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
venture of the United Nations Environment Programme, the International
Labour Organisation, and the World Health Organization. The main
objective of the IPCS is to carry out and disseminate evaluations of
the effects of chemicals on human health and the quality of the
environment. Supporting activities include the development of
epidemiological, experimental laboratory, and risk-assessment methods
that could produce internationally comparable results, and the
development of manpower in the field of toxicology. Other activities
carried out by the IPCS include the development of know-how for coping
with chemical accidents, coordination of laboratory testing and
epidemiological studies, and promotion of research on the mechanisms
of the biological action of chemicals.
WHO Library Cataloguing in Publication Data
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
The World Health Organization welcomes requests for permission to
reproduce or translate its publications, in part or in full.
Applications and enquiries should be addressed to the Office of
Publications, World Health Organization, Geneva, Switzerland, which
will be glad to provide the latest information on any changes made to
the text, plans for new editions, and reprints and translations
already available.
(c) World Health Organization 1995
Publications of the World Health Organization enjoy copyright
protection in accordance with the provisions of Protocol 2 of the
Universal Copyright Convention. All rights reserved. The designations
employed and the presentation of the material in this publication do
not imply the expression of any opinion whatsoever on the part of the
Secretariat of the World Health Organization concerning the legal
status of any country, territory, city or area or of its authorities,
or concerning the delimitation of its frontiers or boundaries. The
mention of specific companies or of certain manufacturers' products
does not imply that they are endorsed or recommended by the World
Health Organization in preference to others of a similar nature that
are not mentioned. Errors and omissions excepted, the names of
proprietary products are distinguished by initial capital letters.
CONTENTS
ENVIRONMENTAL HEALTH CRITERIA FOR 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
Every effort has been made to present information in the criteria
monographs as accurately as possible without unduly delaying their
publication. In the interest of all users of the Environmental Health
Criteria monographs, readers are requested to communicate any errors
that may have occurred to the Director of the International Programme
on Chemical Safety, World Health Organization, Geneva, Switzerland, in
order that they may be included in corrigenda.
* * *
A detailed data profile and a legal file can be obtained from the
International Register of Potentially Toxic Chemicals, Case postale
356, 1219 Châtelaine, Geneva, Switzerland (Telephone No. 9799111).
* * *
This publication was made possible by grant number 5 U01 ES02617-15
from the National Institute of Environmental Health Sciences, National
Institutes of Health, USA, and by financial support from the European
Commission.
Environmental Health Criteria
PREAMBLE
Objectives
In 1973 the WHO Environmental Health Criteria Programme was
initiated with the following objectives:
(i) to assess information on the relationship between exposure to
environmental pollutants and human health, and to provide
guidelines for setting exposure limits;
(ii) to identify new or potential pollutants;
(iii) to identify gaps in knowledge concerning the health effects of
pollutants;
(iv) to promote the harmonization of toxicological and
epidemiological methods in order to have internationally
comparable results.
The first Environmental Health Criteria (EHC) monograph, on
mercury, was published in 1976 and since that time an everincreasing
number of assessments of chemicals and of physical effects have been
produced. In addition, many EHC monographs have been devoted to
evaluating toxicological methodology, e.g., for genetic, neurotoxic,
teratogenic and nephrotoxic effects. Other publications have been
concerned with epidemiological guidelines, evaluation of short-term
tests for carcinogens, biomarkers, effects on the elderly and so
forth.
Since its inauguration the EHC Programme has widened its scope,
and the importance of environmental effects, in addition to health
effects, has been increasingly emphasized in the total evaluation of
chemicals.
The original impetus for the Programme came from World Health
Assembly resolutions and the recommendations of the 1972 UN Conference
on the Human Environment. Subsequently the work became an integral
part of the International Programme on Chemical Safety (IPCS), a
cooperative programme of UNEP, ILO and WHO. In this manner, with the
strong support of the new partners, the importance of occupational
health and environmental effects was fully recognized. The EHC
monographs have become widely established, used and recognized
throughout the world.
The recommendations of the 1992 UN Conference on Environment and
Development and the subsequent establishment of the Intergovernmental
Forum on Chemical Safety with the priorities for action in the six
programme areas of Chapter 19, Agenda 21, all lend further weight to
the need for EHC assessments of the risks of chemicals.
Scope
The criteria monographs are intended to provide critical reviews
on the effect on human health and the environment of chemicals and of
combinations of chemicals and physical and biological agents. As
such, they include and review studies that are of direct relevance for
the evaluation. However, they do not describe every study carried
out. Worldwide data are used and are quoted from original studies,
not from abstracts or reviews. Both published and unpublished reports
are considered and it is incumbent on the authors to assess all the
articles cited in the references. Preference is always given to
published data. Unpublished data are only used when relevant
published data are absent or when they are pivotal to the risk
assessment. A detailed policy statement is available that describes
the procedures used for unpublished proprietary data so that this
information can be used in the evaluation without compromising its
confidential nature (WHO (1990) Revised Guidelines for the Preparation
of Environmental Health Criteria Monographs. PCS/90.69, Geneva, World
Health Organization).
