated equipment, but they are relatively
    insensitive when used for monitoring milder anticoagulation states
    (Tuinman & Van Sittert, 1985; Ross et al., 1992; Travis et al., 1993).
    If possible, specific and more sensitive tests should be used.  The
    most sensitive test, applicable over a wide range of anticoagulation
    states, is the direct detection of descarboxy-prothrombin using a
    monoclonal antibody specifically recognizing the descarboxy form of
    prothrombin (Widdershoven et al., 1987).  Another marker for
    monitoring poor vitamin K status at an early stage is
    descarboxy-osteocalcin (Knapen et al., 1993), but the commercial test
    kits presently available need to be substantially improved and
    simplified before they can be recommended for this purpose in routine
    laboratories.

         Another method was suggested by Park et al. (1986), who
    repeatedly injected 10 mg of vitamin K1 into factory workers who had
    been exposed to brodifacoum. The authors observed that 2-4 h after
    injection the circulating KO/K ratios were significantly elevated even
    18 months after the prothrombin times had returned to normal values.
    This suggests that very low liver concentrations of brodifacoum can be
    detected from the altered KO/K ratio rather than from tests based on
    blood coagulation parameters. Because this method requires vitamin K
    administration shortly before blood sampling, it is only applicable in
    cases of anticoagulant poisoning, and not for the routine control of
    plant workers.

         Methods for the direct detection of coumarin anticoagulants in
    plasma and serum have been reported, all of which are based on the
    extraction of plasma and pre-purification of the sample, followed by
    HPLC analysis with fluorescence detection (Hunter, 1983; Murphy et
    al., 1989; Felice & Murphy, 1989; Felice et al., 1991; O'Bryan &
    Constable, 1991).  However, such facilities will not be available in
    most routine laboratories.  Moreover, the blood sampling should be
    performed within a reasonably short period after ingestion of the
    coumarins, because these drugs are rapidly cleared by the liver.  This
    places severe restrictions on the applicability of these techniques,
    particularly for the second-generation anticoagulants.

         Plasma chlorophacinone determinations were performed in three
    cases of intoxication.  The risk of bleeding was minimal when the
    plasma level was below 1 mg/litre (Burcuoa et al., 1989).

         Brodifacoum was detected in a case of self-ingestion by a
    25-year-old woman who denied any intake of anticoagulants but was in
    need of vitamin K1 treatment for 8 months.  Factor assays revealed a
    marked reduction in the levels of the vitamin K-dependent factors II,
    VII, IX and X and normal levels of factor V and VIII:C (Exner et al.,
    1992).

         The diagnosis in a case of difenacoum intoxication was confirmed
    by analysis of difenacoum in serum (0.6 mg/litre) (Butcher et al.,
    1992).

         Bromadiolone was analysed in the serum of a 27-year-old female
    with a history of bleeding.  She denied any contact with a rodenticide
    but the bromadiolone concentration in serum was 40 mg/litre (Chow et
    al., 1992).

         Hollinger & Pastoor (1993) reported results of comparison of
    plasma brodifacoum concentrations and prothrombin levels over time in
    a case of brodifacoum poisoning.  Brodifacoum was eliminated according
    to a two-compartment mode, with an initial half-life of 18 h and a
    terminal half-life of 24.2 days.  On admission, the brodifacoum level
    was 731 µg/litre.  Treatment with large doses of phytonadione lasted
    for 4 months.

    8.6  Treatment of anticoagulant rodenticide poisoning

         All suspected poisoned patients should receive medical attention
    immediately.  Rapid determination of prothrombin time and search for
    evidence of bleeding is essential and may have to be maintained for
    several weeks.

    8.6.1  Minimizing the absorption

         Gastric lavage or induction of emesis are indicated in all cases
    of superwarfarin rodenticide ingestion if it was recent and the amount
    is possibly lethal or uncertain.  Repeated administration of activated
    charcoal is useful.  Cathartics could also be administered (Sullivan
    et al., 1989; Smolinske et al., 1989; Donovan et al., 1990).

    8.6.2  Specific pharmacological treatment

    8.6.2.1  Vitamin K1 (phytomenadione)

         Vitamin K1 is the specific antidote of choice.  Depending on
    whether the poisoning is due to warfarin or superwarfarin, the dosage
    may differ as well as the duration of treatment.  Dosage is dependent
    on coagulation parameters, mainly prothrombin time.

         If the patient is bleeding severely, 25 mg of vitamin K1
    (phytomenadione) should be given by slow intravenous injection. 
    Prothrombin time should be checked at 3-hourly intervals in severe
    cases and after 8-10 h in less severe cases.  If no improvement
    occurs, vitamin K1 injection should be repeated.  Doses of up to
    125-200 mg/day have been given without adverse effects (Lipton &
    Klass, 1984; Sheen et al., 1994).

         In moderate to minor cases of poisoning, vitamin K1 may be
    given in lower doses.

         After initial parenteral vitamin K1 administration, oral
    treatment can be continued for a prolonged period of time.  Oral
    treatment can also be sufficient in minor cases.

         The major difference between warfarin and second-generation
    rodenticides is that the latter can cause increased bleeding for a
    longer period of time than warfarin, as they have a much longer
    half-life in the body.  Therefore vitamin K1 should be given for
    months rather than weeks.  It is also prudent to monitor prothrombin
    time for some time after cessation of this treatment to ensure that
    there is no regression.

         In warfarin-resistant individuals, 10 times the normal dose of
    warfarin is required to achieve a reduction in the plasma prothrombin
    level.  However, these individuals also respond more strongly to the
    effect of vitamin K (O'Reilly et al., 1963; O'Reilly et al., 1964).

    8.6.2.2  Blood components

         The following products could be considered, subject to
    availability:

    *    whole blood
    *    fresh frozen plasma and fresh blood should be used in cases of
         acute severe bleeding in order to rapidly restore the blood
         clotting factors
    *    factor concentrate may be considered in severe cases, especially
         if the amount of plasma would be too great (volume overload)

    8.6.2.3  Phenobarbital

         In the cases reported by Lipton & Klass (1984) and Jones et al.
    (1984), phenobarbital therapy was included.  It was presumed that
    anticoagulants had been metabolized by the mixed function oxidase
    system based on animal experiments (Bachmann & Sullivan, 1983) and
    that, by inducing this enzyme system, phenobarbital might increase
    their metabolism.

         Robinson & Mac Donald (1966) found that the pharmacological
    activity of warfarin in humans decreased when it was combined with
    phenobarbital.

    8.6.3  Response to therapy

         Patient should be kept in hospital until the prothrombin time has
    remained normal for 3 days.  It is suggested that oral treatment with
    10 mg vitamin K1 twice daily may be necessary for up to 60 days with
    close monitoring of prothrombin time.

         According to Hoffman et al. (1988), factor analysis allows for a
    detailed evaluation of the course of toxicity, and the response to
    therapy.  Monitoring the prothrombin time alone could offer a false
    sense of confidence and delay effective treatment.

    9.  EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD

    9.1  Laboratory experiments

    9.1.1  Microorganisms

         No data are available for the effects on microorganisms in water
    and in soil.

    9.1.2  Aquatic organisms

         The acute toxicity of technical flocoumafen to planktonic algae
    (Selenastrum capricornutum) was determined in a 4-day growth test. 
    The 96-h EC50, based on cell counts on day 4, was calculated to be
    1.1 mg/litre (Pearson & Wallace, 1984).

         Pearson (1984) reported static acute toxicity tests for
    flocoumafen on  Daphnia magna.  The 48-h EC50 values, based on
    immobilization, were 1.4 mg/litre for technical grade flocoumafen and
    280 mg/litre for 0.5% flocoumafen.  Pearson & Wallace (1984) found a
    48-h EC50 for  Daphnia magna of 0.66 mg/litre for technical grade
    flocoumafen.

         Anticoagulants are highly toxic to fish when tested as technical
    formulations (Hill et al., 1976; Pearson & Wallace, 1984) (Table 8).

    9.1.3  Terrestrial organisms

    9.1.3.1  Acute toxicity

         Bird species vary in their susceptibility to anticoagulant
    rodenticides.  The acute oral LD50 of brodifacoum for the mallard
    duck  (Anas platyrhynchos) is 2.0 mg/kg (Ross et al., 1978). 
    Symptoms of poisoning became apparent approximately 7 days after
    dosing and included lethargy, weakness and lack of muscular
    coordination.  Prolonged bleeding occurred from any small wounds and
    extensive bruising and subcutaneous haemorrhage were noted.  Blood was
    observed in the faeces.  Deaths occurred principally during the period
    of 7-14 days after administration.  No deaths occurred more than 4
    weeks after administration.

         The acute toxicity of flocoumafen to the mallard duck appears to
    be dependent on the age of birds.  The LD50 values for 12- and
    18-week-old birds are 24 mg/kg and 94 mg/kg, respectively (Roberts et
    al., 1985b,c).


        Table 8.  Acute toxicity of anticoagulant rodenticides for aquatic organisms (96-h LC50)
                                                                                                                                                

    Rodenticide                       Species                  Stat/flowa     LC50 (mg/litre)         Reference
                                                                                                                                                

    Brodifacoum                     Rainbow trout                flow             0.051          Hill et al. (1976)

    Diphacinone (95%)               Bluegill sunfish                              7.6            Kosmin & Barlow (1976)
                                    Channel catfish                               2.09           Kosmin & Barlow (1976)
                                    Rainbow trout                                 2.82           Kosmin & Barlow (1976)
                                    Pink shrimp                                   > 10           Kosmin & Barlow (1976)
                                    Fiddler crab                                  > 10           Kosmin & Barlow (1976)

    Diphacinone (0.005%)            Bluegill sunfish                              288            Kosmin & Barlow (1976)
                                    Channel catfish                               285            Kosmin & Barlow (1976)
                                    Rainbow trout                                 425            Kosmin & Barlow (1976)
                                    Pink shrimp                                   33             Kosmin & Barlow (1976)
                                    Fiddler crab                                  37             Kosmin & Barlow (1976)

    Warfarin                        Bluegill sunfish                              88             Brorson et al. (1994)

    Flocoumafen (0.5%)              Rainbow trout                static           190            Pearson (1984)

    Flocoumafen (technical grade)   Rainbow trout                static           0.95           Pearson (1984)
                                    Rainbow trout                static           0.32           Pearson & Wallace (1984)
                                    Rainbow trout                static           0.091          Pearson & Wallace (1984)
                                    Common carp                  static           0.22           Pearson & Wallace (1984)

    Bromadiolone                    Rainbow trout                                 1.4            Tomlin (1994)
                                                                                                                                                

    a   stat = static conditions (water unchanged for duration of test)
    

         Sex differences were observed in the acute toxicity for bobwhite
    quail  (Colinus virginianus) of difenacoum.  LD50 values for male
    and female birds were 140 mg/kg body weight and 56 mg/kg body weight,
    respectively (Ross et al., 1980c).

         Birds seem to be relatively tolerant to diphacinone, the LD50
    for mallard duck being 3158 mg/kg (Kosmin & Barlow, 1976).

         The acute toxicity of brodifacoum for various species of birds is
    presented in Table 9.

    Table 9.  The acute toxicity of brodifacoum to birds (Godfrey, 1985)
                                                                        

    Species                                            Acute oral LD50
                                                            (mg/kg)
                                                                        

    Black-backed gull (Larus dominicans)                    < 0.75
    Black-billed gull (L. bulleri)                          < 5.0
    Canada goose (Branta canadensis canadensis)             < 0.75
    Pukeko (Porphyris porphyris melanotus)                    0.95
    Mallard duck (Anas platyrhynchus)                         4.6
    California quail (Lophortyx californica)                  3.3
    Blackbird (Turdus merula)                               > 3
    House sparrow (Passer domesticus)                       > 6
    Hedge sparrow (Prunella modularis occidentalis)         > 3
    Wax-eye (Zosterops lateralis)                           > 6
    Harrier hawk (Circus approximans)                        10.0
    Ring-necked pheasant (Phasianus colchicus)               10.0
    Paradise duck (Tadorna variegata)                      > 20
                                                                        

    9.1.3.2  Primary toxicity

         The subacute (5-day) dietary toxicity (LC50) of difenacoum for
    the bobwhite quail was found to be between 0.25 and 7.00 mg/kg diet
    (Ross et al., 1980a).  The 5-day LC50 of difenacoum for the mallard
    duck was calculated to be 18.9 mg/kg diet (Ross et al., 1980b).

         Similarly to the acute toxicity studies, diphacinone was found to
    be less toxic in 8-day dietary studies.  The LC50 for the bobwhite
    quail was 4485 mg/kg diet and for the mallard duck was > 10 000 mg/kg
    diet (Kosmin & Barlow, 1976).

         White Leghorn hens (4 per group) were fed warfarin,
    coumatetralyl, bromadiolone, difenacoum or brodifacoum in baits as a
    choice to non-poisonous chicken food for 15 days.  No symptoms
    appeared at a total warfarin intake of up to 171 mg/kg body weight. 
    Coumatetralyl and brodifacoum killed all hens after intakes of
    79-137 mg/kg body weight of active ingredient and 7.1-15 mg/kg body
    weight of active ingredient, respectively.  Both bromadiolone and
    difenacoum killed two of the hens, at 5.9 and 15.9 mg/kg body weight
    of  active ingredient for bromadiolone, and at 18.9 and 26.3 mg/kg
    body weight active ingredient for difenacoum within 10-15 days (Lund,
    1981).

         Christopher et al. (1984) dosed male hybrid leghorn chickens with
    anticoagulant rodenticides.  No signs of poisoning were observed in
    chickens fed 183.7 mg warfarin/kg (mean a.i. ingested) for 3 days.  No
    mortality occurred after the ingestion of a total dose of 36.9 mg
    bromadiolone/kg over a 3-day period.  Birds fed a diet containing
    brodifacoum for 3 days ingested a mean of 28.9 mg/kg (active
    ingredient).  One bird died on day 4 while the other five birds
    exposed had died by day 16.

         Two-day no-choice tests in which chukar partridges ( Alectoris
     graeca cypriotes Hartert) were fed 0.005% bromadiolone or difenacoum
    resulted in no mortality.  Ten-day no-choice feeding tests resulted in
    6 out of 12 and 8 out of 12 dead partridges for bromadiolone and
    difenacoum, respectively.  The highest quantity of bait consumed by an
    individual bird without any lethal effects was 411 g of bait with
    bromadiolone (34.8 mg a.i./kg body weight) and 326.2 g of bait with
    difenacoum (29.1 mg a.i./kg body weight) (Krambias & Hoppe, 1987).

    9.1.3.3  Secondary toxicity

         Barn owls  (Tyto alba) were fed rats poisoned with diphacinone,
    chlorophacinone, coumafuryl, difenacoum, bromadiolone or brodifacoum. 
    Five out of six owls died of haemorrhaging after feeding on rats
    killed with brodifacoum after 8 to 11 days.  Sublethal haemorrhaging,
    but no mortality, occurred in owls fed rats killed with difenacoum. 
    One owl died following 10 days of treatment with bromadiolone-poisoned
    rats, while five showed no symptoms.  No abnormalities were observed
    in two owls fed rats killed with diphacinone, coumafuryl or
    chlorophacinone.  Owls that died behaved normally until 24 h or less
    before death, when they became lethargic and stopped eating
    (Mendenhall & Pank, 1980).

         Mice (weighing 35 g) were fed for 1 day (no choice) on a mixture
    containing either 0.005% difenacoum or 0.002% brodifacoum.  The mean
    mass of residue on the day of death in a whole mouse was estimated to
    be 10.17 µg for difenacoum and 15.36 µg for brodifacoum. Difenacoum-
    and brodifacoumpoisoned mice were fed to captive barn owls for
    successive periods of 1, 3 and 6 days.  All of the owls fed on
    difenacoum-poisoned mice survived the treatments and none showed
    external bleeding.  In contrast, four of the six owls fed
    brodifacoum-poisoned mice died within 6-17 days after a 1-day dose. 
    The estimated lethal dose of brodifacoum was 0.15-0.18 mg/kg.  After
    death these owls had 0.63-1.25 mg brodifacoum/kg in their livers
    (Newton et al., 1990).

         Newton et al. (1994) fed barn owls flocoumafen-dosed mice for
    consecutive periods of 1, 3 and 6 days.  Four of the birds survived; 
    the fifth bird died from haemorrhaging 5 days after the final dose. 
    During the feeding trial birds received cumulative flocoumafen doses
    ranging from 0.78 to 1.25 mg/kg.

         Gray et al. (1992) investigated the toxicity of brodifacoum,
    difenacoum and flocoumafen for barn owls fed poisoned mice.  For each
    rodenticide, the owls survived a cumulative dose of at least 1.9 mg/kg
    owl weight over 15 days of treatment.  All owls with cumulative doses
    in excess of 1.9 mg/kg body weight showed multiple treatment-related
    effects.  The three rodenticides had approximately the same order of
    magnitude of toxicity to barn owls.  Gray et al. (1994) developed a
    non-invasive method for monitoring the exposure of these birds to
    second-generation rodenticides by measuring compounds in regurgitated
    owl feed.

         Radvanyi et al. (1988) fed American kestrels (Falco sparverius)
    on meadow voles that had been maintained on 2% chlorophacinone.  Voles
    consumed approximately 53 mg of 2% chlorophacinone (1.14 mg a.i.)
    before dying within 6 days.  No kestrels fed poisoned mice, for up to
    21 consecutive days, died.  Haematomas were observed on the pectoral
    muscles, lungs, liver and heart of exposed birds.

         In a study by Townsend et al. (1981), captive-bred tawny owls
     (Strix aluco) were given warfarin-treated mice for 3 months.  No
    behavioural changes were observed.  Prothrombin levels decreased to
    less than 10-12% of normal but returned to normal after 9 days.

         Townsend et al. (1984) found that daily warfarin intakes
    approaching 0.3 mg/kg body weight or 30 µg of warfarin/day can cause
    the death of list weasels  (Mustela nivalis) by secondary poisoning. 
    Lethal exposure occurred if mice were contaminated with 1.0-1.5 mg
    warfarin/kg, an amount reached following exposure to 0.005 or 0.02%
    sodium warfarin for about 3 days.

    9.2  Field observations

    9.2.1  Primary poisonings

         There are few data available on the non-target hazard of
    anticoagulant rodenticides in field conditions.

         Greig-Smith et al. (1988) reported incidents in England and Wales
    with barn owls and foxes.  There were three incidents in which barn
    owls were found to have residues of the anticoagulant rodenticide
    brodifacoum in their livers.  In two birds, residues were thought to
    represent lethal poisoning (0.61 and 0.29 mg/kg) but in the third the
    level was only 0.099 mg/kg.  There were six separate incidents in
    which dead foxes were found to contain residues of brodifacoum,
    bromadiolone, coumatetralyl, difenacoum or warfarin. Residue levels
    showed a wide range from 0.009 to 0.46 mg/kg tissue.  Three of the
    foxes contained more than one rodenticide, one animal revealing
    residues of difenacoum, bromadiolone and warfarin.

         Poisonings of pheasants and partridges by chlorophacinone used
    against Microtus arvalis have been reported (Giban, 1974).

         Reece et al. (1985) reported that, a few days after bromadiolone
    was placed out for control of rodents, three pea fowl
     (Pavo cristatus), a little raven  (Corvus mellori) and an eastern
    swamp hen  (Porphyrio porphyrio melanotus) died.  Necropsy showed
    that the birds had been in good condition.  One of the pea fowl and
    the swamp hen had massive intra-abdominal haemorrhage without evidence
    of external trauma.  All had severe congestion of the liver and lungs. 
    The birds had negligible gut contents, so analysis for bromadiolone
    could not be performed.

    9.2.2  Secondary poisonings

         Difenacoum and brodifacoum were detected in 15 of a total of 145
    dead barn owls in Britain, and on postmortem the cause of death for
    one owl was diagnosed as rodenticide poisoning (Newton et al., 1990,
    see section 5.1).

         A study of 35 active nests and the radio-telemetry of 34 barn
    owls in an area where brodifacoum baits (0.005%) were used in and
    around farm buildings in New Jersey, USA, showed no adverse effects of
    the rodenticide on the owls studied.  In this study, rats and house
    mice were the target organisms and barn owls were found not to feed on
    these animals to any extent.  Chicks were fledged from at least eight
    nests where poisoned rodents had been available during part of the
    nesting and feeding period, but no rodenticide-induced mortality was
    observed.  Traces of brodifacoum (less than 0.05 mg/kg) were found in
    a barn owl that had been accidentally electrocuted (Hegdal &
    Blaskiewicz, 1984).

         Merson & Byers (1984) monitored eastern screech owls  (Otus asio)
    using radio-transmitters following the use of 0.005% brodifacoum for
    rodent control in a commercial orchard. Brodifacoum was detected in
    screech owl pellets (see section 5.1).  There were no owl deaths which
    could be attributed to brodifacoum poisoning.  Two owls were analysed
    at the end of the study;  one showed signs of haemorrhage and
    contained 0.21 mg brodifacoum/kg and the other owl contained no
    brodifacoum.  In a larger study, Hegdal & Colvin (1988) monitored 38
    eastern screech owls and several other species of owl following
    brodifacoum application in an orchard.  Minimum mortality was 58%
    among screech owls for which more than 20% of the home range was
    treated, as compared  with 17% among those for which less than 10% of
    the home range was treated.  Secondary brodifacoum poisoning was the
    most probable cause of death in six screech owls.  Six radio-equipped
    owls were collected 1-2 months post-treatment, and four contained
    detectable concentrations of brodifacoum.

    10.  EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT

    10.1  Evaluation of human health risks

         Anticoagulants, both hydroxycoumarins and indandiones, known also
    as vitamin K antagonists, are widely used in urban rodent control and
    against rodent pests in agriculture.  They act by inhibiting the
    vitamin K1 epoxide cycle, thereby depleting the active form of
    vitamin K1 necessary for producing blood-clotting factors.

         Since anticoagulant rodenticides are used in general as
    low-concentration bait formulations and have low volatility, increased
    levels in the air are unlikely.  Being only slightly soluble in water
    their use could not be a major source of water contamination. 
    Anticoagulant rodenticides are not intended for direct application to
    growing crops.  Hence no residues in plant foodstuffs are expected.

         The controlled medicinal use of warfarin exposes more people to
    higher concentrations over a longer period than would be expected to
    occur as a result of accidental human exposure due to its use as a
    rodenticide.

         Occupational exposure may occur during manufacture, formulation
    and bait application, but figures indicating the levels of exposure
    are not available.

         Anticoagulant rodenticides are readily absorbed through the
    gastrointestinal tract, skin and respiratory system.  The major route
    of elimination in various species after oral administration is through
    the faeces. The urine is a very minor route of elimination.  The liver
    is the major organ for accumulation and storage of anticoagulants.

         The metabolism pattern of warfarin and indandiones mainly
    involves hydroxylation.  The second-generation hydroxycoumarins are
    found principally as unchanged parent compounds.  Their elimination
    from the liver is slow with a biphasic rate, where a rapid initial
    phase is followed by a prolonged second phase.

         Most of the anticoagulants have high acute toxicity by oral,
    percutaneous and inhalation routes of exposure.  The second-generation
    anticoagulants are more toxic than the first-generation one in the
    sense that a single feeding may be lethal.  Signs of poisoning in all
    species, including humans, are associated with increased bleeding
    tendency.

         Many poisoning incidents (both intentional and unintentional)
    have been reported. A few cases of intoxication from occupational
    exposure to anticoagulants have also been observed.

         The level of prothrombin concentration is a satisfactory guide to
    the severity of acute intoxication and the effectiveness and duration
    of the therapy.  The specific antidote is vitamin K1.  The high
    retention of the second-generation anticoagulants in the liver means
    that any treatment of intoxication should be prolonged to ensure that
    anticoagulant activity does not recur.

         The addition of bittering agents to anticoagulant rodenticides is
    aimed at discouraging human consumption.

         Warfarin has been found to be teratogenic in both rats and
    humans.  Second-generation anticoagulant rodenticides do not
    demonstrate teratogenicity in laboratory animals.  Developmental
    effects in humans are observed when warfarin has been taken as a
    therapeutic agent during pregnancy.  No cases of embryopathy from
    anticoagulants in their use as rodenticides have been reported. 
    Second-generation anticoagulants are not intended to be used as
    therapeutic agents, and the risks associated with, for example,
    warfarin will not apply.

         There is no evidence to suggest that any anticoagulant
    rodenticides are mutagenic, but there are insufficient data available
    on some compounds to demonstrate an absence of mutagenicity.

         There is experimental evidence that coumarin anticoagulants not
    only inhibit the vitamin K cycle in the liver, but also in other
    tissues such as bone.  In humans it has been demonstrated that even
    low levels of vitamin K deficiency form a risk factor for developing
    osteoporosis (Hart et al., 1985; Szulc et al., 1993).  Therefore, it
    appears that long-term exposure to low levels of anticoagulant may
    have an adverse effect on bone metabolism.

    10.2  Evaluation of effects on the environment

         Unlike conventional crop protection products, which must be
    applied over relatively large crop areas, anticoagulant rodenticides
    are applied to discrete sites in the form of low concentration baits.  

         Most of the anticoagulants are stable under normal conditions. 
    They are slightly soluble in water, and as bait formulations their use
    is unlikely to be a source of water contamination.  They appear to
    bind rapidly in the soil, with very slow desorption and no leaching. 
    In general, anticoagulants are highly toxic to aquatic organisms when
    tested as technical material.

         Non-target organisms are potentially at risk in two ways: from
    direct consumption of baits (primary hazard) and through eating
    poisoned rodents (secondary hazard).

         Bird species vary in their susceptibility to anticoagulant
    rodenticides.  Small pellets and whole grain baits are highly
    attractive to birds.  Wax block formulation appear to decrease the
    attractiveness to the birds and so reduce the possibility of poisoning
    incidents.  It is difficult to assess the risks to birds due to direct
    consumption of baits because most of the published studies are
    toxicity trials in laboratory conditions.

         The main cause for poisoning of domestic animals is through
    direct consumption of anticoagulant baits.

         Some of the anticoagulants show a similar range of acute toxicity
    for non-target non-rodent mammals as for target rodents.  The primary
    hazard is usually expressed by the amount of finished bait which must
    be consumed to approach the lethal dose.  The bait concentration of
    second-generation rodenticides is usually around 0.005%, whereas the
    concentration of the first-generation rodenticides in the bait may be
    5 to 10 times higher.  Thus, to reach the toxic or lethal dose,
    non-target animals must consume comparatively large amounts of bait
    with low concentrations of active ingredient.

         Secondary poisoning through the consumption of rats and mice
    killed with anticoagulants may occur in dogs and cats in urban
    situations but is more likely in farm situations.

         Some secondary toxicity laboratory studies with wildlife have
    shown that captive predators could be intoxicated by no-choice feeding
    of anticoagulant-poisoned or dosed prey.  The significance of these
    results in terms of hazard under field conditions is difficult to
    assess because the predators would not be expected to eat only
    poisoned animals.  However, where they occur, predators may take
    poisoned, but not dead, small mammals preferentially.  In areas close
    to baiting, poisoned rodents may represent a high proportion of the
    diet for individual birds.  However, only a few individuals will be
    affected except in situations of very widespread and constant use of
    baits.  Therefore, some kills of owls will be expected but no severe
    population effects. This agrees with observed field effects with small
    numbers of poisoned owls.

    11.  CONCLUSIONS AND RECOMMENDATIONS FOR PROTECTION OF HUMAN HEALTH
         AND THE ENVIRONMENT

    11.1  Conclusions

    a)   Exposure of the general population to anticoagulant rodenticides
         via food and drinking-water is unlikely and does not constitute a
         significant health hazard.

    b)   Poisoning incidents may occur in cases of massive intentional or
         unintentional ingestion or prolonged skin contact during
         manufacture and formulation.

    c)   Anticoagulant rodenticides are relatively persistent in the
         environment, but their specific use as low-concentration bait
         formulations limits the potential for environmental
         contamination.

    d)   Direct and secondary poisoning of birds, domestic and farm   
         animals, and wildlife may occur.

    e)   The major difference between the first- and second-generation
         anticoagulant rodenticides is that the latter have longer body
         retention, resulting in an increased tendency for bleeding over a
         longer period of time.

    f)   The mode of action of anticoagulant rodenticides is known and an
         effective antidote is available, i.e. vitamin K1.

    g)   There is no available evidence that this class of compound is
         mutagenic or carcinogenic.

    h)   Only warfarin has been shown to possess some teratogenic
         potential in both rats and humans.

    11.2  Recommendations for protection of human health and the
          environment

    a)   Exposed workers should receive appropriate biomonitoring and
         health evaluation.

    b)   The inclusion of a bittering agent in formulations at appropriate
         concentrations may reduce accidental ingestion.

    c)   For preventing primary poisoning, baits less attractive to birds
         and domestic animals, as well as pulsating baiting, should be
         used.

    d)   The location of bait placement should be carefully selected.

    e)   Killed rodents should be burned or buried to reduce the risk of
         secondary poisoning of predators.

    f)   Training in the safe handling of rodenticides is essential.

    12.  FURTHER RESEARCH

    a)   Studies of exposed humans are required, particularly with regard
         to possible teratogenic and embryotoxic effects.

    b)   More information is needed in order to evaluate the risks of
         occupational exposure to anticoagulant rodenticides.

    c)   Methods more sensitive than prothrombin time determination need
         to be developed for routinely assessing the absorption and
         effects of anticoagulants.

    d)   More data are required to assess secondary effects on non-target
         organism populations.

    e)   The extent to which second-generation anticoagulant rodenticides
         are transferred across the placenta should be evaluated.

    f)   More information should be collected concerning the tissue
         distribution of anticoagulant rodenticides after their ingestion
         and their effects on physiological processes other than blood
         coagulation, notably calcium and bone metabolism. This is
         particularly necessary for long-term, low-level exposure to these
         compounds.

    13.  PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES

         In the WHO Recommended Classification of Pesticides by Hazard
    (WHO, 1994), anticoagulant rodenticides were classified, according to
    their acute oral LD50, as follows:

    Class Ia (Extremely hazardous)          Oral LD50 (mg/kg)

         Brodifacoum                               0.3
         Bromadiolone                              1.12
         Chlorophacinone                           3.1
         Difenacoum                                1.8
         Difethialone                              0.56
         Diphacinone                               2.3
         Flocoumafen                               0.25

    Class Ib (Highly hazardous)

         Coumachlor                                33
         Coumatetralyl                             16
         Warfarin                                  10

    Class II (Moderately hazardous)

         Pindone                                   50

         A Poison Information Monograph for brodifacoum has been issued
    (IPCS, 1992), and one on warfarin is in preparation.

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    Veenstra GE, Owen DE, & Huckle KR (1991) Metabolic and toxicological
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    RESUME

    1.  Généralités

         Les anticoagulants qui sont décrits dans la présente monographie
    sont ceux que l'on utilise principalement en agriculture et pour la
    destruction des rongeurs en milieu urbain.  La warfarine, premier
    rodenticide anticoagulant à connaître une large utilisation, était à
    l'origine un médicament efficace pour le traitement des
    thromboembolies chez l'homme.

         Selon leur structure chimique, les rodenticides anticoagulants
    peuvent se répartir en deux catégories, les hydroxycoumarines et les
    indanediones, mais leur mode d'action est analogue.

    2.  Propriétés et méthodes d'analyse

         Les rodenticides anticoagulants se présentent sous la forme de
    solides cristallins ou de poudres et sont légèrement solubles dans
    l'eau.  La plupart d'entre eux sont stables dans les conditions
    normales de conservation.

         La plupart des méthodes de dosage des rodenticides anticoagulants
    reposent sur la chromatographie en phase liquide à haute performance.

    3.  Sources d'exposition humaine et environnementale

         Les hydroxycoumarines de la première génération ont commencé à
    être utilisées comme rodenticides vers la fin des années 1940.  Par la
    suite, l'apparition d'une résistance à la warfarine et aux autres
    anticoagulants de la première génération a conduit à mettre au point
    des produits de deuxième génération, plus actifs.  La concentration du
    principe actif dans les appâts varie selon l'efficacité du
    rodenticide.

    4.  Distribution, concentration et exposition dans l'environnement

         Les rodenticides anticoagulants sont principalement utilisés sous
    la forme d'appâts.  Comme ils sont peu volatils, leur concentration
    dans l'air est négligeable.  De même n'étant que légèrement solubles
    dans l'eau, il est peu probable que leur utilisation conduise à la
    contamination des eaux.

         Etant donné que les rodenticides anticoagulants ne sont pas
    destinés à être appliqués directement sur les cultures, il n'y a pas
    lieu de s'attendre à en trouver sous forme de résidus dans les
    aliments d'origine végétale.

         L'exposition aux rodenticides des vertébrés non visés peut se
    produire directement par la consommation d'appâts empoisonnés et
    indirectement par celle de rongeurs contaminés.  Les petits granulés
    et les appâts constitués de grains entiers attirent fortement les
    oiseaux.

         La warfarine est utilisé dans le traitement des thromboembolies.

         Il existe une possibilité d'exposition professionnelle aux
    rodenticides anticoagulants lors de leur fabrication ou formulation ou
    encore lors de la pose d'appâts empoisonnés, mais on ne dispose pas de
    données sur l'ampleur de cette exposition.

    5.  Mode d'action et métabolisme

         Les rodenticides anticoagulants sont des antagonistes de la
    vitamine K.  Leur action est principalement localisée dans le foie où
    plusieurs précurseurs de l'hémostase subissent un processus
    post-traductionnel dépendant de la vitamine K avant d'être convertis
    en zymogènes procoagulants.  L'action proprement dite consiste dans
    l'inhibition de la K1-époxyde-réductase.

         Les rodenticides anticoagulants sont facilement absorbés au
    niveau des voies digestives et pourraient l'être également à travers
    la peau et dans les vois respiratoires.  Après administration par voie
    orale, c'est principalement dans les matières fécales que ces composés
    sont éliminés chez diverses espèces.

         Il est possible que la décomposition métabolique de la warfarine
    et des indanediones chez le rat s'effectue principalement par
    l'intermédiaire d'une hydroxylation.  En revanche, les anti-coagulants
    de la deuxième génération s'éliminent essentiellement tels quels.  Le
    faible taux d'excrétion urinaire empêche d'isoler les métabolites de
    l'urine.

         Le foie est le principal organe où s'accumulent les rodenticides
    anticoagulants.  Cette accumulation a lieu également dans les
    graisses.

    6.  Effets sur les mammifères et les systèmes d'épreuve in vitro

         Chez le rat et la souris, les signes d'intoxication consistent en
    une tendance accrue au saignement.

         La DL50 est très variable, et c'est par la voie orale que les
    composés sont les plus toxiques.  La toxicité est également élevée par
    la voie percutanée et par la voie respiratoire.

         Certains anticoagulants présentent une toxicité aiguë du même
    ordre pour les mammifères non visés que pour les rongeurs à détruire,
    toutefois leur spectre toxique peut varier d'une espèce à l'autre.

         Après administration répétée par voie orale à des rats, les
    principaux effets observés sont ceux qui résultent de l'activité
    anticoagulante.

         On ne possède que peu de données sur les expositions répétées aux
    anticoagulants chez les espèces n'appartenant pas à l'ordre des
    rongeurs.

         Une étude relative aux effets de la warfarine sur le rat a fait
    ressortir une action sur le développement de ces animaux.  En dehors
    de cela, rien n'indique que les anticoagulants aient une action
    tératogène sur les animaux d'expérience.

         Rien n'indique non plus que les rodenticides anticoagulants
    soient mutagènes, toutefois les données relatives aux différents
    composés sont insuffisantes pour qu'on puisse en conclure à l'absence
    de mutagénicité.  La souche, le sexe et le régime alimentaire sont des
    facteurs importants qui influent sur la toxicité des anticoagulants
    chez les rongeurs.  On a fait état d'épisodes d'intoxication chez des
    animaux domestiques qui avaient consommé des appâts additionnés
    d'anticoagulants.  Lorsqu'il y a issue fatale ou du moins un tableau
    clinique grave, c'est généralement qu'il y a eu consommation
    d'anticoagulants de la deuxième génération.  La principale différence
    entre la warfarine et les autres anticoagulants (qu'il s'agisse des
    indanediones ou des hydroxycoumarines de la deuxième génération),
    c'est que ceux-ci persistent plus longtemps dans l'organisme et ont
    par conséquent un effet plus durable que la warfarine.  Dans ces
    conditions, devant une intoxication, il faut poursuivre plus longtemps
    l'administration de l'antidote, c'est-à-dire de la vitamine K1.

    7.  Effets sur l'homme

         De nombreuses intoxications (qu'elles soient intentionnelles on
    non) ont été signalées.  Il y a eu également quelques cas
    d'intoxication imputables à une exposition professionnelle.  En cas
    d'intoxication aiguë par des rodenticides anticoagulants, les
    symptômes vont de l'accroissement de la tendance au saignement en cas
    d'intoxication minime ou modérée, à une hémorragie massive dans les
    cas plus graves.  Les signes cliniques se manifestent un à plusieurs
    jours après l'absorption.

         Chez l'homme, on attribue certaines malformations congénitales à
    des traitements par la warfarine pendant la période de grossesse. 
    Aucune malformation de ce type n'a été observée par suite de
    l'utilisation d'anticoagulants comme rodenticides.

         La concentration de la prothrombine plasmatique donne une
    indication de la gravité de l'intoxication.  Elle est plus sensible
    que des épreuves globales telles que le temps de Quick.  En cas
    d'exposition professionnelle répétée, le dosage direct des traces de
    carboxyprothrombine circulante ou du 2,3-époxyde de la vitamine K peut
    permettre un bilan plus sensible.

         Le traitement d'une intoxication par des anticoagulants doit être
    adapté à la gravité de l'intoxication.  Il est basé sur l'action
    pharmacologique spécifique de la vitamine K1 que l'on administre par
    voie parentérale avec, dans les cas graves, administration simultanée
    de constituants sanguins.  La mesure du temps de Quick permet
    d'apprécier l'efficacité du traitement et de déterminer sa durée.

    8.  Effets sur les autres êtres vivants au laboratoire et dans leur
        milieu naturel

         On peut répartir en deux catégories les effets possibles de
    rodenticides anticoagulants sur les organismes non visés: les effets
    directs (par consommation d'appâts) et les effets indirects (par
    consommation de rongeurs empoisonnés).

         Sous forme de produit technique, les anticoagulants sont
    extrêmement toxiques pour les poissons.  Incorporés à des appâts, il
    est peu probable qu'ils présentent un danger, du fait de leur faible
    solubilité dans l'eau.  C'est pourquoi, à moins d'une erreur de
    manipulation, ils ne devraient pas parvenir jusqu'aux poissons.

         Les différentes espèces d'oiseaux sont d'une sensibilité variable
    aux rodenticides anticoagulants.  Il est difficile d'apprécier les
    risques que représente, pour les oiseaux, la consommation directe
    d'appâts car la plupart des travaux publiés sont des études
    toxicologiques effectuées au laboratoire.  Le caractère attractif des
    appâts constitués de grains entiers pour les petits oiseaux en accroît
    le danger dans la nature.

         Des études de toxicité indirecte effectuées en laboratoire sur
    des animaux sauvages ont montré que des prédateurs captifs pouvaient
    s'intoxiquer si on leur donnait de la nourriture empoisonnée sans
    autre choix ou des proies empoisonnées.  On a également constaté la
    mort de prédateurs dans le milieu naturel.

    9.  Evaluation et conclusion

         Les rodenticides anticoagulants bloquent le mécanisme normal de
    l'hémostase, d'où une tendance accrue au saignement qui peut déboucher
    sur une forte hémorragie.

         Il est peu probable que la population générale puisse être
    exposée involontairement à des rodenticides anticoagulants.

         Il peut y avoir une exposition non négligeable par suite de
    contacts lors de l'activité professionnelle.  Ces contacts peuvent se
    produire au cours des opérations de production et de formulation aussi
    bien que lors de la préparation et de la pose des appâts.

         Les rodenticides anticoagulants sont facilement absorbés au
    niveau des voies digestives ainsi qu'à travers la peau et les voies
    respiratoires.  C'est principalement dans le foie qu'ils sont retenus
    et s'accumulent.  La concentration de prothrombine plasmatique est un
    bon indicateur de la gravité d'une intoxication aiguë et permet
    d'avoir une idée de l'efficacité et de la durée nécessaire du
    traitement.

         L'antidote spécifique est la vitamine K1.

         La principale différence entre les rodenticides anticoagulants de
    première et de deuxième génération tient au fait que ces derniers
    séjournent plus longtemps dans l'organisme et ont donc tendance à
    prolonger l'effet hémorragique.

         La plupart des anticoagulants sont stables dans les conditions
    normales d'utilisation.  Comme ils sont peu solubles dans l'eau et que
    les appâts n'en contiennent qu'une faible quantité, il est peu
    probable qu'ils puissent contaminer les étendues d'eau.  Par ailleurs,
    ils se fixent rapidement aux particules du sol, ne s'en désorbent que
    très lentement et ne sont pas lessivés.

         Aucun organisme non visé ne court de risque d'intoxication
    directe par consommation d'appâts, ni de risque d'intoxication
    indirecte par consommation de rongeurs contaminés.

    RESUMEN

    1.  Generalidades

         Los anticoagulantes descritos en esta monografía son los
    utilizados principalmente en agricultura y en la lucha contra los
    roedores urbanos.  La warfarina, el primer rodenticida anticoagulante
    de uso generalizado, se introdujo como agente eficaz para el
    tratamiento de la tromboembolia en el ser humano.

         De acuerdo con su estructura química, los rodenticidas
    anticoagulantes pueden agruparse en dos categorías, las
    hidroxicumarinas y las indandonas, aunque su mecanismo de acción es
    similar.

    2.  Propiedades y métodos analíticos

         Los rodenticidas anticoagulantes se presentan en forma cristalina
    sólida o en polvo, y son ligeramente solubles en agua.  La mayoría de
    ellos son estables en condiciones de almacenamiento normales.

         La mayoría de los procedimientos para la determinación de los
    rodenticidas anticoagulantes se basan en cromatografía líquida de alta
    resolución.

    3.  Fuentes de exposición humana y ambiental

         Las hidroxicumarinas de primera generación se introdujeron como
    rodenticidas a finales de los años cuarenta.  La aparición de
    resistencia a la warfarina y a otros anticoagulantes de primera
    generación dio lugar a la elaboración de anticoagulantes más potentes,
    de segunda generación.  Las concentraciones de componentes activos en
    los cebos varían en función de la eficacia de los rodenticidas.

    4.  Distribución, niveles y exposición ambientales

         Los rodenticidas anticoagulantes se utilizan principalmente como
    formulaciones para cebo.  Dada su baja volatilidad, las
    concentraciones en el aire son insignificantes.  Como son muy poco
    solubles en agua, es improbable que su uso sea fuente de contaminación
    del agua.

         Como los rodenticidas anticoagulantes no están pensados para su
    aplicación directa a cosechas en pie, no son de prever residuos en
    alimentos vegetales.

         Los vertebrados no destinatarios están expuestos a los
    rodenticidas principalmente por medio del consumo del cebo y de forma
    secundaria por el consumo de roedores envenenados.  Los cebos en
    bolitas y de grano entero son muy atractivos para las aves.

         La warfarina se utiliza como agente terapéutico para la
    tromboembolia.

         Hay un potencial de exposición ocupacional a los rodenticidas
    anticoagulantes durante la fabricación, formulación y aplicación del
    cebo, pero no se dispone de datos sobre los niveles de exposición.

    5.  Modo de acción y metabolismo

         Los rodenticidas anticoagulantes son antagonistas de la vitamina
    K.  Su lugar principal de acción es el hígado, donde varios de los
    precursores de la coagulación de la sangre sufren un procesamiento
    post-traslación dependiente de la vitamina K antes de convertirse en
    los zimógenos procoagulantes respectivos.  Parece que el mecanismo de
    acción es la inhibición de la reductasa epoxídica K1.

         Los rodenticidas anticoagulantes se absorben fácilmente por el
    tracto intestinal, y también pueden absorberse por la piel y el
    sistema respiratorio.  Tras la administración oral, la principal vía
    de eliminación en diversas especies son las heces.

         La degradación metabólica de la warfarina y las indandonas en
    ratas es principalmente la hidroxilación.  Sin embargo, los
    anticoagulantes de segunda generación se eliminan principalmente como
    compuestos inalterados.  El bajo nivel de excreción urinaria impide
    aislar los metabolitos a partir de la orina.

         El hígado es el órgano principal para la acumulación y
    almacenamiento de anticoagulantes rodenticidas.  La acumulación
    también tiene lugar en la grasa.

    6.  Efectos en los mamíferos y en los sistemas de prueba in vitro

         Los signos de envenenamiento en ratas y ratones son los asociados
    a una mayor tendencia a la hemorragia.

         Hay una gran variación en la DL50 de los rodenticidas
    anticoagulantes, siendo máxima la toxicidad por vía oral.  También es
    alta la toxicidad cutánea y por inhalación.

         Los márgenes de toxicidad aguda de algunos anticoagulantes son
    similares en el caso de los mamíferos no destinatarios y de los
    roedores de destino, pero los espectros de toxicidad para los
    anticoagulantes pueden variar entre las especies.

         Tras una administración oral repetida en ratas, los principales
    efectos observados son los asociados a la acción anticoagulante.

         Se dispone de pocos datos sobre la exposición repetida de
    especies distintas de los roedores.

         Un estudio sobre la warfarina en ratas ha indicado efectos sobre
    el desarrollo.  Por lo demás, no hay pruebas convincentes de que los
    anticoagulantes sean teratogénicos en animales de experimentación.

         No hay prueba que sugiera que los rodenticidas anticoagulantes
    son mutagénicos, pero se dispone de datos insuficientes sobre los
    compuestos individuales para demostrar la inexistencia de
    mutagenicidad.  La sobrecarga, el sexo y la alimentación son
    importantes factores modificadores de la toxicidad de los
    anticoagulantes en los roedores.

         Se han dado casos de envenenamiento de animales domésticos tras
    la ingestión de cebos anticoagulantes.  Las muertes y los síndromes
    clínicos graves se deben por lo general a anticoagulantes de segunda
    generación.  La diferencia principal entre la warfarina y los demás
    anticoagulantes (tanto las indandonas como las hidroxicumarinas de
    segunda generación) es que éstos tienen mayor tiempo de retención en
    el organismo y por consiguiente un efecto más prolongado que la
    warfarina.  Por ello, en los casos de envenenamiento, el tratamiento
    antídoto con vitamina K1 debe proseguir durante un periodo más
    largo.

    7.  Efectos en el ser humano

         Se han notificado muchos casos de envenenamiento (tanto
    intencionados como no intencionados).  También se han producido unos
    pocos casos de intoxicación por exposición ocupacional a los
    anticoagulantes.  Los síntomas de intoxicación aguda por rodenticidas
    anticoagulantes van desde una mayor tendencia a la hemorragia en el
    envenenamiento leve o moderado a una hemorragia masiva en casos más
    graves.  Los signos de envenenamiento aparecen con un retraso de uno a
    varios días después de la absorción.

         La warfarina va asociada en el ser humano a la producción de
    malformaciones del desarrollo cuando se toma como agente terapéutico
    durante el embarazo.  No se han notificado casos de defectos de
    desarrollo tras el uso de anticoagulantes como rodenticidas.

         La concentración de protrombina plasmática orienta sobre la
    gravedad de la intoxicación.  Es una indicación más sensible que
    pruebas generales como el tiempo de protrombina.  En la exposición
    ocupacional repetida, la medición directa de cantidades ínfimas de
    descarboxiprotrombina en circulación o de 2,3-epóxido de vitamina K en
    circulación pueden constituir una evaluación más sensible.

         El tratamiento del envenenamiento con anticoagulantes se gradúa
    de acuerdo con la gravedad de la intoxicación.  El tratamiento
    farmacológico específico consiste en la administración parenteral de
    vitamina K1 con administración simultánea, en los casos graves, de
    componentes sanguíneos.  La medición del tiempo de protrombina ayuda a
    determinar la eficacia y la duración de tratamiento necesaria.

    8.  Efectos en otros organismos en el laboratorio y sobre el terreno

         Los posibles efectos de los rodenticidas en organismos no
    destinatarios pueden dividirse en dos categorías: primarios
    (envenenamiento directo por consumo de cebo) y secundario (por consumo
    de roedores envenenados).

         En la forma del producto técnico, los anticoagulantes son muy
    tóxicos para los peces.  Como formulaciones para cebo es improbable
    que planteen riesgos en razón de su baja solubilidad en agua.  Por
    esta razón, a menos que se utilicen de forma indebida, no están al
    alcance de los peces.

         La susceptibilidad de las aves a los rodenticidas anticoagulantes
    es variable.  Es difícil evaluar los riesgos para las aves que entraña
    el consumo directo porque la mayoría de los estudios publicados
    consisten en ensayos de toxicidad en condiciones de laboratorio.  El
    atractivo del cebo de grano integral para las aves pequeñas aumenta el
    riesgo en las condiciones de campo.

         Los estudios en laboratorio de la toxicidad secundaria con la
    fauna silvestre han mostrado que los predadores cautivos pueden
    intoxicarse mediante alimentación sin otra elección con presas que se
    han envenenado con anticoagulante o a las que se ha administrado este
    producto.  Se tiene noticias de algunas muertes de predadores en su
    medio natural.

    9.  Evaluación y conclusión

         Los rodenticidas anticoagulantes perturban los mecanismos
    normales de coagulación de la sangre, determinando una mayor tendencia
    a la hemorragia y, por último, una hemorragia abundante.

         La exposición no intencionada de la población general a los
    rodenticidas anticoagulantes es improbable.

         El contacto ocupacional es una fuente potencial de exposición
    significativa.  Puede tener lugar durante la elaboración y
    formulación, así como durante la preparación y aplicación del cebo.

         Los compuestos de rodenticida anticoagulante se absorben
    fácilmente por el tracto intestinal, y por la piel y el sistema
    respiratorio.  El hígado es el órgano principal de acumulación y
    almacenamiento.  La concentración de protrombina plasmática es una
    buena orientación de la gravedad de la intoxicación aguda y de la
    eficacia y la duración necesaria de la terapia.

         El antídoto específico es la vitamina K1.

         La principal diferencia entre los rodenticidas anticoagulantes de
    la primera generación y los de la segunda es que éstos últimos tienen
    una mayor retención en el organismo y por ello suelen dar lugar a un
    periodo de hemorragia más prolongado.

         La mayoría de los anticoagulantes son estables en condiciones de
    uso normal.  Su baja solubilidad en agua y baja concentración en los
    cebos hace improbable que sean una fuente de contaminación del agua. 
    Parece que se asocian rápidamente a las partículas del suelo, con
    desorción muy lenta y nula propiedad de lixiviación.

         Los organismos no destinatarios pueden correr al riesgo de
    consumir directamente cebos (riesgo primario) y de ingerir roedores
    envenenados (riesgo secundario).
    


    See Also:
       Toxicological Abbreviations