In the evaluation of human health risks, sound human data,
whenever available, are preferred to animal data. Animal and
in vitro studies provide support and are used mainly to supply
evidence missing from human studies. It is mandatory that research on
human subjects is conducted in full accord with ethical principles,
including the provisions of the Helsinki Declaration.
The EHC monographs are intended to assist national and
international authorities in making risk assessments and subsequent
risk management decisions. They represent a thorough evaluation of
risks and are not, in any sense, recommendations for regulation or
standard setting. These latter are the exclusive purview of national
and regional governments.
Content
The layout of EHC monographs for chemicals is outlined below.
* Summary - a review of the salient facts and the risk evaluation
of the chemical
* Identity - physical and chemical properties, analytical methods
* Sources of exposure
* Environmental transport, distribution and transformation
* Environmental levels and human exposure
* Kinetics and metabolism in laboratory animals and humans
* Effects on laboratory mammals and in vitro test systems
* Effects on humans
* Effects on other organisms in the laboratory and field
* Evaluation of human health risks and effects on the environment
* Conclusions and recommendations for protection of human health
and the environment
* Further research
* Previous evaluations by international bodies, e.g., IARC, JECFA,
JMPR
Selection of chemicals
Since the inception of the EHC Programme, the IPCS has organized
meetings of scientists to establish lists of priority chemicals for
subsequent evaluation. Such meetings have been held in: Ispra, Italy,
1980; Oxford, United Kingdom, 1984; Berlin, Germany, 1987; and North
Carolina, USA, 1995. The selection of chemicals has been based on the
following criteria: the existence of scientific evidence that the
substance presents a hazard to human health and/or the environment;
the possible use, persistence, accumulation or degradation of the
substance shows that there may be significant human or environmental
exposure; the size and nature of populations at risk (both human and
other species) and risks for environment; international concern, i.e.
the substance is of major interest to several countries; adequate data
on the hazards are available.
If an EHC monograph is proposed for a chemical not on the
priority list, the IPCS Secretariat consults with the Cooperating
Organizations and all the Participating Institutions before embarking
on the preparation of the monograph.
Procedures
The order of procedures that result in the publication of an EHC
monograph is shown in the flow chart. A designated staff member of
IPCS, responsible for the scientific quality of the document, serves
as Responsible Officer (RO). The IPCS Editor is responsible for
layout and language. The first draft, prepared by consultants or,
more usually, staff from an IPCS Participating Institution, is based
initially on data provided from the International Register of
Potentially Toxic Chemicals, and reference data bases such as Medline
and Toxline.
The draft document, when received by the RO, may require an
initial review by a small panel of experts to determine its scientific
quality and objectivity. Once the RO finds the document acceptable as
a first draft, it is distributed, in its unedited form, to well over
150 EHC contact points throughout the world who are asked to comment
on its completeness and accuracy and, where necessary, provide
additional material. The contact points, usually designated by
governments, may be Participating Institutions, IPCS Focal Points, or
individual scientists known for their particular expertise. Generally
some four months are allowed before the comments are considered by the
RO and author(s). A second draft incorporating comments received and
approved by the Director, IPCS, is then distributed to Task Group
members, who carry out the peer review, at least six weeks before
their meeting.
The Task Group members serve as individual scientists, not as
representatives of any organization, government or industry. Their
function is to evaluate the accuracy, significance and relevance of
the information in the document and to assess the health and
environmental risks from exposure to the chemical. A summary and
recommendations for further research and improved safety aspects are
also required. The composition of the Task Group is dictated by the
range of expertise required for the subject of the meeting and by the
need for a balanced geographical distribution.
The three cooperating organizations of the IPCS recognize the
important role played by nongovernmental organizations.
Representatives from relevant national and international associations
may be invited to join the Task Group as observers. While observers
may provide a valuable contribution to the process, they can only
speak at the invitation of the Chairperson.
Observers do not participate in the final evaluation of the chemical;
this is the sole responsibility of the Task Group members. When the
Task Group considers it to be appropriate, it may meet in camera.
All individuals who as authors, consultants or advisers
participate in the preparation of the EHC monograph must, in addition
to serving in their personal capacity as scientists, inform the RO if
at any time a conflict of interest, whether actual or potential, could
be perceived in their work. They are required to sign a conflict of
interest statement. Such a procedure ensures the transparency and
probity of the process.
When the Task Group has completed its review and the RO is
satisfied as to the scientific correctness and completeness of the
document, it then goes for language editing, reference checking, and
preparation of camera-ready copy. After approval by the Director,
IPCS, the monograph is submitted to the WHO Office of Publications for
printing. At this time a copy of the final draft is sent to the
Chairperson and Rapporteur of the Task Group to check for any errors.
It is accepted that the following criteria should initiate the
updating of an EHC monograph: new data are available that would
substantially change the evaluation; there is public concern for
health or environmental effects of the agent because of greater
exposure; an appreciable time period has elapsed since the last
evaluation.
All Participating Institutions are informed, through the EHC
progress report, of the authors and institutions proposed for the
drafting of the documents. A comprehensive file of all comments
received on drafts of each EHC monograph is maintained and is
available on request. The Chairpersons of Task Groups are briefed
before each meeting on their role and responsibility in ensuring that
these rules are followed.
WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR 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:
* indandiones:
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
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
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
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)
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
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
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
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
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
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
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
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
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
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.
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: